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Application of OsPIL15 gene in regulating and controlling salt tolerance of rice

A salt-tolerance and gene technology, applied in the field of genetic engineering, can solve the problems such as the lack of a transcription factor OsPIL15 and the unclear relationship between the transcription factor OsPIL15 and the response to salt stress, and achieve the effect of improving the application value.

Active Publication Date: 2021-06-11
XINYANG AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the relationship between the transcription factor OsPIL15 and the response to salt stress is not yet clear, and there are no related reports at home and abroad.

Method used

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  • Application of OsPIL15 gene in regulating and controlling salt tolerance of rice
  • Application of OsPIL15 gene in regulating and controlling salt tolerance of rice
  • Application of OsPIL15 gene in regulating and controlling salt tolerance of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Construction of OsPIL15-OE expression vector and OsPIL15-KO expression vector

[0021] Find the OsPIL15 gene (Os01g0286100) through the rice genome annotation database The Rice Annotation Project (https: / / rapdb.dna.affrc.go.jp / ), its coding sequence is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 2 , under the premise of ensuring that the encoded amino acid sequence remains unchanged, the coding sequence of SEQ ID NO:1 is optimized as SEQ ID NO:3, which was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0022] The sequence of SEQ ID NO:3 was connected to the p1305 vector by enzyme-cut ligation to construct the OsPIL15-OE expression vector ( figure 1 ).

[0023] Use the restriction endonucleases KpnI and HindIII of Treasure Bioengineering (Dalian) Co., Ltd. to digest the optimized and synthesized vectors of SEQ ID NO: 3 and p1305 respectively. The 50 μL digestion system is as follows:

[0024]

[0025] Digest overnight at 37°C.

[00...

Embodiment 2

[0035] Example 2 Obtaining and Identification of OsPIL15-OE Overexpression Strains and OsPIL15-KO Knockout Mutants

[0036] The above-mentioned successfully constructed OsPIL15-OE and OsPIL15-KO expression vectors were transformed into Agrobacterium EHA105, and then the Agrobacterium containing the recombinant plasmid was used to infect rice Nipponbare mature embryo callus, according to the method reported by Nishimura et al. (Nishimura et al., 2006) Transgenic rice was screened for resistance to hygromycin and herbicides to obtain transgenic regenerated seedlings.

[0037] Using wild-type Nipponbare WT as a control, the expression level of OsPIL15 in OsPIL15-OE transgenic plants was determined, and three transgenic lines OE-7, OE-8 and OE-11 with higher expression levels of OsPIL15 were selected as OsPIL15-OE overexpression lines. strain.

[0038] Three groups of OsPIL15-OE transgenic plants OE-7, OE-8, and OE-11 were selected from three different lines, and wild-type Nippon...

Embodiment 3

[0043] Example 3 Analysis of salt stress resistance of rice OsPIL15-OE overexpression lines and OsPIL15-KO knockout mutants

[0044] Wild-type Nipponbare (WT), OsPIL15-KO strains (KO-1, KO-3, and KO-7) and OsPIL15-OE strains (OE-7, OE-8, and OE-11) were selected and used in greenhouse hydroponics Methods. Seeds with 10% H 2 o 2 Disinfect and sterilize for 10min, then use distilled water to remove the H on the surface of the seeds 2 o 2 Rinse well, place the seeds evenly in a petri dish with sterilized filter paper, and germinate in the dark for 3 days in a constant temperature incubator at 30±2°C. The germinated seeds were placed in a hydroponic box containing a filter screen and grown in clean water for 7 days, and then gradient culture was carried out with nutrient solution (1 / 4, 1 / 2 strength nutrient solution were cultivated for 3 days, and then full nutrient solution was cultivated). 3d replace once. The nutrient solution contains 1.44mmol·L -1 NH 4 NO 3 , 0.3mmol...

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Abstract

The invention relates to application of an OsPIL15 gene in regulating and controlling salt tolerance of rice. The method comprises the following steps: constructing an OsPIL15-OE overexpression vector and an OsPIL15-KO knockout vector, introducing a japonica rice variety Nipponbare by using an agrobacterium-mediated method, and screening to obtain an OsPIL15 overexpression transgenic line and a knockout mutant; experiments prove that the growth state of the rice mutant with the OsPIL15 gene knocked out is obviously better than that of wild Nipponbare and OsPIL15-OE overexpression plants after the rice mutant is subjected to stress treatment for 7 days by 200mM NaCl salt. According to the invention, gene editing of the OsPIL15 is carried out based on the CRISPR / Cas9 technology, and a simple and effective technical means is provided for rapid creation of a new salt-tolerant rice strain. The OsPIL15 gene has potential application value in improving the salt tolerance of the rice, can be utilized in production by utilizing a molecular improvement technology, and has important practical significance in high yield, stable yield and stress-resistant breeding of the rice.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of the OsPIL15 gene in regulating the salt tolerance of rice. Background technique [0002] In recent years, factors such as global warming and unreasonable agricultural irrigation have continuously expanded the area of ​​saline soil and deepened it, and soil salinization has become the main factor of land degradation. According to the statistics of the Food and Agriculture Organization of the United Nations (FAQ), more than 900 million hectares of land in the world are currently affected by salt damage, and the area affected by salt damage accounts for about 50% of the agricultural area in the world, and the area is increasing year by year (Hossain S. Present scenario of global salt affected soils, its management and importance of salinity research [J]. International Journal of Biological Sciences. 2019, 1(1): 1-3). In China, salinization ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84C07K14/415A01H5/00A01H6/46
CPCC07K14/415C12N15/8218C12N15/8273
Inventor 季新刘娟卫云飞李猛王付娟董丽平杜彦修赵全志
Owner XINYANG AGRI & FORESTRY UNIV
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