DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group II (current claims 10-11, 14, 17-19, 21 & 23-26) and the amino acid sequence of SEQ ID NO: 4 in the reply filed on 7/7/2026 is acknowledged.
Claims 1-4, 6, 8 & 28-30 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 7/7/2026.
The restriction is made FINAL.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Status of Claims
Claims 10-11, 14, 17-19, 21 & 23-26 are under examination on the merits.
Claims 1-4, 6, 8 & 28-30 are withdrawn.
Specification
The abstract of the disclosure is objected to because of undue length. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
The brief description of figure 3 (panel C & D) describes red dots or red highlighting for variant sites (page 13, paragraph 1). The brief description of figure 25 describes a red box (page 19, paragraph 3). However, the figures are provided in black and white. The brief descriptions should be amended to remove reference to color aspects of black and white figures.
Nucleotide and/or Amino Acid Sequence Disclosures
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.831(b). However, this application fails to comply with the requirements of 37 CFR 1.831 through 1.834.
Sequence identifiers are missing from the sequences presented on page 39 (paragraphs 3 & 5), page 40 (paragraphs 4 & 5), page 41 (paragraph 1), page 42 (paragraph 5), and page 43 (paragraphs 3 & 4). See 37 CFR 1.831. In addition, figures 9, 10 and 25 present amino acid sequences longer than 4 residues. These sequences need to be in the sequence listing file and identifier numbers should be provided either in the figures themselves or in the brief description of the figures (page 14, paragraph 5-6; page 19, paragraph 3; and page 19, paragraph 3 respectively). Figures 13 & 15-19 present multiple nucleic acid sequences longer than 10 bases. These sequences need to be in the Sequence listing file and identifier numbers should be provided either in the figures themselves or in the brief description of the figures (page 15, paragraph 3-page 16 paragraph 1; page 16, paragraph 3; page 16, paragraph 4; page 16, paragraph 5; page 17, paragraph 1; and page 17, paragraph 2). Figure 21 presents both nucleic acid sequences longer than 10 bases and amino acid sequences longer than 4 residues. Each sequence needs to be in the Sequence listing file and identifier numbers should be provided either in the figure or in the brief description of the figure (page 17, paragraph 4).
Full compliance with the sequence rules is required in response to this Office action. A complete response to this Office action must include both compliance with the sequence rules and a response to the issues set forth herein. Failure to fully comply with both of these requirements in the time period set forth in this Office action will be held to be non-responsive.
Claim Objections
Claims 10, 14, 17, 18, 21 & 24 are objected to because of the following informalities:
Claim 10 (line 10): “thereof” should be deleted.
Claim 14 (line 1): the abbreviation “GGL” should be written out in full in its first use.
Claim 14 (line 2): “N-terminal” should be replaced with --the N-terminal end--.
Claim 17 (line 6): “that” should be deleted.
Claim 17 (line 7): “has” should be replaced with --having--.
Claim 18 (line 1): the abbreviation “GGL” should be written out in full in its first use.
Claim 18 (line 2): “N-terminal” should be replaced with --the N-terminal end--.
Claim 21 (line 2): the abbreviation “GGL” should be written out in full in its first use.
Claim 24 (line 9): “an” should read --a--.
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 10, 11, 14, 18-19 & 21 are rejected under 35 U.S.C. 101 because the claimed invention is directed to natural phenomena without significantly more. Claims 10, 11, 14 & 18 are drawn to a method for producing a saline-alkali-tolerant plant comprising reducing the expression level of alleles encoding a protein having at least 40% identity to SEQ ID NO: 15. These methods explicitly encompass methods wherein reducing the expression of the alleles is accomplished by natural mutation (claim 11, line 3). Claim(s) 19 & 21 recite a plant or plant material wherein all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15 are mutated.
This judicial exception is not integrated into a practical application because the claims drawn to the method of making the plants encompass natural mutation, which is a process that occurs without human intervention. The claims drawn to the plants do not require any additional application. Plants comprising mutations which lead to reduced activity of the protein encoded by the gene exist naturally.
Xie et al CN115894644A (published 4/4/2023, after the priority date of the instant application. Machine translation of the Description uploaded) describes eight natural variation sites in the coding region of a gene encoding a protein comprising a sequence with 100% identity to instant SEQ ID NO: 4 (paragraph [0129], figure 3 & 4). See alignment below of Xie SEQ ID NO: 1 and instant SEQ ID NO: 4. Xie teaches that the C-terminus of the GC1 protein is a factor that positively regulates the sorghum husk coating trait (paragraph [0188]), and so sorghum comprising natural variations that truncate the C-terminus read on mutations that reduce an activity of the alleles. Xie teaches haplotypes that comprise both a truncation and a serine-glycine substitution in a γ subunit (see eg figure 3 haplotype gc1-a), so an allele comprising a mutated GGL domain is also known to exist naturally.
BMU37403
ID BMU37403 standard; protein; 275 AA.
XX
AC BMU37403;
XX
DT 08-JUN-2023 (first entry)
XX
DE Sorghum bicolor glume coverage 1 (GC1) protein, SEQ:2.
XX
KW G protein gamma subunit; GC1 protein; Glume coverage 1 protein; Plant;
KW crop improvement; gene regulation; plant breeding.
XX
OS Sorghum bicolor; Wheatland.
XX
FH Key Location/Qualifiers
FT Region 78..275
FT /note= "Specifically claimed in claim 1"
XX
CC PN CN115894644-A.
XX
CC PD 04-APR-2023.
XX
CC PF 30-SEP-2021; 2021CN-11158546.
XX
PR 30-SEP-2021; 2021CN-11158546.
XX
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
XX
CC PI Li C, Tang S, Wu Y, Xie P, Xie Q;
XX
DR WPI; 2023-42874Q/039.
DR N-PSDB; BMU37402, BMU37404.
XX
CC PT Use of protein, substance that regulates expression of protein-coding
CC PT gene or activity of protein in regulating, preferably inhibiting or
CC PT improving encrustation traits of plant seeds and cultivating plant,
CC PT preferably Sorghum bicolor.
XX
CC PS Claim 1; SEQ ID NO 2; 46pp; Chinese.
XX
CC The present invention relates to a novel application of an atypical G
CC protein gamma subunit (glume coverage 1 (GC1)) or a substance in
CC regulating the expression of a protein-coding gene or a substance in
CC regulating the activity or content of the protein. The invention further
CC discloses a method for cultivating naked plants, which involves
CC increasing or promoting the activity or content of the protein or/and the
CC expression level of the gene encoding the protein in a plant of interest
CC or knocking out the C-terminus of the protein, thereby obtaining naked
CC plant. The novel application of the invention is for regulating the
CC expression of a gene encoding the protein or a substance for regulating
CC the activity or content of the protein in regulating the encrustation
CC trait of plant seeds and regulating the expression of the protein coding
CC gene or the substance for regulating the activity or the content of the
CC protein in inhibiting the encrustation trait of plant seeds in plant
CC breeding, which has wide application prospect in guiding the breeding and
CC molecular improvement of naked grain varieties of crops.
XX
SQ Sequence 275 AA;
Query Match 100.0%; Score 1174; Length 275;
Best Local Similarity 100.0%;
Matches 198; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 78 MAAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFV 137
Qy 61 GSNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQ 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 138 GSNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQ 197
Qy 121 LKRPAAPSCSCAPRLRKLCCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCACPRCCS 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 198 LKRPAAPSCSCAPRLRKLCCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCACPRCCS 257
Qy 181 CPRCSAACCCAPRCCLCL 198
||||||||||||||||||
Db 258 CPRCSAACCCAPRCCLCL 275
The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the plants do not require any features other than the mutation leading to reduced activity in the alleles. The methods do not require any additional step beyond reducing the expression level, and the step of reducing the expression level can be fulfilled by natural mutation (as explicitly written in claim 11). The claims recite that the growth of the plant under saline-alkali conditions is better than that of a wild-type plant; however, because the method does not require growing in saline-alkali conditions, this wherein clause reads as an intended result. See MPEP 2111.04.
Thus, the claims do not require anything beyond natural mutation leading to plants that are known to exist naturally.
Claim 17 is rejected under 35 U.S.C. 101 because the claimed invention is directed to a mental process without significantly more. The claim(s) recite(s) a method which comprises identifying an offspring plant obtained by a method of selfing or crossing a parent plant.
This judicial exception is not integrated into a practical application because claim 10, which claim 17 depends on, merely requires reducing the expression level of an allele, and the method of claim 10 encompasses natural mutation (see rejection above). Therefore, claim 17 requires only the step of “identifying” an offspring plant of a plant that could be generated by a natural process.
The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claim does not require an active step of selfing or crossing the parent plant or any tangible steps in order to identify the offspring plant. The only step required beyond that of a natural phenomenon (see above) is “identifying” an offspring plant, and identifying a plant is a mental process.
