Application of rice anchoring protein OsANK3 and its encoding gene in regulating seed development

Abstract

The present invention discloses an application of a rice anchoring protein OsANK3 and its encoding gene in regulating seed size and / or cultivating plant varieties with improved seed grain shape, and belongs to the field of plant genetic engineering. The present invention constructs a CRISPR / Cas9-OsANK3 gene knockout vector and transforms rice using an Agrobacterium-mediated method to obtain an OsANK3 gene knockout mutant. The grain length, grain thickness and thousand-grain weight of the OsANK3 gene knockout mutant are significantly increased compared to wild-type seeds. Therefore, the OsANK3 gene participates in the seed size development process as a negative regulatory factor, and can therefore be used in genetic breeding for improving seed grain shape and thousand-grain weight.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an anchoring protein OsANK3 and a coding gene and application thereof. Background Art

[0002] Rice (Oryza sativa) is an important food crop and model organism. Grain shape, as an important trait of rice, plays a vital role in the appearance quality and yield of grains. Grain shape is mainly determined by the size of the husk and the number and size of endosperm cells, including grain length, grain width, grain thickness and aspect ratio. The aspect ratio is a composite trait composed of the ratio of grain length to grain width. Seed size is jointly determined by grain length, grain width, grain thickness and grain fullness, and is the determining factor of 1000-grain weight, an important yield trait. Rice seed size not only affects yield, but also directly affects the aspect ratio and processing quality traits of rice. The final seed size is closely related to the growth and coordinated development of the three components of endosperm, embryo and seed coat, and these growth and development processes are jointly regulated by multiple factors such as intrinsic genetic level, epigenetic level, cellular level and developmental signal environmental factors. Studies have found that multiple regulatory pathways such as ubiquitin-proteasome, G protein signaling, mitogen-activated protein kinase (MAPK) signaling pathway, plant hormones and transcriptional regulatory factors are involved in the regulation of seed size.

[0003] Rice grain shape is one of the important research contents of rice functional genomics. The GS3 gene is widely used in rice breeding. It can improve the appearance quality while increasing the grain quality. By aggregating GS3 alleles and GW8, it was found that the appearance quality of grains can be improved while ensuring the quality. The aggregating breeding research of genes such as GS3, Ghd7, qSW5 and DPE1 successfully selected excellent varieties, which have more advantages in increasing yield and improving quality. The deletion mutant of the grain shape gene GS9 increases the grain length by changing cell division. The aggregation of GS9 and GS3 will obtain longer and thinner grains, indicating that GS9 is a good material for breeding whether alone or in combination with other grain shape genes. In summary, grain shape regulation is not only beneficial to increasing rice yield, but also to improving the appearance quality of rice. Selecting appropriate gene resources for rice breeding and quality improvement can effectively obtain new rice varieties with high yield and high quality. However, the genetic regulatory network of rice appearance quality is complex. Although some genes regulating rice appearance quality have been reported, discovering and cloning more new genes that have the function of regulating rice appearance quality, discovering new regulatory mechanisms, and developing new methods to improve rice appearance quality are still technical problems that need to be solved urgently. Summary of the invention

[0004] The present invention mainly aims at the above technical problems and provides an application of an anchoring protein gene OsANK3 in regulating plant seed shape and weight. This gene is a key regulatory gene for rice seed development and can be used for the cultivation of new high-yield rice varieties.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a use of a rice OsANK3 gene or a protein encoded thereby in regulating rice seed development. The protein sequence encoded by the OsANK3 gene is shown in SEQ ID NO.4.

[0007] In a preferred embodiment, the protein coding region of the OsANK3 gene of the present invention is a nucleotide sequence as shown in SEQ ID NO.3, or a sequence that is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO.3.

[0008] Furthermore, the regulation of rice seed development is to increase seed length, seed thickness and / or thousand-grain weight by reducing the mRNA expression level of the OsANK3 gene or reducing the protein content encoded by the OsANK3 gene.

[0009] In a preferred embodiment, the reduction of the mRNA expression level of the OsANK3 gene or the reduction of the protein content encoded by the OsANK3 gene is achieved by gene knockout, gene silencing and / or gene editing.

