Application of OsATG3 gene in regulation and control of tiller number of rice

Abstract

The invention discloses application of an OsATG3 gene in regulation and control of the tiller number of rice, a nucleotide sequence of an OsATG3 gene coding region is shown as SEQ ID NO.1, and an amino acid sequence coded by the OsATG3 gene is shown as SEQ ID NO.2. The invention further discloses application of the OsATG3 gene in regulation and control of the tiller number of rice. The important role of the OsATG3 gene in rice tillering and yield regulation and control is disclosed for the first time, the OsATG3 is used as a key candidate gene qTN1 for regulating and controlling rice tillering so as to influence the yield, overexpression of the gene can significantly increase the rice tillering number so as to improve the rice yield, a new technical approach is provided for breeding of a new high-yield rice variety, and the application prospect is broad. The method is of great significance to guarantee grain safety.

Description

Technical Field

[0001] This invention relates to molecular breeding of rice, specifically, to a method OsATG3 Application of genes in regulating the number of rice tillers. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, and increasing rice yield is crucial for ensuring food security. Rice yield is mainly determined by three factors: the number of panicles, the number of grains per panicle, and grain weight. The number of panicles is primarily controlled by tillering ability. Tillering is an inherent physiological characteristic of rice, and the number of tillers directly affects the number of effective panicles, thus influencing the final yield. However, excessive tillering leads to excessive plant density, affecting ventilation and light penetration, and increasing the risk of pests and diseases; insufficient tillering prevents the formation of enough effective panicles, resulting in reduced yield. Therefore, how to regulate rice tillering ability is an important research direction in high-yield rice breeding.

[0003] Autophagy is a highly conserved cellular degradation pathway in eukaryotes, playing a crucial role in plant growth, development, and stress responses by recycling intracellular components. Recent studies have revealed that autophagy-related genes are involved in regulating multiple yield-related traits in rice. For example, OsATG8b Gene mutations can lead to smaller grains and increased chalkiness, while overexpression... OsATG8b This can improve grain size and quality (YU J, et al. 2019). However, there are few research reports on the direct regulation of rice tillering ability by autophagy genes and thus its impact on rice yield, and there is a lack of systematic molecular mechanism analysis.

[0004] Currently, rice tillering is mainly regulated through traditional breeding methods or a few known tillering regulatory genes. However, these methods suffer from problems such as low regulatory precision and limited gene resources. Therefore, discovering new tillering regulatory genes and elucidating their molecular mechanisms is of great significance for high-yield rice breeding. Summary of the Invention

[0005] To address the problems of low precision and limited gene resources in current methods for regulating rice tillering, this invention provides a... OsATG3 The application of genes in regulating the number of rice tillers provides new genetic resources and molecular breeding strategies for high-yield rice breeding, and has important application value.