Claim Rejections - 35 USC § 112
Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 11, 14, 18, 21 & 25 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. All dependent claims are included in the rejections below.
Claim 14 (line 2) and claim 18 (line 2) recite “the first exon portion at N-terminal”. It is unclear how to interpret this limitation. Is the “portion” the exon itself, and so the first exon at the N-terminal end of the encoded protein should be knocked out or mutated? Or is the portion part of an exon at the N-terminal? Or is this exon portion required to be part of the GGL domain or GGL-like domain (lines 1-2)? Because the limitation can have multiple distinct interpretations, the claims are indefinite.
Claim 11 (line 3) recites reducing the expression level of all alleles by “chemical induction” and “radiation induction”. Where applicant acts as his or her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term. Process Control Corp. v. HydReclaim Corp., 190 F.3d 1350, 1357, 52 USPQ2d 1029, 1033 (Fed. Cir. 1999). The term “induction” in claim 11 is used by the claim to mean “mutation” or perhaps “reduction”, while the accepted meaning is “activation of expression”. The term is indefinite because the specification does not clearly redefine the term.
Claim 18 (lines 5, 7, 9, 11, 13 & 15), claim 21 (lines 6, 8, 10, 12, 14 & 16), and claim 25 (lines 5, 7, 9, 11, 13 & 15) recite “similar or identical function” to provided sequences. It is unclear if any amino acid sequence with any function that is similar is encompassed or if the claims are limited to amino acid sequence which comprise functions similar to all functions of the provided sequence. It is unclear how different a function can be and still be considered similar. Would an amino acid sequence having the same isoelectric point be encompassed as having a similar function, because it would have the same function in cellular pH? Because the interpretation of a “similar” function is indefinite, the claims are indefinite.
Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 10, 11, 14, 17-19, 21 & 23-26 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
A. Claims 10, 11, 14, 17-19, 21 & 23-26 require a plant comprising knocked out or mutated alleles encoding a protein comprising an amino acid sequence having at least 40% identity or 60% similarity to SEQ ID NO: 15. Claims 10, 11, 14, 17-18 & 24 require reduced expression or non-expression of the alleles, and claims 17, 19 & 21 require that the protein encoded by the alleles has reduced activity or no activity. Claims 14, 18 & 21 further require that a GGL domain or GGL-like domain, or first exon portion at the N-terminal of the alleles is knocked out or mutated. Claims 18 & 21 further require that all alleles of a gene encoding an amino acid sequence having at least 80% identity and similar or identical function to SEQ ID NO: 4 is knocked out or mutated.
The claims broadly require plants comprising alleles with 40% identity to SEQ ID NO: 15 or 80% identity and similar function to SEQ ID NO: 4. Because the claims require that the alleles be knocked out or mutated, the claims require plants comprising endogenous genes over the genus of sequences.
Alleles encoding proteins comprising an amino acid sequence with 40% identity to the 67 amino acid long SEQ ID NO: 15 encompass those encoding proteins comprising sequences with 40 amino acid substitutions relative to SEQ ID NO: 15. Compared to the full-length protein of SEQ ID NO: 4, which comprises 198 amino acids that are not required to be conserved outside of the 67 amino acids of SEQ ID NO: 5, these proteins encompass sequences with just 26 identical amino acids, or 13% identity (26/198) compared to the full length protein of SEQ ID NO: 4. Alleles encoding an amino acid sequence having at least 80% identity to the 198 amino acid-long SEQ ID NO: 4 encompass those encoding proteins comprising sequences with 39 amino acid substitutions relative to SEQ ID NO: 4.
SEQ ID NO: 15 is an amino acid sequence comprising a GGL or GGL-like domain from the sorghum AT1 gene (Table 5). The specification describes GGL domains in homologous genes from 6 species other than sorghum (Table 5, SEQ ID NOs: 86-96), which range in sequence identity from 94% to 43% and range in similarity from 95% to 61% (table 3). Of these, the sequence with the least similarity to instant SEQ ID NO: 15 is SEQ ID NO: 96. See alignment below. Applicant describes truncated variants of homologs of AT1 in these different species (figures 4-6, 9-10, 12). Applicant describes only a single homolog of instant SEQ ID NO: 4 with at least 80% identity, the maize homolog (table 2).
Score
Expect
Method
Identities
Positives
Gaps
55.1 bits(131)
2e-17
Compositional matrix adjust.
28/61(46%)
39/61(63%)
4/61(6%)
Query 5 VDALHREISFLEGE-ISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQQFL 63
V L REI+FLE E + S+EG+ ASRCCKE+ ++V +N DP L P + +S +F
Sbjct 5 VHMLEREITFLEQEELKSVEGLQPASRCCKEIADYVMANADPLL---PSTKKNRRSCRFW 61
Query 64 K 64
K
Sbjct 62 K 6
Proteins comprising as little as 40% identity to SEQ ID NO: 15 are known in the art. Fu et al US 2014/0020135 A1 (published 1/16/2014, hereafter Fu) describes a sorghum homolog (Fu SEQ ID NO: 16) of a rice DEP1 protein comprising a G-protein gamma like domain in the N-terminal portion (paragraph [0144]). This gene is distinct from the gene encoding the rice GS3 protein described in the instant invention (Fu paragraph [0145]; see instant specification page 46, paragraph 5). However, the DEP1 sorghum homolog of Fu comprises a sequence with >40% identity to instant SEQ ID NO: 15 and is therefore encompassed by the instant claims, although no role in saline-alkali tolerance of the DEP1 protein has been described (see alignments below). Although a maize sequence with 80% sequence identity is described in the art (Madappa et al US 2009/0165165 A1, published 6/25/2009, see third alignment below), endogenous genes with alleles encoding protein sequences across the full scope of the claimed sequences are not described in the art.
Sequence 16, US/13982229
Publication No. US20140020135A1
GENERAL INFORMATION
APPLICANT: Fu, Xiangdong
APPLICANT: Wu, Kun
APPLICANT: Qian, Qian
APPLICANT: Zhang, Chengwei
APPLICANT: Liu, Xueying
APPLICANT: Wang, Shuansuo
TITLE OF INVENTION: NOVEL USE OF A DENSE AND ERECT PANICLE 1 GENE IN IMPROVING
TITLE OF INVENTION: NITROGEN UTILIZATION EFFICIENCY
FILE REFERENCE: 9207-68TS
CURRENT APPLICATION NUMBER: US/13/982,229
CURRENT FILING DATE: 2013-07-26
PRIOR APPLICATION NUMBER: PCT/US2012/022930
PRIOR FILING DATE: 2012-01-27
PRIOR APPLICATION NUMBER: CN 201110029759.9
PRIOR FILING DATE: 2011-01-27
NUMBER OF SEQ ID NOS: 19
SEQ ID NO 16
LENGTH: 400
TYPE: PRT
ORGANISM: Sorghum bicolor
Query Match 41.2%; Score 143; Length 400;
Best Local Similarity 50.0%;
Matches 31; Conservative 10; Mismatches 19; Indels 2; Gaps 1;
Qy 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
||| : |:||| ||: |: |:||| || ||||:||||: || | | :| :| :
Db 33 LQLELQILNREIDFLKDELQSLEGVPPVSRSCKEVNEFVGTKQDPLLPI--KKKTHRSCR 90
Qy 61 QF 62
|
Db 91 LF 92
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
58.9 bits(141)
1e-16
Composition-based stats.
31/62(50%)
41/62(66%)
2/62(3%)
Query 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
LQL + L+REI FL+ E+ S+EGV SR CKEV+EFVG+ DP L I+ K +H +
Sbjct 33 LQLELQILNREIDFLKDELQSLEGVPPVSRSCKEVNEFVGTKQDPLLPIK--KKTHRSCR 90
Query 61 QF 62
F
Sbjct 91 LF 92
Sequence 18, US/12316385A
GENERAL INFORMATION
APPLICANT: Monsanto
TITLE OF INVENTION: Transgenic Plants With Enhanced Agronomic Traits
FILE REFERENCE: 38-21(55579)B
CURRENT APPLICATION NUMBER: US/12/316,385A
CURRENT FILING DATE: 2009-01-08
NUMBER OF SEQ ID NOS: 1358
SEQ ID NO 18
LENGTH: 198
TYPE: PRT
ORGANISM: Zea mays
Query Match 80.7%; Score 948; Length 198;
Best Local Similarity 83.5%;
Matches 172; Conservative 1; Mismatches 13; Indels 20; Gaps 5;
Qy 2 AAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVG 61
|||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||
Db 4 AAAPRPKSPPASPDPCGRHRLQLAVDALHREIGFLEGEISSIEGVHAASRCCKEVDEFVG 63
Qy 62 SNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQL 121
||||||||| |:|||||||||||||| |||||||||||| | | |||| |||
Db 64 RNPDPFLTIQQERGSHDQSQQFLKKFRGKSCLSYYLSWIC--------GGGWWCPPPLQL 115
Qy 122 KRPAAPSCSCAPRLRKL-------CCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCA 174
||| |||||||||| || || ||||| ||||| | ||||||||||||||||||
Db 116 KRPPAPSCSCAPRLGKLCSSTASSCCSCCCCRFRVVYA--AAGCGCCAPCPRCSCDCTCA 173
Qy 175 CPRCCS--CPRCSAACCCAPRCCLCL 198
|||||| || |||| ||||||||||
Db 174 CPRCCSCACPMCSAA-CCAPRCCLCL 198
G-protein gamma subunits in plants are highly diverse. In Arabidopsis, G gamma is solely responsible for providing functional selectivity to the G protein heterotrimer (Trusov et al (2012) BMC Research Notes. 5: 608 (published 10/31/2012, hereafter Trusov) page 1, right column, paragraph 1). In plants, C-terminal variability is a unique characteristic of G gamma subunits (Trusov page 2, right column, paragraph 3). Type C G-protein gamma subunits can have long cysteine rich C-terminal domains (Trusov figure 1). These elongated G gamma tails specify G-protein signaling and provide diverse functional selectivity, and removing or adding repetitive elements found in these tails can affect regulation activity, changing from negative to positive in the case of GS3 (Sun et al (2025) The Plant Cell. 37: koaf052 (available 3/15/2025, after the priority date of the instant application); page 11, left column, paragraph 3-4). Known proteins comprising GGL domains in plants therefore have different activities.