[0010] In a preferred embodiment, the CRISPR / Cas9 gene editing system is used to achieve gene knockout of the OsANK3 gene.

[0011] Furthermore, the target sequence and the protospacer sequence adjacent motif recognized by the CRISPR / Cas9 gene editing system are 5'-GAAGACGAGCGTCAAGTCGGAGG-3', wherein the first 20 base sequences GAAGACGAGCGTCAAGTCGG from the 5' end are the target sequence (CRISPR-derived RNA), and the remaining sequence AGG is the protospacer sequence adjacent motif.

[0012] On the other hand, the present invention provides the use of rice OsANK3 gene or the protein encoded by it in rice varieties with improved seed length, grain thickness and / or 1000-grain weight. The protein sequence encoded by the OsANK3 gene is shown in SEQ ID NO.4.

[0013] In a preferred embodiment, the protein coding region of the OsANK3 gene of the present invention is a nucleotide sequence as shown in SEQ ID NO.3, or a sequence that is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO.3.

[0014] Furthermore, the improved seed length or 1000-grain weight is achieved by reducing the mRNA expression level of the OsANK3 gene or reducing the protein content encoded by the OsANK3 gene to increase the seed length, grain thickness and / or 1000-grain weight.

[0015] In a preferred embodiment, the reduction of the mRNA expression level of the OsANK3 gene or the reduction of the protein content encoded by the OsANK3 gene is achieved by gene knockout, gene silencing and / or gene editing.

[0016] In a preferred embodiment, the CRISPR / Cas9 gene editing system is used to achieve gene knockout of the OsANK3 gene.

[0017] Furthermore, the target sequence and the protospacer sequence adjacent motif recognized by the CRISPR / Cas9 are 5'-GAAGACGAGCGTCAAGTCGGAGG-3', wherein the first 20 base sequences GAAGACGAGCGTCAAGTCGG from the 5' end are the target sequence (CRISPR-derived RNA), and the remaining sequence AGG is the protospacer sequence adjacent motif.

[0018] On the other hand, the present invention provides an sgRNA for regulating rice seed development, wherein the targeting sequence (CRISPR-derived RNA) of the sgRNA is GAAGACGAGCGTCAAGTCGG, and the PAM sequence corresponding to the sgRNA is AGG.

[0019] Furthermore, the improved seed length, thickness and / or 1000-grain weight is achieved by reducing the mRNA expression level of the OsANK3 gene or reducing the protein content encoded by the OsANK3 gene to increase the seed length, thickness and / or 1000-grain weight.

[0020] On the other hand, the present invention provides the use of the aforementioned sgRNA in improving grain length, grain thickness and / or thousand-grain weight of high-quality rice seeds.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1) The present invention provides novel genes and proteins capable of simultaneously regulating plant seed shape and 1000-grain weight.

[0023] 2) The present invention provides a novel method for simultaneously regulating plant grain shape and 1000-grain weight, namely, increasing seed length and 1000-grain weight by reducing the expression of the OsANK3 gene or the protein encoded by it.

[0024] 4) The present invention provides a method for breeding plant varieties with improved seed shape and 1000-grain weight, namely, breeding plant varieties with improved seed shape and 1000-grain weight by reducing the expression of OsANK3 gene or the protein encoded by it. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention is described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0026] Figure 1 Schematic diagram of the OsANK3 gene structure and sgRNA target sites.

[0027] Figure 2 Comparison of seed length between wild-type seeds WT and OsANK3 knockout mutant seeds. A is a picture of seed length, and B is a statistical analysis of seed length data.

[0028] Figure 3 Comparison of seed width between wild-type seeds WT and OsANK3 knockout mutant seeds. A is a picture of seed width, and B is a statistical analysis of seed width data.

[0029] Figure 4 Comparison of seed thickness and 1000-grain weight between wild-type seeds WT and OsANK3 knockout mutants. A is the statistical analysis of seed thickness data, and B is the statistical analysis of seed 1000-grain weight data.

