Application of CGR3 gene in regulation and control of cold tolerance of rice

Abstract

Application of a CGR3 gene in regulation and control of rice cold tolerance belongs to the technical field of biology, the mRNA sequence of the CGR3 gene is shown as SEQ ID NO.1, the CGR3 gene is knocked out through CRISPR and other gene targeted editing technical means, a rice mutant with the gene function deleted is obtained, the low-temperature stress tolerance can be remarkably improved, and it is indicated that the CGR3 gene negatively regulates and controls the rice cold tolerance. Therefore, the CGR3 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] This invention belongs to the field of biotechnology, specifically relating to the application of the CGR3 gene in regulating cold tolerance in rice. Background Technology

[0002] Currently, with the intensification of global climate change, the frequency of extreme low-temperature events has increased significantly. Rice, originating in tropical and subtropical regions, is highly sensitive to low temperatures throughout its growth cycle. Therefore, discovering cold-resistant genes and elucidating their molecular mechanisms, deeply understanding their low-temperature sensing and response mechanisms, and further promoting the genetic improvement of cold-resistant rice are crucial for ensuring my country's food security. Cytochrome P450 enzymes (CYP450) are a class of monooxygenases that use heme as a cofactor, named for their characteristic absorption peak at a wavelength of 450 nanometers. They are widely found in all life domains, including eukaryotes, prokaryotes, and some viruses. CYP450 participates in the metabolism of endogenous substances and exogenous substances, including drugs and environmental compounds. Based on the degree of homology in their amino acid sequences, its members are further divided into families, subfamilies, and individual enzymes. Summary of the Invention

[0003] The inventors identified a cold-induced gene in rice, named Cold-induced Genes in Rice 3 (CGR3). The metabolite of the CGR3 gene is CYP450 72A1, and its effects on plant cold tolerance have not yet been reported. Therefore, cloning the CGR3 gene from rice and identifying its function in regulating rice cold tolerance is of great significance for improving rice cold tolerance.

[0004] The purpose of this invention is to provide the application of the CGR3 gene in regulating cold tolerance in rice.

[0005] The CGR3 has any of the following nucleotide sequences:

[0006] (1) The mRNA sequence listed as positions 1-1554 in SEQ ID NO.1;

[0007] (2) A DNA sequence that encodes the same protein as the mRNA sequence described in (1);

[0008] (3) Includes the DNA sequence described in (1) linked with a suitable promoter.

[0009] The cloned CGR3 gene can be used as a probe to screen for the gene or homologous gene of this invention from cDNA and genomic libraries. Similarly, PCR technology can be used to amplify the CGR3 gene and its homologous gene sequences from rice genome, mRNA, and DNA, and then transform this sequence into plants in a certain way to obtain overexpressed transformed plants.

[0010] Genetic engineering techniques such as CRISPR gene-targeted editing were used to knock out or downregulate the expression of the CGR3 gene, resulting in rice mutants with downregulated expression or loss of function. These mutants improved the tolerance to cold stress, indicating that the gene negatively regulates the cold tolerance of rice.

[0011] Knocking out, suppressing, or silencing the expression level of the CGR3 gene in rice can improve the cold tolerance of rice.

[0012] Increasing the CGR3 gene in rice can reduce its cold tolerance.

[0013] The full-length cDNA of the CGR3 gene in this invention was amplified from the rice variety Nipponbare. CGR3 gene knockout mutants (such as...) were obtained using gene editing technology. Figure 1 (As shown). Compared with the wild type, the cold tolerance of the CGR3 gene mutant lines was significantly increased (e.g., Figure 2 (As shown).

[0014] The results of this invention demonstrate that knocking out CGR3 through gene editing using CRISPR / Cas9 technology improves cold tolerance in rice, proving that CGR3 negatively regulates rice cold tolerance. Therefore, rice cold tolerance can be improved by knocking out, inhibiting, or silencing CGR3, and this invention has promising applications in breeding cold-tolerant rice varieties. Attached Figure Description

[0015] Figure 1 A schematic diagram illustrating the target sites, mutation sites, and mutation modes for CGR3 gene editing using CRISPR / Cas9 technology;

[0016] Among them, the exon region of CGR3 has one target site, and the other mutation types include base deletion, insertion and substitution;

[0017] Figure 2 To identify the cold tolerance of WT and CGR3 knockout mutants (CGR3-1, CGR3-2) during the seedling stage;

[0018] Wherein: A represents the growth status of WT, CGR3-1, and CGR3-2 seedlings before cold treatment; B represents the growth status of the three seedlings after cold treatment; C is a comparison table of the survival rates of the three seedlings after recovery. As can be clearly observed from the figure, when the growth status of the three seedlings was similar before cold treatment (A), after cold treatment (B), the growth status of the wild-type seedlings was significantly worse than that of the two mutant seedlings.