The number of G gamma subunits described in plant genomes can differ, from 3 described in Arabidopsis and Setaria italica to 10 described in Glycine max (Tiwari et al (2022) Planta 255: 88 (published 3/19/2022, hereafter Tiwari); table 1). Certain plant lineages comprise extra-large G-proteins and atypical G gamma subunits (Tiwari page 2, left column, paragraph 2). Furthermore, the number of G beta units differs across plants (Tiwari table 1), and the specificity of G-protein signaling may be regulated through tissue- and condition-specific co-expression patterns and interaction specificity of multiple G-protein subunits (Tiwari page 3, left column), suggesting that proteins comprising GGL domains may vary across plants in correspondence with co-expressed subunits.
The instant claims are drawn to the full genus of all plants comprising amino acids with 40% identity to the GGL domain of SEQ ID NO: 15, with no limitation on the C-terminal portion of the protein.
Thus, the claims are drawn to an extremely broad genus of all plants comprising any protein, comprising a sequence with as little as 40% identity to instant SEQ ID NO: 15. The claims do not even require a GGL or GGL-like domain, because the claims which recite this limitation do so in alternative with “the first exon portion at N-terminal”.
Applicant has provided just 14 examples of full-length amino acid sequences within the genus across 7 species (table 2, table 4). Claims 17-19 & 21 require a reduced activity or an activity similar to instant SEQ ID NO: 4; however, Applicant has described activity only of SEQ ID NO: 4 and some of the provided homologs. Proteins with as little as 40% identity to SEQ ID NO: 15 conceivably exist and may or may not have activities not found in the protein of instant SEQ ID NO: 4.
Given the diversity of genes encompassed within the claimed genus and given the few examples provided by Applicant and the description of only homologs of one GGL-domain comprising protein, Applicant has not described or provided examples of plants comprising endogenous genes over the full genus of the claims such that one of ordinary skill in the art would conclude that Applicant was in possession of the plants over the full scope of the claims.
Since the disclosure fails to describe the common attributes that identify members of the genus, and because the genus is highly variant, the domain sequences of SEQ ID NOs: 15 & 86-96 and the protein sequences of SEQ ID NOs: 4 & 74-85 are insufficient to describe the claimed genus.
Hence, Applicant has not, in fact, described plants comprising endogenous alleles encoding proteins with at least 40% identity to SEQ ID NO: 15 or 80% identity to SEQ ID NO: 4 over the full scope of the claims, and the specification fails to provide an adequate written description of the claimed invention. Therefore, given the lack of written description in the specification with regard to the structural and functional characteristics of the claimed compositions, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
B. Claims 10, 11, 14, 17 & 18 are drawn to a method wherein the expression level of all alleles encoding a protein comprising a sequence having at least 40% identity to SEQ ID NO: 15 is reduced. Although claims 14, 17 & 18 further require that the alleles are knocked out or mutated, the claims do not require that the mutation in the alleles is the cause of the reduced expression. Claim 11 explicitly recites that “chemical induction”, “radiation induction”, or “a substance capable of inhibiting the expression of a target gene” may be used to reduce expression level of the alleles.
The instant specification does not define substance. Thus, the claims broadly require any “substance” capable of inhibiting expression of the alleles of the instant claims.
The instant specification describes SEQ ID NO: 15 as an amino acid sequence comprising a GGL or GGL-like domain from the sorghum AT1 gene (Table 5). The specification describes GGL domains in homologous genes from 6 species other than sorghum (Table 5, SEQ ID NOs: 86-96), which range in sequence identity from 94% to 43% and range in similarity from 95% to 61% (table 3).
The specification teaches a method of knocking down the expression of the gene or protein using RNA interferences (page 49, paragraphs 2-3). In this method, the pDS 1301 comprising forward and reverse copies of the GS3 gene introduced into a plant reads on a “substance” capable of inhibiting the expression of a target gene. The specification also teaches CRISPR/Cas9 editing to inhibit expression of the GS3 protein in rice and maize (page 52, paragraph 7-page 55, paragraph 4). Methods of using CRISPR/Cas systems to reduce expression of a gene without gene editing are known (Pan et al (2021) Current Opinion in Plant Biology. 60:101980 available 1/2/2021, hereafter Pan; page 1, right column, paragraph 2 & page 5, left column, paragraph 2-right column, paragraph 1), although they are not described in the instant application. No other “substances” for inhibiting the expression of the alleles are described in the instant specification.
Substances that reduce expression of alleles are known in the art. For example, Pan describes methods of CRISPR based interference for gene repression (page 5, left column, paragraph 2-right column, paragraph 1). Butler et al (2017) ACS Synth. Biol. (2018) 7 (1): 38–45 (published 10/26/2017) describes a method of targeted gene repression that uses chemical epigenetic modifiers capable of binding to a transcription factor and recruiting a corepressor complex to reversibly and specifically repress GFP expression in a cell line (page 39, right column, paragraph 3-page 41, left column, paragraph 4, figure 1, figure 2). Kanazawa et al (2010) The Plant Journal. 65(1): 156-168 (published 10/14/2010) describes gene silencing in plants by targeting double stranded RNA to a gene promoter (abstract).
Genes encoding proteins comprising sequences with 40% identity to SEQ ID NO: 15 or 80% identity to SEQ ID NO: 4 are also known in the art. However, even G-protein gamma subunits comprising sequences with 40% identity to SEQ ID NO: 15 encompasses a broad and diverse gene of proteins.
The number of G gamma subunits described in plant genomes can differ, from 3 described in Arabidopsis and Setaria italica to 10 described in Glycine max (Tiwari et al (2022) Planta 255: 88 (published 3/19/2022, hereafter Tiwari); table 1). Certain plant lineages comprise extra-large G-proteins and atypical G gamma subunits (Tiwari page 2, left column, paragraph 2). Furthermore, the number of G beta units differs across plants (Tiwari table 1), and the specificity of G-protein signaling may be regulated through tissue- and condition-specific co-expression patterns and interaction specificity of multiple G-protein subunits (Tiwari page 3, left column), suggesting that proteins comprising GGL domains may vary across plants in correspondence with co-expressed subunits.
The instant specification does not describe the regulatory elements associated with genes over the full scope of sequences claimed, expression of which is known to vary across genes and plant species. Because many “substances” capable of inhibiting the expression of a target gene rely on specificity to regulatory elements, and neither Applicant nor the prior art have described the regulatory elements across the full genes of claimed alleles, Applicant has not described a common structural feature of substances capable of inhibiting expression of all alleles over the full scope of the claims.
Thus, given the highly diverse genus of substances capable of inhibiting the expression of target genes to reduce expression of alleles with 40% identity to SEQ ID NO: 15, one of ordinary skill in the art would not conclude Applicant was in possession of the full genus of substances required by the claimed method and the method is not fully described by the instant disclosure.
Scope of Enablement
Claims 10-11, 14, 17-19 & 21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for methods for producing a saline-alkali-tolerant plant comprising reducing the expression level of the alleles encoding a protein of SEQ ID NO: 4, or for a plant comprising an amino acid sequence of SEQ ID NO: 4 with reduced or no activity resulting from a knocked out GGL or GGL-like domain and a saline-alkali-tolerant phenotype, does not reasonably provide enablement for methods for producing a saline-alkali-tolerant plant comprising reducing the expression level of alleles encoding proteins comprising amino acid sequences with as little as 40% identity to SEQ ID NO: 15 or plants with reduced or no activity that still have a function GGL domain. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Claims 10, 11, 14 & 17-18 recite a method comprising reducing expression in a plant of all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15, wherein growth of the plant under saline-alkali conditions is better than that of a wild-type plant.
