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Method for regulating and controlling salt tolerance of rice

A technology of salt tolerance and rice, applied in the field of molecular biology and genetic engineering, can solve the problems of sensitivity to BR treatment, unclear action of rice GSK3 kinase, and enlarged osgsk3 leaf angle

Inactive Publication Date: 2021-05-18
HUAIYIN TEACHERS COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the loss-of-function mutant osgsk3 has larger leaf angle, significantly increased tiller number and thousand-grain weight, and is more sensitive to BR treatment
However, so far, it is unclear whether rice GSK3-like kinases play a role in salt stress and whether they are involved in the regulation of rice salt tolerance

Method used

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  • Method for regulating and controlling salt tolerance of rice
  • Method for regulating and controlling salt tolerance of rice
  • Method for regulating and controlling salt tolerance of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Phenotype identification of rice salt-tolerance-related mutant gsk1

[0033] The salt-tolerance-related mutant gsk1 was screened from the rice variety Dongjin T-DNA insertion mutant library. The wild-type and mutant plants grown in an indoor light growth box for 2 weeks were placed in 140mM NaCl solution for 5 days, then cultured in water for 7 days, and the survival rate of the plants was counted. Compared with the wild type, the mutant gsk1 salt tolerance Significantly increased( figure 1 A-B).

[0034] 1. For cloning the mutant gene of the mutant gsk1, design three detection primers P1 (as shown in SEQ ID No.3), P2 (as shown in SEQ ID No.4) and P3 according to the flanking sequence of the T-DNA insertion mutant (as shown in SEQ ID No.5)( figure 2 A). PCR amplification reaction system (10 μL): 2 μL DNA, 1.6 μL dNTP Mixture, 3 μL Buffer, 2 μL Primer, 0.1 μL DNA Polymerase, 1.3 μL ddH 2 O, reaction program: 95°C for 2min; 98°C for 10s, 58°C for 30s, 72°...

Embodiment 2

[0045] Example 2: Expression analysis and subcellular localization of OsGSK1

[0046] Take wild-type roots, stems, leaves, spikes, seeds, and leaf tissues of two-week-old wild-type plants treated with 140mM NaCl, 4°C, 1μMABA and grind them with liquid nitrogen. (CW0559S) to extract total RNA. The reverse transcription kit (PrimeScript Reverse Transcriptase kit) from Dalian Bao Biological Co., Ltd. was used to reverse the cDNA of the total RNA. Using cDNA as a template, SYBR premix Ex TaqTM (TaKaRa, Japan) kit was selected for Real-time PCR, amplified on Bio-Rad fluorescent quantitative PCR instrument CFX96, using 2 -ΔΔCT method for expression analysis. The results showed that OsGSK1 was expressed in a constitutive mode, with the highest expression level in panicles ( Figure 4 A). At the same time, the expression of OsGSK1 was induced by salt, low temperature and ABA ( Figure 4 B).

[0047] The full-length CDS of OsGSK1 (removing the terminator) was cloned into the vect...

Embodiment 3

[0048] Example 3: Screening and identification of OsGSK1 interacting proteins

[0049] The full-length CDS of OsGSK1 was connected to the pGBKT7(BD) vector (purchased from Clontech), and the leaf tissue library treated with salt stress was screened. For the yeast transformation step, refer to the instructions of Clontech. Five VP1- and six OsbZIP72-related positive clones were identified, respectively. The full-length CDS of clone VP1 and OsbZIP72 (as shown in SEQ ID No.10, 11) was connected to pGADT7(AD) vector for yeast co-transformation verification. The results showed that OsGSK1 interacted with VP1 and OsbZIP72 in yeast ( Figure 6 A).

[0050] OsGSK1, VP1 and OsbZIP72 (terminator removed) were fused to the N-terminus (nYFP) and C-terminus (cYFP) of yellow fluorescent protein YFP using the ClonExpress II One Step Cloning Kit (Cat#C112) from Novizan Biotechnology Company, respectively. The positive recombinants were transformed into Agrobacterium, and then infected the ...

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Abstract

The invention discloses a method for regulating and controlling salt tolerance of rice, and belongs to the technical field of molecular biology and genetic engineering. According to the method, the expression of OsGSK1 protein is inhibited in rice so as to promote the improvement of the salt tolerance of rice without changing the main agronomic traits, and then interaction of the OsGSK1 protein with VP1 and OsbZIP72 protein is found by screening the leaf tissue library subjected to salt stress treatment and carrying out yeast co-transformation verification; VP1 and OsbZIP72 are further overexpressed in a wild type rice variety, and a result shows that compared with a control group, the survival rate of a transgenic plant over-expressing VP1 and / or OsbZIP72 under salt stress is remarkably improved, which indicates that overexpression of VP1 and / or OsbZIP72 in rice can regulate and control the salt tolerance of rice. The invention has important application value in rice salt-tolerant molecular breeding.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and more specifically relates to a method for regulating the salt tolerance of rice. Background technique [0002] Rice is the second largest grain in my country and two-thirds of the food in the world. Maintaining and increasing the yield of rice is one of the effective ways to solve the world's food security. However, about one-fifth of the irrigated land in the world is threatened by salinization. The problem of soil salinization is becoming more and more serious, posing a serious threat to world agriculture. Soil salinization seriously limits land use and crop production. . An effective way to solve soil salinization is to cultivate new salt-tolerant crop varieties. Based on the development of modern molecular biology, some genes related to salt stress in rice were identified and cloned, such as DST, OsHAL3, OsHAK21, and the molecular mechanism of rice sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00A01H6/46
CPCC07K14/415C12N9/12C12N15/8218C12N15/8273C12Y207/11
Inventor 刘喜王迪裔新许子怡程行汤佳玉赵亚男
Owner HUAIYIN TEACHERS COLLEGE
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