A gene OsLRK11 for controlling the grain shape of rice seeds, its encoded protein and applications

Abstract

The present invention provides a gene OsLRK11 for controlling the grain shape of rice seeds, belonging to the field of rice genetic engineering. The OsLRK11 has a nucleotide sequence as shown in SEQ ID No.1. The 1000-grain weight, grain length and length-width ratio of the mutant seeds of this gene are significantly improved compared with those of the wild type, and it can be applied to molecular breeding for improving the grain shape of rice seeds. The present invention also provides a coding protein of the gene OsLRK11 for controlling the grain shape of rice seeds and its application.

Description

Technical Field

[0001] The present invention belongs to the field of rice genetic engineering, and particularly relates to a gene OsLRK11 for controlling rice seed grain shape, its encoded protein, and applications thereof. Background Art

[0002] With the increasing population year by year and the continuous reduction of the effective cultivated land area, the future food supply situation remains extremely severe. Therefore, how to utilize limited land resources to increase food production is a major event related to national economy and people's livelihood. Rice (Oryza sativa L.) is one of the most important food crops in the world, and nearly half of the global population takes rice as the staple food. Continuously increasing the rice yield per plant has become an important way to increase the total food production. The rice yield is mainly determined by three factors: the number of effective tillers per plant, the number of grains per panicle, and the 1000-grain weight. The 1000-grain weight is directly affected by the grain shape. The grain shape of rice includes four aspects: grain length, grain width, grain thickness, and length-width ratio, which not only directly affect the 1000-grain weight and thus the rice yield, but also are closely related to the quality of rice. Summary of the Invention

[0003] In order to improve the rice seed grain shape and increase the 1000-grain weight of rice seeds, the present invention provides a gene OsLRK11 for controlling rice seed grain shape. The 1000-grain weight, grain length, and length-width ratio of the mutant grains of this gene are significantly increased compared with the wild type, and it can be applied to molecular breeding for improving rice seed grain shape.

[0004] The present invention also provides an encoded protein of the gene OsLRK11 for controlling rice seed grain shape and its applications.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides a gene OsLRK11 for controlling rice seed grain shape, and the OsLRK11 has a nucleotide sequence as shown in SEQ ID No.1.

[0007] The present invention provides an application of the gene OsLRK11 for controlling rice seed grain shape in rice cross-breeding or safe production of rice yield.

[0008] The present invention provides an encoded protein of the gene OsLRK11 for controlling rice seed grain shape. The encoded protein is named LRK11, and the LRK11 has an amino acid sequence as shown in SEQ ID No.2.

[0009] The present invention provides an application of the encoded protein of the gene OsLRK11 for controlling rice seed grain shape in regulating rice seed grain shape.

[0010] Based on the same inventive concept, the present invention provides a gene for improving the grain shape of rice seeds, and the gene includes (a) or (b):

[0011] (a) having a nucleotide sequence shown in any one of SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5;

[0012] (b) a nucleotide sequence having a homology of more than 75% with the nucleotide sequence in (a).

[0013] Based on the same inventive concept, the present invention provides a gene for improving the grain shape of rice seeds, which is obtained by gene knockout of a gene OsLRK11 for controlling the grain shape of rice seeds as described above.

[0014] Based on the same inventive concept, the present invention provides an application of a gene for improving the grain shape of rice seeds in rice cross breeding or safe production of rice yield.

[0015] Based on the same inventive concept, the present invention provides a target sequence for knocking out a gene OsLRK11 for controlling the grain shape of rice seeds, and the target sequence is composed of nucleotide sequences shown in SEQ ID No.7 and SEQ ID No.8.

[0016] Based on the same inventive concept, the present invention provides an OsU6a sequence for driving gRNA for knocking out a gene OsLRK11 for controlling the grain shape of rice seeds, and the OsU6a sequence is composed of nucleotide sequences shown in SEQ ID No.9 and SEQ ID No.10.

[0017] Based on the same inventive concept, the present invention provides an application of a target sequence for knocking out a gene OsLRK11 for controlling the grain shape of rice seeds or an OsU6a sequence for driving gRNA in rice cross breeding or safe production of rice yield.

[0018] Based on the same inventive concept, the present invention provides a method for cultivating a large-grain rice strain, and the method includes:

[0019] synthesizing a target sequence for knocking out a gene OsLRK11 for controlling the grain shape of rice seeds;

[0020] constructing a CRISPR / CAS9 system expression vector containing the target sequence;

[0021] transforming the CRISPR / CAS9 system expression vector into rice;

[0022] selecting a rice strain with the gene OsLRK11 knocked out, which is a large-grain rice strain.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] For the gene OsLRK11 that controls the grain shape of rice seeds in the present invention, the biological traits of the OsLRK11 gene discovered in the present invention have never been identified in rice. It is a new gene that controls the grain shape of rice. Both the gene and its use in controlling the grain shape of rice seeds are proposed for the first time, and there has been no relevant report before. By constructing a knockout vector of OsLRK11 and transforming rice to knockout the OsLRK11 gene in rice, compared with wild-type rice, although the number of grains per panicle and seed setting rate of the OsLRK11 knockout lines decrease, the seed length, length-width ratio, and 1000-grain weight increase significantly. This shows that OsLRK11 is a gene that controls the size of rice seeds. Brief Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a phenotypic identification diagram of wild type and mutant Irk11; where A is the plant type diagram, B is the grain type diagram, C is the grain width diagram, D is the grain length diagram, E is the caryopsis length diagram, F is the young panicle development diagram, H is the panicle type diagram, and G is the statistical chart of the yield per plant; where WT is wild type Yixiang 1B and Irk11 is the large-grain mutant;