The claims broadly require plants comprising alleles with 40% identity to SEQ ID NO: 15 with reduced expression of the alleles and better growth under saline-alkali conditions.
Alleles encoding proteins comprising an amino acid sequence with 40% identity to the 67 amino acid long SEQ ID NO: 15 encompass those encoding proteins comprising sequences with 40 amino acid substitutions relative to SEQ ID NO: 15. Compared to the full-length protein of SEQ ID NO: 4, which comprises 198 amino acids that are not required to be conserved outside of the 67 amino acids of SEQ ID NO: 5, these proteins encompass sequences with just 26 identical amino acids, or 13% identity (26/198) compared to the full length protein of SEQ ID NO: 4. Alleles encoding an amino acid sequence having at least 80% identity to the 198 amino acid-long SEQ ID NO: 4 encompass those encoding proteins comprising sequences with 39 amino acid substitutions relative to SEQ ID NO: 4.
SEQ ID NO: 15 is an amino acid sequence comprising a GGL or GGL-like domain from the sorghum AT1 gene (Table 5). The specification teaches GGL domains in homologous genes from 6 species other than sorghum (Table 5, SEQ ID NOs: 86-96), which range in sequence identity from 94% to 43% and range in similarity from 95% to 61% (table 3). Of these, the sequence with the least similarity to instant SEQ ID NO: 15 is SEQ ID NO: 96. See alignment below. Applicant teaches truncated variants of homologs of AT1 in these different species (figures 4-6, 9-10, 12). All the sequences comprise homologs to the AT1 protein in sorghum.
Score
Expect
Method
Identities
Positives
Gaps
55.1 bits(131)
2e-17
Compositional matrix adjust.
28/61(46%)
39/61(63%)
4/61(6%)
Query 5 VDALHREISFLEGE-ISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQQFL 63
V L REI+FLE E + S+EG+ ASRCCKE+ ++V +N DP L P + +S +F
Sbjct 5 VHMLEREITFLEQEELKSVEGLQPASRCCKEIADYVMANADPLL---PSTKKNRRSCRFW 61
Query 64 K 64
K
Sbjct 62 K 6
Sequences with at least 40% identity to instant SEQ ID NO: 15 are known in the art. Fu et al US 2014/0020135 A1 (published 1/16/2014, hereafter Fu) teaches a sorghum homolog (Fu SEQ ID NO: 16) of a rice DEP1 protein comprising a G-protein gamma like domain in the N-terminal portion (paragraph [0144]). This gene is distinct from the gene encoding the rice GS3 protein described in the instant invention (Fu paragraph [0145]; see instant specification page 46, paragraph 5). However, the DEP1 sorghum homolog of Fu comprises a sequence with >40% identity to instant SEQ ID NO: 15 and is therefore encompassed in the sequences recited by claim 19, although no role in saline-alkali tolerance of the DEP1 protein has been described (see alignments below). Although a maize sequence with 80% sequence identity is taught by Madappa et al US 2009/0165165 A1 (published 6/25/2009, see third alignment below), endogenous genes with alleles encoding protein sequences across the full scope of the claimed sequences are not taught by prior art references.
Sequence 16, US/13982229
Publication No. US20140020135A1
GENERAL INFORMATION
APPLICANT: Fu, Xiangdong
APPLICANT: Wu, Kun
APPLICANT: Qian, Qian
APPLICANT: Zhang, Chengwei
APPLICANT: Liu, Xueying
APPLICANT: Wang, Shuansuo
TITLE OF INVENTION: NOVEL USE OF A DENSE AND ERECT PANICLE 1 GENE IN IMPROVING
TITLE OF INVENTION: NITROGEN UTILIZATION EFFICIENCY
FILE REFERENCE: 9207-68TS
CURRENT APPLICATION NUMBER: US/13/982,229
CURRENT FILING DATE: 2013-07-26
PRIOR APPLICATION NUMBER: PCT/US2012/022930
PRIOR FILING DATE: 2012-01-27
PRIOR APPLICATION NUMBER: CN 201110029759.9
PRIOR FILING DATE: 2011-01-27
NUMBER OF SEQ ID NOS: 19
SEQ ID NO 16
LENGTH: 400
TYPE: PRT
ORGANISM: Sorghum bicolor
Query Match 41.2%; Score 143; Length 400;
Best Local Similarity 50.0%;
Matches 31; Conservative 10; Mismatches 19; Indels 2; Gaps 1;
Qy 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
||| : |:||| ||: |: |:||| || ||||:||||: || | | :| :| :
Db 33 LQLELQILNREIDFLKDELQSLEGVPPVSRSCKEVNEFVGTKQDPLLPI--KKKTHRSCR 90
Qy 61 QF 62
|
Db 91 LF 9
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
58.9 bits(141)
1e-16
Composition-based stats.
31/62(50%)
41/62(66%)
2/62(3%)
Query 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
LQL + L+REI FL+ E+ S+EGV SR CKEV+EFVG+ DP L I+ K +H +
Sbjct 33 LQLELQILNREIDFLKDELQSLEGVPPVSRSCKEVNEFVGTKQDPLLPIK--KKTHRSCR 90
Query 61 QF 62
F
Sbjct 91 LF 92
Sequence 18, US/12316385A
GENERAL INFORMATION
APPLICANT: Monsanto
TITLE OF INVENTION: Transgenic Plants With Enhanced Agronomic Traits
FILE REFERENCE: 38-21(55579)B
CURRENT APPLICATION NUMBER: US/12/316,385A
CURRENT FILING DATE: 2009-01-08
NUMBER OF SEQ ID NOS: 1358
SEQ ID NO 18
LENGTH: 198
TYPE: PRT
ORGANISM: Zea mays
Query Match 80.7%; Score 948; Length 198;
Best Local Similarity 83.5%;
Matches 172; Conservative 1; Mismatches 13; Indels 20; Gaps 5;
Qy 2 AAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVG 61
|||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||
Db 4 AAAPRPKSPPASPDPCGRHRLQLAVDALHREIGFLEGEISSIEGVHAASRCCKEVDEFVG 63
Qy 62 SNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQL 121
||||||||| |:|||||||||||||| |||||||||||| | | |||| |||
Db 64 RNPDPFLTIQQERGSHDQSQQFLKKFRGKSCLSYYLSWIC--------GGGWWCPPPLQL 115
Qy 122 KRPAAPSCSCAPRLRKL-------CCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCA 174
||| |||||||||| || || ||||| ||||| | ||||||||||||||||||
Db 116 KRPPAPSCSCAPRLGKLCSSTASSCCSCCCCRFRVVYA--AAGCGCCAPCPRCSCDCTCA 173
Qy 175 CPRCCS--CPRCSAACCCAPRCCLCL 198
|||||| || |||| ||||||||||
Db 174 CPRCCSCACPMCSAA-CCAPRCCLCL 198
G-protein gamma subunits in plants are highly diverse. In Arabidopsis, G gamma is solely responsible for providing functional selectivity to the G protein heterotrimer (Trusov et al (2012) BMC Research Notes. 5: 608 (published 10/31/2012, hereafter Trusov) page 1, right column, paragraph 1). In plants, C-terminal variability is a unique characteristic of G gamma subunits (Trusov page 2, right column, paragraph 3). Type C G-protein gamma subunits can have long cysteine rich C-terminal domains (Trusov figure 1). These elongated G gamma tails specify G-protein signaling and provide diverse functional selectivity, and removing or adding repetitive elements found in these tails can affect regulation activity, changing from negative to positive in the case of GS3 (Sun et al (2025) The Plant Cell. 37: (available 3/15/2025, after the priority date of the instant application); page 11, left column, paragraph 3-4). Known proteins comprising GGL domains in plants therefore have different activities.
The number of G gamma subunits described in plant genomes can differ, from 3 described in Arabidopsis and Setaria italica to 10 described in Glycine max (Tiwari et al (2022) Planta 255: 88 (published 3/19/2022, hereafter Tiwari); table 1). Certain plant lineages comprise extra-large G-proteins and atypical G gamma subunits (Tiwari page 2, left column, paragraph 2). Furthermore, the number of G beta units differs across plants (Tiwari table 1), and the specificity of G-protein signaling may be regulated through tissue- and condition-specific co-expression patterns and interaction specificity of multiple G-protein subunits (Tiwari page 3, left column), suggesting that proteins comprising GGL domains may vary across plants in correspondence with co-expressed subunits.
