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

A powdery mildew resistance gene and promoter sequence technology, applied in the field of genetic engineering, can solve the problems of less research on cultivated crops

Inactive Publication Date: 2011-07-13
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 grape VpRFP1 gene promoter

[0022] a. Grape genome DNA was 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 100 μl of 20% PVP and mix well, add an equal volume of chloroform-isoamyl alcohol (24:1), invert the centrifuge tube and mix well, incubate at 65°C for 30min, cool to room temperature, centrifuge at 12000rpm for 10min; take the supernatant and transfer To another clean centrifuge tube, add twice the volume of absolute ethanol or an equal volume of isopropanol, and let stand at room temperature for 15-30min to precipitate DNA; add 1ml of 70% ethanol to a 2ml centrifuge tube, and transfer the DNA into Wash it once; add 1ml of absolute ethanol to a 2ml centrifuge tube, transfer the DNA to it and wash once; pour off the absolute ethanol, leave the DN...

Embodiment 2

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

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

Embodiment 3

[0044] Example 3: Response of grape VpRFP1 gene promoter to biotic and abiotic stress

[0045] The Chinese wild East China grape powdery mildew resistance gene VpRFP1 promoter sequence was connected 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 effect of the promoter on pathogens, pathogen-related signal molecules, high temperature and low temperature were detected. Reactivity of Chinese wild grape powdery mildew resistance gene VpRFP1 using the forward primer 5'-GGG GGATCC GTGGATGTGTTAAATTAAGTGGAGTTTATAGG-3', reverse primer 5'-GGG CTGCAG GGTTGAGTCGAGTCGCCTTCACAGAACGG-3' was used for PCR reaction to obtain the full-length promoter sequence of the powdery mildew resistance gene VpRFP1 of Chinese wild East China grape, which was cloned into the plant transient expression vector pC0390GUS. After the correct sequence of the promoter was verified by sequencing, the r...

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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 a plant disease resistance gene promoter, in particular to the VpRFP1 promoter sequence of the grape powdery mildew resistance transcription factor gene and its application, belonging 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, transmit them to the action site through a series of signal transduction pathways, and then make a defense response to protect the host plant from the pathogen. Plants have different response mechanisms to signals from different pathogenic bacteria, but signal molecules can transfer signal substances to transcription factors and activate them, and the transcription factors can be combined with promoter-specific cis-acting elements to initiate the transcription and expression of target genes. Either form homologous or heterologous dimers, or directly interact with prote...

Claims

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Application Information

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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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