Grape disease resistance related gene vvpub21 and its plant expression vector and application

A disease-resistance-related gene and expression vector technology, which is applied in the field of expression vectors containing VvPUB21 gene, can solve the problem that the role of ubiquitin ligase gene is not very clear, and achieves overcoming the transfer of disease-resistance-related genes, avoiding losses, and improving resistance. Effect

Active Publication Date: 2019-05-24
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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  • Grape disease resistance related gene vvpub21 and its plant expression vector and application
  • Grape disease resistance related gene vvpub21 and its plant expression vector and application
  • Grape disease resistance related gene vvpub21 and its plant expression vector and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Isolation and cloning of grape disease resistance-related gene VvPUB21, the specific method is as follows:

[0028] 1) The total RNA of grape leaves was extracted by SDS / phenol method, and the operation was as follows:

[0029] 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.

[0030] Use liquid nitrogen to pre-cool the mortar, take 0.2g of young grape leaves in the mortar, add an 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 Centrifuge at 12000rpm for 5min at ℃; 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 12000rpm at 4℃ for 15min; draw Transfer the supernatant to another 2.0mL centrifuge tube, ...

Embodiment 2

[0042] The construction of the overexpression vector of the grape disease resistance-related gene VvPUB21 is as follows:

[0043] Grape leaf cDNA was used as a template, and P1 and P2 were used as primers. The underlined part of P1 was the enzyme cutting site Bgl II, and the underlined area of ​​P2 was the enzyme cutting site BstE II. The VvPUB21 gene fragment was amplified by PCR and connected to the pMD19-T vector; the ligation reaction system was: 0.5 μL of pMD19-T vector, 2.5 μL of Solution I, 2.0 μL of the target fragment, mixed well, and reacted at 16°C for 2 hours; The ligation product was transformed into TOP10 competent cells, and the blue-white spot screening was carried out on the LB medium with Amp added, and the positive clones were picked and inoculated into the LB liquid medium for shaking culture, and the plasmid was extracted to obtain the positive plasmid pMD19-T-VvPUB21, which was sent to the company for sequencing detection . Carry out double enzyme digest...

Embodiment 3

[0048] The plant overexpression vector pCAMBIA3301-VvPUB21 is transformed into Agrobacterium, the specific method is as follows:

[0049] 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 liquid LB medium with antibiotics (60mg / L gentamycin, 100mg / L kanamycin), cultur...

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Abstract

The invention discloses a grape disease-resistant related gene VvPUB21, a plant expression vector thereof and application of the grape disease-resistant related gene VvPUB21 and the plant expression vector and belongs to the field of plant gene engineering. The grape disease-resistant related gene VvPUB21, the plant expression vector thereof and the application have the advantages that an open reading frame of the gene VvPUB21 is obtained through cloning, a plant over-expression vector containing the gene VvPUB21 is transferred to leaf blades of Arabidopsis thaliana and grapes, and expression quantity of the gene VvPUB21 is capable of remarkably enhancing high expression of disease resistance of genetically transformed Arabidopsis thaliana and the grape disease-resistant related gene, so that the ability of the gene VvPUB21 to play an important role in enhancement of plant disease resistance is proven, and studies on the gene VvPUB21 are of great significance to breeding new varieties of disease-resistant plants.

Description

technical field [0001] The invention relates to the application of a grape disease resistance-related gene VvPUB21, and also relates to an expression vector containing the VvPUB21 gene and application thereof, 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. , including 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 plant U-box (plant U-box protein, PUB) proteins is far greate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N15/82C12N15/66A01H5/00
CPCC12N9/93C12N15/66C12N15/8205C12N15/8279C12Y603/02019
Inventor 余义和郭大龙李秀珍张会灵杨英军李学强张国海
Owner HENAN UNIV OF SCI & TECH
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