Because the activity and expression profile of GGL-domain comprising proteins is highly variable across plants, one of ordinary skill in the art would have an undue burden to screen different species and varieties of plants to identify alleles across the full scope of the claims, and then one of ordinary skill in the art would be required to screen the plants with reduced expression of these alleles to determine which, if any, grow better under saline-alkaline conditions. The example homologs in 7 species provided by the instant disclosure do not describe the full scope of alleles of G gamma subunit proteins comprised across all species and their diverse activities. Thus, while the methods of claims 10, 11, 14, 17 & 18 are supported over plants that grow better under saline-alkali conditions and comprise reduced expression of alleles encoding SEQ ID NO: 4, they are not supported over the full scope of any plant comprising any allele encoding a protein comprising at least 40% identity to SEQ ID NO: 15 or 80% identity to SEQ ID NO: 4.
Claims 19 & 21 are drawn to a plant wherein all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15 are knocked out or mutated. Claim 21 further requires that the GGL domain or first exon portion at the N-terminal is knocked out or mutated in a sequence having at least 80% identity to SEQ ID NO: 4. Claim 21 does not require that the mutation in the GGL domain of SEQ ID NO: 4 is the same mutation that results in the reduced or no activity of the protein encoded by the alleles. Thus, the claims are broadly drawn to plants comprising a knockout or mutation in all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15.
The claims broadly require plants comprising alleles with 40% identity to SEQ ID NO: 15 or 80% identity and similar function to SEQ ID NO: 4. Because the claims require that the alleles be knocked out or mutated, the claims require plants comprising endogenous genes over the genus of sequences.
Alleles encoding proteins comprising an amino acid sequence with 40% identity to the 67 amino acid long SEQ ID NO: 15 encompass those encoding proteins comprising sequences with 40 amino acid substitutions relative to SEQ ID NO: 15. Compared to the full-length protein of SEQ ID NO: 4, which comprises 198 amino acids that are not required to be conserved outside of the 67 amino acids of SEQ ID NO: 5, these proteins encompass sequences with just 26 identical amino acids, or 13% identity (26/198) compared to the full length protein of SEQ ID NO: 4. Alleles encoding an amino acid sequence having at least 80% identity to the 198 amino acid-long SEQ ID NO: 4 encompass those encoding proteins comprising sequences with 39 amino acid substitutions relative to SEQ ID NO: 4.
SEQ ID NO: 15 is an amino acid sequence comprising a GGL or GGL-like domain from the sorghum AT1 gene (Table 5). The specification teaches GGL domains in homologous genes from 6 species other than sorghum (Table 5, SEQ ID NOs: 86-96), which range in sequence identity from 94% to 43% and range in similarity from 95% to 61% (table 3). Of these, the sequence with the least similarity to instant SEQ ID NO: 15 is SEQ ID NO: 96. See alignment above. Applicant teaches truncated variants of homologs of AT1 in these different species (figures 4-6, 9-10, 12). All the sequences comprise homologs to the AT1 protein in sorghum.
Sequences with at least 40% identity to instant SEQ ID NO: 15 are known in the art. Fu et al US 2014/0020135 A1 (published 1/16/2014, hereafter Fu) teaches a sorghum homolog (Fu SEQ ID NO: 16) of a rice DEP1 protein comprising a G-protein gamma like domain in the N-terminal portion (paragraph [0144]). This gene is distinct from the gene encoding the rice GS3 protein described in the instant invention (Fu paragraph [0145]; see instant specification page 46, paragraph 5). However, the DEP1 sorghum homolog of Fu comprises a sequence with 50% identity to instant SEQ ID NO: 15 and is therefore encompassed in the sequences recited by claim 19, although no role in saline-alkali tolerance of the DEP1 protein has been described (see alignments above).
In addition, the prior art teaches that mutant alleles of proteins comprising sequences with 40% identity to instant SEQ ID NO: 15 do not necessarily have the same activity as SEQ ID NO: 4, and so plants comprising reduced activity or no activity in all such alleles could potentially have a different phenotype than the instant invention. For example, Xie et al CN115894644A (published 4/4/2023, after the priority date of the instant application. Machine translation of the Description uploaded) teaches plant comprising eight natural variations in the coding region of a gene encoding a protein comprising a sequence with 100% identity to instant SEQ ID NO: 4 (paragraph [0129], figure 3 & 4). See alignment below of Xie SEQ ID NO: 1 and instant SEQ ID NO: 4. Xie teaches that the C-terminus of the GC1 protein is a factor that positively regulates the sorghum husk coating trait (paragraph [0188]), and so sorghum comprising natural variations that truncate the C-terminus read on mutations that reduce an activity of the alleles. Xie teaches haplotypes that comprise both a truncation and a serine-glycine substitution in a γ subunit (see eg figure 3 haplotype gc1-a), which reads on an allele comprising a mutated GGL domain and a reduced activity compared to wildtype. Nevertheless, the instant specification teaches mutations such those taught by Xu lead to a phenotype that is not equivalent to the saline-alkali-tolerant phenotype of the instant invention (instant specification page 45, paragraph 3- page 46, paragraph 1).
BMU37403
ID BMU37403 standard; protein; 275 AA.
XX
AC BMU37403;
XX
DT 08-JUN-2023 (first entry)
XX
DE Sorghum bicolor glume coverage 1 (GC1) protein, SEQ:2.
XX
KW G protein gamma subunit; GC1 protein; Glume coverage 1 protein; Plant;
KW crop improvement; gene regulation; plant breeding.
XX
OS Sorghum bicolor; Wheatland.
XX
FH Key Location/Qualifiers
FT Region 78..275
FT /note= "Specifically claimed in claim 1"
XX
CC PN CN115894644-A.
XX
CC PD 04-APR-2023.
XX
CC PF 30-SEP-2021; 2021CN-11158546.
XX
PR 30-SEP-2021; 2021CN-11158546.
XX
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
XX
CC PI Li C, Tang S, Wu Y, Xie P, Xie Q;
XX
DR WPI; 2023-42874Q/039.
DR N-PSDB; BMU37402, BMU37404.
XX
CC PT Use of protein, substance that regulates expression of protein-coding
CC PT gene or activity of protein in regulating, preferably inhibiting or
CC PT improving encrustation traits of plant seeds and cultivating plant,
CC PT preferably Sorghum bicolor.
XX
CC PS Claim 1; SEQ ID NO 2; 46pp; Chinese.
XX
CC The present invention relates to a novel application of an atypical G
CC protein gamma subunit (glume coverage 1 (GC1)) or a substance in
CC regulating the expression of a protein-coding gene or a substance in
CC regulating the activity or content of the protein. The invention further
CC discloses a method for cultivating naked plants, which involves
CC increasing or promoting the activity or content of the protein or/and the
CC expression level of the gene encoding the protein in a plant of interest
CC or knocking out the C-terminus of the protein, thereby obtaining naked
CC plant. The novel application of the invention is for regulating the
CC expression of a gene encoding the protein or a substance for regulating
CC the activity or content of the protein in regulating the encrustation
CC trait of plant seeds and regulating the expression of the protein coding
CC gene or the substance for regulating the activity or the content of the
CC protein in inhibiting the encrustation trait of plant seeds in plant
CC breeding, which has wide application prospect in guiding the breeding and
CC molecular improvement of naked grain varieties of crops.
XX
SQ Sequence 275 AA;
Query Match 100.0%; Score 1174; Length 275;
Best Local Similarity 100.0%;
Matches 198; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 78 MAAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFV 137
Qy 61 GSNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQ 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 138 GSNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQ 197
Qy 121 LKRPAAPSCSCAPRLRKLCCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCACPRCCS 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 198 LKRPAAPSCSCAPRLRKLCCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCACPRCCS 257
Qy 181 CPRCSAACCCAPRCCLCL 198
||||||||||||||||||
Db 258 CPRCSAACCCAPRCCLCL 275
Given the diversity of genes, alleles, and mutations encompassed by the instant claims, one of ordinary skill in the art would be required to screen a large number of plants across many species, comprising an extremely large number of alleles with mutations across the sequence of the gene, and resulting in an unknowably large number of phenotypes, in order to determine a use for plants across the full scope of the claims that do not comprise the saline-alkali-tolerant phenotype of the instant invention. Determining a use for plants over the full scope of the claimed invention would require undue experimentation.
Therefore, the claims may be enabled for plants comprising alleles encoding a protein comprising the sequence of SEQ ID NO: 15 or 4 that have reduced or no activity resulting in saline-alkali-tolerant phenotype, but the claims are not enabled over the full scope of the genus of plants.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 11 & 19 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Fu et al US 10,485,196 B2 (patented 11/26/2019, hereafter Fu 2019).
Fu 2019 discloses plants that do not produce a functional GS3 polypeptide, wherein GS3 comprises Fu 2019 SEQ ID NO: 7 (Fu 2019 claim 6), including plants in which the expression of GS3 is accomplished with an RNA interference construct (Fu 2018 claim 7). Fu 2019 SEQ ID NO: 7 has 79.1% sequence identity to instant SEQ ID NO: 15. See alignment below.
Score
Expect
Method
Identities
Positives
Gaps
115 bits(287)
1e-38
Compositional matrix adjust.
