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Application, expression vector and application of a grape disease resistance-related gene vvpub17

A disease-resistance-related gene and grape technology, which is applied in the field of plant genetic engineering, can solve problems such as the unclear role of ubiquitin ligase genes, and achieve the effects of overcoming the transfer of disease-resistance-related genes, broadening the spectrum of resistance, and improving resistance

Active Publication Date: 2019-06-14
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the role of ubiquitin ligase genes, especially most novel U-box protein genes, in plant disease resistance is still unclear.

Method used

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  • Application, expression vector and application of a grape disease resistance-related gene vvpub17
  • Application, expression vector and application of a grape disease resistance-related gene vvpub17
  • Application, expression vector and application of a grape disease resistance-related gene vvpub17

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The specific method for the isolation and cloning of grape disease resistance-related gene VvPUB17 is as follows:

[0029] 1) Extract total RNA from grape leaves according to the SDS / phenol method

[0030] Prepare extraction solution: Add 850 μL extraction buffer (140 mM LiCl, 10 mM EDTA, 10 mM Tris, 5% (w / v) SDS, 2% (w / v) PVP) and 30 μL β-mercaptoethanol to a 2.0 mL centrifuge tube, fully Mix well and set aside.

[0031] Pre-cool the mortar with liquid nitrogen, put 0.2g of young grape leaves in the mortar, add appropriate amount of liquid nitrogen to fully grind, then pack into 2.0mL centrifuge tubes containing the pre-prepared extract, vortex and mix well, 4°C Centrifuge at 12,000rpm for 5min at 12,000rpm under the same conditions; transfer the supernatant to another 2.0mL centrifuge tube, add an equal volume of chloroform-isoamyl alcohol (24:1), vortex and mix well, and centrifuge at 12,000rpm at 4°C for 15min; Transfer the supernatant to another 2.0mL centrifuge ...

Embodiment 2

[0043] The construction of the overexpression vector of grape disease resistance-related gene VvPUB17 is as follows:

[0044] Use the pMD19-T-VvPUB17 plasmid prepared in Example 1 as a template, and use P1 and P2 as primers to carry out PCR amplification reaction, and the PCR product is subjected to agarose gel electrophoresis to recover the target fragment; the underlined part of P1 is the restriction site Bgl II, the underlined part of P2 is the enzyme cutting site BstE II.

[0045] Forward primer P1: 5'-GGG AGATCT ATGGCCTCCGCTGCGATAGTAT-3' (SEQ ID NO: 10);

[0046] Reverse primer P2: 5'-GGG GGTGACC CTACAAAAACAGGCACTGAAATGGGC-3' (SEQ ID NO: 11).

[0047] Use Bgl II and BstE II to double-digest the above target fragment and the plant expression vector pCAMBIA3301 respectively, recover the target fragment and connect it with the linearized vector; the ligation reaction system is: 5 μL of the linearized vector of pCAMBIA3301, 10 μL of SolutionI, 5 μL of the target fragment,...

Embodiment 3

[0049] The plant overexpression vector pCAMBIA3301-VvPUB17 is transformed into Agrobacterium, the specific method is as follows:

[0050] Place the electric shock cup under ultraviolet light for 20 minutes; take out the competent cells of Agrobacterium GV3101, put them on ice to melt, transfer to the electric shock cup after melting, add 10 μL of plasmid, mix well, and place on ice for 5 minutes; transfer to the electric shock cup Transfer to an electric shock apparatus for electric shock treatment; after treatment, take out the transformation liquid in the electric shock cup into a 1.5mL centrifuge tube, add 1mL LB medium, and incubate at 28°C for 1h; centrifuge briefly in a centrifuge for 15s, and remove part of the supernatant solution, suspended and coated on a plate containing antibiotics (60 mg / L gentamycin, 100 mg / L kanamycin), and cultured at 28°C. Pick a single clone and inoculate it into a liquid LB medium with antibiotics (60mg / L gentamycin, 100mg / L kanamycin), cult...

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Abstract

The invention discloses an application of an anti-disease related gene VvPUB17 of grape, an expression vector and an application thereof and belongs to the field of plant genetic engineering. The application comprises the following steps: separating and cloning the VvPUB17 gene from a grape blade; and converting a plant over-expression vector containing the VvPUB17 gene into arabidopsis and the grape blade. The VvPUB17 gene expression quantity can obviously enhance the anti-disease ability of arabidopsis of genetic transformation and high expression of anti-disease related gene of grape. The VvPUB17 gene plays an important role of enhancing the disease resistance of the plant. Research of the VvPUB17 gene is of important significance of cultivating novel varieties of anti-disease plants.

Description

technical field [0001] The invention relates to the application of a grape disease resistance-related gene VvPUB17 in plant disease resistance, and also relates to an expression vector containing the grape disease resistance-related gene VvPUB17 and its application, belonging to the field of plant genetic engineering. Background technique [0002] Studies in recent years have found that protein ubiquitination is widely involved in the regulation of plant defense. The key enzyme in the process of protein ubiquitination modification is ubiquitin ligase E3, which determines the specific recognition of substrate proteins and involves plant-pathogen interactions. These include early defense responses, gene-to-gene interactions, and induced disease resistance. U-box protein is a novel E3 protein with E3 activity. Studies have found that U-box proteins widely exist in fungi, plants, and animals. Among the eukaryotic U-box proteins that have been identified so far, the number of pl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/82A01H5/00A01H6/00
CPCC12N9/93C12N15/8281C12N15/8282C12N15/8283C12Y603/02019
Inventor 余义和张会灵郭大龙李秀珍杨英军马慧丽李学强张国海
Owner HENAN UNIV OF SCI & TECH
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