Application of CGR2 gene in regulation and control of cold tolerance of rice

Abstract

The invention belongs to the technical field of biology, and discloses application of a CGR2 gene in regulation and control of cold resistance of rice. The invention discloses application of the CGR2 gene in regulation and control of the cold resistance of the rice. The mRNA (messenger ribonucleic acid) sequence of the CGR2 gene is as shown in SEQ ID NO. 1. The CGR2 gene is knocked out through CRISPR (clustered regularly interspaced short palindromic repeats) and other gene targeted editing technical means, a rice mutant with CGR2 gene function deletion is obtained, the cold stress tolerance capacity can be remarkably reduced, and it is indicated that the CGR2 gene positively regulates and controls the rice cold tolerance capacity. Therefore, the CGR2 gene disclosed by the invention has a good application prospect in the aspects of regulating and controlling the cold tolerance of the rice and cultivating cold-tolerant rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of CGR2 gene in regulating the cold tolerance of rice. Background Art

[0002] VQ proteins are a class of proteins unique to plants. They are named VQ proteins because of the amino acid sequence characteristics of "FxxxVQxLTG" (x represents any amino acid) in their domains. VQ proteins have their own unique molecular characteristics. The amino acids are highly conserved only in the VQ domain, while other positions vary greatly. Most VQ genes do not contain introns, and their protein length is less than 300 amino acids. Most VQ proteins are destined to be located in the cell nucleus. The promoters of many VQ genes contain stress or growth-related elements. VQ proteins are a large class of transcriptional regulatory auxiliary factors that regulate plant growth and development, as well as defense responses to biotic and abiotic stresses by interacting with WRKY, MAPK and other proteins and participating in multiple signaling pathways.

[0003] Low temperature and chilling damage is one of the important factors that limit rice yield and quality, seriously affecting food security and people's quality of life in my country and even the world. Revealing the mechanism of rice cold tolerance and thus improving rice cold tolerance is one of the important tasks of rice researchers. Existing research results have shown that multiple VQ proteins play important functions in biotic stress and regulate rice resistance through complex and intertwined transcriptional cascades, but little is known about the regulatory mechanism of VQ proteins in rice abiotic stress, especially in cold signal transduction. Summary of the invention

[0004] The inventors discovered a cold-induced gene in rice, named Cold-induced Genes in Rice 2 (CGR2). Given that the CGR2 gene contains a VQ conserved domain, there is no relevant report on whether it can improve the cold tolerance of rice. Therefore, cloning the CGR2 gene from rice and identifying its function in regulating cold tolerance in rice are of great significance for improving the cold tolerance of rice.

[0005] The purpose of the present invention is to provide application of CGR2 gene in regulating cold tolerance of rice.

[0006] The CGR2 gene has a nucleotide sequence described in any one of the following:

[0007] (1) the mRNA sequence shown in positions 1 to 615 of SEQ ID NO. 1 in the sequence listing;

[0008] (2) a DNA sequence encoding the same protein as the protein encoded by the mRNA sequence described in (1);

[0009] (3) comprising the DNA sequence described in (1) linked to a suitable promoter.

[0010] The cloned CGR2 gene can be used as a probe to screen the gene or homologous gene of the present invention from cDNA and genomic libraries. Similarly, PCR technology can also be used to amplify the CGR2 gene and its homologous gene sequence in the present invention from the rice genome, mRNA and DNA, and this sequence can be transformed into plants in a certain way to obtain overexpression transformed plants. By means of CRISPR gene targeted editing technology and other means, the CGR2 gene is knocked out or down-regulated to obtain a rice mutant with down-regulated expression or functional loss of the CGR2 gene, which reduces the tolerance of rice to low temperature stress, indicating that the CGR2 gene positively regulates the cold tolerance of rice. Therefore, the CGR2 gene can be used to cultivate cold-resistant rice varieties.