[0019] Figure 3 Survival rates of WT and CGR3 knockout mutants (CGR3-1, CGR3-2) seedlings after cold treatment at the seedling stage were statistically analyzed.

[0020] A significance analysis was performed on the survival rate of the restored seedlings, and the survival rate of the wild-type seedlings was 0%, while the survival rate of the mutant seedlings was 100%. Detailed Implementation

[0021] The CGR3 gene of this invention was obtained from the laboratory of the College of Plant Science, Jilin University, located at No. 5333, Xi'an Road, Luyuan, Changchun, Jilin Province, 130062, China. Contact person: Wu Tao.

[0022] Example 1:

[0023] Constructing CGR3 gene mutants using CRISPR-Cas9 technology

[0024] Using the genomic DNA sequence of rice Oryza sativav7.0 from the National Rice Data Center (https: / / www.ricedata.cn / gene / ) as a template, guide RNA (single guide RNA, sgRNA) was designed using the online software CRISPR-P 2.0 (cbi.hzau.edu.cn / CRISPR2), and a suitable sgRNA was selected for subsequent experiments. The target site is located in the first exon, and the target sequence is GGCCCCGCGCCATCACCAGG.

[0025] After synthesizing the target primers corresponding to sgRNA, the corresponding forward and reverse primers were denatured and annealed to form small double-stranded DNA fragments with sticky ends. Then, the target DNA was ligated to the linearized vector pCRISPR-zero using T4 DNA ligase to construct the pCRISPR-zero-sgRNA vector. This vector was transformed into competent *E. coli* cells and cultured overnight on selective medium. Two to three positive clones were selected and sent to Kumei Biotechnology Co., Ltd. for sequencing. The constructed gene-editing vector was transformed into *Agrobacterium* EHA105, and then introduced into rice callus tissue using the *Agrobacterium*-mediated transformation method. After selection culture, differentiation culture, and rooting culture, rice seedlings with the CGR3 gene mutation were obtained. DNA from the knockout mutant was extracted using the CTAB method, and the CGR3 fragment was amplified using polymerase chain reaction (PCR). The PCR primer sequences are as follows:

[0026] primer1: 5´ AAACAGCAACGACGAACTA 3´

[0027] Primer2: 5´ TGAGCTTCTCCTGCCACT 3´

[0028] After obtaining the target fragment, sequencing was performed, and the sequencing results were compared with the wild-type (WT) rice DNA sequence. Four mutation types were obtained. Compared with wild-type WT, CGR3-1 had the 97th and 98th bases replaced by GT (CC), and the 99th-105th bases were deleted; CGR3-2 had one A base inserted after the 105th base; CGR3-3 had one A base inserted after the 105th base; CGR3-4 had the 88th-91st base replaced by TAAT (GGCC), the 93rd base replaced by T (C), the 95th base replaced by G (CCATCACCA), and the 97th-105th bases replaced by GTGGATGGT, all of which led to premature termination of translation.

[0029] Example 2:

[0030] Identification of cold tolerance in seedlings of wild-type (WT) and CGR3 knockout mutants (CGR3-1, CGR3-2)

[0031] Three types of seeds were disinfected with 2.5% NaClO solution for 30 min, then rinsed four times with distilled water. The disinfected seeds were then immersed in distilled water and cultured in a 37℃ constant temperature incubator for 3 days. Germinated seeds were transferred to a hydroponic culture plate and cultured with Kimura nutrient solution under 12-hour light / 12-hour dark, 28℃-day / 25℃-night, and 80% relative humidity conditions until the three-leaf stage (14 days), and photographs were taken. The seeds were then transferred to a 4℃ constant temperature and light incubator for cold treatment for 2-3 days, and finally transferred back to Kimura nutrient solution for recovery treatment for 3 days, with photographs taken and survival rates recorded. Image phenotypes and survival rates were used as indicators of seedling cold stress tolerance. After cold treatment and recovery, the growth status of WT was significantly worse than that of the mutants, and the growth status of the two mutant families was consistent (e.g., ...). Figure 2 As shown), the survival rate of WT was significantly lower than that of mutants (e.g., Figure 3 (As shown in the figure). The results indicate that CGR3 gene editing based on CRISPR / Cas9 technology improved the cold tolerance of rice seedlings.

Claims

1. A CGR3 gene, characterized in that... Its mRNA sequence is shown in SEQ ID NO.

1.

2. The application of the CGR3 gene as described in claim 1 in regulating cold tolerance in rice.

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