[0006] To achieve the above objectives, the present invention provides, in one aspect, a... OsATG3 The application of genes in regulating rice tiller number. OsATG3 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.1. OsATG3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0007] SEQ ID NO.1: ATGCAGGTGAAGCAGAAGGTGTACGAGCTCTACAAGGGGACGGTGGAGCGGGTCACGGGCCCGCGCACCGTGTCGGCCTTCCTCGACAAGGGCGTCCTCTCCGTCCCCGAGTTCATCCTCGCCGGCGACAACCTCGTCTCCAAGTGCCCCACCTGGTCCTGGGAGGCGGGCGATCCGAGCAAGAGGAAGCCGTATCTCCCACCTGACAAGCAATTTCTCGTCACCAGGAACGTGCCTTGCCTAAGACGTGCTGTTTCGCTTGAAGAAGAGTATGATGCAGCAGGAGCTGAGGTAGTTCTCGGTGATGATGAGGACGGTGAAGGCTGGCTTGCCACACATGGAGTGCAAGCATCAAAGCAAGAAGAGGAGGAAGACATACCATCGATGGATACATTGGATATAGGGAAAACTGAAGGGATAAAGTCTATTCCCTCATATTTCAGTGCTGGTAAGAAGGCTGAAGAAGAGGAGGATATACCAGACATGGACACTTATGAAGACTCAGGGAATGATTCAGTGGCTACCGCTCAACCTTCATATTTTGTCGCAGAAGAGCCTGAAGATGACAACATCCTTCGTACTAGGACATATGACGTTAGCATCACATATGACAAATATTACCAAACCCCACGTGTCTGGCTTACCGGATATGATGAGTCAAGAATGCCACTAAAGCCTGAACTTGTGTTTGAAGATATCAGCCAAGATCATGCACGGAAGACGGTGACTATTGAAGACCATCCTCACTTGTCAGCAGGCAAGCATGCTTCAGTGCATCCATGCAAACATGCTGCTGTGATGAAAAAGATCATTGATGTTCTAATGTCTCAAGGAGTTGAACCAGAAGTTGACAAGTACCTTTTCATATTTTTGAAGTTTATGGCCTCAGTCATACCCACTATTGAATATGACTACACTATGGACTTCGATCTGGGCAGCACAAGCAGATGA; SEQ ID NO.2: MQVKQKVYELYKGTVERVTGPRTVSAFLDKGVLSVPEFILAGDNLVSKCPTWSWEAGDPSKRKPYLPPDKQFLVTRNVPCLRRAVSLEEEYDAAGAEVVLGDDEDGEGWLATHGVQASKQEEEEDIPSMDTLDIGKTEGIKSIPSYFSAGKKAEEEED IPDMDTYEDSGNDSVATAQPSYFVAEEPEDDNILRTRTYDVSITYDKYYQTPRVWLTGYDESRMPLKPELVFEDISQDHARKTVTIEDHPHLSAGKHASVHPCKHAAVMKKIIDVLMSQGVEPEVDKYLFIFLKFMASVIPTIEYDYTMDFDLGSTSR.

[0008] Specifically, OsATG3 Genes positively regulate the number of tillers in rice.

[0009] Furthermore, overexpression of rice OsATG3 Genes that increase the number of tillers in rice; or Knock out rice OsATG3 Genes that reduce the number of tillers in rice.

[0010] The second aspect of the present invention provides OsATG3 The method for constructing knockout vectors, which is based on OsATG3 Using genes as targets, CRISPR / Cas9-based sgRNA primer sequences are designed, and DNA fragments containing the sgRNA primer sequences are inserted into the pYLCRISPR / Cas9-MH vector to obtain the target gene.

[0011] Specifically, OsATG3 Gene target 1 is located in exon 2, and its sequence is shown in SEQ ID NO.3. Target 2 is located in exon 3, and its sequence is shown in SEQ ID NO.4.

[0012] Specifically, the sgRNA primers include the sapotg3-1-gRT1 primer as shown in SEQ ID NO.5, the sapotg3-1-OsU3T1 primer as shown in SEQ ID NO.6, the sapotg3-2-gRT1 primer as shown in SEQ ID NO.7, and the sapotg3-2-OsU3T1 primer as shown in SEQ ID NO.8.

[0013] A third aspect of the present invention provides a method for reducing the number of tillers in rice, comprising using a method constructed as described above. OsATG3The steps for converting the knockout vector into rice.

[0014] The fourth aspect of the present invention provides OsATG3 The method for constructing overexpression vectors utilizes primers to amplify the overexpression vector. OsATG3 The encoded region sequence is obtained. OsATG3 The gene fragment is obtained by ligating it into the overexpression vector pCAMBIA1302.

[0015] Specifically, the primer sequences for the overexpression vector are shown in SEQ ID NO.13 and SEQ ID NO.14.

[0016] The fifth aspect of the present invention provides a method for increasing the number of tillers in rice, comprising constructing a method described above. OsATG3 The steps for converting the overexpression vector into rice.

[0017] Through the above technical solution, the present invention achieves the following beneficial effects: 1. This invention discloses for the first time OsATG3 The important role of genes in rice tillering and yield regulation. OsATG3 As a key candidate gene for regulating rice tillering and thus affecting yield. qTN1 Overexpression of this gene can significantly increase the number of rice tillers and thus increase rice yield, providing a new technical approach and important gene resource for the breeding of high-yield rice varieties, which is of great significance for ensuring food security.

