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Super-xerophyte eremosparton songoricum DREB transcription factor EsDREB and encoding gene and application

A technology of transcription factors and genes, applied in the field of plant genetic engineering

Inactive Publication Date: 2012-04-25
XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, there are still a large number of DREB genes and their families in plants to be excavated and studied
In particular, the cloning of the DREB gene from extremely drought-resistant super-xerophytic woody resource plants and breeding for stress resistance have rarely been reported.

Method used

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  • Super-xerophyte eremosparton songoricum DREB transcription factor EsDREB and encoding gene and application
  • Super-xerophyte eremosparton songoricum DREB transcription factor EsDREB and encoding gene and application
  • Super-xerophyte eremosparton songoricum DREB transcription factor EsDREB and encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning of DREB transcription factor EsDREB in Junggar Leafless Bean

[0040] Procurement of plant material:

[0041] The initial plant materials in this experiment were two-week-old Junggar leafless bean seedlings germinated in an indoor light incubator. The Junggar leafless bean seeds were treated with 98% sulfuric acid for 10-15 minutes before sowing, rinsed with water for 5 times, and sown in In a glass petri dish with two layers of filter paper, the temperature is 25°C, 12h / d light culture, distilled water is added every other day, the seedlings are harvested two weeks later and simulated drought stress with 20% polyethylene glycol 6000, and the leafless bean seedlings are treated, treatment 4 Hours later, rinse the plant material with distilled water and immediately freeze it in liquid nitrogen, and then transfer it to a -80°C ultra-low temperature refrigerator for storage;

[0042] Extraction and reverse transcription of total RNA from Junggar leafles...

Embodiment 2

[0070] Example 2: Verification of the transcriptional activation activity of EsDREB transcription factor by yeast one-hybrid method

[0071] The yeast strain used in this experiment is the AH109 strain containing LACZ and HIS double reporter genes preserved in our laboratory, and the positive control plasmid pbridge-AtDREB was cloned and preserved by our laboratory in the early stage; the specific operation is carried out according to the following steps:

[0072] Construction of the EsDREB vector fused with the binding domain (BD):

[0073] Design primers with SalI and PstI restriction sites at the start codon and stop codon of the deduced reading frame of the cloned gene respectively (such as the Y-F and Y-R primers in the above table, the restriction site sequences in the primers are underlined out), then use the plasmid of the EsDREB gene constructed on the pMD18-T carrier as a template to amplify, carry out double digestion and ligation of the recovered PCR amplification ...

Embodiment 3

[0079] Example 3: Analysis of subcellular localization of EsDREB transcription factors

[0080] Firstly design the primer pair Sub-F and Sub-R (see the above table, the sequence of the restriction site is underlined) without a stop codon without a restriction site, and use the EsDREB gene that has been constructed into pMD19-T- The plasmid was amplified as a template, and the PCR product was recovered and identified by sequencing. At the same time, the target fragment was cut with Bam HI and Sal I and pBI221 was empty, recovered, ligated, transformed, screened, and the fusion expression vector pBI221-GFP-EsDREB was constructed. Using the empty pBI221-GFP as a control, the onion epidermal cells were bombarded by gene gun method, cultured at 28°C for 24 hours, and observed by fluorescence microscope. The identified fusion expression vector pBI221-GFP-EsDREB was transformed into onion epidermal cells by gene gun transformation method , and an empty pBI221-GFP vector was used as a...

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Abstract

The invention provides an eremosparton songoricum DREB transcription factor EsDREB gene and an application thereof; the protein has an amino acid sequence as shown in SEQ ID No 3; the length of the gene encoding region is 624 bp, and 207 amino acids are encoded; a subcellular localization experiment shows that the gene is localized in a nucleus; and a yeast one-hybrid experiment demonstrates that the gene as a transcription factor has the activity of transcription activation. Meanwhile, the yeast transforming the gene has higher survival rates than a control of non-transgenic yeast in drought, cold, heat stress, which indicates that the DREB transcription factor EsDREB of a desert plant of eremosparton songoricum has abiotic stress resistance. The gene of the invention is applicable to research of crop abiotic stress transgenic breeding.

Description

technical field [0001] The invention relates to a DREB transcription factor EsDREB and an encoding gene of a super-xerophyte Junggar leafless bean. The application of the gene in improving the resistance of yeast to drought resistance, low temperature resistance and high temperature stress belongs to the technical field of plant genetic engineering. Background technique [0002] Drought is the main factor limiting plant growth and crop yield, resulting in more than 50% crop yield reduction every year. Plant drought tolerance is mostly a quantitative trait controlled by polygenes. The use of conventional breeding methods to improve drought resistance of crops is limited by the long cycle and the lack of excellent germplasm resources. In recent years, studies on transcriptomics, proteomics and gene expression regulation have initially revealed the molecular mechanism of plant drought stress. At present, the use of drought stress-related genes to improve the drought resistance...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N1/19A01H5/00
Inventor 李小双张道远杨红兰裴金玲蔡明张远明
Owner XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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