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Application of cotton GbDREB gene in resisting verticillium wilt

A technology for resistance to Verticillium wilt and Verticillium wilt, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as changes and few reports of disease resistance

Active Publication Date: 2017-02-22
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As in normal times, the activation of downstream genes and DREB2A The gene is not related, but if a negative regulatory sequence is missing, it will lead to DREB2A change
[0007] In general, there are many research reports on the DREB family in plant stress, but there are few reports on disease resistance.

Method used

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  • Application of cotton GbDREB gene in resisting verticillium wilt
  • Application of cotton GbDREB gene in resisting verticillium wilt
  • Application of cotton GbDREB gene in resisting verticillium wilt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Based on previous studies, the inventors found that after being infected by Verticillium wilt, cotton GbDREB Significant changes have taken place in gene expression, therefore, the inventors based on sea island cotton (Xinhai 15) to GbDREB Genes were analyzed in detail, and the specific process is briefly described below.

[0042] 1. Sea Island cotton GbDREB Gene acquisition

[0043] First, plant cotton, extract the RNA of cotton seedlings, and perform reverse transcription to obtain cDNA;

[0044] For the RNA extraction steps, refer to the EASYspin Plus Plant RNA Rapid Extraction Kit, and the reverse transcription steps are as follows:

[0045] Total RNA, 1000 / RNA concentration;

[0046] Oligo dT18 (10 μM), 1 μL;

[0047] Random primers, 1 μL;

[0048] wxya 2 O (RNase-free) made up to 13 μL;

[0049] In a PCR machine, run at 65°C for 5 minutes, and quickly put it on ice for 2 minutes;

[0050] Add the following reagents to the above reaction system:

[0051] ...

Embodiment 2

[0091] In the aforementioned Example 1, for the gene GbDREBBased on the understanding of the basic characteristics, the inventors further analyzed the tissue expression pattern of this gene in cotton tissue under natural conditions and the tissue expression after stress. The relevant experiments are briefly introduced as follows.

[0092] 1. Natural (wild-type) genes GbDREB tissue expression pattern

[0093] The root, stem, and leaf tissues of Xinhai 15 were collected separately, RNA was extracted, and cDNA was obtained using a reverse transcription kit, and the following quantitative PCR primers were used for qPCR amplification:

[0094] QRT-PCR-GbDREBF: 5'-CTACAGGAGAGTATGTGGATGAGGATG-3',

[0095] QRT-PCR-GbDREBR: 5'-CGTCGTCATCGGAAGGTATCAAG-3';

[0096] Ubiquitin 7 (UB7) was selected as an internal reference gene, and the primer sequences were designed as follows:

[0097] UBQ7F: 5'-GAAGGCATTCCACCTGACCAAC-3',

[0098] UBQ7R: 5'-CTTGACCTTTCTTCTTCTTGTGCTTG-3';

[0099] qR...

Embodiment 3

[0125] Further, the inventors used VIGS technology to construct a VIGS interference vector to gene GbDREB Conduct silence. After observing the phenotypic changes of cotton against Verticillium dahliae infection after gene silencing, the results further proved that the gene GbDREB It is highly related to Verticillium wilt resistance, and the related experiments are briefly introduced as follows.

[0126] (1) Construction of VIGS interference carrier

[0127] in gene GbDREB Design primers for non-conserved segments:

[0128] F: 5'-GCGGATCCATTTTTCTAATAATGCTGCTGTAAG-3',

[0129] R: 5'-GGGGTACCGCTGCAACTTAATACCACCAAT-3';

[0130] The principle of primer design is: add an enzyme to the upstream primer Kpn Restriction site and protective base of I, add enzyme to downstream primer Bam Restriction sites and protective bases of HI;

[0131] PCR amplified target sequence (about 200 bp);

[0132] Then, after recovering the PCR amplification product and the empty vector pTRV2, u...

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Abstract

The invention belongs to the technical field of gene cotton resistance breeding, and in particular relates to an application of a cotton GbDREB gene in resisting cotton verticillium wilt. The GbDREB gene belongs to DREB A4 members and AP2 family members of sea island cotton. The gene is expressed in roots, stems and leaves of cotton, and when the cotton is infected by verticillium wilt germs, the expression amount of the gene is significantly improved. Based upon application experiments, it is shown that a new plant body shows obviously different characteristics on verticillium wilt infection when the gene is silenced. On the basis of the research result, a certain application foundation can be laid for conducting molecular mechanism researches on verticillium wilt resistance and for brewing new verticillium wilt resistant plant varieties.

Description

technical field [0001] The application belongs to the technical field of cotton resistance breeding, and specifically relates to cotton GbDREB Patent application for the application of genes in cotton resistance to Verticillium wilt. Background technique [0002] Cotton verticillium wilt is transmitted by Verticillium dahliae through soil, and eventually causes a fungal disease of vascular bundles, which has very serious damage. It has many characteristics such as a wide distribution range, many types of hosts and a long survival time. It can cause a large reduction in cotton production or even a complete crop failure in severe cases. [0003] The reason why plants can resist the invasion of pathogenic microorganisms is because the natural immune system of plants plays a role. So that plants can resist the invasion of pathogenic microorganisms. Resistance to this regulation is very common. There is also an immune response that can be stimulated by effector proteins, whic...

Claims

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Application Information

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IPC IPC(8): C12N15/29
CPCC07K14/415
Inventor 蔡应繁刘鑫龙璐孙全王微娜张骁
Owner HENAN UNIVERSITY
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