[0018] 2. Cloned by this invention OsATG3 It has great application potential and prospects for improving rice tillering and yield traits. Attached Figure Description

[0019] Figure 1 This is a schematic diagram showing the location of rice tillering gene loci; Figure 2 yes qTN1 The distribution of candidate genes; Figure 3 This is a heatmap of gene expression levels of seven candidate genes under high-nitrogen and low-nitrogen conditions; Figure 4 This is a graph showing the expression analysis of 7 candidate genes in 12 high-tillering and 12 low-tillering varieties; Figure 5 yes OsATG3 Gene editing sequencing results; Figure 6 yes OsATG3 Gene expression levels in wild-type and overexpression plants; Figure 7 It is a wild-type Japanese sunflower. OsATG3 Photos of mutant and overexpressing plants; Figure 8 It is a wild-type Japanese sunflower. OsATG3 The number of tillers in mutant and overexpression plants; Figure 9 The agronomic traits of wild-type Nipponbare and overexpressing plants were investigated, in the following order: number of tillers, number of grains per spike, thousand-grain weight, yield per plant, and yield per plot. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0021] Example 1: Genome-wide association analysis and candidate gene identification in rice Tiller number was determined in 312 core rice varieties, including indica and japonica rice. Genome-wide association analysis identified a significant locus located on chromosome 1, named [locus name missing]. qTN1 Subsequently, linkage disequilibrium analysis located the gene locus controlling rice tillering to between 5.17 and 5.77 Mb on chromosome 1. Figure 1 Using a strict significance threshold () P <1 × 10 -7 The study identified 34 SNPs significantly associated with tiller number. Only two of these SNPs resulted in missense mutations, affecting a transposon-related gene and a known grain size regulator, respectively. OSH15 Therefore, they are unlikely to be key sites regulating tillering. The remaining regulatory SNPs were mapped to seven annotated genes, which were identified as... qTN1 Candidate genes ( Figure 2 To identify the candidate gene, heatmap analysis of gene expression levels under high-nitrogen and low-nitrogen conditions revealed that under low-nitrogen conditions, gene IV (… LOC_Os01g10290 The expression level is relatively high. Figure 3 Subsequently, expression analysis of these 7 genes was performed on 12 high-tillering and 12 low-tillering varieties. Only one gene, IV (…), was found to be… LOC_Os01g10290 Significant differences were observed in the expression of these two types of rice varieties. Figure 4 This suggests that the target gene is very likely... LOC_Os01g10290 . LOC_Os01g10290 It encodes an autophagy protein 3, therefore, the gene is named... OsATG3 . OsATG3 The gene is located on chromosome 1 of the rice genome, and its gene number is [gene number missing]. Os01g0200000 (RAP-DB naming rules), or LOC_Os01g10290 (MSU naming rules). OsATG3 The coding region of the gene has a nucleotide sequence length of 951 bp, as shown in SEQ ID NO.1. OsATG3 The gene encodes an amino acid sequence of 316 amino acids, as shown in SEQ ID NO.2. The protein size is approximately 35.6 kDa.

[0022] Example 2 OsATG3 Construction of knockout and overexpression vectors and their transformation in rice 2.1 OsATG3 Knockout vector construction: Targets were screened using the CRISPR target prediction website (http: / / crispr.dbcls.jp / ) to ensure that the targets were located in conserved functional domains and had a low risk of off-target effects. Based on the website's prediction results, suitable target sequences were selected. OsATG3 The gene target 1 is located in exon 2, and its sequence SEQ ID NO.3 is: CTGACAAGCAATTTCTCGTCACCAGG. OsATG3 The gene target 2 is located in exon 3, and its sequence SEQ ID NO.4 is: CTTCAAGCGAAACAGCACGTCTTAGG.

[0023] sgRNA primers were synthesized using the CRISPR primer design platform (http: / / skl.scau.edu.cn / primerdesign / vector / ). The SEQ ID NO. 5 sequence of the osatg3-1-gRT1 primer is: CTGACAAGCAATTTCTCGTCACCAGGgttttagagctagaaat; the SEQ ID NO. 6 sequence of the osatg3-1-OsU3T1 primer is: CCTGGTGACGAGAAATTGCTTGTCAGTgccacggatcatctgc; the SEQ ID NO. 7 sequence of the osatg3-2-gRT1 primer is: CTTCAAGCGAAACAGCACGTCTTAGGgttttagagctagaaat; and the SEQ ID NO. 8 sequence of the osatg3-2-OsU3T1 primer is: CCTAAGACGTGCTGTTTCGCTTGAAGTgccacggatcatctgc.