[0030] Figure 5 Comparison of seed length between wild-type WT and OsANK3 knockout mutant seeds after husking. A is a picture of husked seed length, and B is a statistical analysis of husked seed length data.

[0031] Figure 6 Comparison of seed width between wild-type seeds WT and OsANK3 knockout mutant after husking. A is a picture of seed width after husking, and B is a statistical analysis of seed width after husking.

[0032] Figure 7 Comparison of grain thickness and 1000-grain weight of wild-type seeds WT and OsANK3 knockout mutant seeds after husking. A is the statistical analysis of grain thickness of husked seeds, and B is the statistical analysis of 1000-grain weight of husked seeds.

[0033] Figure 8 Scanning electron microscopy observation of the glumes of wild type WT and OsANK3 gene knockout mutant.

[0034] Fig. 9 Statistical analysis of the size and number of glumes in the wild type WT and OsANK3 knockout mutant. A is the number of longitudinal glumes cells, B is the number of transverse glumes cells, C is the longitudinal glumes cell length, and D is the transverse glumes cell length. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention.Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.The experimental methods used in the following examples are conventional methods unless otherwise specified.The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0036] The rice plant material used in the present invention is Zhonghua 11, which was planted in the experimental field of Xinyang Normal University. The OsANK3 gene sequence, CDS sequence and amino acid sequence of the present invention are as follows:

[0037] 1. OsANK3 genome sequence (SEQ ID NO.2)

