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Application of arabidopsis transcription factor AT5G59820 gene in cultivation of disease-resistant transgenic plants

A technology of AT5G59820, transgenic plants, applied in the field of genetic engineering, can solve problems such as secondary pollution and ecological hazards

Active Publication Date: 2021-05-14
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the control of crop diseases and insect pests mainly adopts chemical agents or biological control methods, but these two common methods will cause secondary pollution, or some other ecological hazards

Method used

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  • Application of arabidopsis transcription factor AT5G59820 gene in cultivation of disease-resistant transgenic plants
  • Application of arabidopsis transcription factor AT5G59820 gene in cultivation of disease-resistant transgenic plants
  • Application of arabidopsis transcription factor AT5G59820 gene in cultivation of disease-resistant transgenic plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1 Acquisition of Arabidopsis thaliana transcription factor AT5G59820 gene sequence and construction of plant expression vector.

[0013] Login to the Arabidopsis Information Resource Database website

[0014] https: / / www.arabidopsis.org / servlets / TairObject?id=en type=sequence&id=125264, find the sequence of transcription factor AT5G59820 gene (SEQ ID No. 1), and design PCR amplification primers according to the CDS of AT5G59820 gene,

[0015] AT5G59820-F:5'-ATGGTTGCGATATCGGAGATC-3'

[0016] AT5G59820-R:5'-ATAAACTGTTCTTCCAAGCTC-3'

[0017] The wild-type Arabidopsis Col-0 was cultivated, and the Arabidopsis thaliana leaves grown in short-day for 3 weeks were used as materials, and the total plant RNA was extracted by TRIzol method.

[0018] The specific steps for RNA extraction are as follows: Grind and pulverize Arabidopsis leaves in a liquid nitrogen environment. After the liquid nitrogen is completely volatilized, the samples are dispensed into a centrifuge ...

Embodiment 2

[0021] Example 2 Obtaining of transgenic Arabidopsis plants

[0022] First, the plants were cultivated to the flowering stage, and then transformed with the Agrobacterium dipping method. After the seeds were collected, the transgenic plants were screened.

[0023] The method of plant cultivation:

[0024] Take enough Arabidopsis seeds into centrifuge tubes for surface sterilization. First, use 75% ethanol for surface disinfection for 30s, rinse once with sterile water, then use 1% NaClO for surface disinfection for 5-8 minutes, then rinse with sterile water three times; after surface disinfection, the treated seeds are sown in 1 / 2MS culture on the base plate. The spotted plates were sealed with parafilm and placed in the dark at 4°C. After 2-4 days, the plates were placed in a plant light incubator (light cycle of 12h light / 12h dark). Eight days after seed germination, Arabidopsis seedlings were transferred to nutrient soil, grown under the same light conditions, and water...

Embodiment 3

[0029] Example 3 Verification of transgenic Arabidopsis

[0030] The genomic DNA was extracted from the leaves of the transgenic plants and verified by PCR to confirm that the AT5G59820-flag DNA fragment was inserted into the Arabidopsis genome.

[0031] Extraction of genomic DNA:

[0032] Take 50 mg of normal growing Arabidopsis thaliana leaves to be tested and grind, add 400 μL of plant genomic DNA extract (200 mM Tris-Cl pH7.4, 250 mM NaCl, 25 mM EDTA, 1% SDS), vortex and mix, and then centrifuge at high speed for 5 min. Aspirate the supernatant and add an equal volume of isopropanol, precipitate for 30 min at room temperature, discard the supernatant after high-speed centrifugation for 5 min, wash the precipitate twice with 70% ethanol, and dissolve the precipitate in 20 μL of nuclease-free ddH 2 O.

[0033] PCR amplification using specific primers of the pGWB11 vector:

[0034] PCR was performed using the genomic DNA extracted above as a template. The system included 4...

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Abstract

The invention relates to an application of arabidopsis thaliana transcription factor AT5G59820 gene in cultivation of disease-resistant transgenic plants, the CDS sequence of the arabidopsis thaliana transcription factor AT5G59820 gene is constructed to the upstream with an FLAG protein tag through the Gateway technology, the arabidopsis thaliana plant is transformed, screened and identified, and finally the transgenic plants are obtained. The screened plants are analyzed and found to have the effect of enhancing the resistance of arabidopsis thaliana to tomato pseudomonas syringae subspecies, that is, the gene affects the disease resistance phenotype of the plants, and homologous genes in crops possibly have the same or similar functions, so that the gene has important significance in production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to the application of Arabidopsis thaliana transcription factor AT5G59820 gene in cultivating disease-resistant transgenic plants. Background technique [0002] As a country with a large population, my country has a huge demand for food every year. The yield of crops in agricultural production will be affected by various factors, leading to yield reduction, among which pests and diseases are one of the important factors affecting global agricultural production. At present, the control of crop diseases and insect pests mainly adopts chemical or biological control methods, but these two common methods will cause secondary pollution or some other ecological hazards. Therefore, it is very necessary to use modern biotechnology to mine broad-spectrum resistance genes and provide guidance for traditional breeding techniques. Now some broad-spectrum disease resistance genes have been ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N1/21A01H5/00A01H6/20
CPCC07K14/415C12N15/8281C07K2319/43
Inventor 潘巧娜崔北米加利·约翰·洛克
Owner XUZHOU NORMAL UNIVERSITY
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