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Application of protein AtbHLH122 of arabidopsis thaliana and coding gene of protein AtbHLH122 for regulating and controlling stress tolerance of plants

A technology of plant stress tolerance and encoding genes, applied to the application field of Arabidopsis protein AtbHLH122 and its encoding genes in regulating plant stress tolerance, can solve problems such as unclear understanding and complex biological processes

Inactive Publication Date: 2013-09-25
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large number of members of the plant bHLH family and the complexity of the biological processes involved, the understanding of them is still not very clear.

Method used

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  • Application of protein AtbHLH122 of arabidopsis thaliana and coding gene of protein AtbHLH122 for regulating and controlling stress tolerance of plants
  • Application of protein AtbHLH122 of arabidopsis thaliana and coding gene of protein AtbHLH122 for regulating and controlling stress tolerance of plants
  • Application of protein AtbHLH122 of arabidopsis thaliana and coding gene of protein AtbHLH122 for regulating and controlling stress tolerance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1, the acquisition of protein AtbHLH122 in Arabidopsis thaliana and its coding gene and recombinant vector

[0027] Total RNA was extracted from wild-type Arabidopsis thaliana ecotype Col-0 (Arabidopsis thaliana ecotype Col-0) plants, and cDNA was obtained by reverse transcription. Using the cDNA as a template, the upstream primer 5'- CACC ATGGAATCAGAATTCCAGCA-3' and the downstream primer 5'-AGTTTCTAACAAAAGAAAATAAACT-3' were amplified by PCR, and a DNA fragment of about 1.5kb was recovered and purified, and cloned into the vector pENTR by TOPO TM / D-TOPO (purchased from Invitrogen, catalog number K2400-20) at the multiple cloning site, the recombinant vector pENTR-AtbHLH122 was obtained; it was confirmed by sequencing that the recombinant vector pENTR-AtbHLH122 was in the vector pENTR TM The DNA fragment shown in the 141st-1625th position of Sequence 2 of the Sequence Listing was inserted at the multiple cloning site of / D-TOPO.

[0028] Sequence Listing 2 is ...

Embodiment 2

[0030] Example 2. Acquisition and Stress Tolerance Identification of Transgenic Arabidopsis Plants Overexpressing Gene AtbHLH122

[0031] 1. Construction of recombinant Agrobacterium

[0032] The recombinant vector pMDC32-AtbHLH122 prepared in Example 1 was used to transform Agrobacterium tumefaciens GV3301 to obtain a recombinant Agrobacterium tumefaciens containing the recombinant vector pMDC32-AtbHLH122, named X.

[0033]The empty vector pMDC32 was used to transform Agrobacterium tumefaciens GV3301, and the recombinant Agrobacterium tumefaciens containing the recombinant vector pMDC32 was obtained, which was named CK.

[0034] 2. Obtaining transgenic Arabidopsis

[0035] The recombinant Agrobacterium X and CK were respectively transformed into wild-type Arabidopsis thaliana ecotype Col-0 (WT for short) by flower bud soaking method, and harvested T 0 generation seed; the T 0 , T1, T 2 Generation seeds were screened with 1 / 2MS medium containing 35 μg / L hygromycin to obtai...

Embodiment 3

[0073] Embodiment 3, bhlh122 mutant strain is sensitive to salt and osmotic stress

[0074] 1. Obtaining of bhlh122 mutant strain

[0075] The SALK_049022C mutant strain was purchased from ABRC (Arabidopsis Biological Resource Center), and it was confirmed by PCR that the mutant strain was on the first exon of the At1g51140 gene of the wild type Arabidopsis thaliana Col-0 (Arabidopsis thaliana Columbia) Inserted T-DNA. According to the method in step 3 of Example 2, it was confirmed that the gene bHLH122 was not expressed in the mutant strain, and the mutant strain was named bhlh122.

[0076] 2. Salt tolerance identification of bhlh122 mutant strain

[0077] After sowing the mutant bhlh122 and the wild-type Arabidopsis WT at the same time and harvesting the seeds, the mature seeds of the mutant bhlh122 and WT plants from the same batch were sterilized (the seeds were washed with 75% ethanol for 30 seconds, washed once with sterile water, and 1.5 % sodium hypochlorite soluti...

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Abstract

The invention discloses application of protein AtbHLH122 of Arabidopsis thaliana and a coding gene of the protein AtbHLH122 for regulating and controlling the stress tolerance of plants. The amino acid sequence of the protein is shown in a sequence 1 of a sequence table and can be used for regulating and controlling the stress tolerance of the plants, or substances and methods, capable of adjusting the expression level of the protein shown in the sequence 1 of the sequence table, can be used for regulating and controlling the stress tolerance of the plants. Shown by experiments, after the coding gene of the protein AtbHLH122, which is shown in a sequence 2 of the sequence table, is over-expressed in Arabidopsis thaliana, a transgenic Arabidopsis thaliana plant is obtained, the survival rate after drought treatment, the germination rate and leafy rate after 140mM NaCl treatment and the root length after 250mM mannitol treatment of the transgenic Arabidopsis thaliana plant are remarkably lower than those of the wild plants, and furthermore, after the treatments, gene AtbHLH122 knockout mutants are less than those of the wild plants; and thus, the protein AtbHLH122 shown in the sequence 1 of the sequence table has the function of regulating and controlling the stress tolerance of the plants and has an important significance on the aspects of stress tolerance research and breeding of the plants.

Description

technical field [0001] The invention relates to the application of protein AtbHLH122 in Arabidopsis thaliana and its coding gene in regulating stress tolerance of plants. Background technique [0002] Drought and salt stress are major environmental factors limiting crop yields today. During long-term evolution, plants have developed a variety of mechanisms to cope with environmental changes and various stresses, including morphological, physiological and biochemical changes. With the development of biotechnology, the molecular mechanism of plant adaptation to environmental stress has been widely concerned and studied, and the differential expression of stress response genes plays an important role in plant stress resistance. Stress response genes can be roughly divided into two groups according to their functions: the first group mainly includes some soluble enzymes, LEA (the Late Embryogenesis Abundant Proteins) proteins, aquaporins, molecular chaperones and enzymes used t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 李文学刘文文邰欢欢谢传晓
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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