Application of arabidopsis transcription factor AT3G46090 gene in cultivation of disease-resistant transgenic plants

A technology of AT3G46090, transgenic plants, applied in the field of genetic engineering, can solve the problems of secondary pollution and ecological harm, and achieve the effect of enhancing disease resistance

Active Publication Date: 2021-05-28
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 c...

Method used

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  • Application of arabidopsis transcription factor AT3G46090 gene in cultivation of disease-resistant transgenic plants
  • Application of arabidopsis transcription factor AT3G46090 gene in cultivation of disease-resistant transgenic plants
  • Application of arabidopsis transcription factor AT3G46090 gene in cultivation of disease-resistant transgenic plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1 Obtaining the Gene Sequence of the Arabidopsis Transcription Factor AT3G46090 and Construction of the Plant Expression Vector.

[0013] Log in to the Arabidopsis information resource database website https: / / www.arabidopsis.org / servlets / TairObject? type=sequence&id=30046, find the sequence (SEQ ID No.1) of the transcription factor AT3G46090 gene, design PCR amplification primers according to the CDS of the AT3G46090 gene,

[0014] AT3G46090-F:5'-ATGGTTGCGAGAAGTGAGGAA-3'

[0015] AT3G46090-R:5'-TTAACTCCAAGAAATCGTTCT-3'

[0016] The wild-type Arabidopsis Col-0 was cultivated, and the leaves of Arabidopsis thaliana grown for 3 weeks under short-day sunlight were taken as materials. The total plant RNA was extracted by TRIzol method, and the cDNA was obtained by reverse transcription PCR, and then the target sequence was amplified using the cDNA as a template.

[0017] The specific steps for extracting RNA are as follows: Grind the leaves of Arabidopsis thaliana...

Embodiment 2

[0020] The acquisition of embodiment 2 transgenic Arabidopsis plants

[0021] Firstly, the plants are cultivated to the flowering stage, and then transformed with the Agrobacterium dipping method, and the transgenic plants are screened after the seeds are harvested.

[0022] The method of plant cultivation:

[0023] Take enough Arabidopsis seeds and put them into centrifuge tubes for surface disinfection. First surface disinfection with 75% ethanol for 30s, rinse once with sterile water, then surface disinfection with 1% NaClO for 5-8min, rinse with sterile water three times; after surface disinfection is completed, sow the treated seeds in 1 / 2MS culture base plate. Seal the spot plate with parafilm and place it in the dark at 4°C. After 2-4 days, the plates were placed in a plant light incubator (the light cycle was 12h light / 12h dark). Eight days after the seeds germinated, the Arabidopsis seedlings were transferred to nutrient soil, grown under the same light conditions...

Embodiment 3

[0028] Example 3 Verification of transgenic Arabidopsis

[0029] Genomic DNA was extracted from the leaves of transgenic plants and verified by PCR to confirm that the AT3G46090-flag DNA fragment was inserted into the Arabidopsis genome.

[0030] Genomic DNA extraction:

[0031] Take 50 mg of normal growing Arabidopsis leaves to be tested and add 400 μL of plant genomic DNA extract (200 mM Tris-Cl pH7.4, 250 mM NaCl, 25 mM EDTA, 1% SDS) after grinding, vortex and mix well, and centrifuge at high speed for 5 min. Aspirate the supernatant and add an equal volume of isopropanol, precipitate at room temperature for 30 minutes, discard the supernatant after high-speed centrifugation for 5 minutes, wash the precipitate twice with 70% ethanol, and dissolve the precipitate in 20 μL of nuclease-free ddH 2 O.

[0032] Use the specific primers of pGWB11 vector for PCR amplification:

[0033] PCR was performed using the above-mentioned extracted genomic DNA as a template, and the syste...

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Abstract

The invention provides the application of an Arabidopsis thaliana transcription factor, namely the AT3G46090 gene in cultivation of disease-resistant transgenic plants. The CDS sequence of the Arabidopsis thaliana AT3G46090 gene is constructed to upper stream with an FLAG protein tag through the Gateway technology, Arabidopsis thaliana plants are transformed, screened and identified, and finally, the transgenic plants are obtained. The screened plants are analyzed, and it is found that the AT3G46090 gene has the effect of enhancing the resistance of the plants to the pseudomonas syringae tomato subspecies, the expression of the gene affects the disease resistance phenotype of the plants, and homologous genes in crops possibly have the same function, so the gene has important significance in production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the application of Arabidopsis thaliana transcription factor AT3G46090 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 agents or biological control methods, but these two common methods will cause secondary pollution or other ecological hazards. Therefore, it is necessary to use modern biotechnology to mine broad-spectrum resistance genes to provide guidance for traditional breeding techniques. Now some broad-spectrum disease resistance genes have ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H6/20
CPCC07K14/415C12N15/8281
Inventor 潘巧娜崔北米加利·约翰·洛克
Owner XUZHOU NORMAL UNIVERSITY
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