WO2024037338A1 - 一种植株籽粒大小调控基因及其应用 - Google Patents

一种植株籽粒大小调控基因及其应用 Download PDF

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WO2024037338A1
WO2024037338A1 PCT/CN2023/110679 CN2023110679W WO2024037338A1 WO 2024037338 A1 WO2024037338 A1 WO 2024037338A1 CN 2023110679 W CN2023110679 W CN 2023110679W WO 2024037338 A1 WO2024037338 A1 WO 2024037338A1
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gene
nucleotide sequence
sequence
plant
mutation
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PCT/CN2023/110679
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French (fr)
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马力耕
曹颖
吴静
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首都师范大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the invention belongs to the field of plant biotechnology breeding, and specifically relates to a PCFS family gene and a method for obtaining plants with improved agronomic traits by mutating the gene.
  • Plant grain size is a key trait in agricultural production, which directly affects crop yields. Larger grains of staple food crops such as rice, wheat, and corn can increase grain yields. Larger grains of oil crops such as rape and soybeans can increase oil production. , vital to promoting agricultural production.
  • crops are often affected by harsh environments, such as drought, salt stress, pests and diseases, etc. Therefore, selecting varieties with strong resistance to drought, salt, diseases and pests, etc. can effectively improve the quality of plants. resistance and adaptability to achieve high yields. Rapeseed has many branches and the pods on the branches mature late. Machines often cannot harvest the seeds on the side branches. How to obtain a reasonable plant type is also a topic of great concern in crop breeding. It can be seen from the above that whether it is larger grains, fewer branches, or increased resistance to osmotic stress, they are all beneficial traits in agriculture and are very important for increasing crop yields and developing modern agriculture.
  • the PCFS (Pcf11-SIMILAR PROTEIN) homologous gene was first reported in yeast.
  • the gene name in yeast is Pcf11 (protein 1 of CF I).
  • Pcf11 protein 1 of CF I
  • the relevant genes can be found in plants. Homologous proteins.
  • the PCFS family In higher plants, the PCFS family generally has two or more homologous genes. Prior to this, little attention was paid to the gene functions of the PCFS family in plants.
  • the inventor of this application used CRISPR/Cas9 technology to edit the PCFS family genes of Arabidopsis, rape, corn, soybean, rice and other plants.
  • the embodiments of the present application provide a gene and its mutant nucleotide sequence, which is characterized in that plants containing the gene mutation have enlarged grains, and the nucleotide sequence of the gene is selected from the following group of sequences: one:
  • the full length of the nucleotide sequence of any one of (a) to (f) is at least 85%, 90%, 95% or above sequence similarity, and the mutation in the plant has the ability to change the grain of the plant.
  • the embodiments of the present application provide a gene and its mutant nucleotide sequence. After mutation, the gene has the phenotype of larger and/or heavier plant grains.
  • the nucleotide sequence of the gene is selected from the following group of sequences: one:
  • the above-mentioned genes provided in the examples of this application can be isolated from various plants. Those skilled in the art should know that The genes described in this application also include homologous genes that are highly homologous to the nucleotide sequence or protein sequence of the gene and have the same function of increasing grain size and/or weight after mutation.
  • the homologous genes include DNA sequences that can hybridize to the nucleotide sequences of the genes disclosed in the examples of this application under stringent conditions. "Stringent conditions" used herein are well known, including hybridization in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH 6.4) and 1mM EDTA.
  • the hybridization temperature is preferably 53°C-60°C, and the hybridization time It is preferably 12-16 hours, and then washed with a washing solution containing 0.5 ⁇ SSC and 0.1% SDS.
  • the washing temperature is preferably 62°C-68°C, and the washing time is 15-60 minutes.
  • Homologous genes also include at least 80%, 85%, 90%, 95%, 98%, 99% or higher sequence similarity to the DNA sequence shown in the genes disclosed in the embodiments of this application, or are identical to those implemented in this application.
  • the amino acid sequences of functional genes disclosed in Examples have at least 80%, 85%, 90%, 95%, 98%, 99% or higher sequence similarity, and have the function of making plant grains larger and/or heavier after mutation.
  • DNA sequences can be isolated from any plant.
  • the protein amino acid sequence encoded by the DNA sequence of the PCFS family gene has a complete N-terminal, and the amino acid sequence of the N-terminal is Y...L X ELT X N X KP X IT X LTI X A...E ...Q X LP X LYLLDSIVKN X G XX Y ... F ... L XX VF XX AY...M XX LF stands for any amino acid,... stands for more than any three random amino acid sequences, and the remaining letters stand for specific amino acids).
  • the percentage of sequence similarity can be obtained through well-known bioinformatics algorithms, including Myers and Miller algorithms, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul's algorithm, which is well known to those skilled in the art.
  • SNP single nucleotide polymorphism
  • the mutation described in the embodiments of this application refers to the substitution, deletion and/or addition of one or more nucleotides on the nucleotide sequence of the gene of this application.
  • the gene has the function of improving agronomic traits after mutation, including Make the plant grains heavier and/or larger.
  • pcfs4 gene number is AT4G04885, nucleotide sequence is shown as SEQ ID NO: 7 or 8
  • single gene mutation or pcfs2 and pcfs4 double mutations also have the ability to increase tolerance. Regulatory functions such as reversal and/or reduction of branch number.
  • the mutations described in the embodiments of this application may be point mutations, or DNA deletion or insertion mutations.
  • the mutations can be obtained by physical mutagenesis, chemical mutagenesis or gene editing.
  • Chemical mutagenesis methods include mutagenesis caused by treatment with mutagens such as EMS; gene editing methods include but are not limited to ZFN, TALEN and /or gene editing methods such as CRISPR/Cas9.
  • the target sequence used by the CRISPR/Cas9 gene editing method is selected from one of the following groups of sequences:
  • sequence is SEQ ID NO: 4, 5, 7, 8, 13, 14, 16, 17, 22, 23, 25, 26, 28, 29, 34, 35, 37, 38, 40, 42, 43 , 44, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73 or 74 consistent with 5'-Nx -NGG-3' sequence is a regularly arranged fragment, where N represents any one of A, G, C and T, 14 ⁇ X ⁇ 30, and X is an integer, and Nx represents X consecutive nucleotides; or
  • the target sequence for Arabidopsis plants is as shown in any one of SEQ ID NO:76-79
  • the target sequence for rapeseed plants is as shown in SEQ ID NO: Any one of 80-83 is shown
  • the target sequence for soybean plants is shown in any one of SEQ ID NO: 84-87
  • the target sequence for rice plants is shown in any one of SEQ ID NO: 88-91
  • the target sequence for corn plants is shown in any one of SEQ ID NO: 92-95.
  • nucleotide sequence of the gene mutant described in the embodiment of the present application it is characterized in that the nucleotide sequence of the gene mutant is as shown in any one of SEQ ID NO: 97, 98, 100-105.
  • the embodiments of the present application also disclose a method for regulating agronomic traits of plants, which is characterized in that the grains of the plants become larger and heavier by mutating the following genes of the plants, and the nucleotide sequence of the genes is selected from the following group of sequences: one:
  • (f) has at least 85%, 90%, 95% or more sequence similarity with the full length of any of the nucleotide sequences described in (a)-(f), and has the ability to change the grain of the plant after mutation in the plant A large functional nucleotide sequence;
  • the method for regulating agronomic traits of plants disclosed in the embodiments of the present application is characterized by making the plant grains larger and/or heavier by mutating the following genes of the plants, and the nucleotide sequence of the genes is selected from the following group: One of the sequences:
  • the genes can be isolated and obtained from various plants.
  • the genes used also include genes that are highly homologous to the nucleotide sequence or protein sequence of the gene, and have the same effect of larger grains after mutation. Homologous genes with variable weight function.
  • the homologous genes include DNA sequences that can hybridize to the nucleotide sequences of the genes disclosed in the examples of this application under stringent conditions.
  • “Stringent conditions” used herein are well known, including hybridization in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH 6.4) and 1mM EDTA.
  • the temperature of the hybridization is preferably 53°C-60°C, and the hybridization time It is preferably 12-16 hours, and then washed with a washing solution containing 0.5 ⁇ SSC and 0.1% SDS.
  • the washing temperature is preferably 62°C-68°C, and the washing time is 15-60 minutes.
