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Plant drought intimidation inducible expression promoter GaPROP5CS and application thereof

An expression cassette and molecular technology, applied in the field of plant drought stress-inducible expression promoter GaPROP5CS, can solve the problems of delayed growth of transgenic crops, excess exogenous genes, consumption of crop energy, etc., and achieve the effect of improving expression and accumulation level.

Inactive Publication Date: 2014-01-01
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these promoters have high expression efficiency, they can be expressed at different times and in different parts, which will cause the overexpression of foreign genes and consume a lot of energy of the crops, and even cause transgenic crops to appear under normal circumstances. severely retarded growth

Method used

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  • Plant drought intimidation inducible expression promoter GaPROP5CS and application thereof
  • Plant drought intimidation inducible expression promoter GaPROP5CS and application thereof
  • Plant drought intimidation inducible expression promoter GaPROP5CS and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1, Acquisition of Plant Drought Stress Inducible Expression Promoter GaPROP5C

[0039] 1. Primer design

[0040] The full-length cDNA of the Asian cotton GaP5CS gene is 2827bp, which can encode a protein composed of 716 amino acids (Wang Yurong, Zhang Xueyan, etc., Construction and identification of a uniform full-length cDNA library for the whole growth period of Asian cotton (Gossypium arboreum L.), Chinese Agricultural Sciences: 2009, 42 (4)), through amino acid sequence comparison, it was found that there is a highly conserved region in the protein encoded by the GaP5CS gene, which is related to the function of the enzyme. According to the cDNA sequence of the Asian cotton GaP5CS gene, primers were designed to amplify its promoter. The specific primer sequences are as follows:

[0041] Specific primers:

[0042] SP1: 5'-CCCACTGCTAATCTTCCATCA-3';

[0043] SP2: 5'-GAAGGGACAAAGCCACATCTC-3';

[0044] SP3: 5'-GGCGACTCGAAGACGATGATG-3';

[0045] Random primers...

Embodiment 2

[0055] Example 2. Construction of Plant Drought Stress Inducible Expression Promoter Expression Vector (pCBI-GaPROP5CS)

[0056] 1. Construction of intermediate vector pCBI

[0057] The vector pBI121 was digested with BamHI and EcoRI, and the 2.0kb GUS gene fragment was recovered, connected to the backbone fragment of the vector pCAMBIA2300 that had been digested with BamHI and EcoRI, to obtain the intermediate vector pCBI, which was digested ( figure 2 ) and sequencing confirmed that the intermediate vector pCBI ( image 3 ) is the GUS gene fragment connected between the BamHI and EcoRI sites of the vector pCAMBIA2300. The T-DNA region of the recombinant vector pCBI contains expression cassette+GUS+NOS.

[0058] 2. Construction of recombinant expression vector containing promoter GaPROP5CS and GUS gene

[0059] The recombinant vector PropMD18-T obtained in Example 1 was double digested with PstI and BamHI, and the 0.8kb DNA fragment in the digested product was connected t...

Embodiment 3

[0065] Example 3, Transient expression analysis of plant drought stress-inducible expression promoter (GaPROP5CS)

[0066] According to the PDS-1000 / HE Biolistic gene gun bombardment operation method of BioRad Company, the recombinant vector pCBI-GaPROP5CS (with the vector pBI121 and the vector pCBI as the control) was transformed into the inner epidermis of the onion cultured by hyperosmosis by gene gun method, and the onion inner epidermis after bombardment After the epidermis was cultured in MS medium at 37°C in the dark for 1 day, a part of the onion inner epidermis containing the vector pCBI-GaPROP5CS (or vector pBI121, or vector pCBI) was transferred to MS medium with a concentration of PEG6000 of 15% (150g / L) Cultivate in the dark at 37°C for 24 hours (that is, drought stress-induced culture); the other part continues to culture in MS medium without PEG6000 at 37°C in the dark for 24 hours (that is, without drought stress-induced culture); the onion inner epidermis GUS ...

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Abstract

The invention discloses a plant drought intimidation inducible expression promoter GaPROP5CS and the application thereof. The promoter GaPROP5CS is derived from Asiatic cotton (Gossypium arboreum L.), and specifically can be a DNA molecule consisting of a nucleotide sequence shown in a sequence 1 in a sequence table. Particle gun transformation test to inner skin of an onion shows that the induced activity of the promoter GaPROP5CS in the condition of the drought intimidation of 15 percent PEG6000 is equal with the activity of promoter CaMV35S in pBI121; and agrobacterium transformation test to tobacco shows that the induced activity of the promoter GaPROP5CS in the condition of the drought intimidation of water break is equal with the activity of the promoter CaMV35S in pBI121, and is higher than the activity of drought intimidation inducible promoter rd29A. By adopting the promoter, the expression level and the accumulation level of foreign gene can be improved under adversities of drought intimidation and the like, and the promoter provides a theoretical basis and gene resources for breeding new drought-resistant varieties, and has a great application prospect.

Description

technical field [0001] The invention relates to a plant drought stress inducible expression type promoter GaPROP5CS and application thereof. Background technique [0002] The various life manifestations of organisms are the result of the orderly expression of genes. The orderly expression of genes in time and space is inseparable from the accurate expression regulation of genes. According to the order in which gene regulation occurs in the same event, it can be divided into transcriptional regulation, post-transcriptional regulation, translational regulation, and protein processing regulation. Among them, gene regulation at the transcriptional level is very important, and it is also a hot issue in current molecular biology research. It mainly involves the interaction of RNA polymerase, cis-regulatory elements and trans-acting factors. The promoter is an important cis-acting element, which is a DNA sequence located in the upstream region of the 5' end of the structural gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/84C12N1/21A01H5/00
Inventor 李付广曾小林张雪妍
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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