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Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter

A promoter and gene technology, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2015-05-20
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the promoters induced by stresses such as high temperature, drought and high concentration of salt in cotton.

Method used

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  • Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter
  • Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter
  • Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Promoter cloning and sequence analysis of heat shock transcription factor GhHSF39 gene

[0036] (1) PCR amplification of the target fragment

[0037] The primers were designed according to the published sequence of the promoter region of the heat shock transcription factor GhHSF39 gene of Gossypium raimondii, and the size of the amplified product was 1711 bp.

[0038] GhHSF39-pro F (SEQ ID NO. 2): 5’-TAAAAATAAAATTCGGTTCGAGTAAATG-3’

[0039] GhHSF39-pro R (SEQ ID NO.3): 5’-AGTCAAGAACGGCGGCGGACCAACCTCG-3’

[0040] The DNA of Gossypium hirsutum gene was extracted and diluted to 100ng / μl as a template, and PCR amplification was carried out using the above primers.

[0041] (2) Recovery of target fragments and construction of intermediate vectors

[0042] Recover the target DNA fragments by agarose gel electrophoresis ( figure 1 ), using Kangwei Century's rapid agarose gel DNA recovery kit. For specific operation steps, see the kit instructions.

[0043] Such as figure 1 As sh...

Embodiment 2

[0047] Example 2: Expression of GhHSF39 gene driven by pghhsf39 promoter under different stresses in cotton analysis

[0048] 2.1 Adversity stress treatment and RNA extraction of cotton seedlings

[0049] (1) Treatment of cotton seedlings under different adversities

[0050] Take upland cotton Coker312 at the five-leaf stage, use 42℃ environment to simulate heat stress, treat the whole plant for one hour, then recover at 30℃ for four hours, collect the leaves in time periods; culture with 200mM mannitol solution, pH 8.8 MS The base and 100mM sodium chloride solution simulate drought stress, alkali stress, and salt stress respectively. The treatment lasts for three hours, and the roots are collected in time periods. All collected materials are immediately stored in liquid nitrogen for RNA extraction.

[0051] (2) Extract RNA from the above materials

[0052] Take the tissue materials of the control group and various experimental groups, and use the polysaccharide polyphenol plant tot...

Embodiment 3

[0065] Example 3: Construction of the recombinant vector pEarleygate101-pghhsf39::GhHSF39-YFP and tobacco instant Time conversion analysis

[0066] 3.1 Construction of recombinant vector pEarleygate101-pghhsf39::GhHSF39-YFP

[0067] (1) CDS sequence connecting promoter pghhsf39 and gene GhHSF39

[0068] According to the CDS sequence of promoter pghhsf39 and gene GhHSF39, design primers:

[0069] pghhsf39FStuⅠ(SEQ ID NO.8)5’-AAAAGGCCTTTTTAAAAATAAAATTCGGTTCGAGTAAATG-3’

[0070] pghhsf39R (SEQ ID NO. 9) 5’-CCGGAATTCCGGTTGAGACTTCGTTTTCACTTCAC-3’

[0071] GhHSF39CDS F (SEQ ID NO. 10) 5’-CCGGAATTCCGGATGGAAGGAGTTGTAGTAAAAGAAGAG-3’

[0072] GhHSF39CDS R SpeⅠ(SEQ ID NO.11) 5’-CGGACTAGTCCGTGGATTTGACCTGAGATAACCC-3’

[0073] The CDS of the gene GhHSF39 was cloned from cotton cDNA using GhHSF39CDS F, GhHSF39CDS R SpeI, and the specific method refers to Example 1. Then using the overlap PCR method, using primers pghhsf39F StuI, pghhsf39R, GhHSF39F, GhHSF39R SpeI, connect the CDS of pghhsf39 and GhHS...

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PUM

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Abstract

The invention belongs to the fields of genetic engineering and molecular breeding and discloses a promoter of cotton heat shock transcription factor GhHsf 39 genes and application of the promoter. The nucleotide sequence of the promoter is shown as SEQ ID NO:1. When a plant is subjected to abiotic stress, the expression of target genes can be regulated by pghhsf39. When the pghhsf39 sequence cloned from upland cotton is subjected to different abiotic stress stimulus, the expression quantity of the GhHSF 39 genes can be increased. The pghhsf39 promoter disclosed by the invention is connected with the target genes so as to form an expression cassette, and transgenic plants subjected to adversity stress to regulate the expression of the target genes can be obtained. The invented pghhsf39 provides an important promoter element for researching plant stress resistance and disease resistance. Therefore, the promoter has wide application values.

Description

Technical field [0001] The invention relates to a gene promoter in the technical field of genetic engineering and its application, belonging to the field of biotechnology, and specifically relates to a promoter of cotton heat shock transcription factor gene GhHsf39 and its application. Background technique [0002] The vast majority of plants cannot choose their own growth conditions, so they need to adjust various physiological activities according to changing environmental conditions to complete the necessary life processes. The regulation of gene expression is mainly through the promoter to realize the change of the gene at the transcription level, so as to realize the regulation of plant growth and metabolism. A promoter is a nucleotide sequence located in a specific region upstream of a gene. It generally contains a variety of cis-acting elements. These cis-acting elements work in concert with various trans-acting factors to regulate RNA polymerase at a specific time and Di...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/10C12N15/84C12N1/21A01H5/00
Inventor 左开井王俊邓婷
Owner SHANGHAI JIAO TONG UNIV
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