  • Homologous genes also include DNA sequences that have at least 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with the PCFS family genes disclosed in the examples of this application, and have the same sequence after mutation. DNA sequences responsible for increasing grain size and/or weight can be isolated from any plant. More specifically, the protein amino acid sequence encoded by the DNA sequence of the PCFS family gene has a complete N-terminal, and the amino acid sequence of the N-terminal is Y...L X ELT X N X KP X IT X LTI X A...E ...Q X LP X LYLLDSIVKN X G XX Y ... F ...
  • L XX VF XX AY...M XX LF stands for any amino acid,... stands for more than any three random amino acid sequences, and the remaining letters stand for specific amino acids).
  • the percentage of sequence similarity can be obtained through well-known bioinformatics algorithms, including Myers and Miller algorithms, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul's algorithm, which is well known to those skilled in the art.
  • the mutation refers to substitution, deletion and/or addition of one or more nucleotides on the nucleotide sequence of the gene disclosed in the present application
  • the gene mutation has the function of improving agronomic traits, including making plant grains heavier and/or larger.
  • pcfs4 gene number is AT4G04885, nucleotide sequence is shown in SEQ ID NO: 7 or 8) single gene mutation
  • the double gene mutation of pcfs2 and pcfs4 also has the regulatory function of increasing stress tolerance and/or reducing the number of branches.
  • the mutations described in the embodiments of this application may be point mutations, or DNA deletion or insertion mutations.
  • the mutations can be obtained by physical mutagenesis, chemical mutagenesis or gene editing.
  • Chemical mutagenesis methods include mutagenesis caused by treatment with mutagens such as EMS; gene editing methods include but are not limited to ZFN, TALEN and /or gene editing methods such as CRISPR/Cas9.
  • the target sequence used is selected from any one of the following groups:
  • sequence is SEQ ID NO: 4, 5, 7, 8, 13, 14, 16, 17, 22, 23, 25, 26, 28, 29, 34, 35, 37, 38, 40, 42, 43 , 44, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73 or 74 consistent with 5'-Nx -NGG-3' sequence is a regularly arranged fragment, where N represents any one of A, G, C and T, 14 ⁇ X ⁇ 30, and X is an integer, and Nx represents X consecutive nucleotides; or
  • the target sequence for Arabidopsis plants is as shown in any one of SEQ ID NO:76-79
  • the target sequence for rapeseed plants is as shown in SEQ ID NO: Any one of 80-83 is shown
  • the target sequence for soybean plants is shown in any one of SEQ ID NO: 84-87
  • the target sequence for rice plants is shown in any one of SEQ ID NO: 88-91
  • the target sequence for corn plants is shown in any one of SEQ ID NO: 92-95.
  • nucleotide sequence of the gene mutant described in the embodiment of the present application it is characterized in that the nucleotide sequence of the gene mutant is as shown in any one of SEQ ID NO: 97, 98, 100-105.
  • the embodiments of the present application also disclose a plant cell, tissue, organ or product that is not used as propagation material, characterized in that the plant cell, tissue, organ or product contains any of the gene mutant nuclei described above in the present application. nucleotide sequence.
  • the embodiments of this application also disclose any of the aforementioned methods and the application of the mutant materials obtained in breeding.
  • the application in breeding refers to obtaining the gene mutant through gene mutation or by crossing with the gene mutant material of the present application, so that the plant will have heavier and/or larger grains. Phenotype.
  • This application provides a plant grain size regulatory gene, which belongs to the PCFS family of genes.
  • the N-terminal of the amino acid sequence of the protein encoded by this gene has a conserved domain.
  • the sequence of this domain is Y ... L N X KP X IT X LTI X A ... E ... Q X LP L XX I...L...IH ( X represents any amino acid, ... represents more than any three random amino acid sequences, and the remaining letters represent specific amino acids).
  • the PCFS gene mutation provided in this application can make the grains of the plant larger, thereby increasing the yield of the plant and providing new genetic resources for high-yield crop breeding.
  • the PCFS family gene provided in this application can be used as a gene to control crop grain size, improve yield and quality, and be used in the improvement of crop varieties, helping to breed new crop varieties with high-quality traits.
  • the gene of this application can also be used in molecular marker technology to serve practical production applications such as large-grain and high-yield crop breeding.
  • the PCFS gene provided in this application has homologous genes in many plants such as Arabidopsis, rape, soybean, corn, and rice, and can be used not only for the breeding of the aforementioned plants, but also for the cultivation of new varieties of other plants.
  • Figure 1 is a diagram of the gene editing vector.
  • U6promoter represents the U6 promoter
  • U6terminal represents the terminator
  • target represents the gene editing target.
  • Figure 2 shows the PCR detection results of Arabidopsis gene-edited mutants.
  • Figure 3 shows the branching phenotypes and statistical results of different mutants of Arabidopsis PCFS family genes.
  • Figure 3A is a picture of the number of branches of the wild type and mutants grown for 50 days.
  • Figure 3B is a quantitative statistical chart of the number of branches. The abscissa is the wild type and each mutant, and the ordinate is the ratio of the number of branches in different plants.
  • Figure 4 shows the statistical results of grain phenotype and thousand-grain weight of different mutants of Arabidopsis PCFS family genes.
  • Figure 4A is a picture of mature seeds of the wild type and mutants taken under a dissecting microscope.
  • Figure 4B is a quantitative statistical chart of the thousand-grain weight. The abscissa is the wild type and each mutant, and the ordinate is the thousand-grain weight of the seeds. Analysis of significant differences. The mutants are significantly different from the wild type.
  • Figure 5 shows the phenotypes and statistical results of different mutants of Arabidopsis PCFS family genes under 300mM mannitol treatment.
  • Figure 5A is a phenotypic diagram after growing for 6 days in normal medium and then moving to 300mM mannitol medium for 8 days.
  • Figure 5B is a quantitative statistical diagram of cotyledon yellowing. The abscissa is the wild type and each mutant, and the ordinate is It is the ratio of cotyledon colors of different plants.
  • Figure 6 shows the grain phenotypes after PCFS family gene mutations in different crops.
  • Figure 6A is a comparison of the grain size of wild type rapeseed and its pcfs mutant
  • Figure 6B is a comparison of the grain size of wild type soybean and its pcfs mutant
  • Figure 6C is a comparison of the grain size of wild type rice and its pcfs mutant after shelling Figure
  • Figure 6D is a comparison of the grain sizes of wild-type maize and its pcfs mutant.
  • WT means wild type, and mutant means mutant.
  • Figure 7 shows the statistics of the 1,000-grain weight of PCFS family genes in different crops.
  • Figure 7A is the statistical result of the 1000-grain weight of the rapeseed wild type and its pcfs mutant;
  • Figure 7B is the statistical result of the 100-grain weight of the soybean wild type and its pcfs mutant;
  • Figure 7C is the statistical result of the 1000-grain weight of the rice wild type and its pcfs mutant;
  • Figure 7D is the statistical results of the 100-kernel weight of corn wild type and its pcfs mutant.
  • the protein sequence of yeast Pcf11 was used to compare homologous proteins in Arabidopsis on the Phytozome website (after entering the website, click Tools, click BLAST, enter the protein sequence of yeast Pcf11, and select the species Arabidopsis thaliana, click go), there are four homologous proteins in Arabidopsis, namely PCFS1, PCFS2, PCFS4 and PCFS5. Download the corresponding gene sequence, CDS sequence and protein sequence respectively from the TAIR website. Among them, the gene numbers corresponding to PCFS1, PCFS2, PCFS4 and PCFS5 are respectively AT1G66500, AT2G36480, AT4G04885 and AT5G43620.
  • the inventor plans to obtain Arabidopsis mutants of the above genes through CRISPR/Cas9 gene editing.
  • PAM defaults after entering the website, select U6 for snoRNA promoter, select 19 for guide sequence length, enter Arabidopsis thaliana, gene number or gene sequence, and click submit).
  • PAM defaults after entering the website, select U6 for snoRNA promoter, select 19 for guide sequence length, enter Arabidopsis thaliana, gene number or gene sequence, and click submit).
  • Such target editing is efficient and the purpose is to construct mutant plants.
  • a total of 4 targets were selected to target four PCFS family genes. The specific information of the 4 targets is shown in Table 1.
  • the method of constructing the target into the gene editing vector is as follows:
  • BsaI enzyme cutting sites on the gene editing vector, which can be digested with BsaI endonuclease. Use the enzyme cutting and connection system and conditions in Table 3. Since there are unpaired ends of the vector and the 4 gene synthesis fragments, Nucleotide fragments, using T4 ligase, can connect four gene synthesis fragments into the vector in a specific order based on the principle of complementary base pairing.