[0027] Figure 2 It is a statistical chart of agronomic traits; where A is the plant height statistical chart, B is the panicle length statistical chart, C is the number of filled grains per panicle statistical chart, D is the grain length statistical chart, E is the grain width statistical chart, and F is the 1000-grain weight statistical chart; where WT is wild type Yixiang 1B and Irk11 is the large-grain mutant;

[0028] Figure 3 It is a map-based cloning diagram of OsLRK11; where A is the initial gene mapping and Mutmap resequencing Manhattan diagram, B is the OsLRK11 gene structure analysis diagram, C is the LRK11 protein domain analysis diagram, and D is the protein structure prediction diagram; where WT is wild type Yixiang 1B and Irk11 is the large-grain mutant;

[0029] Figure 4 It is a phenotypic diagram of the T2 generation of the OsLRK11 knockout line; where A is the plant phenotype of the OsLRK11 knockout line and B is the grain phenotype diagram of the OsLRK11 knockout line; where WT is wild type Yixiang 1B, and K0-1, K0-2, and K0-3 are large-grain mutants;

[0030] Figure 5 It is a statistical chart of the agronomic traits of the T2 generation of the OsLRK11 knockout line; among them, A is the statistical chart of plant height, B is the statistical chart of grain length, C is the statistical chart of grain width, D is the statistical chart of 1000-grain weight, E is the statistical chart of tillering, F is the statistical chart of seed setting rate, G is the statistical chart of the number of primary branches, H is the statistical chart of the number of secondary branches, I is the statistical chart of panicle length; among them, WT is the wild-type Yixiang 1B, and K0 is the large-grain mutant;

[0031] Figure 6 It is a diagram for cytological (paraffin section and scanning electron microscopy) and related gene expression pattern analysis; among them, A is the electron microscopy observation diagram of the outer epidermis cells of the lemma, bar = 200 μm, B is the comparison diagram of the number and length of longitudinal cells of the outer epidermis of the lemma (the data of the wild-type WT is adjusted to 1), C is the observation diagram of the paraffin section of the glume cells, D is the quantitative expression diagram of genes related to cell proliferation, E is the quantitative expression diagram of genes related to the cell cycle; among them, WT is the wild-type Yixiang 1B, and Irk11 is the large-grain mutant;

[0032] Figure 7 It is an evolutionary analysis diagram of LRK11; among them, A is the phylogenetic tree, B is the gene structure diagram of the rice RLK family, C is the conserved sequence diagram of the rice RLK family genes, D is the statistical chart of the functional domains of the rice RLK family, E is the transmembrane domain analysis diagram of OsLRK11, F is the conserved domain analysis diagram of the OsLRK11 gene;

[0033] Figure 8 It is a transcriptome analysis diagram; among them, A is the statistical chart of the number of differentially expressed genes, B is the heat map of differentially expressed genes, C is the statistical chart of the G0 functional classification of differentially expressed genes, D is the heat map of genes related to the MAPK pathway and plant hormone pathway, E is the relative quantitative detection diagram of genes related to the MAPK pathway; among them, WT is the wild-type Yixiang 1B, and Irkll is the large-grain mutant. Detailed implementation manners

[0034] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0035] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0036] Unless otherwise specified, all kinds of raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0037] The following will combine examples and experimental data to elaborate in detail on a gene OsLRK11 for controlling rice seed grain shape, its encoded protein, and its applications in the present application.

[0038] The genetic background of the present invention's research is the hybrid rice backbone parent Yixiang 1B in China. A large-grain rice mutant was obtained by screening in an ethyl methanesulfonate (EMS) mutagenesis library and named bg11 (big grain 11. For convenience of record later, the mutant was named Irk11 with the OsLRK11 gene annotation). Genetic analysis found that this large-grain mutant trait is controlled by a single recessive nuclear gene. Further research found that the 1000-grain weight, grain length, and length-width ratio of the mutant seeds are significantly increased compared with the wild type, but traits such as plant height and the number of filled grains per panicle also decreased to a certain extent.

[0039] The gene Os11g0173432 (named OsLRK11) encodes a leucine-rich repeat receptor kinase, and its main biological function is to regulate the MPKK4-MAPKK6 cascade reaction. At present, no research reports on the cloning of the OsLRK11 gene and its involvement in controlling rice seed size have been found.

[0040] Example 1

[0041] The present invention provides the grain phenotype identification and genetic analysis of the large-grain mutant Irk11 and the wild-type parental lines Yixiang 1B and Zhonghua 11. By analyzing the upstream and downstream genes of the OsLRK11 gene and the related regulatory signaling pathways involved, the mechanism of action of OsLRK11 in controlling seed size is clarified, laying a theoretical foundation for enriching the molecular mechanism of cereal crop seed size.

