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Application of rice gene OsNF-YC5 in improving salt tolerance of rice

A technology of osnf-yc5 and rice, applied in the field of plant genetic engineering technology and rice molecular breeding, to achieve the effect of improving tolerance

Active Publication Date: 2020-12-08
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The OsNF-YC5 gene in rice encodes a subunit of NF-Y transcription factor, but the specific function of OsNF-YC5 in rice has not yet been reported. This patent reports for the first time that OsNF has been modified by genetic engineering methods such as CRISPR-Cas9 gene targeted editing technology. Knock out or down-regulate the expression of the YC5 gene, and find that it can enhance the salt-tolerant ability of rice, which can provide new genes and methods for cultivating salt-tolerant rice varieties

Method used

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  • Application of rice gene OsNF-YC5 in improving salt tolerance of rice

Examples

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Embodiment 1

[0023] Example 1 CRISPR-Cas9 knockout vector construction

[0024] (1) Design the knockout target: the full-length sequence of the OsNF-YC5 gene is 6203bp, containing 6 exons, as shown; then use the online tool CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), select two knockout target sites in the 3rd and 6th exons of the OsNF-YC5 gene, see figure 2 , and respectively connected to the adapter corresponding to the promoter selected by the target point, the base sequence of the forward primer of the first target point adapter is SEQ ID NO.4: 5'-GCCGCTCGACCTCAGGAATGCGG-3; the base sequence of the first target point adapter reverse primer Base sequence SEQ ID NO. 5: 5'-AAACCCGCATTCCTGAGGTCGAG-3'. Base sequence of the second target linker forward primer SEQ ID NO.6: 5'-GTTGTCTTGATCCTTGCAAGCGGG-3'; second target linker reverse primer base sequence SEQ ID NO.7: 5'-AAACCCCCGCTTGCAA GGATCAAG A-3'.

[0025] (2) Target adapter preparation: Dissolve and dilute the target adapter fo...

Embodiment 2

[0038] Example 2 OsNF-YC5 rice mutants obtained

[0039] The successfully constructed pYLCRISPR / Cas9-OsNF-YC5-sgRNA dual-target vector was transformed into Agrobacterium EHA105 by freeze-thaw method, referring to the Hiei method (Hiei Y, Ohta S, Komari T, et al. Efficient transformation of rice (Oryza sativa L .) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA [J]. The Plant Journal, 1994, 6(2): 271-282.), using Agrobacterium-mediated transformation of rice (Jing Line 17, JX17 ) callus, obtained regenerated rice seedlings through resistant callus screening, differentiation, rooting and other processes, planted in the field, and then obtained transgenic positive rice seedlings through PCR amplification of hygromycin phosphotransferase gene, using primer SEQ ID No.14 and primer SEQ ID NO.15 were used to amplify the transgenic positive rice genomic DNA by PCR to obtain the DNA sequence containing target 1, and the transgenic positive rice genomic DN...

Embodiment 3

[0044] Example 3 Identification of Salt Resistance Phenotype of OsNF-YC5 Rice Mutants

[0045] Wild-type rice (Jing Line 17) and three different mutant types of OsNF-YC5 rice mutant T 2 Substitute seeds were sterilized with 3% sodium hypochlorite for 30 minutes, then rinsed with water several times, and then soaked in water at 28°C for 2 days. The seeds were placed in an incubator at 37°C overnight after whitening. The wild-type and transgenic seedlings were cultured in an incubator with Kimura B hydroponic nutrient solution. The specific culture conditions are: long daylight conditions (16h light / / 8h dark), the temperature is 30°C during the day, 30°C at night, and the relative humidity is 60-70%. The wild-type and transgenic plants (two leaves and one heart) grown for 15 days were treated with 200mM NaCl for 48h, and then recovered with normal hydroponic nutrient solution for one month, and then the survival rate of wild-type and transgenic plants was counted, and the grow...

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Abstract

The invention relates to the technical field of plant genetic engineering and rice molecular breeding, and particularly relates to application of a rice gene OsNF-YC5 in improving salt tolerance of rice. The rice gene OsNF-YC5 is knocked out or the expression of the rice gene OsNF-YC5 is down-regulated, so that the salt tolerance of the rice is improved, or a rice variety with high salt toleranceis bred; and the genomic DNA sequence of the rice gene OsNF-YC5 is shown as SEQ ID NO. 1. According to the application, the gene OsNF-YC5 can be knocked out or the expression of the gene OsNF-YC5 is down-regulated by genetic engineering means such as a CRISPR gene targeting editing technology, and a rice mutant with the down-regulated expression or function deletion of the gene is obtained, so that the tolerance of high salt stress is remarkably improved, and as a result, the gene can be applied to breeding of salt-tolerant rice varieties.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering technology and rice molecular breeding technology; in particular, it relates to the application of rice gene OsNF-YC5 in improving rice salt tolerance. Background technique [0002] According to reports, there are about 1 billion hm in the world 2 of land is affected by salinization. In my country, the area of ​​salinized land is about 36 million hm 2 , the area of ​​salinized cultivated land is about 7.6 million hm 2 , accounting for about 4.88% of the country's usable land area, and the saline-alkali cultivated land is still increasing rapidly every year. Excess sodium (Na + ) etc. will cause osmotic stress, ion stress and oxidative stress to plants. Osmotic stress causes water loss and internal dehydration, inhibits cell expansion and cell division, and absorbs nutrients, while ionic and oxidative stress causes ion toxicity and metabolic disorders, affects protein synthesis, enzyme...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8273C12N15/8216
Inventor 廖鹏飞王乐权李惠李绍波王鑫朱友林
Owner NANCHANG UNIV
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