  • Table 1 List of PCFS family information in Arabidopsis thaliana
  • the gene editing vector has a hygromycin resistance gene in the plant.
  • the seeds of the transgenic plant that is, the transgenic T 1 generation, are recovered and spread on a plant medium containing hygromycin. Plants that can grow normally are considered successfully transgenic plants. Take a small leaf of a successfully transgenic plant, extract DNA, and use the primers in Table 4 below to specifically amplify the PCFS family genes by PCR. When the PCR amplification product is electrophoresed, the agarose gel shows that the position is different from that of the wild type. The plants with the type of strips are the mutant plants expected by this application. The specific electrophoresis results are shown in Figure 2.
  • the obtained transgenic T 1 -generation mutant plants were crossed with the wild type. Because the transgenic site is heterozygous, the obtained hybrid Some of the 1st generation plants of cross F contain the transgene insertion and some do not. Use the primers in Table 5 below to perform PCR specific amplification of the Cas gene on the transgenic vector, and determine whether the vector has been screened out based on the presence or absence of the band. Those with no amplified bands are plants whose vectors have been screened out. At this time, the PCFS gene of the plant is in a heterozygous state. After another generation of selfing, pcfs-related mutants whose vectors have been screened out can be obtained.
  • pcfs1 is a deletion of nucleotide sequence 178-886 on the DNA sequence of PCFS1.
  • the nucleotide sequence of the mutated gene is shown in SEQ ID NO:96. This mutation causes the 60th amino acid of the encoded protein to begin to change. A stop codon is encountered at position 64.
  • pcfs2 is a deletion of the 104-293 nucleotide sequence on the DNA sequence of PCFS2, and at the same time, a 6 bp nucleotide is inserted.
  • the mutated gene nucleotide sequence is shown in SEQ ID NO:97. This mutation leads to the 35th position of the encoded protein. The amino acid at position 46 begins to change and a stop codon is encountered at position 46.
  • pcfs4 is a deletion of the 867-918th nucleotide sequence in the PCFS4 DNA sequence.
  • the mutated gene nucleotide sequence is shown in SEQ ID NO:98. This mutation causes the 206th amino acid of the encoded protein to begin to change. A stop codon is encountered at position 210.
  • pcfs5 is a deletion of nucleotide sequence 178-886 on the DNA sequence of PCFS5.
  • the nucleotide sequence of the mutated gene is shown in SEQ ID NO:99. This mutation causes the 60th amino acid of the encoded protein to begin to change. A stop codon is encountered at position 64.
  • the above results indicate that the mutation affects the normal expression of the gene protein, making the expressed protein polypeptide shorter and
  • the pcfs1 and pcfs5 mutants did not have the phenotype of larger and heavier grains or fewer branches.
  • N-termini Some PCFS family members lack the N-terminus, but the N-termini of members with complete N-termini are also conserved.
  • the amino acid sequence of the N-terminus is Y...L X ELT X N X KP X IT X LTI X A...E... Q X LP X LYLLDSIVKN X G XX Y ... F ... L XX VF XX AY ...M XX LF out of the complete N-terminal plant species (100% match).
  • PCFS2 and PCFS4 possess N-terminal conserved sequences, which show the phenotype of larger grains after mutation; PCFS1 and PCFS5 lack the N-terminus, and their mutations are the same as wild type. There is no obvious difference. Therefore, for gene editing in crops such as rape, soybean, corn, and rice, the inventors preferentially select to knock out PCFS family members with intact N-termini.
  • Arabidopsis and rapeseed are both Brassicaceae plants.
  • Source protein after entering the website, click Tools, then click Blast, and follow the prompts to enter the name of the Arabidopsis PCFS family gene to get the homologous sequence in rapeseed).
  • Brassica napus is an allodiploid, with a total of 8 PCFS families.
  • Homologous proteins 6 of which have complete The N-terminals are BnaA04G0223600WE, BnaA05G0075100WE, BnaA09G0222400WE, BnaC04G0102200WE, BnaC04G0535800WE and BnaC09G0254600WE respectively.
  • Their genomic DNA sequences are as follows: SEQ ID NO: 13, 16, 22, 25, 2 8 and 34, the CDS sequences are as follows: SEQ ID NOs: 14, 17, 23, 26, 29, and 35, and their protein amino acid sequences are shown in SEQ ID NOs: 15, 18, 24, 27, 30, and 36 respectively.
  • the target sequences and their corresponding sequence numbers are shown in Table 6 below.
  • double mutants BnaA05G0075100WE and BnaA09G0222400WE were obtained.
  • the 108-109 nucleotide position of the DNA sequence of the BnaA09G0222400WE gene of this mutant is deleted.
  • the nucleotide sequence of the mutated gene is shown in SEQ ID NO: 100.
  • the mutation causes the protein sequence to start at amino acid 37. A change occurred, a stop codon was encountered at amino acid position 78, and a nucleotide was inserted at position 110 of the DNA sequence of the BnaC09G0254600WE gene.
  • the mutated gene nucleotide sequence is shown in SEQ ID NO: 101. This mutation causes The protein sequence began to change at amino acid position 37, and encountered a stop codon at amino acid position 42, indicating that the mutation affected the normal expression of the gene protein, making the expressed protein polypeptide shorter and inhibiting the normal function of the protein.
  • the grain size of the wild type and the mutant was observed, and it was found that the mutant had a phenotype of larger grains (as shown in Figure 6A), and the thousand-grain weight increased by 20% compared to the wild type (as shown in Figure 7A).
  • Table 6 List of PCFS family information in rapeseed
  • the gene numbers of PCFS family homologous genes in soybean, corn, and rice are shown in Table 7 below.
  • the complete N-terminal gene numbers in soybean are Glyma.03G191200, Glyma.10G066300, Glyma.10G251100, Glyma.19G191800, Glyma.19G191900, Glyma.20G142500.
  • the gene sequence, CDS sequence and protein sequence were obtained from the website SoyBase (https ://www.soybase.org/).
  • the genomic DNA sequences are shown in SEQ ID NO: 37, 40, 43, 49, 52 and 55 respectively, and the CDS sequences are shown in 38, 41, 44, 50, 53 and 56 respectively.
  • the protein amino acid sequences are shown in SEQ ID NO: 39, 42, 45, 51, 54 and 57 respectively.
  • the gene editing target sites designed in this application for PCFS family homologous genes in soybeans are shown in SEQ ID NO: 84-87 (Table 7).
  • the PCFS family homologous genes in rice are LOC_Os08g08830 and LOC_Os09g39270, and their gene sequences, CDS sequences and protein sequences are obtained from the website Phytozome (https://phytozome-next.jgi.doe.gov/). Its genomic DNA sequence is shown in SEQ ID NO: 58 and 61 respectively, its CDS sequence is shown in SEQ ID NO: 59 and 62 respectively, and its protein amino acid sequence is shown in SEQ ID NO: 60 and 63 respectively.
  • the gene editing target sites designed in this application for homologous genes of the rice PCFS family are shown in SEQ ID NO: 88-91 (Table 7).
  • the PCFS family homologous genes in corn are Zm00001d000023, Zm00001d005350, Zm00001d019856, and Zm00001d049442. Their gene sequences, CDS sequences, and protein sequences are obtained from the website Phytozome (https://phytozome-next.jgi.doe.gov/). Its genomic DNA sequence is shown in SEQ ID NO:64, 67, 70, and 73, its CDS sequence is shown in SEQ ID NO: 65, 68, 71, and 74, respectively, and its protein amino acid sequence is shown in SEQ ID NO :66, 69, 72, 75.
  • the gene editing target sites designed in this application for the corn PCFS family homologous genes are shown in SEQ ID NO: 92-95 (Table 7).
  • mutant plants of the above crops were obtained.
  • a mutant of the Glyma.10G251100 gene was obtained in soybean.
  • the 193rd nucleotide of the mutant's DNA sequence was deleted.
  • the nucleotide sequence of the mutated gene is shown in SEQ ID NO:102. This mutation resulted in a protein sequence The change begins at amino acid position 65, and a stop codon is encountered at amino acid position 80; a mutant of the LOC_Os08g08830 gene was obtained in rice.
  • the nucleotides 203-4604 of the mutant's DNA sequence were deleted, and its mutated
  • the nucleotide sequence of the gene is shown in SEQ ID NO: 103.
