Application of OsBBX24 gene in regulation and control of heading period of rice

Abstract

The invention relates to the technical field of biology, in particular to application of an OsBBX24 gene in rice heading period regulation and control. According to the invention, the OsBBX24 gene is subjected to CRISPR gene editing, such that transgenic rice is obtained. Compared with a receptor rice DJ, the transgenic rice OsBBX24 gene knockout mutant shows a heading period postponed phenotype, and the OsBBX24 plays an important role in the process of regulating and controlling the heading period of rice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to... OsBBX24 Application of genes in the regulation of rice heading stage. Background Technology

[0002] The timing of rice heading in rice production is essentially a reflection of the synergy between the rice's growth and development rhythm and environmental conditions. Heading too early can limit yield due to insufficient photosynthetic products (the "source"). Heading too late can lead to reduced yield or even crop failure due to damage to the "sink" (decreased seed setting rate and thousand-grain weight) or adverse weather conditions. Timely heading maximizes the coordination of the "source" (leaf photosynthetic capacity), "sink" (number of grains per panicle), and "flow" (material transport), while mitigating natural disasters, thus achieving high, stable, and high-quality yields. Therefore, accurate prediction and control of the heading period are crucial technical aspects for ensuring a bumper harvest in rice production.

[0003] The present invention relates to OsBBX24 Belonging to the category of proteins containing a B-box domain, B-box zinc finger proteins (BBX) are an important class of transcription factors that play a crucial "fine-tuning" role in multiple aspects of rice, including disease resistance, stress resistance (such as drought resistance), and growth and development. This invention discovers that... OsBBX24 After the gene was knocked out by CRISPR gene editing, compared with the recipient rice DJ, OsBBX24 Two different lines of the knockout mutant exhibited a delayed heading phenotype, proving that... OsBBX24 Genes play a crucial role in regulating the heading stage of rice, and subsequent development of genes is also important. OsBBX24 Research on this and its downstream genes regulating heading time will be of great significance for fine-tuning the heading time of rice. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide... OsBBX24 Application of genes in regulating the heading stage of rice. This involves... OsBBX24 After gene knockout, OsBBX24 A significant decrease in protein expression can alter the heading stage of rice.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides OsBBX24 The application of genes in the regulation of rice heading stage, the aforementioned OsBBX24 The nucleotide sequence encoded by the gene is shown in SEQ ID NO:1.

[0006] Preferably, the OsBBX24 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:3.

[0007] Preferably, the regulation is achieved by knocking out OsBBX24 Genes or reduce this OsBBX24 The expression level of genes can delay the heading period of rice.

[0008] Preferably, the knockout is performed using CRISPR / Cas9 gene editing technology. OsBBX24 Genes are knocked out.

[0009] Preferably, the OsBBX24 The nucleotide sequences of the gene knockout target sites are shown in SEQ ID NO:4~5.

[0010] The present invention also provides a rice OsBBX24 Gene knockout mutant, wherein the rice mutant is a gene knockout mutant containing the gene knockout mutant. OsBBX24 Mutants in genes.

[0011] Preferably, the rice OsBBX24 Gene knockout mutants are produced by modifying the gene knockout mutants described above. OsBBX24 Gene editing enables OsBBX24 The nucleotide sequence of the gene is mutated, and thus... OsBBX24 The alteration of the protein encoded by the gene is thus achieved; The mutation includes OsBBX24 The substitution, deletion, and / or addition of one or more nucleotides in a gene.

[0012] This invention also provides a method for regulating the heading stage of rice, comprising the following steps: (1) According to the above OsBBX24 Design CRISPR / Cas9 editing vectors for gene target sites; (2) The CRISPR / Cas9 editing vector was infected into rice plants by Agrobacterium, and then the T0 generation positive plants with functional defect mutations were screened according to the designed primers. The T0 generation positive plants showed delayed heading.

[0013] Preferably, the target sites of the CRISPR / Cas9 editing vector in step (1) are as shown in SEQ ID NO:4~5.

[0014] Preferably, the nucleotide sequence of the primer in step (2) is shown in SEQ ID NO:6~7.

