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Arabidopsis thaliana clubroot infection candidate gene AT2G35930 and application thereof

An AT2G35930, candidate gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as irreparable, and achieve the effect of promoting root swelling and good application prospects

Active Publication Date: 2021-03-05
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Coevolution cannot fix such perturbations

Method used

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  • Arabidopsis thaliana clubroot infection candidate gene AT2G35930 and application thereof
  • Arabidopsis thaliana clubroot infection candidate gene AT2G35930 and application thereof
  • Arabidopsis thaliana clubroot infection candidate gene AT2G35930 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 real-time fluorescent quantitative PCR

[0027] 1. Plant a sufficient amount of Columbia-type wild Arabidopsis, grow for about 3 weeks, and inoculate the pathogen as the treatment group. (concrete steps see following embodiment 5).

[0028] 2. Real-time fluorescent quantitative PCR

[0029] Real-time fluorescent quantitative PCR analysis of the changes in the expression of AT2G35930 at 24h and 48h after the pathogen infects Arabidopsis: at least 10 wild-type Arabidopsis plants of the treatment (24h and 48h after inoculation of the pathogen) and the control group were mixed and collected, and washed. After the roots were marked, they were quickly fixed in liquid nitrogen. After all the samples were collected, total RNA was extracted and cDNA was synthesized by reverse transcription, and then analyzed by qRT-PCR. Primers used in qRT-PCR analysis were designed by Primer Premier 6, as shown in Table 1. The reaction system is 15 μL: 7.5 μL of SYBR Green Maste...

Embodiment 2

[0033] Example 2 Construction of AT2G35930 overexpression vector

[0034] 1. Double digestion of pBI121 vector

[0035] BamHI and XbalⅠ double enzyme digestion, electrophoresis separation of large fragment bands, tapping gel recovery;

[0036] 2. RNA extraction by Trizol method

[0037]Sampling wild-type Arabidopsis root tissue, RNA extraction by Trizol method: calculate the number of samples, prepare the corresponding mortar and pestle and small iron spoon, wrap in tin foil and bake at 180°C for 4-5h; 1.5mL centrifuge tube (RNAFree), RNAFree The gun and tip, liquid nitrogen, centrifuge tube plate, 121 ℃, high-pressure steam sterilization for 40min. Take the corresponding number of centrifuge tubes and make a number; add 1mL Trizol to each tube in the fume hood, put it on ice; take the sample from liquid nitrogen into a pre-cooled mortar, add liquid nitrogen to grind 3-5 times to powder, and transfer into Trizol , fully shake and vortex; add 200mL chloroform in the fume hoo...

Embodiment 3

[0046] Example 3 Arabidopsis thaliana flower dipping method transformation and screening of positive transformants

[0047] 1. Transformation of Arabidopsis thaliana by soaking flowers

[0048] The above-mentioned bacteria solution verified by sequencing and the Agrobacterium GV3101 bacteria solution of the pBI121 empty vector plasmid were used as the mother solution, and the flower soaking method was used to transform Arabidopsis thaliana. In liquid LB of rifampicin (50mg / L), shake the bacteria at 28°C and 200rpm for about 30h until the OD600 is 1.2; centrifuge at 8000rpm for 10min to obtain the Agrobacterium precipitate, 200mL 5% (mass fraction) sucrose suspension; add Silwet- 77 to a final concentration of 200μL / L, shake at 200rpm at 28°C for 2min; remove the open flowers and siliques of wild-type Arabidopsis thaliana, soak the flower buds in the bacterial solution for 30sec, blot the excess bacterial solution, and culture in the dark at 25°C for 24h, and then culture norma...

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Abstract

The invention discloses an arabidopsis thaliana clubroot infection candidate gene AT2G35930 and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the candidate gene AT2G35930 is a nucleotide sequence shown in SEQ ID No. 1. The T-DNA insertion mutant material of the gene is more resistant to clubroot than Columbia type wild arabidopsisthaliana, AT2G35930 with an enhanced promoter is transformed into arabidopsis thaliana through agrobacterium infection, an AT2G35930 overexpression arabidopsis thaliana strain is obtained, the resultshows that overexpression of AT2G35930 can cause that arabidopsis thaliana is more susceptible to clubroot, and the phenotype is specifically generated due to the fact that the gene participates in supporting growth and development of pathogens at the roots of plants. The gene is closely related to clubroot, the gene and homologous genes of the gene in other cruciferae plants can be applied to cruciferae plant breeding, and good application prospects are achieved.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to an Arabidopsis clubroot susceptibility candidate gene AT2G35930 and its application. Background technique [0002] Arabidopsis thaliana is a model plant of Brassicaceae, which has important research significance in basic plant science. Plasmodiophora brassicae (Plasmodiophora brassicae) belongs to the kingdom Protists, Plasmodiophora brassicae phylum, Brassicae Plasmodiomycetes class, Brassicae Plasmodiophora order, Brassicae Plasmodiophora genus, the Brassicaceae caused by it Plant clubroot (clubroot) occurs exclusively in the roots of cruciferous plants, causing abnormal changes in plant hormones at different disease stages, and eventually causing abnormal expansion of plant roots. The abnormally enlarged roots cannot absorb water and nutrients. In severe cases, the diseased plants The whole plant dies. Cruciferous clubroot is widely distributed i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/00
CPCC07K14/415C12N15/8282
Inventor 余小林高莹莹章艺赵坤宋建伟
Owner ZHEJIANG UNIV
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