  • This mutation causes the deletion of amino acids 71-449 in the protein sequence; a mutant with simultaneous mutations of Zm00001d000023 and Zm00001d049442 was obtained in corn.
  • the DNA sequence of the Zm00001d000023 gene of this mutant The 1318-1609 nucleotide sites of the gene are deleted.
  • the nucleotide sequence of the mutated gene is shown in SEQ ID NO:104. This mutation causes the protein sequence to start to change at amino acid position 103 and terminate at amino acid position 126. codon, and at the same time, nucleotide positions 1789-2081 of the DNA sequence of the Zm00001d049442 gene are deleted.
  • the nucleotide sequence of the mutated gene is shown in SEQ ID NO: 105, causing the protein sequence to start to change at amino acid position 103.
  • a stop codon is encountered at amino acid position 116.
  • the N-terminal complete PCFS family genes have the function of regulating grain size in crops such as rape, rice, soybeans, and corn. In rapeseed and corn, the N-terminal complete PCFS family genes mutate and the grains change. Large, its 1,000/100 grain weight increases by about 20%; in rice and soybeans, the grains also become larger after the N-terminal complete PCFS family gene mutation, and the 1,000/100 grain weight increases by about 10%. This example further proves that the complete N-terminal PCFS family gene has the function of regulating plant grain size.
  • inhibiting the normal function of the gene protein through mutation and other technical means By inhibiting the normal function of the gene protein described in this application includes affecting the normal function of the gene protein through mutation, and also includes affecting the regulatory factors of the gene protein, thereby inhibiting the N-terminus of the application.
  • the normal function of the complete PCFS family genes results in the phenotype of larger plant grains.

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Abstract

公开了一种植株籽粒大小调控基因及其应用，属于生物技术领域。通过基因编辑的方式，获得不同作物中的PCFS基因突变体，并通过实验及统计分析发现所述突变体具有植株籽粒变大的表型，部分突变体具有耐胁迫能力提高的表型。所提供的基因、突变体及其应用方法，有助于提高作物产量和改善品质，增强植株对逆境的抗性，为培育具有大粒重、抗逆性强的植物新品种提供了基因资源和技术支持，对作物农艺性状的改良和高产、强抗逆性分子育种工作具有重要的意义和应用价值。

Description

一种植株籽粒大小调控基因及其应用
本申请要求于2022年8月19日提交中国专利局、优先权号为202210999291.4、发明名称为“一种植株籽粒大小调控基因及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于植物生物技术育种领域，具体涉及一种PCFS家族基因，及通过突变该基因来获得农艺性状改良植株的方法。
背景技术
人口持续增长与耕地面积不断减少的现状，对全球粮食安全提出了严峻的挑战。如何通过生物技术的手段，挖掘优质基因资源，应用于作物育种，从而获得高产量、高质量、利于机械化耕作的作物品种，是全人类的重要课题。
植物籽粒大小是农业生产中重点关注的性状，直接影响农作物的产量，水稻、小麦、玉米等主粮作物的籽粒变大可以提高粮食产量，油菜，大豆等油料作物的籽粒变大可以提高产油量，对于促进农业生产至关重要。此外，农作物在生长的过程中，常常受到恶劣的环境的影响，比如干旱、盐胁迫、病虫灾害等，因此，选取抗旱、抗盐、抗病虫害等抗性强的品种，可以有效提高植物的抗性和适应力，以达到高产的目的。对于油菜具有的分枝多且分枝上的果荚成熟晚，机器常常不能收获到侧枝上籽粒的问题，如何获得合理株型也是作物育种中十分关注的课题。由以上可知，不论是籽粒变大，分枝变少，还是对渗透胁迫的抗性增强，都是农业上的有利性状，对于提高作物产量、发展现代化农业非常重要。
PCFS(Pcf11-SIMILAR PROTEIN)同源基因最早报道是在酵母中，酵母中的基因名称为Pcf11(protein 1 of CF I)，使用酵母Pcf11的蛋白序列进行基因比对，可在植物中找到相关的同源蛋白。在高等植物中，PCFS家族一般有两个或两个以上的同源基因。在此之前，很少有人关注植物中PCFS家族的基因功能。本申请的发明人在探索PCFS家族的基因功能时，通过CRISPR/Cas9技术对拟南芥、油菜、玉米、大豆、水稻等植株的PCFS家族基因进行编辑，拿到突变体后，出乎意料的发现N端完整的PCFS家族基因的突变，可以调控植株的籽粒大小，使植株的籽粒变大变重，同时部分PCFS基因突变还有增强植株对逆境胁迫的耐受性、减少分枝数的功能。本申请实施例所提供的基因、突变体及其应用方法，有助于提高作物产量和改善品质，增强植株对逆境的抗性，为培育具有大粒重、抗逆性强的植物新品种提供了基因资源和技术支持，对作物农艺性状的改良和高产、强抗逆性分子育种工作具有重要的意义和应用价值。
发明内容
本文提到的所有参考文献都通过引用并入本文。除非有相反指明,本文所用的所有技术和科学术语都具有与本申请所属领域普通技术人员通常所理解的相同的含义。除非有相反指明,本文所使用的或提到的技术是本领域普通技术人员公知的标准技术。材料、方法和例子仅作阐述用,而非加以限制。
本申请实施例提供了一种基因及其突变体核苷酸序列，其特征在于，含有所述基因突变的植株，其籽粒变大，所述基因的核苷酸序列选自下列组的序列之一：
(a)拟南芥中基因号为AT2G36480或AT4G04885的核苷酸序列；
(b)油菜中基因号为BnaA04G0223600WE、BnaA05G0075100WE、BnaA09G0222400WE、BnaC04G0102200WE、BnaC04G0535800WE或BnaC09G0254600WE的核苷酸序列；
(c)大豆中基因号为Glyma.03G191200、Glyma.10G066300、Glyma.10G251100、Glyma.19G191800、Glyma.19G191900或Glyma.20G142500的核苷酸序列；
(d)水稻中基因号为LOC_Os08g08830或LOC_Os09g39270的核苷酸序列；
(e)玉米中基因号为Zm00001d000023、Zm00001d005350、Zm00001d019856或Zm00001d049442的核苷酸序列；
(f)在严谨条件下能够与(a)-(e)之任一所述核苷酸序列杂交的核苷酸序列；
(g)与(a)-(f)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且在植株中突变后具有使植株籽粒变大功能的核苷酸序列；或
(h)与(a)-(g)之任一所述核苷酸序列互补的核苷酸序列。
本申请实施例提供了一种基因及其突变体核苷酸序列，所述基因突变后具有植株籽粒变大和/或变重的表型，所述基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：4、5、7、8、13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、62、64、65、67、68、70、71、73或74所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：6、9、15、18、24、27、30、36、39、42、45、51、54、57、60、63、66、69、72或75所示的核苷酸序列；