[0015] The present invention has the following technical effects and advantages: This invention isolates and clones from rice. OsBBX24 This invention, through the construction of a CRISPR / Cas9 mutant, first demonstrated that this gene is involved in the regulation of heading stage in rice; this invention experimentally proves that the gene in wild-type rice...OsBBX24 Gene knockdown yields transgenic rice with a delayed heading date. Compared to the recipient rice, transgenic rice exhibits [a certain characteristic / adjustment]. OsBBX24 Gene expression is reduced. Therefore, OsBBX24 This invention relates to genes involved in rice heading date, laying a theoretical foundation for breeding transgenic plants that can regulate heading date delay. It is of significant theoretical importance for further elucidating the molecular mechanisms by which plants regulate heading date and for breeding new crop varieties with delayed heading dates through genetic engineering. Attached Figure Description

[0016] Figure 1 Wild-type DJ background obtained using CRISPR / Cas9 technology OsBBX24 The mutation types of the mutants are named respectively. osbbx24 -L1 and osbbx24 -L2, both of which are loss-of-function mutants.

[0017] Figure 2 homozygous mutant osbbx24 -L1、 osbbx24 -L2 and wild-type DJ heading phenotypes were cultured under greenhouse long-day (LD) conditions (14 h light / 10 h dark, 22℃-30℃).

[0018] Figure 3 homozygous mutant osbbx24 -L1、 osbbx24 -Statistical chart of heading period for L2 and wild-type DJ. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] The experimental material used in this study was the commonly used japonica rice variety Dongjing (DJ).

[0022] The CRISPR / Cas9 vectors used in the following examples were kindly provided by the laboratory of Liu Yaoguang at South China Agricultural University.

[0023] Example 1 Rice heading stage response genes OsBBX24 Cloning The inventors of this invention isolated and cloned a rice gene that regulates the heading period from the rice variety DJ. OsBBX24 .

[0024] SEQ ID NO:1 OsBBX24 The nucleotide sequence of the gene's genome is shown below: SEQ ID NO:2 OsBBX24 Gene CDS sequence ATGAGGATCCAGTGCGACGCGTGCGAGGCCGCGGCGGCCACGGTGGTGTGCTGCGCGGACGAGGCGGCGCTGTGCGCGCGCTGCGACGTCGAGATCCACGCCGCCAACAAGCTCGCCAGCAAGCACCAGCGCCTCCCGCTCGACGCCGCGCTCCCCGCCGCCCTCCCGCGCTGCGACGTCTGCCAGGAGAAGGCGGCGTTCATCTTCTGCGTGGAGGACAGGGCGCTCTTCTGCCGGGACTGCGACGAGCCCATCCACGTCCCGGGGACGCTCTCCGGCAACCACCAGCGCTACCTCACCACCGGCATCCGCGTCGGGTTCAGCTCCGTCTGTAGCGCCAACGCCGACCACCTCCCGCCGCCAGCGCCCAAGGGGAACTCCAAGCCGCCGGCAAGCGGCATCGCTGCTGCTGCTGCTCCCAAGCCGGCCGTGTCCGCGGCGGCGCAGGAGGTGCCGTCGTCACCGTTCTTGCCGCCGTCGGGCTGGGCCGTCGAGGATCTCCTGCAGCTCTCCGACTACGAGTCCAGCGACAAGAAGGGCTCTCCTATTGGGTTCAAGGATCTGGAGTGGCTCGATGACATCGACCTGTTCCATGTCCAGTCGCCGGCCAAGGGAGGCAGCACGGCGGCGGAGGTGCCTGAGCTCTTCGCCTCGCCGCAGCCAGCGAGCAACATGGGGCTCTACAAGGCGAGCGGTGCACGCCAAAGCAAGAAGCCACGGGTGGAGATACCCGATGACGACGAGGACTTCTTCATCGTTCCTGATCTTGGATGA. SEQ ID NO:3 OsBBX24 Protein amino acid sequence MRIQCDACEAAAATVVCCADEAALCARCDVEIHAANKLASKHQRLPLDAALPAALPRCDVCQEKAAFIFCVEDRALFCRDCDEPIHVPGTLSGNHQRYLTTGIRVGFSSVCSANADHLPPPAPKGNSKP PASGIAAAAPKPAVSAAAQEVPSSPFLPPSGWAVEDLLQLSDYESSDKKGSPIGFKDLEWLDDIDLFHVQSPAKGGSTAAEVPELFASPQPASNMGLYKASGARQSKKPRVEIPDDDEDFFIVPDLG. Example 2 OsBBX24 Construction of missing mutant rice (1) Construction of vector and recombinant bacteria Suitable targets were screened on the E-CRISPR website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) using the DNA sequence shown in SEQ ID NO:1. Based on the score and target location, targets located in... OsBBX24 The exons CGACGAGCCCATCCACGTCC (SEQ ID NO:4) and GAGGGGGGTACATCGGCATG (SEQ ID NO:5) are the two CRISPR / Cas9 targets. Following the guidelines in the literature (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants. Mol Plant 8: 1274-1284), the pCRISPR / Cas9 plasmid was constructed, ultimately yielding the vector pCRISPR / Cas9- OsBBX24 The plasmid pCRISPR / Cas9- OsBBX24 Transformed into Agrobacterium EHA105 competent cells, recombinant Agrobacterium pCRISPR / Cas9- OsBBX24 .

