Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof

A promoter sequence and transcription factor technology, applied in the field of genetic engineering, can solve problems such as less research on cultivated crops

Inactive Publication Date: 2012-11-14
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In plants, the research on ring zinc finger family genes is limited to the model plant Arabidopsis, and there are few studies on cultivated crops, especially on fruit trees.

Method used

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  • Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof
  • Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof
  • Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Cloning and sequence analysis of the promoter of VpRFP1 gene in grape

[0022] a. The genomic DNA of grapes is extracted using the CTAB method. Take 0.2-0.5g of young leaves and place them in a mortar, add liquid nitrogen and grind them into powder, quickly put the powder into a 2ml centrifuge tube, add 500μl CTAB buffer, and then Add 100μl of 20% PVP and mix well, add an equal volume of chloroform-isoamyl alcohol (24:1), invert the centrifuge tube to mix, incubate at 65°C for 30min, cool to room temperature, centrifuge at 12000rpm for 10min; take the supernatant and transfer Add double volume of absolute ethanol or equal volume of isopropanol to another clean centrifuge tube, and let stand at room temperature for 15-30 minutes to precipitate DNA; add 1ml 70% ethanol to a 2ml centrifuge tube to transfer DNA into Wash once; add 1ml of absolute ethanol to a 2ml centrifuge tube, transfer the DNA into it and wash once; pour off the absolute ethanol, leave the DNA at...

Embodiment 2

[0042] Example 2: Response of grape VpRFP1 gene to pathogens

[0043] SDS / phenol method was used to extract total RNA from leaves at different stages after inoculation with pathogenic bacteria, according to PrimeScript TM RT-PCR Kit instructions for reverse transcription, the reverse transcription products of 7 different periods are used as Real-time PCR templates to detect the response of the Chinese wild East China grape powdery mildew resistance gene VpRFP1 to pathogens in the resistant strains, Real- time PCR is based on TAKARA’s SYBR PremixEx TM To operate the TaqII kit, the reaction system is: template 1μl, SYBR Mix 12.5μl, forward and reverse primers 1μl each, sterilized distilled water to make up to 25μl, the reaction procedure is: 95℃5min; 95℃30S, 68℃30S, 72 ℃30S, 30 cycles; 72℃5min; 4℃10min, VpRFP1 forward primer is 5'-GCAAACAGTCCCACAAGTC-3', reverse primer is 5'-CTGAACAACACCCACCACT-3', VpGAPDH is used as internal reference gene, forward primer is 5 '-TTCACTGACAAGGAC...

Embodiment 3

[0044] Example 3: Response of VpRFP1 Gene Promoter of Grape to Biological and Abiotic Stress

[0045] The VpRFP1 promoter sequence of the Chinese wild East China grape powdery mildew resistance gene was ligated to the plant transient expression vector pC0390GUS, and the activity of the Chinese wild East China grape powdery mildew resistance gene VpRFP1 promoter and the promoter's activity against pathogens, pathogen-related signal molecules, high temperature and low temperature The reactivity, using the forward primer 5'-GGG of the promoter sequence of the powdery mildew resistance gene VpRFP1 of Chinese wild East China grape GGATCC GTGGATGTGTTAAATTAAGTGGAGTTTATAGG-3’, reverse primer 5’-GGG CTGCAG GGTTGAGTCGAGTCGCCTTCACAGAACGG-3' was subjected to PCR reaction to obtain the full length of the promoter sequence of the powdery mildew resistance gene VpRFP1 of Chinese wild East China grapes, which was cloned into the plant transient expression vector pC0390GUS. After the correct pro...

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Abstract

The invention relates to a grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof. In the sequence, a full-length 1249bp promoter sequence of a Chinese wild vitis pseudoreticulata powdery mildew transcription factor gene VpRFP1 is cloned by adopting chromosomal walking in combination with a nested polymerase chain reaction (PCR) technology, wherein the sequence comprises 103 TATA-boxes and 27 CAAT-boxes, has 8 elements related to plant defense reaction, 14 photoreaction elements, 5 plant hormone response elements, and 14 other unknown elements. By adopting an agrobacterium tumefaciens-mediated instantaneously converted nicotiana benthamiana leaves analyzing method, the function of the Chinese wild vitis pseudoreticulata powdery mildew transcription factor gene VpRFP1 promoter proves that: the promoter has stronger promotion activity, can actively respond to pathogen invasion and disease resistant signal substances, and can improve the disease resistance and high temperature resistance of wheat, rice, potato, corn, soybean, rape and other plants through a transgenic method.

Description

Technical field [0001] The invention relates to the cloning of plant disease resistance gene promoters, in particular to the promoter sequence of the grape powdery mildew resistance transcription factor gene VpRFP1 and its application, and belongs to the technical field of genetic engineering. Background technique [0002] When a plant is attacked by a pathogen, it can respond to external environmental signals and transmit it to the site of action through a series of signal transduction pathways, and then make a defensive response to protect the host plant from the pathogen. Plants have different response mechanisms to the signals of different pathogens, but signal molecules can transmit signal substances to transcription factors and activate them. The transcription factor and promoter-specific cis-acting elements are combined to initiate the transcription and expression of target genes. Either form homodimers or heterodimers, or directly interact with proteins to cause a series ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/68C12N15/82
Inventor 王跃进余义和徐伟荣李树秀徐炎
Owner NORTHWEST A & F UNIV
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