(c)在严谨条件下能够与(a)或(b)中所述序列杂交的核苷酸序列；
(d)与(a)-(c)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且突变后具有使植株籽粒变大和/或变重功能的核苷酸序列；或
(e)与(a)-(d)之任一所述序列互补的核苷酸序列。
本申请实施例中提供的上述基因可从各种植物中分离获得。本领域技术人员应该知晓， 本申请所述的基因还包括与该基因的核苷酸序列或蛋白序列高度同源，并且突变后具有同样的籽粒变大和/或变重功能的同源基因。所述同源基因包括在严谨条件下能够与本申请实施例所公开的基因的核苷酸序列杂交的DNA序列。本文中使用的“严谨条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和1mM EDTA的杂交液中杂交，所述杂交的温度优选是53℃-60℃，杂交时间优选为12-16小时，然后用含0.5×SSC和0.1％SDS的洗涤液洗涤，洗涤温度优选为62℃-68℃，洗涤时间为15-60分钟。
同源基因还包括与本申请实施例所公开的基因所示的DNA序列有至少80％、85％、90％、95％、98％、99％或更高序列相似性，或者与本申请实施例所公开的功能基因的氨基酸序列有至少80％、85％、90％、95％、98％、99％或更高序列相似性，且突变后具有使植株籽粒变大和/或变重功能的DNA序列，可以从任何植物中分离获得。更具体地，所述PCFS家族基因DNA序列编码的蛋白氨基酸序列具有完整的N端，所述N端的氨基酸序列为Y...LXELTXNXKPXITXLTIXA...E...QXLPXLYLLDSIVKNXGXXY...F...LXXVFXXAY...MXXLFXTWXXVF...LXXI...L...IH(X代表任一氨基酸，...代表超过任意三个随机氨基酸序列，其余字母代表特定氨基酸)。其中，序列相似性的百分比可以通过公知的生物信息学算法来获得，包括Myers和Miller算法、Needleman-Wunsch全局比对法、Smith-Waterman局部比对法、Pearson和Lipman相似性搜索法、Karlin和Altschul的算法，这对于本领域技术人员来说是公知的。
本领域技术人员应该知晓，同一种植物不同品种间的同一基因存在单核苷酸多样性(single nucleotide polymorphism,SNP)，即同一基因的核苷酸序列往往存在个别碱基的差异，但同一作物品种数量很多，发明人不可能进行一一列举，本申请仅提供了不同作物中具有代表性的品种的序列。因此，本领域技术人员应该知悉，不同品种来源的与本申请所保护基因及其核苷酸序列存在SNP的核苷酸序列也在本申请的保护范围内。
本申请实施例所述的突变是指在本申请基因的核苷酸序列上进行取代、缺失和/或添加一个或多个核苷酸获得，所述基因突变后具有改善农艺性状的功能，包括使植株籽粒变重和/或变大，其中pcfs4(基因编号为AT4G04885，核苷酸序列如SEQ ID NO:7或8所示)单基因突变或是pcfs2和pcfs4双突之后，还具有增加耐逆性和/或减少分枝数等的调控功能。本申请实施例所述的突变可以是点突变，也可以是DNA缺失或***突变。所述突变可以通过物理诱变、化学诱变或基因编辑的方式获得,化学诱变的方法包括用EMS等诱变剂处理所导致的诱变；基因编辑的方法包括但不限于ZFN、TALEN和/或CRISPR/Cas9等基因编辑方法。
更具体地，在通过CRISPR/Cas9基因编辑方法获得本申请实施例中基因突变体核苷酸序列时，所述CRISPR/Cas9基因编辑方法所用的靶点序列选自下列组的序列之一:
(a)序列为SEQ ID NO：4、5、7、8、13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、62、64、65、67、68、70、71、73或74所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段，其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸；或
(b)与(a)所述的多核苷酸互补的多核苷酸。
更具体地，在使用CRISPR/Cas9技术进行基因编辑时，针对拟南芥植株的靶点序列如SEQ ID NO:76-79之任一所示，针对油菜植株的靶点序列如SEQ ID NO:80-83之任一所示，针对大豆植株的靶点序列如SEQ ID NO:84-87之任一所示，针对水稻植株的靶点序列如SEQ ID NO:88-91之任一所示，针对玉米植株的靶点序列如SEQ ID NO:92-95之任一所示。
根据本申请实施例所述的基因突变体核苷酸序列，其特征在于，所述基因突变体的核苷酸序列如SEQ ID NO:97、98、100-105之任一所示。
本申请实施例还公开了一种调控植株农艺性状的方法，其特征在于通过突变植株的下述基因，使植株籽粒变大变重，所述基因的核苷酸序列选自下列组的序列之一：
(a)拟南芥中基因号为AT2G36480或AT4G04885的核苷酸序列；
(b)油菜中基因号为BnaA04G0223600WE、BnaA05G0075100WE、BnaA09G0222400WE、BnaC04G0102200WE、BnaC04G0535800WE或BnaC09G0254600WE的核苷酸序列；
(c)大豆中基因号为Glyma.03G191200、Glyma.10G066300、Glyma.10G251100、Glyma.19G191800、Glyma.19G191900或Glyma.20G142500的核苷酸序列；
(d)水稻中基因号为LOC_Os08g08830或LOC_Os09g39270的核苷酸序列；或玉米中基因号为Zm00001d000023、Zm00001d005350、Zm00001d019856或Zm00001d049442的核苷酸序列；
(e)在严谨条件下能够与(a)-(e)之任一所述核苷酸序列杂交的DNA序列；或
(f)与(a)-(f)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且在植株中突变后具有使植株籽粒变大功能的核苷酸序列；或
(g)与(a)-(f)之任一所述核苷酸序列互补的DNA序列。
具体地，本申请实施例所公开的调控植株农艺性状的方法，其特征在于通过突变植株的下述基因，使植株籽粒变大和/或变重，所述基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：4、5、7、8、13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、62、64、65、67、68、70、71、73或74所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：6、9、15、18、24、27、30、36、39、42、45、51、54、57、60、63、66、69、72或75所示的核苷酸序列；
(c)在严谨条件下能够与(a)或(b)中所述序列杂交的核苷酸序列；
(d)与(a)-(c)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且在植株中突变后具有使植株籽粒变大功能的核苷酸序列；或
(e)与(a)-(d)之任一所述序列互补的DNA序列。
本申请实施例中提供的调控植株农艺性状的方法，其中所述的基因可从各种植物中分离获得。本领域技术人员应该知晓，本申请所述的调控植株农艺性状的方法中，所使用的基因还包括与该基因的核苷酸序列或蛋白序列高度同源，并且突变后具有同样的籽粒变大变重功能的同源基因。所述同源基因包括在严谨条件下能够与本申请实施例所公开的基因的核苷酸序列杂交的DNA序列。本文中使用的“严谨条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和1mM EDTA的杂交液中杂交，所述杂交的温度优选是53℃-60℃，杂交时间优选为12-16小时，然后用含0.5×SSC和0.1％SDS的洗涤液洗涤，洗涤温度优选为62℃-68℃，洗涤时间为15-60分钟。
同源基因还包括与本申请实施例所公开的PCFS家族基因所示的DNA序列有至少80％、85％、90％、95％、98％或99％序列相似性，且突变后具有同样的籽粒变大和/或变重功能的DNA序列，可以从任何植物中分离获得。更具体地，所述PCFS家族基因DNA序列编码的蛋白氨基酸序列具有完整的N端，所述N端的氨基酸序列为Y...LXELTXNXKPXITXLTIXA...E...QXLPXLYLLDSIVKNXGXXY...F...LXXVFXXAY...MXXLFXTWXXVF...LXXI...L...IH(X代表任一氨基酸，...代表超过任意三个随机氨基酸序列，其余字母代表特定氨基酸)。其中，序列相似性的百分比可以通过公知的生物信息学算法来获得，包括Myers和Miller算法、Needleman-Wunsch全局比对法、Smith-Waterman局部比对法、Pearson和Lipman相似性搜索法、Karlin和Altschul的算法，这对于本领域技术人员来说是公知的。