53/67(79%)
58/67(86%)
0/67(0%)
Query 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
LQLAVDALHREI FLEGEI+SIEG+HAASRCC+EVDEF+G PDPF+TI EK SHD S
Sbjct 24 LQLAVDALHREIGFLEGEINSIEGIHAASRCCREVDEFIGRTPDPFITISSEKRSHDHSH 83
Query 61 QFLKKFR 67
FLKKFR
Sbjct 84 HFLKKFR 90
Fu 2019 discloses a method comprising reducing or abolishing the expression of a nucleic acid sequence encoding a GS3 polypeptide (Fu 2019 Claim 14).
The plant of Fu 2019 claim 6 which does not produce a functional GS3 polypeptide anticipates instant claim 19, a plant wherein all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to instant SEQ ID NO: 15 is mutated, resulting in that the protein of the alleles has reduced or no activity. The method of reducing or abolishing the expression of a nucleic acid sequence encoding a GS3 polypeptide reads on instant SEQ ID NO: 10. Although Fu 2019 does not teach that growth of the plant is better than that of a wild-type plant under saline-alkali conditions, the method of instant claim 10 does not require a step of growing a plant under saline-alkali conditions. Therefore, the wherein clause has been interpreted as an intended result of the actively recited steps of the method. See MPEP 2111.04.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 10, 11, 17, 19, & 23-26 are rejected under 35 U.S.C. 103 as being unpatentable over Fu et al US 10,485,196 B2 (patented 11/26/2019, hereafter Fu 2019).
Claims 10, 11 & 17 are drawn to a method of reducing the expression level of all alleles encoding a protein comprising an amino acid sequence at least 40% identity to SEQ ID NO: 15. Claim 19 is drawn to a plant wherein all alleles encoding such a protein have been knocked out or mutated so that the protein has reduced or no activity. Claim 24 is drawn to a method comprising propagating a parental seed to harvest an offspring seed wherein all alleles have been knocked out or mutated so that the protein is not expressed or has a reduced expression level. Claims 23, 25 & 26 are drawn to a method comprising hybridizing a first parent plant with a second parent plant where in both parent plants all alleles encoding a protein are knocked out or mutated.
The teachings of Fu 2019 are presented above. Additionally, Fu 2019 teaches that hybrid rice varieties have heterosis, which has boosted grain yield (column 1, lines 34-36). Fu 2018 teaches a method for creating novel rice varieties by crossing plants of the invention followed by generations of selection. Fu 2019 teaches that it may be desired to choose second plants to cross which themselves exhibit one or more selected desirable characteristics (column 33, lines 54-67). Fu 2019 teaches a NIL with the gs3 allele (column 35, lines 46-52). Fu 2018 describes pyramiding the GW7TFA and gs3 alleles and developing high-yielding indica hybrid rice varieties (column 40, lines 48-56).
Fu 2019 envisions the invention to include bioenergy crops such as sweet sorghum (column 24, lines 24-37), and sorghum is a preferred plant (column 24, lines 41-45).
Fu teaches a homolog of OsGS3 in sorghum Fu 2019 SEQ ID NO: 101 (table 11), which has 100% sequence identity to instant SEQ ID NO: 4. See alignment below.
Score
Expect
Method
Identities
Positives
Gaps
379 bits(974)
2e-141
Compositional matrix adjust.
198/198(100%)
198/198(100%)
0/198(0%)
Query 1 MAAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFV 60
MAAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFV
Sbjct 1 MAAAPRPKSPPASPDPCGRHRLQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFV 60
Query 61 GSNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQ 120
GSNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQ
Sbjct 61 GSNPDPFLTIQPEKGSHDQSQQFLKKFRAKSCLSYYLSWICCCGGGGGGGSGGWCPPSLQ 120
Query 121 LKRPAAPSCSCAPRLRKLCCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCACPRCCS 180
LKRPAAPSCSCAPRLRKLCCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCACPRCCS
Sbjct 121 LKRPAAPSCSCAPRLRKLCCCCCCCRCRVVYAGGAGGCGCCAPCPRCSCDCTCACPRCCS 180
Query 181 CPRCSAACCCAPRCCLCL 198
CPRCSAACCCAPRCCLCL
Sbjct 181 CPRCSAACCCAPRCCLCL 198
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Fu 2019 of crossing rice with a mutated GS3 gene to select a second rice plant which comprises alleles of the GS3 protein that are knocked out or mutated and the harvesting seed for further backcrossing. One of ordinary skill in the art would have been motivated to select a first and second parent with mutated GS3 alleles, because Fu teaches that selecting second plants to cross with a desirable trait may be advantageous. One of ordinary skill in the art would have had reasonable expectation of success, because rice varieties and NILS comprising mutations at the GS3 locus were known in the art prior to the filing of the instant application. Thus, a method of hybridizing a first and second parent plant and harvesting seed (instant claim 23) would have been obvious, including wherein one of the parent plants is an inbred line (instant claim 26).
Before the filing of the instant application, it would have been obvious to one of skill in the art to generate a sorghum plant with reduced expression of GS3 according to the method in rice described by Fu. One of ordinary skill in the art would have been motivated to reduce expression of GS3 in sorghum, because Fu 2019 teaches sorghum to be a preferred plant for the invention. One of ordinary skill in the art would have had reasonable expectation of success, because Fu 2019 identifies GS3 homologs in sorghum. Claim 25, wherein all alleles encoding a protein comprising a sequence with at least 80% identity to SEQ ID NO: 4 is mutated is therefore obvious.
The plant in which expression is reduced by RNA interference makes obvious a method of reducing expression by RNAi (instant claims 10 & 11) and reads on the plant of instant claim 19.
Finally, the plants comprising two mutated alleles encoding GS3 protein read on plants wherein the wild-type protein is not expressed. Thus, the method of breeding NILs taught by Fu 2019 reads on a method comprising propagating a parental seed to harvest an offspring wherein all alleles have been mutated so that the protein is not expressed or has a reduced expression (instant claim 24) and also reads on a method which comprises reducing expression of the alleles and further identifying an offspring plant obtained by selfing a parent plant comprising a mutation of the alleles wherein the mutation has a reduced activity (claim 17).
Claims 10, 11, 17, 19 & 23-26 are obvious over Fu 2019.
Claim(s) 10 & 11 are rejected under 35 U.S.C. 103 as being unpatentable over Guo et al US 2016/0002648 A1 (published 1/7/2016, hereafter Guo) in view of GenBank sequence FJ797616.1 (available 11/18/2009).
Claims 10 & 11 are drawn to a method for producing a saline-alkali-tolerant plant comprising reducing the expression level of all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15.
Guo teaches a nucleotide sequence of the sorghum PCYS1 gene (Guo SEQ ID NO: 195, table 1 page 10) encoding a sorghum polypeptide with 100% sequence identity to instant SEQ ID NO: 4 and comprising a sequence with 100% sequence identity to instant SEQ ID NO: 15. See alignments below.
Sequence 195, US/14771528
GENERAL INFORMATION
APPLICANT: Pioneer Hi-Bred International, Inc.
