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Method for identifying disease resistant variety of cruciferous crop

A technology of cruciferous crops and cruciferous, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problems of long time spent and inadaptability to modern production and construction.

Inactive Publication Date: 2011-05-25
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that it takes a long time and is not suitable for the growing needs of modern agricultural production and construction.

Method used

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  • Method for identifying disease resistant variety of cruciferous crop
  • Method for identifying disease resistant variety of cruciferous crop
  • Method for identifying disease resistant variety of cruciferous crop

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Cloning of XC_0542 Gene and Construction of Expression Vector pBI0542

[0054] According to the gene sequence of XC_0542, design primers:

[0055] Forward primer: F, 5′-GGGTCTAGAATGGAGATCAAGAAAGCGCAGTCC-3′,

[0056] Reverse primer: R, 5'-GGGGGATCCTCAGCCGGAGCGCGGCGGGTC-3'.

[0057] Using the total DNA of Brassicaceae black rot fungus as a template, the full-length sequence of the gene was amplified by PCR, cloned into pBI121, and the expression vector pBI0542 was constructed and verified by restriction enzyme digestion. Please refer to figure 1 . The sequence was subcloned into pK18mob and verified by sequencing. The sequence was consistent with the sequence of XC_0542 gene (GeneID: 3381550).

[0058] figure 1 Electrophoretic pattern for PCR clone XC_0542 gene, M: 0321 standard DNA (fragment size from large to small: 3kb, 2kb, 1.5kb, 1.2kb, 1.0kb, 0.9kb, 0.8kb, 0.7kb, 0.6kb, 0.5 kb, 0.4kb, 0.3kb, 0.2kb, 0.1kb, ); Lane 1: PCR cloned fragment of XC_0542 gene.

Embodiment 2

[0059] Embodiment 2, introducing expression vector pBI0542 into Agrobacterium

[0060] The triparental pBI0542 was introduced into Agrobacterium tumefaciens EHA105, and the obtained triparental zygote was named EHA105 / pBI0542. For the PCR verification of the triparental zygote, please refer to figure 2 and image 3 .

[0061] figure 2 Restriction electrophoresis pattern of pBI0542 expression vector, M: 0321 standard DNA (fragment sizes from large to small: 3kb, 2kb, 1.5kb, 1.2kb, 1.0kb, 0.9kb, 0.8kb, 0.7kb, 0.6kb, 0.5kb, 0.4kb, 0.3kb, 0.2kb, 0.1kb, ); Lane 1: XbaI / BamHI fragment of gene clone pBI0542.

[0062] image 3 It is the PCR verification electrophoretic pattern of pBI0542 in EHA105, M: 0321 standard DNA (fragment sizes from large to small are: 3kb, 2kb, 1.5kb, 1.2kb, 1.0kb, 0.9kb, 0.8kb, 0.7kb, 0.6kb, 0.5kb, 0.4kb, 0.3kb, 0.2kb, 0.1kb, ); Lane 1: PCR verified fragment of pBI0542 in EHA105.

Embodiment 3

[0064] Agrobacterium Hypersensitivity Reaction (HR) Test

[0065] The plant materials used are cauliflower, A is a disease-resistant variety, and B is a disease-susceptible variety. Healthy cauliflower leaves were selected for testing.

[0066] (1) Inoculate Agrobacterium into LB medium supplemented with corresponding antibiotics and culture overnight at 28°C.

[0067] (2)OD 600 When the temperature is 0.4 to 0.5, collect 1 mL of the bacterial solution in a 1.5 mL EP tube, centrifuge at 5000 rpm for 5 min, pour off the supernatant, and use 2 mL of induction medium (10 mM MgCl 2 , 5mM MES pH5.6, 150μM AS) resuspended, induced at 28°C for 5h.

[0068] (3) Adjust OD with induction medium 600 to 0.4 to 0.5, the wild-type Xcc 8004 of the cruciferous black rot fungus was used as a positive control (such as Figure 4 Middle A1 and B1), EHA105 / pBI121 as negative control (such as Figure 4 in A3 and B3). Use a needle-free syringe to draw the bacterial solution and infuse it into...

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Abstract

The invention discloses a method for identifying a disease resistant variety of a cruciferous crop. The method comprises the following steps of: finding non-toxic genes in a xanthomonascampestris pv. campestris 8004 strain genome; constructing a series of identification strains by using the non-toxic genes in the 8004 strain genome; and injecting agrobacterium tumfaciens suspension at least carrying the non-toxic genes into leaf veins of cruciferous tested hosts, and detecting the anaphylactic reaction of the tested hosts. The method can quickly identify the disease resistant variety of the cruciferous crop, can provide related information of the disease resistant genes of the cruciferous crop, lays a foundation for quickly separating and cloning the disease resistant genes, and has remarkable immeasurable significance for disease resistance and breeding of the cruciferous crop and improvement on yield and quality of cruciferous vegetables.

Description

technical field [0001] The invention relates to a method for identifying disease-resistant varieties of crops, in particular to a method for identifying disease-resistant varieties of cruciferous crops. Background technique [0002] The pathogenic bacteria of cruciferous vegetable black rot is Brassicaceae black rot (Xcc, Xanthomonas campestris pv. campestris), also known as Xanthomonas campestris pv. Black rot of cruciferous vegetables mainly occurs in cruciferous vegetable crops such as Chinese cabbage, radish, Chinese cabbage, mustard greens, green cauliflower, cabbage flower, kohlrabi and rape. The yield and quality of this type of vegetables cause great economic losses. Planting disease-resistant varieties of crops is one of the effective measures to deal with crop diseases, and identifying crop disease-resistant varieties resources is the basis for disease-resistant breeding. [0003] The most important biological pathogens are fungi, followed by viruses, bacteria, n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 何勇强姜伯乐唐纪良岑卫健姜伟刘娇黄俊锭
Owner GUANGXI UNIV
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