本申请实施例所述调控植株农艺性状的方法中，其中所述的突变是指在本申请所披露基因的核苷酸序列上进行取代、缺失和/或添加一个或多个核苷酸获得，所述基因突变后具有改善农艺性状的功能，包括使植株籽粒变重和/或变大，其中pcfs4(基因号为AT4G04885，核苷酸序列如SEQ ID NO:7或8所示)单基因突变或是pcfs2和pcfs4双基因突变之后，还具有增加耐逆性和/或减少分枝数等的调控功能。本申请实施例所述的突变可以是点突变，也可以是DNA缺失或***突变。所述突变可以通过物理诱变、化学诱变或基因编辑的方式获得,化学诱变的方法包括用EMS等诱变剂处理所导致的诱变；基因编辑的方法包括但不限于ZFN、TALEN和/或CRISPR/Cas9等基因编辑方法。
更具体地，在使用CRISPR/Cas9技术进行植株农艺性状调控时，所用的靶点序列选自下列组的任一序列之一:
(a)序列为SEQ ID NO：4、5、7、8、13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、62、64、65、67、68、70、71、73或74所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段，其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸；或
(b)与(a)所述的多核苷酸互补的多核苷酸。
更具体地，在使用CRISPR/Cas9技术进行基因编辑时，针对拟南芥植株的靶点序列如SEQ ID NO:76-79之任一所示，针对油菜植株的靶点序列如SEQ ID NO:80-83之任一所示，针对大豆植株的靶点序列如SEQ ID NO:84-87之任一所示，针对水稻植株的靶点序列如SEQ ID NO:88-91之任一所示，针对玉米植株的靶点序列如SEQ ID NO:92-95之任一所示。
根据本申请实施例所述的基因突变体核苷酸序列，其特征在于，所述基因突变体的核苷酸序列如SEQ ID NO:97、98、100-105之任一所示。
本申请实施例还公开了一种非作为繁殖材料的植物细胞、组织、器官或产品，其特征在于，所述植物细胞、组织、器官或产品含有本申请前述任一所述的基因突变体核苷酸序列。
本申请实施例还公开了前述任一所述的方法、及其获得的突变体材料在育种中的应用。具体地，所述在育种中的应用是指通过基因突变的方式，或是通过与本申请基因突变体材料杂交的方式，获得该基因突变体，使植株呈现籽粒变重和/或变大的表型。
与相关技术相比,本申请具有如下有益效果:
1)本申请提供了一种植株籽粒大小调控基因,该基因属于PCFS家族基因，该基因编码的蛋白氨基酸序列的N端具有保守结构域，该结构域的序列为Y...LXELTXNXKPXITXLTIXA...E...QXLPXLYLLDSIVKNXGXXY...F...LXXVFXXAY...MXXLFXTWXXVF...LXXI...L...IH(X代表任一氨基酸，...代表超过任意三个随机氨基酸序列，其余字母代表特定氨基酸)。
2)本申请所提供的PCFS基因突变后可使植株的籽粒变大,从而增加植株的产量,为作物的高产育种提供了新的基因资源。
3)本申请提供的PCFS家族基因可以作为控制作物籽粒大小、提高产量和品质的一个基因,应用于作物品种的改良,有助于选育出优质性状的作物新品种。同时,本申请基因还可用于分子标记技术,为作物大粒高产育种等实际生产应用服务。
4)本申请提供的PCFS基因在拟南芥、油菜、大豆、玉米、水稻等众多植物中具有同源基因,不仅可以用于前述植物的育种,还可用于其它植物的新品种培育。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图，其中:
图1是基因编辑载体图。其中U6promoter表示U6启动子，U6terminal表示终止子，target表示基因编辑靶点。
图2是拟南芥基因编辑突变体的PCR检测结果。
图3是拟南芥PCFS家族基因不同突变体的分枝表型及统计结果。图3A为生长50天的野生型及突变体分枝数量图片，图3B为分枝数量量化统计图，横坐标是野生型及各个突变体，纵坐标为不同植株分枝个数的比例。
图4是拟南芥PCFS家族基因不同突变体籽粒表型及千粒重统计结果。图4A为野生型及突变体成熟后的籽粒在解剖镜下所拍的图片，图4B为千粒重的量化统计图，横坐标是野生型及各个突变体，纵坐标为籽粒的千粒重，差异显著分析显示突变体与野生型极显著。
图5是拟南芥PCFS家族基因不同突变体在300mM甘露醇处理下的表型及统计结果。图5A为在正常培养基下生长6天后移到300mM甘露醇培养基上生长8天后的表型图，图5B为子叶变黄的量化统计图，横坐标是野生型及各个突变体，纵坐标是不同植株子叶颜色的比例。
图6是不同作物PCFS家族基因突变后的籽粒表型。图6A是油菜野生型与其pcfs突变体的籽粒大小对比图；图6B是野生型大豆与其pcfs突变体的籽粒大小对比图；图6C是水稻野生型与其pcfs突变体的籽粒脱壳后的大小对比图；图6D是野生型玉米与其pcfs突变体的籽粒大小对比图。WT表示野生型，mutant表示突变体。
图7是不同作物PCFS家族基因突变后的籽粒千粒重统计。图7A是油菜野生型与其pcfs突变体的籽粒千粒重统计结果；图7B是大豆野生型与其pcfs突变体的籽粒百粒重统计结果；图7C是水稻野生型与其pcfs突变体的籽粒千粒重统计结果；图7D是玉米野生型与其pcfs突变体的籽粒百粒重统计结果。
具体实施方式
以下实施例用于说明本申请,但不用来限制本申请的范围。在不背离本申请精神和实质的情况下,对本申请方法、步骤或条件所作的修改或替换,均属于本申请的范围。若未特别指明,实施例中所用的材料、生物化学试剂均为常规市售试剂,实施例中所用的技术手段为本领域技术人员熟知常规手段。
实施例1、构建拟南芥PCFS突变体植物
为了研究植物中PCFS家族基因的功能，利用酵母Pcf11的蛋白序列，在Phytozome网站上比对拟南芥中的同源蛋白(进入网站后点击Tools，点击BLAST，输入酵母Pcf11的蛋白序列，选择物种Arabidopsis thaliana，点击go)，拟南芥中的同源蛋白共有4个，分别为PCFS1、PCFS2、PCFS4和PCFS5。在TAIR网站上分别下载相应的基因序列、CDS序列和蛋白序列。其中PCFS1、PCFS2、PCFS4和PCFS5对应的基因号依次分别为AT1G66500、AT2G36480、AT4G04885、AT5G43620，其基因组DNA序列依次分别如SEQ ID NO:1、4、7、10所示,其CDS(编码区序列)序列依次分别如SEQ ID NO:2、5、8、11所示，其蛋白氨基酸序列依次分别如SEQ ID NO:3、6、9、12所示。
发明人拟通过CRISPR/Cas9基因编辑的方式获得上述基因的拟南芥突变体。先利用CRISPR-P V2.0网站预测基因编辑的高效靶点(进入网站后PAM默认，snoRNA promoter选U6，guide sequence length选19，输入Arabidopsis thaliana、基因号或者基因序列，点击submit)，在给出的预测靶点中选取分高且靶点位于外显子上的靶点，这样的靶点编辑高效，其目的是构建突变体植株。本实施例一共选取了4个靶点靶向四个PCFS家族基因，4个靶点的具体信息如表1所示。将这4个靶点一起构建到PHEE401E载体(该载体购买自addgene(https://www.addgene.org/)，该载体信息参考文章：Wang ZP,Xing HL,Dong L,et al.Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation.Genome Biol.2015；16(1):144.)，转化到大肠杆菌和农杆菌中，利用农杆菌浸花法将载体转化到Col-0背景的野生型拟南芥中。
具体地，将所述靶点构建到基因编辑载体的方法如下：
1)针对下表1中的拟南芥PCFS家族的四个基因，选择四个靶点，人工合成4条核苷酸产物，如下表2所示，4条核苷酸产物分别包含四个靶点信息，其核苷酸序列分别如SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109所示，其中靶点在表2中用下划线标注。
2)基因编辑载体上有2个BsaⅠ酶切位点，可用BsaⅠ内切酶酶切，使用表3的酶切连接体系及条件，由于载体和4条基因合成片段的5’端有未配对的核苷酸片段，利用T4连接酶，根据碱基互补配对的原则可以将4条基因合成片段按照特定的顺序连入载体。
表1：拟南芥中PCFS家族信息列表

表2：基因合成片段

表3：酶切连接体系及条件
基因编辑载体在植物中具有潮霉素抗性基因，收回转基因植株的籽粒，即转基因T1代，将其铺在有潮霉素的植物培养基上，能正常生长的为转基因成功的植株。取一小片转基因成功植物的叶片，提取DNA，利用下表4中的引物对PCFS家族基因进行PCR特异扩增，在对PCR扩增产物进行电泳时，琼脂糖凝胶上表现为位置不同于野生型的条带的植株为本申请所预期的突变体植株，具体电泳结果如图2所示。
表4：鉴定拟南芥突变体引物
将获得的转基因T1代突变体植株与野生型杂交，因为转基因位点是杂合，所以获得的杂 交F1代植株部分含有转基因***，部分不含有。利用下表5中的引物对转基因载体上的Cas基因进行PCR特异扩增，根据条带的有无判断载体是否被筛出。没有扩增条带的为载体被筛掉的植株，此时植株的PCFS基因为杂合状态，再自交1代则可以获得载体被筛掉的pcfs相关突变体。
表5：鉴定载体是否筛出植物体外引物