TITLE OF INVENTION: GENES FOR IMPROVING NUTRIENT UPDATE AND ABIOTIC STRESS TOLERANCE
TITLE OF INVENTION: IN PLANTS
FILE REFERENCE: 4125-WO-PCT
CURRENT APPLICATION NUMBER: US/14/771,528
CURRENT FILING DATE: 2015-08-31
PRIOR APPLICATION NUMBER: 61/776,363
PRIOR FILING DATE: 2013-03-11
NUMBER OF SEQ ID NOS: 5105
SEQ ID NO 195
LENGTH: 597
TYPE: DNA
ORGANISM: Sorghum bicolor
Length: 597
Score: 1174.00 Matches: 198
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 100.0% Indels: 0
Gaps: 0
US-19-103-381-4 (1-198) x US-61-776-363-195 (1-597)
Qy 1 MetAlaAlaAlaProArgProLysSerProProAlaSerProAspProCysGlyArgHis 20
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 ATGGCGGCGGCGCCGAGGCCCAAGTCGCCGCCTGCCTCGCCGGACCCCTGCGGCCGCCAC 60
Qy 21 ArgLeuGlnLeuAlaValAspAlaLeuHisArgGluIleSerPheLeuGluGlyGluIle 40
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 CGCCTCCAGCTCGCCGTCGACGCGCTCCACCGGGAGATCAGCTTCCTCGAGGGCGAAATA 120
Qy 41 SerSerIleGluGlyValHisAlaAlaSerArgCysCysLysGluValAspGluPheVal 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 AGTTCCATTGAGGGGGTCCACGCTGCCTCCAGATGCTGCAAAGAGGTTGATGAGTTTGTT 180
Qy 61 GlySerAsnProAspProPheLeuThrIleGlnProGluLysGlySerHisAspGlnSer 80
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GGAAGTAATCCGGATCCATTCCTAACGATTCAGCCAGAGAAAGGAAGCCATGATCAATCT 240
Qy 81 GlnGlnPheLeuLysLysPheArgAlaLysSerCysLeuSerTyrTyrLeuSerTrpIle 100
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 CAGCAGTTTCTGAAGAAGTTCCGAGCAAAGAGCTGCCTGAGCTACTACCTCTCGTGGATC 300
Qy 101 CysCysCysGlyGlyGlyGlyGlyGlyGlySerGlyGlyTrpCysProProSerLeuGln 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 TGCTGCTGCGGCGGTGGCGGTGGCGGTGGCAGTGGCGGGTGGTGCCCGCCGTCGCTGCAG 360
Qy 121 LeuLysArgProAlaAlaProSerCysSerCysAlaProArgLeuArgLysLeuCysCys 140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 CTCAAGAGGCCAGCGGCGCCGAGCTGCTCCTGCGCGCCGCGGCTGAGGAAGCTCTGCTGC 420
Qy 141 CysCysCysCysCysArgCysArgValValTyrAlaGlyGlyAlaGlyGlyCysGlyCys 160
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 TGCTGTTGCTGCTGCCGGTGCCGCGTGGTGTACGCCGGCGGCGCCGGCGGCTGCGGGTGC 480
Qy 161 CysAlaProCysProArgCysSerCysAspCysThrCysAlaCysProArgCysCysSer 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 TGCGCGCCGTGCCCGCGCTGCTCGTGCGACTGCACCTGCGCCTGCCCGCGCTGCTGCTCC 540
Qy 181 CysProArgCysSerAlaAlaCysCysCysAlaProArgCysCysLeuCysLeu 198
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 TGCCCCAGGTGCAGCGCCGCGTGCTGCTGCGCCCCGCGCTGCTGCCTGTGCCTA 594
Length: 597
Score: 347.00 Matches: 67
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 100.0% Indels: 0
Gaps: 0
US-19-103-381-15 (1-67) x US-61-776-363-195 (1-597)
Qy 1 LeuGlnLeuAlaValAspAlaLeuHisArgGluIleSerPheLeuGluGlyGluIleSer 20
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 64 CTCCAGCTCGCCGTCGACGCGCTCCACCGGGAGATCAGCTTCCTCGAGGGCGAAATAAGT 123
Qy 21 SerIleGluGlyValHisAlaAlaSerArgCysCysLysGluValAspGluPheValGly 40
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 124 TCCATTGAGGGGGTCCACGCTGCCTCCAGATGCTGCAAAGAGGTTGATGAGTTTGTTGGA 183
Qy 41 SerAsnProAspProPheLeuThrIleGlnProGluLysGlySerHisAspGlnSerGln 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 184 AGTAATCCGGATCCATTCCTAACGATTCAGCCAGAGAAAGGAAGCCATGATCAATCTCAG 243
Qy 61 GlnPheLeuLysLysPheArg 67
|||||||||||||||||||||
Db 244 CAGTTTCTGAAGAAGTTCCGA 264
Guo teaches a method of transformation of maize with sorghum genomic clones containing nutrient uptake/stress tolerance genes (paragraph [0233, 0251]) as well as antisense sequences of the nutrient uptake/stress tolerance gene sequences (paragraph [0250]). Guo teaches that antisense suppression comprises an expression cassette designed to express an RNA molecule complementary to all or a part of a messenger RNA encoding the polypeptide, overexpression of which can result in reduced expression of the native gene (paragraph [0137]) and can be used to inhibit the expression of multiple proteins in the same plant (paragraph [0138]).
Guo teaches a plant comprising an expression cassette comprising a polynucleotide of Guo SEQ ID NO: 195, linked in sense or anti-sense orientation to a promoter (Guo claim 4), as well as a method of introducing the expression cassette into a sorghum or maize plant cell, culturing under growing conditions, and regenerating (Guo claim 12). Guo envisions transgenic seed of the plants (Guo claim 8).
Guo suggests, in Guo claim 13, a method of introducing a complement of the nucleotide sequence of Guo SEQ ID NO: 195 into a plant cell to inhibit expression of mRNA having the sequence of Guo SEQ ID NO: 195.
Guo does not teach that maize comprises an allele encoding a protein comprising a sequence having at least 40% identity to SEQ ID NO: 15.
GenBank sequence FJ797616.1 teaches a maize mRNA for a GS3-like protein from line B73 comprising a sequence with 94% sequence identity to instant SEQ ID NO: 15. See alignment below.
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
Frame
131 bits(329)
6e-39
Compositional matrix adjust.
63/67(94%)
64/67(95%)
0/67(0%)
+2
Query 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
LQLAVDALHREI FLEGEISSIEGVHAASRCCKEVDEFVG NPDPFLTIQ E+GSHDQSQ
Sbjct 101 LQLAVDALHREIGFLEGEISSIEGVHAASRCCKEVDEFVGRNPDPFLTIQQERGSHDQSQ 280
Query 61 QFLKKFR 67
QFLKKFR
Sbjct 281 QFLKKFR 301
Before the time of filing of the instant application, it would have been obvious to one of skill in the art to use a maize plant from line B73 for the method taught by Guo. Guo is silent as to the maize genotype used, but one of ordinary skill in the art would have been motivated to substitute one maize line for another as simple substitution. One of ordinary skill in the art would have had reasonable expectation of success because Guo did not rely on a specific maize background for the method taught.
Guo is silent as to whether the growth of the plant with reduced expression is better than that of a wild-type plant in saline-alkali conditions; however, claims 10 and 11 do not require a step of growing the plant under saline-alkali conditions, so this “wherein” clause reads on an intended result of a process step positively recited. See MPEP 2111.04. Because a reduction in the expression of the alleles encoding the protein are sufficient to confer the saline-alkali tolerant phenotype, and because Guo teaches that the antisense method can target multiple proteins, the method of Guo to reduce the expression of the alleles encoding the protein homologous to instant SEQ ID NO: 4 reads on the production of a plant which would have better growth under saline-alkali conditions.
Because B73 maize plants comprise alleles for proteins comprising a sequence with >40% identity to instant SEQ ID NO: 15, the method of Guo to reduce expression of the gene of Guo SEQ ID NO: 195 in maize reads on a method of instant claim 10 and the complement nucleotide sequence introduced into the plant cell reads on at least a “substance capable of inhibiting the expression of a target gene” (instant claim 11, lines 3-4).
Claim(s) 10, 11, 14, 17, 19 & 24 are rejected under 35 U.S.C. 103 as being unpatentable over Fu et al US 2014/0020135 A1 (published 1/16/2014, hereafter Fu).
Claim 19 is drawn to a plant wherein all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15 are knocked out or mutated, resulting in that the protein has a reduced activity or no activity. Claim 24 is drawn to a method comprising propagating a parental seed to harvest an offspring seed wherein all alleles encoding a protein comprising a sequence having at least 40% identity to SEQ ID NO: 15 have been knocked out or mutated so that the protein has a reduced expression level. Claims 10, 11, 14 & 17 are drawn to a method comprising reducing the expression level of all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15 wherein the growth of the plant is better than that of a wild-type plant under saline-alkali conditions.
Fu teaches a sorghum homolog (Fu SEQ ID NO: 16) of a rice DEP1 protein comprising a G-protein gamma like domain in the N-terminal portion (paragraph [0144]). This gene is distinct from the gene encoding the rice GS3 protein (Fu paragraph [0145]). The DEP1 sorghum homolog of Fu comprises a sequence with >40% identity to instant SEQ ID NO: 15; see alignment below.
Sequence 16, US/13982229
Publication No. US20140020135A1
GENERAL INFORMATION
APPLICANT: Fu, Xiangdong
APPLICANT: Wu, Kun
APPLICANT: Qian, Qian
APPLICANT: Zhang, Chengwei
APPLICANT: Liu, Xueying
APPLICANT: Wang, Shuansuo
TITLE OF INVENTION: NOVEL USE OF A DENSE AND ERECT PANICLE 1 GENE IN IMPROVING
TITLE OF INVENTION: NITROGEN UTILIZATION EFFICIENCY
FILE REFERENCE: 9207-68TS
CURRENT APPLICATION NUMBER: US/13/982,229
CURRENT FILING DATE: 2013-07-26
PRIOR APPLICATION NUMBER: PCT/US2012/022930
PRIOR FILING DATE: 2012-01-27
PRIOR APPLICATION NUMBER: CN 201110029759.9
PRIOR FILING DATE: 2011-01-27
NUMBER OF SEQ ID NOS: 19
SEQ ID NO 16
LENGTH: 400
TYPE: PRT
ORGANISM: Sorghum bicolor
Query Match 41.2%; Score 143; Length 400;
Best Local Similarity 50.0%;
Matches 31; Conservative 10; Mismatches 19; Indels 2; Gaps 1;
Qy 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
||| : |:||| ||: |: |:||| || ||||:||||: || | | :| :| :
Db 33 LQLELQILNREIDFLKDELQSLEGVPPVSRSCKEVNEFVGTKQDPLLPI--KKKTHRSCR 90
Qy 61 QF 62
|
Db 91 LF 9
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
58.9 bits(141)
1e-16
Composition-based stats.