[0042] (1) Test materials

[0043] 1) The rice variety under test was the japonica rice variety Zhonghua 11, which is rice (Oryza sativa subsp. Japonica. ZH11). The Zhonghua 11 japonica rice variety was preserved in the Genetic Research Laboratory of the Rice Research Institute of Sichuan Agricultural University. Before planting the newly harvested seeds of each season, they were first disinfected with 1.5% hydrogen peroxide (H2O2) by mass percentage concentration for 20 minutes, soaked in oxygen-supplied ddH2O (containing 1% H2O2) at 37°C for 16 hours, and then the seeds were placed in an incubator at 37°C for germination culture, changing the water two to three times a day. After seeing the seeds showing white tips, they were transferred to a 28°C environment for culture. When the rice buds grew to 1-2 cm long, they were sown on a window screen cloth and cultured in a rice nutrient solution until the two-leaf and one-heart stage, and then the rice seedlings were transplanted into a rice cultivation field for management, observation, and trait statistics.

[0044] 2) The large-grain mutant Irk11 was a grain-type mutant screened from a mutant library constructed by EMS mutagenesis with the indica rice maintainer variety Yixiang 1B as the background by the inventors. This mutant was backcrossed with the wild-type Yixiang 1B for multiple generations, and the large-grain mutant trait could be stably inherited.

[0045] (2) Test methods

[0046] 2.1 Reagents and vectors used

[0047] Plasmid CRISPR / Cas9::P35S-H was purchased from Hangzhou Baige Biotechnology Co., Ltd.; Agrobacterium competent EHA105 was purchased from Beijing Tsingke Biotechnology Co., Ltd.; Escherichia coli (E. coli) DH5α competent was purchased from Shanghai Sangon Biotech Co., Ltd.; Plasmid Extraction Kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd.: Plasmid Mini Kit DC201, FastPure EndoFree PlasmidMaxi Kit DC202; NAprep Pure Total Plant RNA Extraction Kit was purchased from Beijing TransGen Biotech Co., Ltd.; Reverse Transcription Kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd. II QRT SuperMix for qPCR(+gDNAwiper); qPCR Kit and Recombinase were purchased from Nanjing Novozymes Biotechnology Co., Ltd.: AceQ qPCR SYBR Green Master Mix (without ROX), II One Step Cloning Kit;

[0048] 2.2 Investigation of agronomic traits

[0049] At the mature stage of rice, 10 normally developed individual plants were randomly selected from each plot of wild type, mutant, control group, and gene knockout experimental group (constructed by the method of Example 4) growing in the field, and agronomic traits such as plant height, grain length, grain width, number of primary branches, number of secondary branches, tiller number, main panicle length, and 1000-grain weight were investigated respectively. The final index reference value of each trait index was the average value of 10 individual plants.

[0050] As Figure 1 .A-G shows, a mutant Irk11 with significantly increased grain weight and significantly reduced spikelet number was identified in the EMS mutant library of Yixiang 1B. As Figure 2 A-F shows, compared with the wild type, grain weight-related traits such as grain length and 1000-grain weight of the mutant Irk11 were significantly increased (increased by 62.8% and 26.7% respectively), but its plant height and spikelet number were significantly reduced (-9.8%, -31.2%). Among them, there was no significant difference in the number of primary branches, but an incidental trait of long awns appeared.

[0051] 2.3 Genetic analysis experiment of large grain mutant

[0052] Both the wild type Yixiang 1B and the large grain mutant Irk11 were planted in the rice experimental field of Qingpu Garden, Wenjiang Campus, Sichuan Agricultural University. The wild type Yixiang 1B and the mutant Irk11 were reciprocally crossed respectively, and a genetic segregation population was constructed for counting the phenotypes of BC1F1 generation and the segregation ratio of BC1F2 generation population.

[0053] The BC1F1 generation was obtained by reciprocal crossing of the wild type Yixiang 1B and the mutant Irk11. The grain size of the BC1F1 generation plants was similar to that of the wild type. After chi-square test, the results (see Table 1) showed that the segregation ratio of wild type and large grain phenotypes in the BC1F2 generation was 3:1, which was consistent with the segregation ratio of Mendelian single recessive gene, indicating that the large grain trait of the mutant Irk11 was controlled by a single recessive nuclear gene.

[0054] Table 1 Results of genetic analysis experiment of large grain variant Irk11

[0055]

[0056] In Table 1, 02428 is a japonica rice variety.

[0057] Example 2

[0058] Observation and statistics of agronomic traits of the wild type Yixiang 1B, the large grain mutant Irk11, and the CR / SPR / Cas9-OsLRK11 knockout lines K0-1, K0-2, K0-3 of the present invention and the wild type ZH11.

[0059] In the summers of 2019 and in the spring and summer of 2020, wild-type Yixiang B, large-grain mutant Irk11, transgenic CRISPR / Cas9 receptor material Zhonghua 11, and CRISPR / Cas9 positive homozygous lines (constructed using the method of Example 4) were planted in Lingshui, Hainan and Wenjiang according to a randomized block design, with 3 replicates in rows of 10 plants each. At maturity, 5 normally growing individual plants were randomly selected from each plot to investigate the relevant main agronomic traits, with three replicates. A Wanshen SC-G automatic seed analyzer and a thousand-grain weight analyzer were used to examine the grain shape traits, and the specific method was carried out in accordance with the "China Rice Resource Evaluation Standard".