[0024] First, a first round of PCR amplification was performed. Using the U3 vector as a template, DNA fragments a1 and a2 were obtained by PCR amplification with primers B1' using satag3-1-gRT1 and satag3-2-gRT1, respectively. DNA fragments b1 and b2 were obtained by PCR amplification with primers B2 using satag3-1-OsU3T1 and satag3-2-OsU3T1, respectively. The PCR products were recovered by agarose gel electrophoresis. The sequence of primer B1' (SEQ ID NO. 9) is: TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG, and the sequence of primer B2 (SEQ ID NO. 10) is: AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC. Next, a second round of PCR amplification was performed. Using the products from the first round of amplification as templates, DNA fragments a1 and b1, and a2 and b2 were mixed and used as templates for PCR amplification with primers B1' and B2 to obtain DNA fragments A1 and A2. The PCR products A1 and A2 were then recovered by agarose gel electrophoresis.

[0025] DNA fragments A1 and A2 were inserted into the pYLCRISPR / Cas9-MH vector using the Golden Gate cloning method. First, the circular pYLCRISPR / Cas9-MH plasmid vector was digested with BstB1, and the linearized pYLCRISPR / Cas9-MH vector was recovered by agarose gel electrophoresis. Then, the DNA fragments were ligated into the linearized vector using T4 DNA ligase. The reaction products were transformed into competent *E. coli* cells, and positive colonies were selected by kanamycin resistance screening. Single colonies were then selected for culture by shaking and Sanger sequencing verification. The sequencing primer MH-F sequence (SEQ ID NO. 11) was CGGTGTCATCTATGTTACTAG, and the sequencing primer MH-R sequence (SEQ ID NO. 12) was CCGACATAGATGCAATAACTTC. Finally, it was confirmed that DNA fragments A1 and A2 were correctly constructed into the pYLCRISPR / Cas9-MH vector.

[0026] 2.2 OsATG3 Construction of overexpression vectors: according to OsATG3 Based on the coding region sequence, overexpression vector primers were designed. The OE-F primer sequence (SEQ ID NO. 13) is: ttcgagctcagatctggtaccATGCAGGTGAAGCAGAAGGTG, and the OE-R primer sequence (SEQ ID NO. 14) is: acgggggactctagtggatccTCATCTGCTTGTGCTGCCC. PCR amplification was then performed. OsATG3The coding region sequence was obtained by glue recovery. OsATG3 The DNA fragment was obtained. The pCAMBIA1302 vector was digested with Kpn1 and BamH1 enzymes, and the linearized pCAMBIA1302 vector was obtained by gel purification. Homologous recombinase was used to... OsATG3 Homologous recombination of gene fragments was performed into the overexpression vector pCAMBIA1302. The reaction products were transformed into competent *E. coli* cells, and positive colonies were selected by kanamycin resistance screening. Single colonies were then selected for culture by shaking and Sanger sequencing for verification, ultimately confirming the positive results. OsATG3 The vector was correctly constructed into the pCAMBIA1302 vector.

[0027] 2.3 OsATG3 Rice transformation with knockout and overexpression vectors Using the japonica rice variety Nipponbare as the recipient, the constructed pYLCRISPR / Cas9-MH vector was transformed into rice callus tissue via Agrobacterium-mediated transformation (strain EHA105). Positive transgenic plants were screened using hygromycin (50 mg / L), and 20 T0 generation transgenic plants were obtained. Rice transformation was performed by Wuhan Boyuan Company. Preliminary PCR detection of the pYLCRISPR / Cas9-MH vector revealed the following sequence: Cas9-F sequence (SEQ ID NO. 15): GTCGCCTACCACGAGAAGTA; Cas9-R sequence (SEQ ID NO. 16): GTGAGGTCCTGGTGGTGCTC. The constructed pCAMBIA1302 vector was also transformed into rice callus tissue via Agrobacterium-mediated transformation. Positive transgenic plants were screened using hygromycin, and 20 T0 generation transgenic plants were obtained. Rice transformation was performed by Wuhan Boyuan Company.