[0038] >LOC_Os03g63480

[0039] AAACCAAGCCTCTGGTTGGTCGCACTCACTCACTCGCATCCTCTCCTCTCAAATACTCC

[0040] TCCACCTTATCATCATCACCACCACTAACAACAACGCCACTGCCTGAACCAAGAACAC

[0041] TGCCCTCCTCCACACGACATGGCTTCTCAAGGTCAAAAGAACTGATTTGCTGTTTATTT

[0042] CTTCAATTCTTTCTTTCTTGTTTGATTTGCTCCGGCTCAATTCTTTCTTGATACTGATAAGA

[0043] GACGACATGAGTAATTGTGTGCGCAGAAGAGAAGACGAGCGTCAAGTCGGAGGAGGC

[0044] CTCGTCGGCGGCGGAGGAGCAGCCACCTCAAGCTGCAGCTCCGCCGCCGCGCAGGGGT

[0045] GTGCCCGCCGGCCAACCCCTTCGACTTCTCAACCATGATGAACTTGCTCAATGTAAGCTT

[0046] ACCTCGCTGCCGACTTATTTCACTAATTGCCTACTCGTTTCAGCGTAATTTCCTCAACTA

[0047] TATTTTGGGTATAATCTCCTCTTGAACCTAAGTTACCCGATAATTAATTAGCAGCT

[0048] ACCTGTTCAAGATTGAGTTAATTAAGCAACTCACTATATAACCAACCAGGTGTTGTTGC

[0049] TTATTAGCTGAGATGGAAGCTGGATTGATTAATTATACAGTTCATGCAATTACTAGTAA

[0050] TAATTAGTGAGTCCATTGATGAATGATGAGCAGAAAAAATAATTAGATGAATAAT

[0051] CTGCATGGTAATTTGTTTGTTTGGAGCAGGATCCGAGCATAAAGGAGATGGCGGAGCA

[0052] GATCGCCAAGGACCCGGCGTTCACGGAGATGGCGGAGCAGCTGCAGAAGACGGTGCA

[0053] GTCGCCGCCGGCGCGGGGGCGGCGCAGGAGCTGCGGCGGCGGCGGCGCCGGCGCT

[0054] GGACCCGAGCAAGTACGTGTCGACGATGCAGCAGCTGATGCAGAACCCGCAGTTCGTG

[0055] GCGATGGCGGAGCGGCTGGGCAGCGCGCTGATGCAGGACCCGGCCATGTCGTCCATGC

[0056] TTGGCGGCCTCACCAACCCCGCCCACAAGGAGCAGCTCGAGGCCCGCATCGCCCGCAT

[0057] GAAGGACGACCCCTCCCTCAAGCCCATCCTCGACGAGATCGAGAACGGCGGCCCCGCC

[0058] GCCATGATGAAGTAAGCATAGTTGCAATTGGCGCCATGGCTGGTTGAGTTCATCGTCGT

[0059] CGTCGTCGTCGATCTCGAAAGCAAATGAGCAATGGCAATACTTGTTGCTTCCTTCAGGT

[0060] ATTGGAACGACCCTGAAGCGCTGCAGAAGTTCGGGAGGGCGATGGGCGTCGGCCCGTC

[0061] GGGCGAGGGCGCGGCGGCGGCCGGCGGCGAGCACGAGGAGGCGGAGGAAGAGGGCG

[0062] GGGAGGAGGGCGAGTACGAGGACGAGTCCGTCATCCACCACACTGCCAGCGTCGGCG

[0063] ATGTCGAGGTCTCTAAATCAATTCTCACTTCTATTACTGCCTACTCTCTCCTTTTCAAGA

[0064] CAAGATGGTCAAAATCAAACCATTTTAAATTTAACTAAATTATAGACAAATATAGTAA

[0065] TATTTATAATACTAAATTAGTTTTTTTCTTGATTATTTATAGAGTAATATTTACAATACA

[0066] AAATCAATAATTGAATATATTCTCATAATAATATTTGTTTAGGATTGAATTGTTATTAT

[0067] TTTTTCTATAAACTTGTTTAAATCTAAAGCTGTTTCACTTTGATTAAATTTAAAACGTT

[0068] TTATAATCTGAAATCAGAGGGAGTACTAATTATGGCGCATTTGCTCAAGTAAAATCTGCA

[0069] TCTTTTCCTTCATCTTCAACACGCATTGGCATCGTTCAGGGCCTGAAGAAAGCATTGGA

[0070] GGAAGGCGTGGACAAGGACGAGGAGGATTCGGAAGGGAGGAGAGGCCTGCATTTCGC

[0071] TTGCGGCTATGGCGAGGTAAGCATCATGCATGCAGTTCTCAGTGAACATCTTGTTCAGT

[0072] TATCTGATCGATTCGTTGCTTGTGTTTGCGCAGCTGAAATGCGCGCAGGTTCTCCTGGA

[0073] GGCCGGCGCGGCCGTGGACGCGGTGGACAAGAACAAGAACACGGCGCTGCACTACGC

[0074] CGCCGGCTACGGCCGCAAGGACTGCGTCGCCCTCCTGCTCGACCACGGCGCCGCCGTG

[0075] TAAGTCCATCGCATCTGCAATTCTGCATGCATATGCCCATGCCATCCATCAATGGACAT

[0076] CTGACTGAAACTAACCCGGTTTGGTTATGCAATGTGCAGGACGGTGCAGAACCTGGAC

[0077] GGGAAGACGGCCATCGACGTGGCGAAACTCAACAACCAGGAGGAAGTCCTCAAGCTG

[0078] CTCGAGAAGCACGCCTTCGTATAGAATGAAACAATCGAGGCCAGGCCAGGCCAGGCC

[0079] AGCCACACTCCTCTCTCTCAATTCAGAGACAAATGCTGCATTTCTGCTCTGCATATGCT

[0080] CCTGTTCATCCGCAAATGAGTACCTGTATGCTTCTGTATGTATCGAAGAGTGCACTAGA

[0081] TTATTTTTTATTTCACTTGGGAGCAAGGTTAAGGTGAGATTGGATTGCTATTCTATTCAT

[0082] CCTTCCAGAGTTTTGTTTAATGAATAAGGAAGAAAAGTAAATTTCATTTCAGTGTTTAA

[0083] CTGTTTATGTCTGAATTTGACATCTCTCATTGGATTTGGATCCGTCAAAATGTTGGACTT

[0084] TTAGGCCTTGTTTAGATGCCTTGGTAA

[0085] 2. CDS sequence of OsANK3 (SEQ ID NO.3)