[0060] The results are as Figure 5 . shown in A-I. The knockout homozygous mutants showed phenotypes similar to those of the mutants: such as a significant increase in grain length and thousand-grain weight (an increase of 52.6% and 28.3% respectively), a significant decrease in plant height, but no significant decrease in seed setting rate.

[0061] Example 3

[0062] Mapping and cloning experiment of the candidate gene of the large-grain mutant Irk11 of the present invention

[0063] I) Experimental materials

[0064] The large-grain mutant / rk11, wild-type Yixiang 1B, and 02428 were all provided by the Genetic Research Laboratory of the Rice Research Institute of Sichuan Agricultural University.

[0065] II) Experimental methods

[0066] (1) Population construction

[0067] The mutant Irk11 was crossed with the japonica rice variety 02428 to construct an F1 population, and self-crossed to obtain an F2 population for genetic mapping. The mutant Irk11 was backcrossed with Yixiang1B to construct a BC1F3 population for Mutmap sequencing for fine mapping of the gene.

[0068] (2) Near-isogenic pool construction

[0069] The F1 generation obtained by crossing Irk11 with 02428 was then self-crossed to obtain the F2 segregation population, and the bulk segregation analysis (BSA) method was used for gene mapping and analysis. First, randomly select the leaves of 10 individual plants of Irk11 and 02428 respectively, and extract DNA by mixing the leaves of every 10 plants equally to construct pools, obtaining 2 parental DNA pools for screening polymorphic molecular markers between the parents. Then, select the leaves of 10 individual plants with large grain phenotypes and the leaves of 10 individual plants with normal grain phenotypes similar to the wild type in the F2 segregation population obtained by crossing the mutant Irk11 with 02428, and extract DNA by mixing the leaves of every 10 plants equally to obtain a dominant pool and a recessive pool respectively for analyzing the linkage relationship between the mutant traits and chromosomes. Finally, select the leaves of 120 individual plants with large grain mutant phenotypes in the F2 population obtained by crossing the mutant Irk11 with 02428, and extract DNA from individual plants using the improved CTAB method for gene mapping.

[0070] (3) Synthesis of mapping primers and gene mapping

[0071] First, 512 pairs of SSR primers evenly distributed on 12 rice chromosomes and stored in our laboratory (for specific sequences, see http: / / www.gramene.org / bd / markers) were used for PCR amplification, and then agarose gel electrophoresis was used to screen out 32 pairs of primers with polymorphisms between the genomes of Irk11 and 02428; subsequently, the 32 pairs of screened polymorphic primers were used to detect the dominant pool and the recessive pool, as well as 120 recessive individual plants in the F2 population constructed by Irk11 and 02428 for preliminary gene mapping; within the initially mapped interval, Inde1 primers were designed based on the differences between the nucleotide sequences of the target regions of indica rice variety 9311 and japonica rice variety Nipponbare published on the website (http: / / www.gramene.org).

[0072] Among them, the PCR reaction system (20 μL): Taq enzyme (5 U / μL) 0.2 μL, Primer (10 mmol / L) 2 μL, dNTP (10 mmol / L) 0.3 μL, DNA template (50 - 200 ng / μL) 2 μL, 10× Buffer (25 mM) 2 μL, ddH2O 13.5 μL. PCR reaction program: 95°C / 5 min; 95°C / 30 s, 55°C / 30 s, 72°C / 1 min, steps 2 - 4, 34 cycles; 72°C / 10 min, 12°C / 1 min.

[0073] Electrophorese the PCR amplification products in a 2.5% (7.5 g agarose dissolved in 300 ml ddH20) agarose gel at a constant voltage of 180 V - 200 V for about 30 min - 45 min, and image and save the record using a gel scanning imager (Bio-rad Gel Doc 2000).

[0074] (4) Fine mapping and gene prediction of candidate genes

[0075] To further quickly and accurately find the mutant gene, 30 large-grain mutant phenotype single plants and 30 single plants with a grain type similar to the wild type were randomly selected from the BCF3 population generated by backcrossing Irk11 with wild-type Yixiang 1B. Equal amounts of DNA were used to construct a mutant pool and a wild-type pool respectively for MutMap whole-genome resequencing. Using the wild-type pool genomic sequence as a control group, the high-throughput sequencing results of the mutant's whole genome were analyzed and interpreted, with a sequencing depth of 30 layers. The sequencing data was completely aligned with the published Nipponbare reference genome (MSU Osa1 Release 7 Annotation) using the SOAP2 software, and short sequence fragments at specific chromosomal positions were screened according to the initial mapping interval. Data analysis software such as SOAPsnp, SOAPsv, and SOAPindeI were used to analyze and interpret the single nucleotide polymorphisms (SNPs), SVs, and InDels between the mutant and the Nipponbare reference group. Six SNPs and two SVs were specifically found in the mutant / rk11 within the mapping interval. By calculating the ΔSNP index, high ΔSNP index readings with F2-read ≥ 15 and continuous distribution were selected. As Figure 2 shown, a scatter plot of SNP sites on 12 chromosomes was obtained through data analysis. A high ΔSNP index score with continuous distribution appeared on the short arm of chromosome 11, which was consistent with our initial mapping interval. Thus, the interval where our candidate gene is located was determined to be between 2 M and 4 M in physical distance on the short arm of chromosome 11, and there are no reported genes related to the mutant trait. Therefore, the research results indicate that OsLRK11 is a new gene controlling seed size.