[0025] (2) pCRISPR / Cas9- OsBBX24 Transformed rice The genetic transformation of rice was completed by Wuhan Boyuan Biotechnology Co., Ltd., which obtained CRISPR / Cas9- under DJ background. OsBBX24 T0 generation transgenic lines, i.e., rice OsBBX24 T0 generation of mutant.

[0026] Example 3 Genetically modified rice CRISPR / Cas9- OsBBX24 T0 generation plant identification Take the rice described in Example 2 OsBBX24 DNA was extracted from leaves of mutant generation T0. Using the extracted DNA as a template, [the following was used]... OsBBX24 Gene-specific primers were used for PCR amplification. The primer sequences were F (SEQ ID NO:6): ATGAGGATCCAGTGCGACGCGT; R (SEQ ID NO:7): TCATCCAAGATCAGGAACG. The PCR reaction program was: 94°C pre-denaturation for 2 min; 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min. The PCR products were then subjected to Sanger sequencing to check for mutations at the target site.

[0027] Leaf DNA was extracted using the CTAB method. Identification results showed that the final identified DNA was... OsBBX24 Two independent transgenic plants with gene mutations (namely) osbbx24 -L1 and osbbx24 -L2), compared with the DNA sequence, the mutation sites are as follows Figure 1 As shown. Among them osbbx24 -L1 lacks a G base, making OsBBX24 Frameshift mutations lead to loss of function. osbbx24 The insertion of 19 bp bases into -L2 also leads to a frameshift mutation, resulting in the loss of its function.

[0028] Example 4 OsBBX24 Statistics on the heading period of mutant plants The cultivation conditions were a greenhouse long-day LD (14 h light / 10 h dark, 22℃-30℃), and wild-type DJ and the one obtained in Example 2 were used. osbbx24 -L1、 ​ -L2 mutant materials were photographed when they showed signs of heading, and their phenotypes were as follows: ​ As shown, where ​ In ​ -L1 indicates ​Mutant strain 1, ​ In ​ -L2 indicates ​ Mutant line 2, where DJ represents wild-type DJ. Statistics on DJ and... ​ -L1、 ​ -L2 heading period, such as ​ As shown. Under long-day conditions in a greenhouse, the heading period of DJ is approximately 130 days, while ​ The mutant's heading period is approximately 140 days, which indicates that... ​ The loss of function leads to a delay in the heading stage of rice.

[0029] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. OsBBX24 The application of genes in the regulation of rice heading stage is characterized by, The OsBBX24 The nucleotide sequence encoded by the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The OsBBX24 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

3.

3. The application according to claim 1, characterized in that, The regulation is achieved by knocking out OsBBX24 Genes or reduce this OsBBX24 The expression level of genes can delay the heading period of rice.

4. The application according to claim 3, characterized in that, The knockout refers to the use of CRISPR / Cas9 gene editing technology to... OsBBX24 Genes are knocked out.

5. The application according to claim 4, characterized in that, The OsBBX24 The nucleotide sequences of the gene knockout target sites are shown in SEQ ID NO:4~5.

6. A type of rice OsBBX24 Gene knockout mutants are characterized by, The rice mutant is a knockout variant comprising the content described in claim 1. OsBBX24 Mutants in genes.

7. The rice according to claim 6 OsBBX24 Gene knockout mutants are characterized by, The rice OsBBX24 Gene knockout mutants are produced by modifying the gene knockout mutants described above. OsBBX24 Gene editing enables OsBBX24 The nucleotide sequence of the gene is mutated, and thus... OsBBX24 The alteration of the protein encoded by the gene is thus achieved; The mutation includes OsBBX24 The substitution, deletion, and / or addition of one or more nucleotides in a gene.

8. A method for regulating the heading stage of rice, characterized in that, Includes the following steps: (1) As described in claim 1 OsBBX24 Design CRISPR / Cas9 editing vectors for gene target sites; (2) The CRISPR / Cas9 editing vector was infected into rice plants by Agrobacterium, and then the T0 generation positive plants with functional defect mutations were screened according to the designed primers. The T0 generation positive plants showed delayed heading.

9. The method according to claim 8, characterized in that, The target sites of the CRISPR / Cas9 editing vector in step (1) are shown in SEQ ID NO:4~5.

10. The method according to claim 8, characterized in that, The nucleotide sequences of the primers used in step (2) are shown in SEQ ID NO: 6~7.

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