[0011] The application of the CGR2 gene in regulating the cold tolerance of rice is specifically: increasing the expression level of the CGR2 gene in rice to improve the cold tolerance of rice; knocking out, inhibiting or silencing the CGR2 gene in rice to weaken the cold tolerance of rice.

[0012] The full-length cDNA of the CGR2 gene of the present invention is amplified from the rice variety kitaake. A CGR2 gene knockout mutant was obtained by gene editing technology, and the cold tolerance of the CGR2 gene mutant strain was significantly reduced compared with the wild type. The results of the present invention show that in rice, knocking out the CGR2 gene by gene editing with CRISPR / Cas9 technology reduces the cold tolerance of rice, proving that the CGR2 gene positively regulates the cold tolerance of rice. Therefore, the cold tolerance of rice can be improved by overexpressing the CGR2 gene, which has good application prospects in breeding cold-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the mutation type of CGR2 gene mutant plants cgr2-1 and cgr2-2;

[0014] Figure 2 The photos and comparison table are for the identification of cold tolerance of wild type (WT) and mutants (cgr2-1, cgr2-2) at the seedling stage;

[0015] Figure 3 The following are photos and a comparison table showing the identification of cold tolerance of the wild type (WT) and mutants (cgr2-1, cgr2-2) at the bud stage. DETAILED DESCRIPTION

[0016] The present invention is described below with reference to the accompanying drawings.

[0017] The CGR2 gene of the present invention is from the College of Plant Sciences of Jilin University, address: No. 5333, Xi'an Road, Lvyuan, Changchun City, Jilin Province, zip code: 130062.

[0018] Example 1: Construction of CGR2 gene mutant

[0019] In order to further clarify the function of the CGR2 gene in cold stress response, the inventors constructed a mutant based on CRISPR / Cas9. By designing single guide RNAs (sgRNAs) and cloning them into the pYLCRISPR / Cas9Pubi-MH vector, these vectors were then transformed into rice callus using Agrobacterium tumefaciens (EHA105). The present invention selected two homozygous mutant families (cgr2-1 and cgr2-2). Compared with the wild-type kitaake, a C was inserted before the cgr2-1 target site, and 2bp bases were replaced in the target site, in which the 10th base G was replaced by C and the 20th base A was replaced by T. A base C was inserted into the cgr2-2 target site, all of which resulted in amino acid frameshifts in the translated protein (such as Figure 1 shown).

[0020] Example 2: Identification of cold tolerance of wild type (WT) and mutants (cgr2-1, cgr2-2) at the seedling stage

[0021] The WT, cgr2-1, and cgr2-2 plants were hydroponically cultured with Kimura nutrient solution until the rice seedlings reached the three-leaf stage, and then transferred to a 4°C low-temperature incubator for 3 days. After 7 days of recovery to normal growth conditions, the survival rate was counted and photographed. The seedling survival rate was used as a measure of cold stress tolerance. The results showed that after cold treatment and recovery, the survival rate of WT was significantly higher than that of mutants (such as Figure 2 shown).

[0022] Example 3: Identification of cold tolerance of wild type (WT) and mutants (cgr2-1, cgr2-2) at the bud stage

[0023] 100 seeds of WT, cgr2-1 and cgr2-2 were soaked for 3 days (in a 28℃ incubator with 12 hours of light and 12 hours of dark). 25 seeds with the same germination length were selected and placed in a 4℃ incubator for low temperature treatment for 21 days. After returning to normal growth conditions for 3 days, the survival rate was counted and photographed. The survival rate was used as a measure of cold stress tolerance. The results showed that after cold treatment and recovery, the survival rate of WT was significantly higher than that of mutants (such as Figure 3 shown).

Claims

1. A CGR2 gene, characterized in that The mRNA sequence is shown in SEQ ID NO.

1.

2. Use of the CGR2 gene according to claim 1 in regulating the cold tolerance of rice.

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