[0086] >LOC_Os03g63480.1

[0087] ATGGCTTCTCAAGAAGAGAAGACGAGCGTCAAGTCGGAGGAGGCCTCGTCGGCGGCG

[0088] GAGGAGCAGCCACCTCAAGCTGCAGCTCCGCCGCCGCGCAGGGGTGTGCCGCCGGCCA

[0089] ACCCCTTCGACTTCTCAACCATGATGAACTTGCTCAATGATCCGAGCATAAAGGAGAT

[0090] GGCGGAGCAGATCGCCAAGGACCCGGCGTTCACGGAGATGGCGGAGCAGCTGCAGAA

[0091] GACGGTGCAGTCGCCGCCGGCGCGGGGGGCGGCGCAGGAGGCTGCGGCGGCGGCGGC

[0092] GCCGGCGCTGGACCCGAGCAAGTACGTGTCGACGATGCAGCAGCTGATGCAGAACCCG

[0093] CAGTTCGTGGCGATGGCGGAGCGGCTGGGCAGCGCGCTGATGCAGGACCCGGCCATGT

[0094] CGTCCATGCTTGGCGGCCTCACCAACCCCGCCCAAGGAGCAGCTCGAGGCCCGCAT

[0095] CGCCCGCATGAAGGACGACCCCTCCCTCAAGCCCATCCTCGACGAGATCGAGAACGGC

[0096] GGCCCCGCCGCCATGATGAAGTATTGGAACGACCCTGAAGCGCTGCAGAAGTTCGGGA

[0097] GGGCGATGGGCGTCGGCCCGTCGGGCGAGGCGCGGCGGCGGCCGGCGGCGAGCACG

[0098] AGGAGGCGGAGGAAGGGCGGGGAGGGCGAGTCCGTCATC

[0099] CACCACACTGCCAGCGTCGGCGATGTCGAGGGCCTGAAGAAAGCATTGGAGGAAGGC

[0100] GTGGACAAGGACGAGGAGGATTCGGAAGGGAGGCCTGCATTTCGCTTGCGGC

[0101] TATGGCGAGCTGAAATGCGCGCAGGTTCTCCTGGAGGCCGGCGCGGCCGTGGACGCGG

[0102] TGGACAAGAACAAGAACACGGCGCTGCACTACGCCGCCGGCTACGGCCGCAAGGACT

[0103] GCGTCGCCCTCCTGCTCGACCACGGCGCCGCCGTGACGGTGCAGAACCTGGACGGGAA

[0104] GACGGCCATCGACGTGGCGAAACTCAACAACCAGGAAGTCCTCAAGCTGCTCGA

[0105] GAAGCACGCCTTCGTATAGOsANK3 transcript (SEQ ID NO.4)