[0076] The analysis results of Mutmap whole-genome sequencing data were combined with the mutant phenotype of Irk11, and the indica rice databases in the rice genome annotation websites (http: / / rice.plantbiology.msu.ed / cgi-bin) and (http: / / plants.ensembl.org / index.html). According to the gene function annotation, screening was carried out within the mapped interval. Further analysis showed that most of these SNP sites were intergenic, intronic or synonymous mutations. Only one SNP site was located in the first exon of the gene Os11g0173432, which was a non-synonymous mutation. To confirm the reliability of the Mutmap resequencing results, primers were designed for three SNP sites and the deletion fragment given by the analysis company, and PCR amplification and Sanger sequencing were carried out in wild-type and mutant plants respectively. The results showed that the single-base mutation of the non-synonymous SNP occurred in wild-type and mutant plants, which was consistent with the results given by the analysis company. At the 714th base in the CDS region of this protein-coding gene, G (guanine) was converted to A (thymine), resulting in the 238th amino acid encoded changing from M (methionine) to I (isoleucine). This base mutation might disrupt the normal coding sequence of the protein, leading to the disruption of protein function (as shown in Figure 3 .A-D), so the gene Os11g0173432 was listed as a candidate gene for the Irk11 mutant.

[0077] Example 4

[0078] CRISPR / CAS9 (gene knockout) experiment on the candidate gene of the large-grain mutant / rk11 1. Construction of the CRISPR / Cas9-OsLRK11 gene knockout vector

[0079] Using the cDNA of mutant Irk11, Yixiang 1B, and Zhonghua 11 as templates to amplify this gene respectively, it was found that the amino acid sequences in the coding regions of this gene in indica rice Yixiang 1B and japonica rice Nip were highly homologous, indicating that the protein encoded by this gene might perform similar biological functions in indica rice Yixiang 1B and japonica rice Zhonghua 11.

[0080] Using the nucleotide sequence of the OsLRK11 gene (SEQ ID NO.6) in the japonica rice variety Zhonghua 11 (ZH11) as a template, a specific region was selected, and 1 independent knockout target site was designed. Using the BWA(V)H-CAS9 BGK03 gene knockout vector, referring to the kit (Hangzhou Baige Biotechnology Co., Ltd.), the CRISPR / CAS9-OsLRK11 vector was constructed. The specific construction process is as follows:

[0081] (1). Design and synthesize the following linker primers to form the gRNA target sequence:

[0082] F: 5'-GTTTTAGACAATATGATAGCTCATGTT-3' (SEQ ID NO.7),

[0083] R: 5'-CAAAATACATGAGCTATCATATTGTCA-3' (SEQ ID NO.8);

[0084] OsU6a promoter for knockout

[0085] F: 5'-AACTTATAAACCGCGCGCT-3' (SEQ ID NO.9)

[0086] R: 5'-TTGAATATTTGGGCGCGCGA-3' (SEQ ID NO.10)

[0087] (2), Preparation of primer dimers

[0088] Dissolve the primer pairs synthesized in step (1) in water to 10 μM. After mixing according to the following reaction system, heat at 95 °C for 3 min in a PCR instrument, and then slowly cool to 20 °C at about 0.2 °C / second to obtain primer dimers. The reaction system is as follows: 18 μL of Ageal Buffer, 1 μL of each gRNA target primer, add ddH2O to make up to 20 μL.

[0089] (3), Construct the primer dimers into the BWA(V)H vector.

[0090] Mix each component of the reaction system on ice. After mixing, react at 20 °C for 1 hour and then transform Escherichia coli for standby to obtain an expression vector containing elements such as a promoter, target sequence, and gRNA. The reaction system: 2 μL of BWA(V)H vector, 1 μL of Oligo dimer, 1 μL of enzyme mixture, add ddH2O to make up to 10 μL.

[0091] 2. Escherichia coli transformation

[0092] (1), Take out a tube of prepared Escherichia coli competent cells from the -80 °C refrigerator and place them on ice to melt;

[0093] (2), Add 100 μL of competent cell suspension to every 10 μL of ligation product, mix well and place on ice for 30 min;

[0094] (3), Heat shock at 42 °C for 30 s, quickly take out and immediately place on ice for 2 min;

[0095] (4), Add 500 L of LB liquid medium without antibiotics and culture at 37 °C and 200 rpm for 1 hour to obtain an activated bacterial solution:

[0096] (5) Centrifuge the activated bacterial liquid at 5000 rpm for 1 min, pour off most of the supernatant under sterile conditions, gently pipette and mix the precipitate, aspirate 80 μL, and transfer and spread the bacterial liquid onto an LB screening plate containing kanamycin on a super clean bench;

[0097] (6) Place the LB solid medium plate coated with the bacterial liquid face up for about 10 min. After the bacterial liquid is completely absorbed by the LB solid medium, invert the coated medium plate and incubate it overnight at 37 °C in an incubator;

[0098] (7) Pick single colonies and perform PCR detection using P-OsLRK11. The P-OsLRK11 primer pair is:

[0099] P-OsLRK11 K0-F: 5′-CCCAGTCACGACGTTGTAAA-3’ (SEQ ID NO.11),

[0100] The PGR reaction program: 98 °C / 3 min; 95 °C / 15 s, 57 °C / 15 s, 72 °C / 30 s, 35 cycles; 72 °C / 10 min, 4 °C / 1 min.