通过以上实验，获得了PCFS家族的相关突变体，分别命名为pcfs1，pcfs2，pcfs4，pcfs5。pcfs1为PCFS1的DNA序列上178-886位核苷酸序列缺失，其突变后的基因核苷酸序列如SEQ ID NO:96所示，该突变导致编码蛋白的第60位氨基酸开始发生改变，在64位碰到终止密码子。pcfs2为PCFS2的DNA序列上104-293位核苷酸序列缺失，同时***6bp核苷酸，其突变后的基因核苷酸序列如SEQ ID NO:97所示，该突变导致编码蛋白的第35位氨基酸开始发生改变，在46位碰到终止密码子。pcfs4为PCFS4的DNA序列上867-918位核苷酸序列缺失，其突变后的基因核苷酸序列如SEQ ID NO:98所示，该突变导致编码蛋白的第206位氨基酸开始发生改变，在210位碰到终止密码子。pcfs5为PCFS5的DNA序列上178-886位核苷酸序列缺失，其突变后的基因核苷酸序列如SEQ ID NO:99所示，该突变导致编码蛋白的第60位氨基酸开始发生改变，在64位碰到终止密码子。前述结果说明所述突变影响了所述基因蛋白的正常表达，使得其表达的蛋白多肽变短，抑制了蛋白的正常功能。
实施例2、观察拟南芥分枝表型及籽粒大小的表型
将同批次收到的野生型和突变体籽粒分别用15％的次氯酸钠溶液消毒10分钟，用灭过菌的双蒸水洗2遍，然后点在1/2MS植物培养基上，放进光照培养箱，在长日照(16小时光照21℃/8小时黑暗19℃)的条件下生长2周，然后将萌发的幼苗移到土里，放进温室，培养条件不变，2天浇一次水。
等到拟南芥抽薹后3个星期，株型完全确定，统计分枝数目。由图3可知，pcfs2突变体的分枝个数相比于野生型没有明显变化，而pcfs4突变体的分枝个数相比于野生型是减少的，双突的表型与pcfs4相似。本实验说明pcfs4基因突变还有减少植株分枝数的功能。
等到拟南芥籽粒完全黄透后收获籽粒，利用干燥球干燥两天后，在显微镜下观察籽粒大小及利用千分之一天平称量千粒重。由图4A可知，pcfs2、pcfs4及pcfs2pcfs4双突突变体的籽粒都比野生型的籽粒大。由图4B可知，pcfs2和pcfs4的千粒重相比野生型也都是增加 的，增加的幅度分别为21％和22％，双突的籽粒变大表型加剧，千粒重提高了70％。
与此相比，pcfs1和pcfs5的突变体，二者都没有籽粒变大变重、或是分枝数变少的表型。
实施例3、观察拟南芥抗逆表型
将同批次收到的野生型和突变体籽粒用15％的次氯酸钠溶液消毒10分钟，用灭过菌的双蒸水洗2遍，然后点在竖直1/2MS植物培养基上，放进光照培养箱，在全日照(24小时光照21℃)的条件下生长6天，而后转到含有300mM甘露醇(模拟渗透胁迫，强度约为-1.0MPa)的水平1/2MS植物培养基上生长8天，统计子叶变黄的比例。由图5可知，pcfs2的抗性相对野生型没有明显差异，而pcfs4的存活率明显提高，同时pcfs2pcfs4双突的存活率大幅度提升。本实验说明pcfs4基因突变还具有耐胁迫的功能，而pcfs2和pcfs4同时突变的植株，由于其功能冗余，具有更强的耐胁迫性。
实施例4、不同作物中PCFS家族同源序列比对
由不同植物PCFS家族成员的氨基酸序列比对的结果可以知道，拟南芥，油菜，大豆，玉米，水稻等植物PCFS家族成员的C端的锌指结构域保守，拥有L...CXXC...E...H...EXQXXCXLCXE...W...IXHXXC(X代表任一氨基酸，...代表超过任意三个随机氨基酸序列，其余字母代表特定氨基酸)保守结构域，(以上序列在给出的植物物种中100％匹配)。运用SMART网站(http://smart.embl-heidelberg.de/)和InterProScan网站(http://www.ebi.ac.uk/interpro/search/sequence/)都可以预测到以上所提物种的锌指结构域。部分PCFS家族成员缺失N端，但拥有完整N端的成员的N端也是保守的，N端的氨基酸序列为Y...LXELTXNXKPXITXLTIXA...E...QXLPXLYLLDSIVKNXGXXY...F...LXXVFXXAY...MXXLFXTWXXVF...LXXI...L...IH(以上序列在给出的N端完整植物物种中100％匹配)。
通过拟南芥的前述实验结果及基因序列的结构分析，发现PCFS2和PCFS4拥有N端保守序列，其突变后表现出籽粒变大的表型；PCFS1和PCFS5缺失N端，其突变后和野生型相比没有明显差异。因此，在油菜、大豆、玉米、水稻等作物中的基因编辑，发明人优先选择敲除拥有完整N端的PCFS家族成员。
实施例5、构建油菜PCFS突变体植物
拟南芥和油菜都是十字花科的植物，利用拟南芥PCFS家族的蛋白序列，在BnTIR(http://yanglab.hzau.edu.cn/BnTIR/download)网站上比对油菜中的同源蛋白(进入网站后点击Tools，然后点击Blast，按照提示输入拟南芥PCFS家族基因名称就能得到在油菜中的同源序列)，甘蓝型油菜是异源二倍体，共有8个PCFS家族同源蛋白，其中6个拥有完整 的N端，分别为BnaA04G0223600WE、BnaA05G0075100WE、BnaA09G0222400WE、BnaC04G0102200WE、BnaC04G0535800WE和BnaC09G0254600WE，其基因组DNA序列依次分别如SEQ ID NO:13、16、22、25、28、34所示，其CDS序列依次分别如SEQ ID NO:14、17、23、26、29、35所示，其蛋白氨基酸序列依次分别如SEQ ID NO:15、18、24、27、30、36所示。
发明人利用基因中的高度同源序列设计4个靶点靶向这6个基因，其靶点序列及其对应的序列编号如下表6所示。经转基因筛选，获得了BnaA05G0075100WE和BnaA09G0222400WE的双突突变体。该突变体的BnaA09G0222400WE基因的DNA序列的108-109位核苷酸位点缺失，其突变后的基因核苷酸序列如SEQ ID NO:100所示，所述突变导致蛋白序列在37位氨基酸开始发生改变，在78位氨基酸碰到终止密码子，同时BnaC09G0254600WE基因的DNA序列的110位***1个核苷酸，其突变后的基因核苷酸序列如SEQ ID NO:101所示，该突变导致蛋白序列在37位氨基酸开始发生改变，在42位氨基酸碰到终止密码子，说明所述突变影响了所述基因蛋白的正常表达，使得其表达的蛋白多肽变短，抑制了蛋白的正常功能。对野生型和突变体的籽粒大小进行观察，发现突变体具有籽粒变大的表型(如图6A所示)，相比野生型的千粒重提高了20％(如图7A所示)。
表6：油菜中PCFS家族信息列表
实施例6、分别构建大豆、玉米、水稻PCFS突变体植物
大豆、玉米、水稻中的PCFS家族同源基因的基因编号分别如下表7所示。其中大豆中N端完整的基因编号分别为Glyma.03G191200、Glyma.10G066300、Glyma.10G251100、Glyma.19G191800、Glyma.19G191900、Glyma.20G142500，其基因序列、CDS序列和蛋白序列获取自网站SoyBase(https://www.soybase.org/)。其基因组DNA序列依次分别如SEQ ID NO:37、40、43、49、52和55所示，其CDS序列分别依次如38、41、44、50、53和56所示， 其蛋白氨基酸序列依次分别如SEQ ID NO:39、42、45、51、54和57所示。本申请针对大豆中的PCFS家族同源基因设计的基因编辑靶位点分别如SEQ ID NO:84-87所示(表7)。
水稻中的PCFS家族同源基因分别为LOC_Os08g08830和LOC_Os09g39270，其基因序列、CDS序列和蛋白序列获取网站Phytozome(https://phytozome-next.jgi.doe.gov/)。其基因组DNA序列分别如SEQ ID NO：58、61所示，其CDS序列分别如SEQ ID NO:59、62所示，其蛋白氨基酸序列分别如SEQ ID NO:60、63所示。本申请针对水稻PCFS家族同源基因设计的基因编辑靶位点分别如SEQ ID NO:88-91所示(表7)。
玉米中的PCFS家族同源基因分别为Zm00001d000023、Zm00001d005350、Zm00001d019856、Zm00001d049442，其基因序列、CDS序列和蛋白序列获取网站Phytozome(https://phytozome-next.jgi.doe.gov/)。其基因组DNA序列分别如SEQ ID NO:64、67、70、73所示，其CDS序列依次分别如SEQ ID NO:65、68、71、74所示，其蛋白氨基酸序列依次分别如SEQ ID NO:66、69、72、75所示。本申请针对玉米PCFS家族同源基因设计的基因编辑靶位点分别如SEQ ID NO:92-95所示(表7)。
表7：作物中PCFS家族信息列表

经过转基因筛选，获得了上述作物的突变体植株。在大豆中获得Glyma.10G251100基因发生突变的突变体，该突变体的DNA序列上193位核苷酸缺失，突变后的基因核苷酸序列如SEQ ID NO:102所示，该突变导致蛋白序列在65位氨基酸开始发生改变，在80位氨基酸碰到终止密码子；在水稻中获得LOC_Os08g08830基因发生突变的突变体，该突变体的DNA序列上203-4604位核苷酸缺失，其突变后的基因核苷酸序列如SEQ ID NO:103所示，该突变导致蛋白序列上71-449位氨基酸缺失；在玉米中获得Zm00001d000023和Zm00001d049442同时发生突变的突变体，该突变体的Zm00001d000023基因的DNA序列的1318-1609位核苷酸位点缺失，该突变后的基因核苷酸序列如SEQ ID NO:104所示，该突变导致蛋白序列在103位氨基酸开始发生改变，在126位氨基酸碰到终止密码子，同时Zm00001d049442基因的DNA序列的1789-2081位核苷酸位点缺失，该突变后的基因核苷酸序列如SEQ ID NO:105所示，导致蛋白序列在103位氨基酸开始发生改变，在116位氨基酸碰到终止密码子。前述实验结果表明，通过突变的方式使得本申请基因蛋白的表达受到了影响，包括表达的蛋白多肽变短，从而抑制了本申请基因蛋白的正常功能。
