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VdUDG gene and application thereof in reducing pathogenicity of verticillium dahliae

A technology of Verticillium dahliae and coding gene, applied in the biological field

Inactive Publication Date: 2015-04-01
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since no effective control agents and disease-resistant varieties have been developed so far, it is called the "cancer" of cotton. Therefore, how to control the rampant damage of Verticillium wilt has become a key issue for the sustainable development of cotton production.

Method used

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  • VdUDG gene and application thereof in reducing pathogenicity of verticillium dahliae
  • VdUDG gene and application thereof in reducing pathogenicity of verticillium dahliae
  • VdUDG gene and application thereof in reducing pathogenicity of verticillium dahliae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1, Preparation of Agrobacterium pRF-HU2::Dgene

[0079] 1. Linearization of plasmids

[0080] PacI and Nt.BbvCI double digestion pRF-HU2 to obtain two linearized fragments of the vector pRF-HU2.

[0081] 2. Obtaining the knockout homology arm

[0082] (1) Primer design and synthesis

[0083] The primers shown in Table 1 were designed and synthesized.

[0084]Table 1 Primer Sequence

[0085]

[0086]

[0087] (2) PCR amplification

[0088] Using the genome DNA of Verticillium dahliae wild-type strain V592 of the Verticillium dahliae strain of cotton as a template, and VdUDGup-s and VdUDGup-a as primers, PCR amplification was performed to obtain a PCR amplification product, which was named as Left homology arm fragment;

[0089] The genomic DNA of Verticillium dahliae (V.dahliae) wild-type strain V592 of cotton verticillium dahliae strain was used as a template, and VdUDGdown-s and VdUDGdown-a were used as primers for PCR amplification to obtain a PCR ...

Embodiment 2

[0096] Example 2, Functional Verification of Verticillium dahliae VdUDG Knockout Mutant

[0097] The construction strategy of the knockout vector is shown in image 3 shown.

[0098] 1. Acquisition of conidia of Verticillium dahliae

[0099] First, a number of cultured Verticillium dahliae wild-type strain V592 was beaten and inoculated in a 1L Erlenmeyer flask containing 250mLRA medium at 200r min -1 , Shake culture on a horizontal shaker at 26°C for 5 days, filter the culture with sterilized microporous filter cloth into a sterile 50mL centrifuge tube; centrifuge at 14000Xg, 4°C for 40min; discard the supernatant; wash with 10mLddHO 2 0 resuspended, centrifuged at 14000Xg, 4°C for 40min; discarded the supernatant; washed with 5mL ddH 2 0 was resuspended; the spore concentration was measured using an optical microscope and a hemocytometer; then centrifuged at 14000Xg at 8°C for 30 minutes; the precipitated spores were resuspended with 10% glycerol, and the final spore conc...

Embodiment 3

[0152] Embodiment 3, the phenotype of complementary mutant

[0153] 1. Construction of Complementary Vectors

[0154] (1) Using the genomic DNA of the wild-type strain V592 as a template, VdUDG-s and VdUDG-a as primers for PCR amplification, the PCR amplification product, that is, the full-length Vdudg gene, from the start codon ATG to the stop codon GAG, the size is 1243bp. Water was used as a template to carry out the above experiments as a negative control.

[0155] The agarose gel electrophoresis of the PCR amplified fragment Figure 9 shown.

[0156] Figure 9 Middle, M: marker2000; 1: full-length Vdudg gene; 2: negative control.

[0157] (2) SmaI and XmaI double-enzyme-digest the PCR amplification product to obtain the target gene fragment; SmaI and XmaI double-enzyme-digest pSULPH-mut-RG#PB to obtain a large vector fragment; connect the gene fragment to the large vector fragment to obtain a recombinant plasmid , name it VdUDG::pSULPH-mut-RG#PB. The VdUDG::pSULPH-...

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Abstract

The invention discloses a VdUDG gene and an application thereof in reducing the pathogenicity of verticillium dahliae. The protein disclosed by the invention is (1) protein shown by SEQ ID No.15; or (2) protein obtained by substituting and / or losing and / or adding one or multiple amino acid residues of the amino acid sequence shown by SEQ ID No.15 and having the same function. The invention proves that the VdUDG gene is an essential factor during the formation of microsclerotia and plays an important role in the processes of verticillium dahliae pathopoiesia, microsclerotia formation and spore production. The invention promotes study on the pathogenesis of cotton verticillium dahliae and finally provides a theoretical basis to developing a new strategy of preventing and controlling verticillium wilt.

Description

technical field [0001] The invention relates to a VdUDG gene and its application in reducing the pathogenicity of Verticillium dahliae, belonging to the field of biotechnology. Background technique [0002] Cotton verticillium wilt is a soil-borne disease of the vascular system caused by Verticillium dahliae, which has caused serious harm to cotton production. Since 1935, cotton verticillium wilt was introduced into my country with the introduction of cotton seeds, and then gradually spread across the country. Especially since the 1990s, cotton Verticillium wilt has spread very rapidly in my country. Among them, cotton Verticillium wilt was the most serious in 1993, with an incidence area of ​​2.6667 million hm2. Continuous outbreaks across the country have caused serious losses. Cotton Verticillium wilt has posed a great threat to my country's cotton production and sustainable development. [0003] Cotton verticillium wilt is caused by fungi of the genus Verticillium in th...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/80C12N1/15
CPCC12N9/2497C12Y302/02027
Inventor 高峰张燕玲张婷
Owner SHIHEZI UNIVERSITY
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