[0101] (8) Pick positive clones into 5 ml of LB culture medium containing kanamycin (50 mg / L), culture at 37 °C and 200 rpm for about 16 h, preserve the bacterial liquid and extract the plasmid.

[0102] 3. Plasmid extraction

[0103] Extract the Escherichia coli plasmid according to the product instruction manual of OMEGA Plasmid Extraction Kit, collect the extracted plasmid DNA into a clean centrifuge tube, and store it at -20 °C.

[0104] 4. Determination and sequence analysis of plasmid sequences

[0105] Send the positive clone plasmid to Chengdu Qingke Technology Co., Ltd. for sequencing. Use DNAMAN software to perform sequence alignment on the sequencing results to confirm the correctness of the gRNA sequence, and name the positive clone plasmid CRISPR / Cas9-OsLRK11.

[0106] 5. Agrobacterium transformation

[0107] (1) Agrobacterium chemical transformation method

[0108] According to one plasmid: When 50 μL of competent cells are taken out from -80°C, quickly thaw them in the palm of the hand; add 0.4 - 1 μg of the constructed CRISPR / Cas9-OsLRK11 plasmid to 50 μL of competent cells, and place them on ice for 30 min; freeze in liquid nitrogen for 2 min; incubate in a 37°C water bath for 2 min to melt the cells; immediately add 5 times the volume of antibiotic-free LB liquid medium, and culture on a shaker at 28°C and 170 rpm for 2 - 3 h; centrifuge at 7000 rpm for 2 minutes, and suspend the cells in 100 μL of LB liquid medium; spread them on a rifampicin and kanamycin double-resistant medium, and culture at 28°C for 2 - 3 days; perform colony PCR detection of the agrobacterium with the hygromycin molecular marker P-OsLRK11 primer. For the positive agrobacterium monoclonal that can amplify the target band, add glycerol as a cryoprotectant and store it at -80°C for later use.

[0109] (2) Transformation of rice by agrobacterium-mediated method

[0110] (a) Induction of callus: First, disinfect Nipponbare seeds with 75% alcohol for 1 minute, rinse them 3 times with sterile water, then rinse them with 40% sodium hypochlorite for 30 min, and then rinse them 5 times with sterile water. Place them in a petri dish with filter paper to drain, and inoculate them on NMB medium with forceps. Culture at 28°C under light conditions for 7 days. Subculture every 7 days. After subculturing 2 - 3 times, pick out the good callus grown from the seeds and subculture them on NMB medium. Culture at 28°C under dark conditions for 4 days.

[0111] (b) Activation of agrobacterium strain: Add 30 μL of agrobacterium stored at -80°C in (1) to 3 mL of YEP liquid medium containing rifampicin and kanamycin, and culture with shaking at 28°C for 14 h; then take 1 mL of it and transfer it to 50 mL of YEP liquid medium containing rifampicin and kanamycin, and culture with shaking at 28°C for another 4 h to obtain the activated agrobacterium liquid.

[0112] (c) Co-culture transformation: Centrifuge the activated liquid in (b) at 5000 rpm to collect the bacteria, resuspend the bacteria in 30 mL of AAM liquid medium containing 100 μM / L acetosyringone, immerse the pre-picked callus in (a) in the bacterial liquid for 20 min, suck out the excess bacterial liquid, spread it evenly on the co-culture solid medium, and culture in the dark at 28°C for 2 d.

[0113] (d) Callus decontamination culture and callus resistance screening: Rinse the callus after 2 d of co-culture with sterile water until the water is clear, then sterilize it by shaking in sterile water containing cephalosporin (500 mg / L) for 30 min. Thoroughly dry the callus with sterile filter paper or absorbent paper, and then inoculate it on the selection medium and culture for about 3 weeks.

[0114] (e) Differentiation and rooting of transgenic plants: Inoculate the newly grown resistant callus in (d) onto the differentiation medium and culture it under light for 1 - 2 months. Then transfer the seedlings about 3 cm tall to the rooting medium for rooting culture. When the seedlings grow to about 10 cm, take the leaves to extract DNA, use the p-OsLRK11 primer pair to amplify the full-length DNA of the target gene, and finally obtain 3 transgenic positive plants. Name the 5 transgenic positive plants respectively as: K0-1, K0-2, K0-3;

[0115] (f) Hardening off: After hardening off the positive transgenic plants indoors for 2 - 3 days, transplant them to the field.