不同作物的籽粒表型及其千粒重或百粒重统计结果如图6和图7所示。从图中可以看出，N端完整的PCFS家族基因在油菜、水稻、大豆、玉米等作物中具有调控籽粒大小的功能的，在油菜和玉米中，N端完整的PCFS家族基因突变后籽粒变大，其千/百粒重提高了20％左右；在水稻和大豆中，N端完整的PCFS家族基因突变后其籽粒也变大，千/百粒重提高了10％左右。本实施例进一步证明了N端完整的PCFS家族基因具有植株籽粒大小调控的功能，通过突变等技术手段抑制该基因蛋白的正常功能后，可以使植株的籽粒变大，从而获得高产的作物品种。本领域技术人员应该知晓，本申请所述的抑制该基因蛋白的正常功能，包括通过突变的方式影响基因蛋白的正常功能，也包括通过影响所述基因蛋白的调控因子，从而抑制本申请N端 完整的PCFS家族基因的正常功能，获得植株籽粒变大的表型。虽然，上文中已经用一般性说明、具体实施方式及试验，对本申请实施例作了详尽的描述，但在本申请基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本申请精神的基础上所做的这些修改或改进，均属于本申请要求保护的范围。

Claims (17)

  1. 一种基因突变体核苷酸序列，含有该基因突变的植株籽粒变大，所述基因的核苷酸序列选自下列组的序列之一：
    (a)油菜中基因号为BnaA04G0223600WE、BnaA05G0075100WE、BnaA09G0222400WE、BnaC04G0102200WE、BnaC04G0535800WE或BnaC09G0254600WE的核苷酸序列；
    (b)大豆中基因号为Glyma.03G191200、Glyma.10G066300、Glyma.10G251100、Glyma.19G191800、Glyma.19G191900或Glyma.20G142500的核苷酸序列；
    (c)水稻中基因号为LOC_Os08g08830或LOC_Os09g39270的核苷酸序列；或
    (d)玉米中基因号为Zm00001d000023、Zm00001d005350、Zm00001d019856或Zm00001d049442的核苷酸序列；或
    (e)拟南芥中基因号为AT2G36480或AT4G04885的核苷酸序列；
    (f)在严谨条件下能够与(a)-(e)之任一所述核苷酸序列杂交的核苷酸序列；
    (g)与(a)-(f)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且在植株中突变后具有使植株籽粒变大功能的核苷酸序列；或
    (h)与(a)-(g)之任一所述核苷酸序列互补的核苷酸序列。
  2. 根据权利要求1所述的基因突变体核苷酸序列，其中所述的基因的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、62、64、65、67、68、70、71、73、74、4、5、7或8所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：15、18、24、27、30、36、39、42、45、51、54、57、60、63、66、69、72、75、6或9所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述核苷酸序列杂交的DNA序列；
    (d)与(a)-(c)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且突变后具有使植株籽粒变大功能的核苷酸序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  3. 根据权利要求1或2所述的基因突变体核苷酸序列，所述的基因突变包括在该基因的核苷酸序列上进行取代、缺失和/或添加一个或多个核苷酸。
  4. 根据权利要求1-3之任一所述的基因突变体核苷酸序列，所述的突变通过物理诱变、化学诱变、ZFN、TALEN或CRISPR/Cas9等技术获得。
  5. 根据权利要求4所述的基因突变体核苷酸序列，其中所述的CRISPR/Cas9技术所用的靶点序列选自下列组的序列之一:
    (a)序列为SEQ ID NO：4、5、7、8、13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、62、64、65、67、68、70、71、73或74所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段，其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸；或
    (b)与(a)所述的多核苷酸互补的多核苷酸。
  6. 根据权利要求5所述的基因突变体核苷酸序列，其中所述的CRISPR/Cas9技术所用的靶点序列如SEQ ID NO:76-79、80-95之任一所示的序列。
  7. 根据权利要求1-6之任一所述的基因突变体核苷酸序列，所述基因突变体的核苷酸序列如SEQ ID NO:97、98、100-105之任一所示。
  8. 一种调控植株农艺性状的方法，通过突变植株的PCFS家族基因，使植株籽粒变大，所述基因的核苷酸序列选自下列组的序列之一：
    (a)油菜中基因号为BnaA04G0223600WE、BnaA05G0075100WE、BnaA09G0222400WE、BnaC04G0102200WE、BnaC04G0535800WE或BnaC09G0254600WE的核苷酸序列；
    (b)大豆中基因号为Glyma.03G191200、Glyma.10G066300、Glyma.10G251100、Glyma.19G191800、Glyma.19G191900或Glyma.20G142500的核苷酸序列；
    (c)水稻中基因号为LOC_Os08g08830或LOC_Os09g39270的核苷酸序列；或
    (d)玉米中基因位置号为Zm00001d000023、Zm00001d005350、Zm00001d019856 或Zm00001d049442的核苷酸序列；
    (e)拟南芥中基因号为AT2G36480或AT4G04885的核苷酸序列；
    (f)在严谨条件下能够与(a)-(e)之任一所述核苷酸序列杂交的DNA序列；
    (g)与(a)-(f)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且在植株中突变后具有使植株籽粒变大功能的核苷酸序列；或
    (h)与(a)-(g)之任一所述核苷酸序列互补的DNA序列。
  9. 根据权利要求8所述的方法，所述基因的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、62、64、65、67、68、70、71、73、74、4、5、7或8所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：15、18、24、27、30、36、39、42、45、51、54、57、60、63、66、69、72、75、6或9所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列杂交的DNA序列；或
    (d)与(a)-(c)之任一所述核苷酸序列的全长至少有85％、90％、95％或以上序列相似性，且突变后具有使植株籽粒变大功能的核苷酸序列；
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  10. 根据权利要求8或9所述的方法，所述基因的突变包括在该基因的核苷酸序列上进行取代、缺失和/或添加一个或多个核苷酸。
  11. 根据权利要求8-10之任一所述的方法，所述突变通过物理诱变、化学诱变、ZFN、TALEN或CRISPR/Cas9等技术获得。
  12. 根据权利要求11所述的方法，其中所述的CRISPR/Cas9技术所用的靶点序列选自下列组的序列之一:
    (a)序列为SEQ ID NO：4、5、7、8、13、14、16、17、22、23、25、26、28、29、34、35、37、38、40、42、43、44、49、50、52、53、55、56、58、59、61、 62、64、65、67、68、70、71、73或74所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段，其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸；
    (b)与(a)所述的多核苷酸互补的多核苷酸。
  13. 根据权利要求12所述的方法，其中所述的CRISPR/Cas9技术所用的靶点序列如SEQ ID NO:76-79、80-95之任一所示的序列。
  14. 根据权利要求8-13之任一所述的方法，所述基因突变后的核苷酸序列如SEQ ID NO:97、98、100-105之任一所示。
  15. 一种非作为繁殖材料的植物细胞、组织、器官或产品，所述植物细胞、组织、器官或产品含有权利要求1-7之任一所述的基因突变体核苷酸序列。
  16. 权利要求8-14之任一所述的方法、及其获得的突变体材料在育种中的应用。
  17. 根据权利要求16所述的应用，其中所述的在育种中的应用是指通过基因突变或与突变体材料杂交的方式，使植株获得该基因突变体，籽粒呈现变大的表型。
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