[0028] Example 3 OsATG3 Identification and Phenotypic Analysis of Mutants and Overexpressing Plants 3.1 Gene Editing Detection: Genomic DNA was extracted from T0 generation transgenic plants, and PCR amplification and sequencing were performed using primers near the target site. OsATG3 The sequencing primers OsATG3-F sequence (SEQ ID NO. 17) is: ATTGCTGGGTTTGGGTTTGG, and the OsATG3-R sequence (SEQ ID NO. 18) is: CAACCTTGCACTCCATGTGT. PCR sequencing results showed... osatg3-1 The target site contains a 2bp base deletion ( Figure 5 ). osatg3-2 The target site contains a 1 bp base deletion ( Figure 5 The T0 generation transgenic mutant was self-crossed to obtain the T1 generation, which was then further validated by sequencing. OsATG3Gene knockout results in homozygous mutant lines.

[0029] 3.2 OsATG3 Identification of overexpression plants RNA was extracted from leaves of wild-type and transgenic plants and reverse transcribed into cDNA. The qRT-PCR primers (SEQ ID NO. 19: AGGTGAAGCAGAAGGTGTAC) and (SEQ ID NO. 20: CAGGACCAGGTGGGGCACTT) were used for quantitative real-time PCR. qRT-PCR results showed that, compared to the wild-type, the overexpressing plants... OsATG3 Expression levels significantly increased ( Figure 6 ).

[0030] 3.3 OsATG3 Phenotypic determination of gene knockout mutants and overexpression plants: Wild-type Nipponbare was cultivated under summer field conditions in Yangzhou. OsATG3 Mutant and overexpression plants were photographed under low nitrogen management. The phenotypes of the rice plants were then analyzed. Figure 7 ) and tiller number statistics revealed that compared with the wild type, OsATG3 The mutant had a significantly reduced number of tillers, while the overexpressing plants had a significantly increased number of tillers. Figure 8 Further investigation into the agronomical sex transformation of wild-type and overexpression plants revealed that, compared with wild-type Nipponbare, the number of grains per ear and the weight of 1000 grains in overexpression plants showed no significant changes, but the yield per plant and the yield per plot were significantly increased. Figure 9 ).

[0031] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0032] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0033] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A kind OsATG3 The application of genes in regulating the number of rice tillers is characterized by, OsATG3 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.

1. OsATG3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, OsATG3 Genes positively regulate the number of tillers in rice.

3. The application according to claim 2, characterized in that, Overexpression of rice OsATG3 Genes that increase the number of tillers in rice; or Knock out rice OsATG3 Genes that reduce the number of tillers in rice.

4. OsATG3 The method for constructing a knockout vector is characterized by, by OsATG3 Using genes as targets, CRISPR / Cas9-based sgRNA primer sequences are designed, and DNA fragments containing the sgRNA primer sequences are inserted into the pYLCRISPR / Cas9-MH vector to obtain the target gene.

5. The construction method according to claim 4, characterized in that, OsATG3 Gene target 1 is located in exon 2, and its sequence is shown in SEQ ID NO.

3. Target 2 is located in exon 3, and its sequence is shown in SEQ ID NO.

4.

6. The construction method according to claim 4, characterized in that, sgRNA primers include the sapotg3-1-gRT1 primer shown in SEQ ID NO.5, the sapotg3-1-OsU3T1 primer shown in SEQ ID NO.6, the sapotg3-2-gRT1 primer shown in SEQ ID NO.7, and the sapotg3-2-OsU3T1 primer shown in SEQ ID NO.

8.

7. A method for reducing the number of tillers in rice, characterized in that, The method constructed using any one of claims 4 to 6 OsATG3 The knockout vector was transferred into rice.

8. OsATG3 A method for constructing an overexpression vector, characterized in that, Amplification using overexpression vector primers OsATG3 The encoded region sequence is obtained. OsATG3 The gene fragment is obtained by ligating it into the overexpression vector pCAMBIA1302.

9. The construction method according to claim 8, characterized in that, The primer sequences for the overexpression vector are shown in SEQ ID NO.13 and SEQ ID NO.

14.

10. A method for increasing the number of tillers in rice, characterized in that, The method constructed using claim 8 or 9 OsATG3 The overexpression vector was transformed into rice.

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