[0106] >LOC_Os03g63480.1

[0107] MASQEEKTSVKSEEASSAAEEQPPQAAAPPPRRGVPPANPFDFSTMMNLLNDPSIKEMAE

[0108] QIAKDPAFTEMAEQLQKTVQSPPARGAAQEAAAAAAPALDPSKYVSTMQQLMQNPQFVA

[0109] MAERLGSALMQDPAMSSMLGGLTNPAHKEQLEARIARMKDDPSLKPILDEIENGGPAAM

[0110] MKYWNDPEALQKFGRAMGVGPSGEGAAAAGGEHEEAEEEGGEEGEYEDESVIHHTASVG

[0111] DVEGLKKALEEGVDKDEEDSEGRRGLHFACGYGELKCAQVLLEAGAAVDAVDKNKNTA

[0112] LHYAAGYGRKDCVALLLDHGAAVTVQNLDGKTAIDVAKLNNQEEVLKLLEKHAFV

[0113] Example 1 Construction of OsANK3 gene knockout vector

[0114] Primer design for OsANK3 gene target site: Download the OsANK3 reference genome sequence and CDS sequence from the MSU website (Rice Genome Annotation Project). Based on the protospacer adjacent motif (PAM) recognized by the CRISPR / Cas9 system, target sites were designed near the start codon and conserved domain of the OsANK3 gene (see the schematic diagram of the target site structure for details). Figure 1), the target sequence is SEQ ID NO.1:5'-GAAGACGAGCGTCAAGTCGG-3'. Through three PCR amplifications, the 20-base target site of the target gene was integrated into the U6 promoter. The U6 promoter integrated with the exogenous fragment was connected to the pCXUN-Cas9 vector using a ligase. The connection system is: 3μl Amm ligase + 1μl vector plasmid + 1μl exogenous fragment, 37°C, 50min. The connected recombinant vector was transformed into DH5α Escherichia coli competent cells by chemical transformation, propagated and shaken, and colony PCR positive detection was performed. The PCR reaction system: 4μl connected plasmid, 2μl 10×buffer, 0.35μl dNTP, 0.2μl F primer, 0.2μl R primer, 0.1μl r-Taq + 13.15μl ddH 2 O, colony PCR program was: 95℃10min, 94℃30sec, 55℃30sec, 72℃1min, 32cycles, 72℃7min, 25℃1min. Samples with target bands were sent to Shanghai Bioengineering for sequencing. The sequencing primer was CRXU-R: 5'-TGTGTGGAATTGTGAGCGGATA-3'. Plasmids were extracted from clones with correct sequencing, thus completing the construction of the recombinant vector.

[0115] Example 2 Obtaining OsANK3 gene knockout mutants

[0116] 1. Transformation of OsANK3 gene knockout vector into Agrobacterium EHA105

[0117] Take 1 μl of OsANK3 gene knockout vector and put it into 20 μl of EHA105 competent medium. After electroporation, add 200 μl of non-resistant LB medium into a 0.5 ml centrifuge tube and revive at 28°C for 45 minutes. After revival, spread it on a culture dish containing Kan antibiotic resistance and culture it at 28°C for 48 hours. Pick 8 single clones and streak them on a new culture dish containing Kan antibiotic resistance, culture it for 24 hours, and use it for infection experiments.

[0118] 2. Agrobacterium-mediated genetic transformation of rice

[0119] After the mature seeds are shelled and disinfected in a clean bench, they are cultured in the dark at 26°C on an induction medium for 30-40 days. The induced calli are expanded and cultured on the subculture medium for 2-3 times, and each dark culture lasts about 15 days. Bright yellow and hard calli are selected for infection experiments, immersed in Agrobacterium solution for 30 minutes, and cultured at 19°C for 2 days. The calli are washed several times with sterile water and transferred to a screening medium with a screening marker antibiotic to obtain resistant calli. After 2-3 rounds of screening, each lasting about 15 days, the resistant calli are transferred to a pre-differentiation medium for culture for 7 days, and differentiated and regenerated into plants on a differentiation medium under light conditions.

[0120] 3. Identification of OsANK3 gene knockout transgenic plants

[0121] The CTAB method was used to extract rice leaf genomic DNA. The primers were synthesized on both sides with the target site as the center. The sequences were F: 5'-GACATGGCTTCTCAAGGTC-3', R: 5'-AAACGAGTAGGCAATTAGTG-3'. PCR amplification was performed on each transgenic plant to amplify the target site. The reaction system was: 4μl DNA template, 2μl 10× buffer, 0.35μl dNTP, 0.2μl F primer, 0.2μl R primer, 0.1μl Ex-Taq, 13.15μl ddH 2 O; PCR program: 95℃10min, 94℃30sec, 55℃30sec, 72℃40sec, 30cycles, 72℃7min, 25℃1min. The amplified target fragment was sent to a biological company for sequencing. The target site sequence of the transgenic plant was analyzed by sequence alignment to identify the mutation type. As a result, three mutant strains were obtained, named KO2, KO6, and KO7. Their mutation types are shown in Figure 1 .

