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. There

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

Example Embodiment

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

[0079] 1. Linearization of plasmid

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

[0081] 2. Obtaining homology arms by knocking out

[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 genomic DNA of the wild-type strain V592 of Verticillium dahliae (V.dahliae) as a template, and VdUDGup-s and VdUDGup-a as primers for PCR amplification, the PCR amplification product was obtained, which was named Left homology arm fragment;

[0089] Using the genomic DNA of the cotton Verticillium wilt strain V.dahliae wild-type strain V592 as a template, and VdUDGdown-s and VdUDGdown-a as primers for PCR amplification, the PCR amplification product was obtained, which was named Fragment of the right homology arm.

[0090] Three, ...

Example Embodiment

[0096] Example 2. Functional verification of VdUDG knockout mutants of Verticillium dahliae

[0097] The construction strategy diagram of knockout vector is shown as image 3 Shown.

[0098] 1. Acquisition of Conidia of Verticillium dahliae

[0099] Firstly, the cultivated wild-type Verticillium dahliae strain V592 was beaten into several cakes, and inoculated into a 1L Erlenmeyer flask containing 250mL RA medium at 200 r·min -1 Shake culture on a horizontal shaker at 26℃ for 5 days, filter the culture with sterilized microporous filter cloth into a sterile 50mL centrifuge tube; centrifuge at 14000Xg, 4℃ for 40min; discard the supernatant; use 10mLddH 2 0 Resuspend, centrifuge at 14000Xg, 4℃ for 40min; discard the supernatant; use 5mL ddH 2 0 Resuspension; use an optical microscope and a hemocytometer to determine the spore concentration; then centrifuge at 14000Xg at 8°C for 30 minutes; resuspend the precipitated spores with 10% glycerol, and the final spore concentration will be 1×1...

Example Embodiment

[0152] Example 3. Phenotype of complementary mutant

[0153] 1. Construction of complementary vector

[0154] (1) Using the genomic DNA of wild-type strain V592 as a template, and VdUDG-s and VdUDG-a as primers for PCR amplification, the PCR amplification product is obtained, namely the full-length Vdudg gene, from the start codon ATG to the stop codon GAG, the size is 1243bp. Use water as a template to perform the above experiment as a negative control.

[0155] The agarose gel electrophoresis of the PCR amplified fragments is as Picture 9 Shown.

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

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

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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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