Gossypium barbadense DREB transcription factor gene and application thereof

A transcription factor, island technology, applied in applications, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2011-03-09
曲延英 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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Method used

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  • Gossypium barbadense DREB transcription factor gene and application thereof
  • Gossypium barbadense DREB transcription factor gene and application thereof
  • Gossypium barbadense DREB transcription factor gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cloning of XHDREB gene of sea island cotton

[0039] 1. Cultivation of plant materials

[0040] Sea-island cotton (Gossypium barbadense L.) Xinhai 16, soak the seeds in sterilized distilled water, place them in a 37°C incubator for 48 hours, wash them with sterilized distilled water after they become white, squeeze out the seed coats and place them in 1 / 2 MS In the culture medium, cultivate under the conditions of 25°C, light intensity 2500 Lux and 12h photoperiod. One week later, the leaves were picked from the sterile seedlings and stored in liquid nitrogen to extract genomic DNA and RNA.

[0041] 2. Extraction of sea island cotton genomic DNA

[0042] The extraction method refers to the CTAB method, and the specific steps are as follows:

[0043] Take 3-5 g of leaves, grind them in liquid nitrogen, and put them into a 1.5ml centrifuge tube; add 800 μL of DNA extraction buffer (preheated at 65°C) and 50 μL of β-mercaptoethanol to the 1.5ml centrifuge tube, ...

Embodiment 2

[0066] Example 2 Construction of pC-DREB plant expression vector

[0067] see figure 2 , the XHDREB gene fragment and the expression vector pCAMBIA2300-35SOCS were digested with KI and Pst I endonucleases, and the excised XHDREB gene was inserted between the CaMV35S promoter and the OCS terminator of the pCAMBIA2300-35SOCS expression vector to construct a plant Expression vector pC-DREB. The plant expression vector pCAMBIA2300-35SOCS is constructed on the basis of pCAMBIA2300, with a 35S promoter and multiple enzyme cutting sites added.

[0068] Double Enzyme Digested Sea Island Cotton DREB Gene Fragment

[0069] Using primers DR-1 and DR-2 designed with KI and Pst I restriction sites, PCR amplification was performed on the sea island cotton cDNA library, and the amplified product was recovered to obtain the XHDREB gene fragment with KI and Pst I restriction sites . Then use KI, Pst I to carry out double enzyme digestion on the recovered gene fragment, and the enzyme dige...

Embodiment 3

[0077] Example 3 Obtaining Transgenic XHDREB Gene Tobacco Using Agrobacterium-Mediated Transformation

[0078] 1. Preparation of Agrobacterium Competent Cells

[0079] Pick a single colony of EHA105 and inoculate it in 5mL of YEB liquid medium (containing streptomycin 100mg / mL, rifampicin 100mg / mL), culture overnight at 28°C, 250rpm; transfer 2mL of the culture into 50mL of YEB liquid medium, and continue to cultivate until the OD600 value is 0.6; transfer the bacterial solution to a sterile centrifuge tube, ice-bath for 30 minutes, and centrifuge at 5000 rpm for 5 minutes; Aliquot 200 μL of the tube into sterile 1.5mL microcentrifuge tubes and keep at -70°C for use.

[0080] 2. Freeze-thaw method to transfer recombinant plasmid PC-XHDREB into Agrobacterium

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Abstract

The invention relates to the field of plant genetic engineering, and provides a gossypium barbadense DREB transcription factor gene which has the nucleotide sequence shown as the SEQ ID NO:1. The gossypium barbadense DREB transcription factor gene is constructed by the steps of constructing a pC-XHDREB plant expression vector, transforming the tobacco by the agrobactrium tumefaciens to obtain the XHDREB gene transformed tobacco, and verifying the XHDREB gene transformed tobacco by the drought simulation experiment. The tobacco has the capacities of drought resistance, saline resistance and cold resistance due to the over-expression of the XHDREB.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a sea-island cotton DREB transcription factor gene, a plant expression vector constructed by it and its application in the development of drought-tolerant transgenic plants. Background technique [0002] Transcription factor (transcription factor, TF), also known as a trans-acting factor, refers to a protein that can specifically bind to a cis-acting element in the promoter region of a eukaryotic gene. activating or repressing the transcription of certain genes. According to different binding methods, transcription factors can be divided into two categories: one is universal transcription factors, and the other is specific transcription factors. Universal transcription factors can bind to the TATA box of the core sequence of the promoter and activate all genes. The transcription of specific transcription factors, combined with other regulatory elements on the DNA sequenc...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N5/10C07K14/415A01H5/00
Inventor 曲延英陈全家张艳妮
Owner 曲延英
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