31/62(50%)
41/62(66%)
2/62(3%)
Query 1 LQLAVDALHREISFLEGEISSIEGVHAASRCCKEVDEFVGSNPDPFLTIQPEKGSHDQSQ 60
LQL + L+REI FL+ E+ S+EGV SR CKEV+EFVG+ DP L I+ K +H +
Sbjct 33 LQLELQILNREIDFLKDELQSLEGVPPVSRSCKEVNEFVGTKQDPLLPIK--KKTHRSCR 90
Query 61 QF 62
F
Sbjct 91 LF 92
Fu teaches a qngr9 allele in rice that confers a lodging resistance and tolerance to low nitrogen phenotype (paragraph [0294-0295]). Fu teaches introgressing the qngr9 alleles into a NIL in an otherwise homogenous QZL2 background (paragraphs [0292]). Fu teaches RILs bread from a cross between NJ6xQZL2 inbred for six generations and a near isogenic recombinant inbred line constructed by backcrossing for three generations, and that one of the lines behaved in a similar manner to QZL2 (paragraph [0292]). The qngr9 allele was mapped to the gene Dense Erect Panicle 1 (DEP1), and the dep1 allele cDNA lacks 696 bp at the 3’ end of the gene corresponding to 231 fewer amino acids at the C end due to a premature stop codon (paragraphs [0299-0301]).
Nitrogen Utilization Efficiency (NUE) is defined by Fu as the ability to absorb, assimilate, or use nitrogen; Fu teaches that in embodiments of the invention, NUE is increased in a plant under low nitrogen conditions compared with a control plant that does not express the dep1 polypeptide (paragraph [0055]). Fu teaches that nitrogen is known to be generally low or deficient in saline soils and alkaline soils with low soil organic matter (paragraph [0071]).
Fu teaches a method of transforming rice with vector constructs expressing the DEP1 homolog from wheat; transformed plants had a similar phenotype to rice dep1, including increased grain and semi-dwarf (paragraph [0312]). Fu also teaches a method of using RNAi to knockdown wheat DEP1 expression, resulting in an increase in the length of the ear and a reduced number of spikelets (paragraph [0313]). Overexpression of the rice dep1 gene in maize led to a semi-dwarf plant type and increased photosynthetic efficiency (paragraph [0314]).
Fu suggests that in embodiments of the invention, a dep1 polypeptide comprises one or more of the OSR, GGL, first VWFC or transmembrane domains (paragraph [0152]), including embodiments wherein the dep1 polypeptide comprises only a portion of the OSR domain (paragraph [0158]). Fu teaches that the GGL domain is in the N-terminal portion of the polypeptide (amino acids 28-82 of Fu SEQ ID NO: 19) and the OSR domain is located with amino acid 1-20 of Fu SEQ ID NO: 19 (paragraph [0144]). Fu teaches a sorghum variant with an amino acid insertion at the 13th amino acid residue (Figure 22, paragraph [0311]) and suggests embodiments wherein the plant used in the instant invention is sorghum (paragraph [0019]).
Fu teaches that the polypeptides result in an increase in Nitrogen Use Efficiency and a semi-dwarf phenotype (paragraph [0175]); Fu teaches a motivation in improve Nitrogen Use Efficiency because plants utilize only a small amount of the nitrogen fertilizer applied, and nitrogen not utilized by plants is released into the environment causing atmospheric pollution and eutrophication of rivers and lacks. There is a need to develop plants that have increased yield at a given input of nitrogen fertilizer to raise crop productivity while conserving environmental quality (paragraphs [0003-0005]).
Fu teaches that the sequence encoding the dep1 polypeptide can be naturally occurring (paragraph [0169]).
Fu teaches that the expression of genes involved in uptake and assimilation of ammonium in roots was higher in plants comprising the ngr9 allele compared to wildtype in a nitrogen deficient environment and higher above-ground nitrogen per plant (paragraphs [0296-0298]).
Fu does not teach that growth of the plant with the reduced expression of the DEP1 protein is better than that of a wild-type plant under saline-alkali conditions.
Before the filing date of the instant application, one of ordinary skill in the art would have been motivated to modify the method of Fu of reducing expression of the DEP1 protein in wheat or rice to reduce expression or activity of the DEP1 homolog in sorghum. One of ordinary skill in the art would have been motivated to modify sorghum, because the mutation of the DEP1 gene leads to higher Nitrogen Use Efficiency and less environmental pollution. One of ordinary skill in the art would have had reasonable expectation of success, because Fu identified a homologous gene including natural variants of the gene in sorghum.
The loss of alleles encoding the wild-type protein reads on reduced expression of alleles encoding the protein with an amino acid sequence comprising at least 40% sequence identity to instant SEQ ID NO: 15; therefore, the method of Fu for producing inbred lines over multiple generations reads on a method of propagating a parental seed to harvest an offspring seed (instant claim 24). The sorghum plant with a mutated DEP1 protein reads on a plant wherein all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to Seq ID NO: 15 are mutated resulting in the protein having a reduced activity or no activity (claim 19).
Applying Fu’s methods of RNAi suppression to sorghum reads on instant claims 10 & 11. Fu suggests embodiments wherein the encoded polypeptide only comprises part of the OSR domain. Plants comprising genes encoding only a portion of the OSR domain read on mutations wherein the GGL-like domain is mutated or partially deleted (instant claim 14). Furthermore, Fu describes a sorghum variant of the gene that comprises a mutation in the GGL domain, and so mutations that reduce expression of the protein in that sorghum background, even if the GGL domain remained functional, would read on instant claim 14. Fu’s methods of generating inbred lines with mutated alleles with reduced activity, when applied to sorghum, makes obvious claim 17, because collecting the seed of the inbred lines requires identifying a seed as being an offspring plant obtained by selfing a parent plant comprising a mutation of the alleles resulting in reduced activity.
Although claim 10 recites that the growth of the saline-alkali-tolerant plant is better under saline-alkali conditions that that of a wild-type plant, there is no active step of growing the plant under saline-alkali conditions. Thus, this wherein clause is interpreted as an intended result. A "‘whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.’" Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003)). See MPEP 2111.04.
Claim(s) 23 & 26 are rejected under 35 U.S.C. 103 as being unpatentable over Fu as applied to claims 10, 11, 14, 17, 19 & 24 above, and further in view of Knoll et al (May-June 2018) Sugar Tech. 20(3):261–274 (published 1/29/2018, hereafter Knoll).
Claims 23 & 26 are drawn to a method comprising hybridizing a first parent plant with a second parent plant wherein in both parent plants all alleles encoding a protein comprising an amino acid sequence having at least 40% identity to SEQ ID NO: 15 are knocked out or mutated and harvesting seeds of the hybrid plant.
The teachings of Fu are presented above. Fu teaches that there are multiple naturally occurring amino acid variations in sorghum SbDEP1 protein (paragraph [0311]). Although Fu teaches a method of hybridizing plants in order to generate NIL, Fu does not specifically teach hybridizing two plants wherein both parent plants comprise all alleles knocked out or mutated.
Knoll teaches a method of producing a hybrid sweet sorghum plant by crossing an inbred cultivar with a short-statured male-sterile parent (page 261, right column, paragraph 2; page 263, right column, paragraph 2). Knoll teaches that heterosis for panicle yield could be observed in hybrid sweet sorghum in some plants (page 267, left column, paragraph 2). Although heterosis was influenced by environmental conditions (page 271, left column, paragraph 1), Knoll teaches that hybrid sweet sorghum produces large quantities of seed compared to inbred lines (page 272, left column).
Before the time of filing of the instant application, it would have been obvious to modify the method of Fu of hybridizing a plant carrying a dep1 mutation to hybridize two lines of sorghum, each carrying a mutated dep1 gene. One of ordinary skill in the art would have been motivated to cross two different parents each carrying a dep1 mutation, because hybrid sorghum produces large quantities of seed compared to an inbred line. One of ordinary skill in the art would have had reasonable expectation of success, because methods of hybridizing sorghum were known and practiced in the art prior to the filing of the instant application.
Alternatively, Fu teaches that sorghum lines comprise mutations in the DEP1 protein naturally. Instant claim 23 does not require that the mutation in the protein encoding SEQ ID NO: 15 result in a non-functional protein. Thus, any hybridization breeding process between two sorghum lines comprising a mutation as taught by Fu (figure 22) reads on the method of instant claim 23.
Knoll teaches hybridization of inbred sorghum lines, and measuring the quantity of seed of hybrid plants reads on harvesting seeds of the hybrid plant. Thus, claims 23 & 26, in addition to claims 10, 11, 14, 17, 19 & 24 are obvious over Fu and Knoll.
Conclusion
No claims are allowed.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
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