[0116] 6. Detection of transgenic rice

[0117] (1) PCR amplification

[0118] Extract the DNA of the positive transgenic plants obtained in step 5 by using the improved CTAB method, and use the P-OsLRK11 primer pair to amplify the full-length sequence of the knockout target gene in the transgenic plants. The size of the PCR product fragment is 845 bp. The P-OsLRK11 primer pair is as follows:

[0119] OsLRK11 K0-F: 5′-ACATCGCAATCTAGTTC-3′ (SEQ ID NO.12)

[0120] OsLRK11 K0-R: 5′-CTTGGTAGCCACCTCCT-3′ (SEQ ID NO.13)

[0121] Among them, the PCR reaction system (25 μL): Tap enzyme (5 U / μL) 0.5 μL, Primer (10 mmol / L) 2 μL, dNTP (2.5 mmol / L) 0.5 μL, DNA (20 - 100 ng / μL) 2 μL, 2×Buffer (25 mM) 12.5 μL, ddH2O 7.5 μL. The PCR reaction program is: 95°C for 5 min; 95°C for 30 s, 56°C for 5 s, 72°C for 2.5 min, 30 cycles; 72°C for 10 min, 12°C for 1 min.

[0122] (2) Recovery and sequencing of PCR products

[0123] After the PCR amplification reaction is completed, add bromophenol blue indicator to the product, electrophorese it in 2% agarose, and use the Tiangen PCR product recovery kit for recovery and preservation. The reaction system is specifically as follows:

[0124] 1) After the fragment is completely separated, quickly cut the target band with a small knife under ultraviolet light and put it into a new EP tube.

[0125] 2) Weigh the gel block on an electronic balance. According to the ratio of adding 1 ml of Binding Buffer per 1 g of gel, add an appropriate amount of Binding Buffer and water bath in a 60 °C water bath for 10 min until the gel block is completely dissolved. During this period, gently invert it every 2 min.

[0126] 3) Insert the HiBind DNA column into a 2-ml collection tube.

[0127] 4) Transfer the gel mixture to the HiBind DNA column and centrifuge at 10000 g / min for 1 min.

[0128] 5) Discard the filtrate, reinstall the column back into the collection tube (a HiBind column can hold 700 μL of solution at a time), and repeat steps 4 - 5.

[0129] 6) Reinstall the column back into the collection tube, add 300 μL of Bind Buffer, centrifuge at 10000 xg / min for 1 min, and discard the filtrate.

[0130] 7) Reinstall the column back into the collection tube, add 700 μL of SPW Wash Buffer, centrifuge at 10000 xg / min for 1 min, and discard the bottom liquid (the SPW Wash Buffer is diluted with absolute ethanol first).

[0131] 8) Repeat step 7 once.

[0132] 9) Discard the filtrate, reinstall the column back into the collection tube, and centrifuge at 13000 g for 2 min with no sample.

[0133] 10) Reinstall the column into a sterilized 1.5-ml EP tube, add 40 μL of ElutionBuffer heated in a 65 °C water bath, let it stand at room temperature for 2 min, centrifuge at 13000 g / min for 2 min to elute the DNA, then perform gel electrophoresis, load the Maker, analyze the integrity and concentration of the purified DNA, and send it to Chengdu Qingke Technology Co., Ltd. for sequencing after detection.

[0134] Results (see Figure 4 .A - B) All three independent transgenic positive plant lines showed the phenotype of large-seeded mutants. Compared with the negative control, it was found that single-base mutations occurred in the CDS coding region of the OsLRK11 gene in the three transgenic plants (see SEQ ID NO.3 - SEQ ID NO.5). Through the knockout experiment of the OsLRK11 gene, it was shown that the OsLRK11 gene is the gene controlling the seed size phenotype; it was also proved that OsLRK11 is the gene controlling the large-seeded phenotype of the mutant LRK11.

[0135] 7. Using the method described in Example 2, the transgenic knockout lines K0-1, K0-2, K0-3 and the control variety Zhonghua 11 were subjected to seed measurement and statistics.

[0136] The results (see Figure 5 .A-I) showed that similar to the large-grain trait of the mutant LRK11, compared with the control Zhonghua 11, the grain length and 1000-grain weight of the seeds of the transgenic lines K0-1, K0-2, K0-3 with the OsLRK11 gene knocked out were significantly increased compared with the negative control Zhonghua 11 (ZH11). This indicated that editing the CDS coding region of the OsLRK11 gene (relative to the mutation site of the mutant LRK11), including one or several additions, substitutions, and deletions, could also obtain the large-grain phenotype of the mutant LRK11; it showed that the OsLRK11 gene was a gene related to controlling seed size, and knocking out this gene could increase grain length and 1000-grain weight.

[0137] Example 5

[0138] Cytology (paraffin section and scanning electron microscopy) and analysis of related gene expression patterns

[0139] 1) Steps for paraffin section:

[0140] (1) Select rice glumes at the booting stage, immerse them in 50% FAA fixative, use a vacuum concentrator to evacuate until the materials sink to the bottom, then replace with fresh FAA fixative, and seal in a 4°C refrigerator for overnight fixation;

[0141] (2) The fixed materials are successively placed in solutions of different concentrations of alcohol, H2O, and 10% HF for softening:

[0142] (3) Gradually immerse the softened materials in alcohol for dehydration;

[0143] (4) Make the materials transparent;

[0144] (5) Impregnate with wax: Prepare broken wax in advance and intermittently put a small amount of broken wax into the well-transparent materials;

[0145] (6) Prepare a paper boat in advance, pour the melted paraffin and the materials together into the pre-folded paper boat, then place it on ice to solidify, and store it in a 4°C refrigerator after solidification. It can be taken out the next day to trim the wax for standby;

[0146] (7) Section: Cut the wax block with the sample fixed into relatively thin wax slices;

