Grape pathogen inducible promoter separating method and application in breeding for disease resistance

A separation method and promoter technology, applied in the field of plant genetic engineering, can solve the problems of wine quality decline, wine quality decline, failure to identify and isolate grape pathogenic bacteria-induced promoters, etc., to achieve the effect of giving full play to the advantages of transgenics and reducing negative effects

Inactive Publication Date: 2011-07-13
NORTHWEST A & F UNIV
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  • Application Information

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Problems solved by technology

However, Ferreira et al pointed out in "Trends in Biotechnology" 2004 22: 168-173 "Improving the disease resistance of transgenic grapes without changing the quality of wine" that excessive accumulation of disease process-related proteins in grape fruits leads to serious decline in wine qualit

Method used

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  • Grape pathogen inducible promoter separating method and application in breeding for disease resistance
  • Grape pathogen inducible promoter separating method and application in breeding for disease resistance
  • Grape pathogen inducible promoter separating method and application in breeding for disease resistance

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Effect test

Embodiment 1

[0020] Embodiment 1: the isolation method of grape VpWRKY1 and VpWRKY2 gene promoter

[0021] a. Grape VpWRKY1 and VpWRKY2 gene semi-quantitative reverse transcription PCR detection sampling:

[0022] First, the grape leaves are inoculated with powdery mildew. When the new shoots grow to 25-30cm long and the new leaves grow to 5-6cm wide, select 3 young leaves on each new shoot, and spray sterile water in advance to make them There are tiny droplets on the leaves, but there is no obvious dew point. Pick the leaves with the same powdery mildew symptoms from the diseased grapes in the field, and press the selected leaves for more than 5 seconds. After pressing, bag them for 24 hours to moisturize To facilitate the germination of powdery mildew spores, the leaves were collected at 0, 6, 12, 24, 48, 72, 96, and 120 hours after inoculation and stored in liquid nitrogen in the field, and the leaves treated with deionized water were used as controls;

[0023] b. SDS / phenol method wa...

Embodiment 2

[0056] Example 2: Construction and application of promoter recombinant vector

[0057] a. Recombinant vector construction, in order to construct the GUS reporter recombinant vector, redesign the primers with restriction sites XbaI and NcoI to amplify only the part of the promoter fragment that does not overlap with the gene, and name them PvpWRKY1 and PvpWRKY2 respectively. The 35S promoter of pCAMBIA1301 was replaced by the promoter, so that GUS was under the control of the cloned promoter, and a promoter::GUS recombinant expression vector was constructed. In addition, the 35S promoter of pCAMBIA1301 was digested with BamHI and BglII, and the vector was reconnected by complementary enzymes Create a GUS vector without 35S, named w / o35S::GUS, pCAMBIA1301 as a positive control, that is, 35 constitutively activates GUS expression;

[0058] PvpWRKY1::GUS upstream primer: gggtctagaCTATTAGGAGGAGTTGGTTG

[0059] PvpWRKY1::GUS downstream primer: gggccatggACTCACCAAGTGAAGATCAA

[0060...

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Abstract

The invention discloses a promoter quick cloning method. In the method, two grape pathogen inducible promoters are obtained by cloning to construct a promoter:: a reporter gene fused vector, wherein expression of two promoter start reporter genes in grape leaves is induced by blumeria graminis, thus the method provides a pathogen inducible promoter cloning method for the grape disease resistance gene engineering and successfully applies the pathogen inducible promoter to breeding for disease resistance of grapes. Both the two cloned inducible promoters can accurately regulate exogenous genes when pathogen invades, so that the exogenous genes can be promoted to express only under a pathogen induced condition; in the breeding for disease resistance of grape, only the reporter gene promoted by the promoter in recombinant vector is converted into an arbitrary disease resistance gene, so that the grape can be converted into a transgenic plant with pathogen induced expression exogenous disease resistance gene, the adverse influence of a constitutive promoter on transgenic grape in the breeding of disease resistance gene engineering can be reduced, and the advantage of transgenic grape can be played to the greatest extent.

Description

technical field [0001] The invention relates to a method for separating two inducible promoters derived from grape pathogenic bacteria, construction of a recombinant expression vector and application in transgenic grapes, belonging to the field of plant genetic engineering. Background technique [0002] Promoter is an important DNA sequence that regulates the binding of transcription factors in the process of gene expression. Together with transcription factors, it determines the time, type, level and tissue organ specificity of downstream gene expression. According to their mode of action and function in genetic engineering, promoters are usually divided into three categories: tissue-specific promoters, constitutive promoters and inducible promoters. [0003] Tissue-specific promoters, such promoters only promote the expression of transgenes in specific organs or tissues, and are more effective than constitutive promoters in the study of directional changes in tissue and or...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12Q1/68
Inventor 王跃进李慧娥朱自果周起谢小青
Owner NORTHWEST A & F UNIV
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