[0122] Example 3 Observation and statistics of seed enlargement phenotype of OsANK3 gene knockout mutant

[0123] The wild-type WT and OsANK knockout mutants were observed throughout the growth and development stages. It was found that after the OsANK3 gene was knocked out, the grain length of the OsANK3 mutant seeds increased significantly compared with the wild-type seeds, showing a phenotype that the OsANK3 knockout mutant seeds were significantly larger than the wild-type seeds. The grain length, grain width, grain thickness and thousand-grain weight of the seeds were further statistically analyzed. The results were plotted into a chart using Sigma Plot software, and the differences were marked, p<0.05(*), p<0.01(**). The statistical results showed that the grain length of the OsANK3 knockout mutant seeds ( Figure 2 , Figure 5 ), grain thickness ( Figure 4 A and Figure 7 A) and thousand-grain weight ( Figure 4 B and Figure 7 B) were significantly increased compared with wild-type seeds, while the seed width ( Figure 3 , Figure 6 The above results showed that OsANK3 gene was involved in the development of seed size and negatively regulated seed length, seed thickness and 1000-grain weight.

[0124] Example 3 Scanning electron microscope observation and statistics of husk cell size and number

[0125] The rice husk was fixed with 2.5% glutaraldehyde solution and stored in a 4°C refrigerator. The fixative was washed three times with 0.01M PBS solution and then dehydrated with gradient ethanol. The dried sample was glued to the sample stage, the sample stage was gold-sprayed with an ion sputtering instrument, and then the sample was observed with a cold field emission scanning electron microscope. The seeds of each strain were photographed at 25 times and 200 times ( Figure 8 ), the longitudinal and transverse cell numbers of the husk were counted at 25 times, and the longitudinal and transverse cell lengths of the husk were counted at 200 times ( Fig. 9 ), and the results were plotted into graphs using Sigma Plot software, with differences marked at p<0.05(*), p<0.01(**). The statistical results showed that the number and size of longitudinal cells in seeds of OsANK3 knockout mutants were significantly increased compared with those of wild type WT. Based on the above results, OsANK3 gene may be involved in the growth and development process of longitudinal cell division and expansion in rice seeds.

[0126] Based on the above results, it is confirmed that OsANK3 is a gene that regulates plant seed shape and 1000-grain weight, and can therefore be used in genetic breeding to improve seed shape and 1000-grain weight.

[0127] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the present invention.

Claims

1. Application of rice OsANK3 gene in regulating rice seed development, wherein the protein sequence encoded by the OsANK3 gene is shown in SEQ ID NO.4, The regulation of rice seed development is to increase seed length, seed thickness and / or thousand-grain weight by reducing the expression level of the OsANK3 gene.

2. Application of a protein encoded by the rice OsANK3 gene in regulating rice seed development, wherein the protein sequence encoded by the OsANK3 gene is shown in SEQ ID NO.4, The method for regulating rice seed development is to increase seed length, seed thickness and / or thousand-grain weight by reducing the protein content encoded by the OsANK3 gene.

3. Application of the rice OsANK3 gene in breeding rice varieties with improved seed length, grain thickness and / or 1000-grain weight, wherein the protein sequence encoded by the OsANK3 gene is shown in SEQ ID NO.4, and the breeding of rice varieties with improved seed length, grain thickness and / or 1000-grain weight is to increase the seed length, grain thickness and / or 1000-grain weight by reducing the expression level of the OsANK3 gene.

4. Use of the protein encoded by the rice OsANK3 gene in breeding rice varieties with improved seed length, grain thickness and / or 1000-grain weight, wherein the protein sequence encoded by the OsANK3 gene is shown in SEQ ID NO.4, and the breeding of rice varieties with improved seed length, grain thickness and / or 1000-grain weight is to increase the seed length, grain thickness and / or 1000-grain weight by reducing the content of the protein encoded by the OsANK3 gene.

5. A sgRNA for regulating rice seed development, wherein the targeting sequence of the sgRNA is SEQ ID NO.

1.

6. A method for improving rice seed shape and thousand-grain weight, characterized in that: The method increases seed length, seed thickness and / or thousand-grain weight by reducing the expression level of the OsANK3 gene or reducing the content of the protein encoded by the OsANK3 gene. The protein sequence encoded by the OsANK3 gene is shown in SEQ ID NO.

4.

7. The method according to claim 6, characterized in that The reduction of the expression level of the OsANK3 gene or the reduction of the protein content encoded by the OsANK3 gene is achieved by gene knockout or gene silencing.

8. Use of the sgRNA according to claim 5 in improving rice seed length, grain thickness and / or thousand-grain weight.

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