[0147] (8) The baked slides are successively subjected to steps such as dewaxing, rehydration, staining, and dehydration;

[0148] (9) Prepare neutral resin in advance for mounting the slides;

[0149] 2) Preparation of Scanning Electron Microscope Samples:

[0150] (1) Sample Preparation and Fixation: Select the young panicle tissues of wild type and mutants at the primordium differentiation stage during the booting stage, place them in pre-cooled 2.5% glutaraldehyde for fixation, and fix at 4°C for more than 2 hours or overnight;

[0151] (2) Gradient Dehydration of Samples: Prepare 50%, 70%, 85%, 95%, and 100% ethanol solutions. Place the young panicles in centrifuge tubes and dehydrate them with different gradients of ethanol for 15 minutes each. Finally, dehydrate with 100% ethanol three times;

[0152] (3) Supercritical Fluid Drying;

[0153] (4) Sample Gold Spraying Treatment;

[0154] (5) Observe using a scanning electron microscope.

[0155] 3) Gene Expression Analysis

[0156] To detect the expression levels of related genes in mutants and wild types, we sampled different tissue parts of mutants and wild types at different developmental stages, and detected the changes in the expression level of OsLRK11 by qRT-PCR method. The qualified total RNA extracted by Trizol was used to synthesize single-stranded cDNA with a quantitative reverse transcription kit (TaKaRa Biotechnology CO., LTD, Japan). Referring to the instructions of the SYBR Green quantitative kit (BioBad, USA) and the CFX96 real-time system instrument, qPCR quantification was performed on the target genes (classical grain type genes (GS3, GS5, GW8, SRS5, GL7, etc.), spikelet number genes (LAX1, Gn1a, SPL14, RCN1, etc.)). We set three replicates for the samples to be detected and the rice internal reference gene ACTIN respectively. The algorithm is the 2 -ΔΔCT method;

[0157] As Figure 6 . A-E shows, the number and size of cells on the lemma epidermis of wild type and mutant Irk11 were observed by scanning electron microscope ( Figure 6 A), and the results showed that there was no significant difference in the number of mutant cells, but the cells became longer and the area increased, resulting in an increase in the grain length, grain width, and grain weight of Irk11; quantitative detection of cell cycle-related genes showed that genes related to cell proliferation were significantly increased in Irk11, while the expression levels of cell cycle-related genes showed no significant difference.

[0158] Example 6

[0159] Mechanism Analysis of OsLRK11 Gene (Phylogenetic Tree Analysis and Transcriptome Testing)

[0160] 1) Phylogenetic tree analysis

[0161] To explore the homology of the LRK11 amino acid sequence, we used the NCBI database to align the amino acid sequences and selected the top 25 amino acid sequences with higher homology to construct a phylogenetic tree ( Figure 7 .A). It can be seen that LRK11 has a relatively high homology with some LRK receptor protein kinases in rice. According to the analysis results of the comprehensive bioinformatics analysis website SMART (http: / / smart.embl-heidelberg.de / ), it was found that there is an LRR domain at positions 121-143 at the N-terminus of LRK11, an unknown domain at positions 156-172, a total of 6 LRR domains in the middle of the amino acids (positions 414-607), and a transmembrane domain at the C-terminus ( Figure 7 .B-E).

[0162] 2) Transcriptome determination

[0163] Total RNA was extracted from the young panicles (YP: 4 cm) of ZH11 and OsLRK11-K0 plants respectively and sent to Shanghai OE Biotech Co., Ltd. for transcriptome sequencing. After mRNA purification and library construction, transcriptome sequencing was carried out, and GO and KEGG enrichment analyses were performed on differentially expressed genes to find the regulatory pathways involved in differentially expressed genes, especially focusing on genes related to grain weight and spikelet number per panicle. The sequencing results showed ( Figure 8 .A-E) that there were a total of 836 differentially expressed genes, among which 681 genes were up-regulated and 151 genes were significantly down-regulated or silenced. Interestingly, through the combined analysis of GO functional analysis and KEGG enrichment of these differentially expressed genes, the differentially expressed genes were mainly concentrated in two pathways: plant hormone signal transduction (mainly BR and auxin responses) and MAPK signaling pathway.

[0164] Finally, it should also be noted that the term "comprising", ";", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0165] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0166] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Application of knocking out the gene OsLRK11 that controls rice seed grain shape in improving rice seed grain shape or 1000-grain weight, characterized in that, The nucleotide sequence of the OsLRK11 is shown as SEQ ID No.1, and the rice variety is Yixiang 1B.

2. Use of a target sequence for knocking out the gene OsLRK11 in improving the grain shape or 1000-grain weight of rice seeds, characterized in that, The target sequence consists of the nucleotide sequences shown by SEQ ID No.7 and SEQ ID No.8; The nucleotide sequence of the OsLRK11 is shown as SEQ ID No.

1.

3. A method for cultivating a large-grain rice strain, characterized in that, The method includes: Synthesizing a target sequence for knocking out the gene OsLRK11; Constructing a CRISPR / CAS9 system expression vector containing the target sequence; Transforming the CRISPR / CAS9 system expression vector into rice; Select OsLRK11 The rice line with the OsLRK11 gene knocked out is the large-grain rice line; The nucleotide sequence of the OsLRK11 is shown as SEQ ID No.1.

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