Application of CGR1 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 CGR1 gene in regulation and control of rice cold resistance. The invention discloses application of the CGR1 gene in regulation and control of rice cold resistance. The mRNA (messenger ribonucleic acid) sequence of the CGR1 gene is as shown in SEQ ID NO. 1. The CGR1 gene is knocked out through CRISPR (clustered regularly interspaced short palindromic repeats) and other gene targeted editing technical means, a rice mutant with CGR1 gene function deletion is obtained, the cold stress tolerance capacity can be remarkably reduced, and it is indicated that the CGR1 gene positively regulates and controls the rice cold tolerance capacity. Therefore, the CGR1 gene disclosed by the invention has a good application prospect in the aspects of regulating and controlling the cold resistance of the rice and cultivating cold-resistant rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of CGR1 gene in regulating the cold tolerance of rice. Background Art

[0002] With the intensification of global climate change, rice cultivation faces the threat of extreme low temperature weather, which has a serious impact on rice growth and yield. Therefore, improving rice tolerance to low temperatures has become one of the research focuses of rice researchers. Through genomics, molecular biology and other technical means, scientists have identified multiple genes related to rice cold tolerance, such as COLD1, CTB4a, HAN1, OsSRO1c, OsERF52, OsWRKY63, etc., and analyzed the molecular mechanism of these genes regulating rice cold tolerance. Although some rice cold stress response genes have been identified, most genes have not been used in breeding practice. The reasons are: 1) cold tolerance phenotypes are often accompanied by adverse growth and development traits; 2) there are differences in the identification results of natural cold damage in the field and simulated cold damage in the room; 3) the same gene has different cold tolerance effects under different genetic backgrounds; 4) the cold tolerance effect of a single gene is not strong; 5) excellent alleles have not been fully explored, etc. Therefore, identifying more cold-tolerant genes or gene modules with important breeding application value, exploring excellent alleles, and using these genes for genetic improvement of cold tolerance are the core content and key technologies for improving the cold tolerance of rice. Summary of the invention

[0003] The inventors discovered a cold-induced gene in rice, named Cold-induced Gene in Rice 1 (CGR1). The purpose of the present invention is to provide an application of the CGR1 gene in regulating the cold tolerance of rice.

[0004] The CGR1 gene has a nucleotide sequence described in any one of the following:

[0005] (1) the mRNA sequence shown in positions 1 to 414 of SEQ ID NO. 1 in the sequence listing;

[0006] (2) a DNA sequence encoding the same protein as the protein encoded by the mRNA sequence described in (1);

[0007] (3) comprising the DNA sequence described in (1) linked to a suitable promoter.

[0008] The cloned CGR1 gene can be used as a probe to screen the CGR1 gene or homologous gene of the present invention from cDNA and genomic libraries. Similarly, PCR technology can be used to amplify the CGR1 gene and its homologous gene sequence of 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.

[0009] Through genetic engineering methods such as CRISPR and other gene targeted editing technologies, the CGR1 gene was knocked out or downregulated, and a rice mutant with downregulated expression or functional loss of the CGR1 gene was obtained. This mutant had reduced tolerance to low temperature stress, indicating that the CGR1 gene positively regulates the cold tolerance of rice.

[0010] The application of the CGR1 gene in regulating the cold tolerance of rice is specifically: increasing the expression level of the CGR1 gene in rice to improve the cold tolerance of rice; knocking out, inhibiting or silencing the CGR1 gene in rice to weaken the cold tolerance of rice.

[0011] The full-length cDNA of the CGR1 gene of the present invention is amplified from the rice variety kitaake. The CGR1 gene knockout mutant was obtained by gene editing technology, and the cold tolerance of the CGR1 gene mutant strain was significantly reduced compared with the wild type. The results of the present invention show that in rice, the cold tolerance of rice is reduced by knocking out the CGR1 gene through gene editing with CRISPR / Cas9 technology, which proves that the CGR1 gene positively regulates the cold tolerance of rice. Therefore, the cold tolerance of rice can be improved by overexpressing the CGR1 gene, which has good application prospects in cultivating new cold-resistant rice varieties and improving rice yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the mutation pattern of CGR1 gene knockout mutant (cgr1);

[0013] Figure 2 The photos and comparison table are for the cold tolerance identification of wild type (WT) and CGR1 gene knockout mutant (cgr1) at the seedling stage;

[0014] Figure 3 The following are photos and a comparison table showing the identification of cold tolerance of the wild type (WT) and CGR1 gene knockout mutant (cgr1) at the bud stage. DETAILED DESCRIPTION

[0015] The present invention is described below with reference to the accompanying drawings.

[0016] The CGR1 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.

[0017] Example 1: Mutation type of CGR1 gene knockout mutant (cgr1)

[0018] In order to study the function of the CGR1 gene, the pCRISPR-zero-CGR1 vector was constructed and the CGR1 gene knockout mutant plants were obtained using CRISPR-Cas9 technology. The DNA of the knockout mutant was extracted using the CTAB method, and the CGR1 fragment was amplified using the polymerase chain reaction (PCR) technique. After sequencing, it was compared with the wild-type (WT) rice DNA sequence. The present invention selected two homozygous mutant systems (cgr1-1, cgr 1-2). Compared with the wild-type Kitaake, sur1-1 lacked 1bp base, and sur1-2 inserted a base T (such as Figure 1 ), both of which lead to premature termination of translation after amino acid frameshift.

[0019] Example 2: Identification of cold tolerance of wild type (WT) and CGR1 knockout mutant (cgr1) at seedling stage

[0020] WT, cgr1-1, and cgr1-2 seedlings were hydroponically cultured with Kimura nutrient solution until the three-leaf stage, transferred to a 4°C low-temperature incubator for 3 days of cold treatment, and restored to normal growth conditions for 7 days. The survival rate was counted and photographed. The survival rate was used as a measure of seedling cold stress tolerance. After cold treatment and recovery, the survival rate of WT was significantly higher than that of mutants (such as Figure 2 The results showed that CRISPR / Cas9-based editing of the CGR1 gene reduced the cold tolerance of rice seedlings.

[0021] Example 3: Identification of cold tolerance of wild type (WT) and CGR1 knockout mutant (cgr1) at bud stage

[0022] The seeds of WT, cgr1-1, and cgr1-2 were soaked in water for 3 days, and 25 seeds with consistent germination were selected and cold-treated in a 4°C low-temperature incubator 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. After cold treatment and recovery, the survival rate of WT was significantly higher than that of mutants (such as Figure 3 The results showed that CRISPR / Cas9 technology could be used to edit the CGR1 gene, which reduced the cold tolerance of rice seedlings.

Claims

1. A CGR1 gene, characterized in that The mRNA sequence is shown in SEQ ID NO.

1.

2. Use of the CGR1 gene according to claim 1 in regulating cold tolerance of rice.

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