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Pathogenicity related protein of Verticillium dahliae in cotton and its coding gene, application and mutant

A technology of cotton Verticillium dahliae and pathogenic related genes, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of lack of antigenic materials, lagging of disease-resistant breeding, loss of variety resistance, etc., and achieve good application potential Effect

Inactive Publication Date: 2018-06-08
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The main reasons for the outbreak of Verticillium wilt in cotton are as follows: continuous cropping in the field leads to the continuous accumulation of pathogenic bacteria in the soil; Hysteresis, rapid loss of variety resistance
In addition, the pathogenic mechanism of this pathogen is complex, and the interaction mechanism between the pathogen and the host is still unclear, which is also the basis and difficulty of research in this field

Method used

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  • Pathogenicity related protein of Verticillium dahliae in cotton and its coding gene, application and mutant
  • Pathogenicity related protein of Verticillium dahliae in cotton and its coding gene, application and mutant
  • Pathogenicity related protein of Verticillium dahliae in cotton and its coding gene, application and mutant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 Construction of Verticillium dahliae T-DNA insertion mutant library of cotton

[0030] Cotton Verticillium dahliae Vd080 isolated from a severely diseased field in Xinji, Hebei, as the starting strain, will carry

[0031] For the Agrobacterium pCTHyg binary vector, the Agrobacterium-mediated transformation (ATMT) method was used to construct a T-DNA insertion mutant library with a capacity of 3000 carrying hygromycin (HPH) resistance selection markers. Whether there is a hygromycin label is an important basis for screening positive transformants. Use hygromycin-specific primers Hyg-F / Hyg-R (Hyg-F: 5′-GCCAAGCTTGCATGCCTGCAGGTC-3′; Hyg-R: 5′-GCGCTCGAG

[0032] TCCTCTAGAAAAGAA GGATTAC-3′) amplified to obtain a specific band of 895bp is a positive transformant ( figure 1 ). The Southern hybridization technique was used to detect the copy number of T-DNA insertion in the mutant (Figure 2).

Embodiment 2

[0033] The acquisition of embodiment 2 low pathogenicity mutant T1027 bacterial strain

[0034] The pathogenicity of cotton Verticillium dahliae mutants was determined by using the upland cotton susceptible variety Jimian 11 as the identification host, and using the method of quantitative inoculation in vermiculite sandy soil with bottomless paper pots. After two rounds of primary screening and re-screening, the mutant T1027 with significantly reduced pathogenicity was obtained. Compared with the wild-type relative disease index of 50, the relative disease index of T1027 was only 8.7, and the pathogenicity was greatly reduced. see comparison image 3 .

Embodiment 3

[0035] Example 3 Mutant T1027 biological shape analysis

[0036] 3.1 Observation of colony morphology and determination of growth rate

[0037] Take the wild-type strain Vd080 as a control, drop 5 μL of mutant spore suspension with a concentration of 1×107 / mL in the center of the PDA medium, and repeat 5 times for each strain, and culture at a constant temperature of 25°C for 10 days, and record the number of colonies growth form. And at 5d, 7d, 9d and 11d, adopt the cross method to measure the colony growth diameter respectively, and calculate the growth rate.

[0038] The results showed that compared with the wild-type Vd080, the sclerotium production of the mutant T1027 decreased sharply, and the synthesis of melanin also decreased significantly. But the growth rate of the two tends to be the same, both are 4.5mm / d.

[0039] 3.2 Determination of spore production and crude toxin content

[0040] Taking the wild-type strain Vd080 as a control, drop 5 μL of the mutant spor...

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Abstract

The invention relates to the field of the pathogenesis mechanism of Verticillium dahliae in cotton and specifically relates to a pathogenicity related protein of Verticillium dahliae in cotton and itscoding gene, application and mutant. The coding gene has a nucleotide sequence shown in the formula of SEQ ID No. 2. The low pathogenicity mutant T1027 is screened and has a growth rate the basicallysame to that of the wild body and a sporulation capability and secretion of crude toxins significantly lower than those of the wild body. An identification result shows that the T-DNA-inserted gene can code dextranase. The mutant T1027 of Verticillium dahliae in cotton is an ideal strain for researching the Verticillium dahlia molecular pathogenesis. The expression and modification of the gene and protein can be used as target sites for the design and screening of antifungal agents and have the good potential for application.

Description

technical field [0001] The invention relates to the field of pathogenic mechanism of Verticillium dahliae of cotton, in particular to pathogenicity-related protein of Verticillium dahliae of cotton and its coding gene, application and mutant. Background technique [0002] Cotton verticillium wilt (Verticillium Wilt) is the primary disease affecting the high quality and high yield of cotton (Gossypium hirsutum L.) in major cotton producing areas in the world. [0003] The main reasons for the outbreak of Verticillium wilt in cotton are as follows: continuous cropping in the field leads to the continuous accumulation of pathogenic bacteria in the soil; Lag, variety resistance loss quickly. In addition, the pathogenic mechanism of the pathogen is complex, and the interaction mechanism between the pathogen and the host is still unclear, which is also the basis and difficulty of research in this field. To reveal its pathogenic mechanism in detail, it is necessary to isolate and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/46C12N15/56C12N1/14C12R1/645
CPCC12N9/2454C12Y302/01011
Inventor 朱荷琴李志芳冯自力冯鸿杰师勇强赵丽红魏锋
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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