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Construction of the CRE/loxP Recombination Activation System for Analysising the Function of Stress Tolerance Genes and Promoters

Author: ZhuYing
Tutor: CuiJiZhe
School: Harbin Normal University
Course: Biochemistry and Molecular Biology
Keywords: CRE / loxP recombination activating system Na ~ / H ~ antiporter protein gene Inducible promoter Green Fluorescent Protein Arabidopsis
CLC: S336
Type: Master's thesis
Year: 2010
Downloads: 79
Quote: 2
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Abstract


Abiotic stress seriously affect the normal growth and development of plants, the stress-inducible promoter-driven resilience gene expression in transgenic plants to cultivate resilience of new crop varieties. Cloning of a new stress-inducible promoter and stress resistance genes, the inducible promoter successfully applied to transgenic plants, gene expression regulation resilience is the direction of the development of genetic engineering of plant defense, but how objective and scientific evaluation of the promoter and The function of the target gene is critical. But for the resilience gene and identification of promoter function and expression the regulation analysis system has its limitations. In this regard, the inducible promoter the (AtRD29A AtHsp18.2 promoter) and salt-tolerant the gene (TvNHX1 and AtNHX1) test materials, construct CRE / loxP-based carrier recombination activating system; using CRE / loxP recombination system GAL4-VP16/UAS two-factor trans-activation system interaction, expression of the principle of structural gene with a reporter gene GFP to time and space synchronization detection NaCl-induced transgenic Arabidopsis GFP expression and plant phenotypic traits can quickly identification of promoter and structural gene, and to establish a set of suitable analysis resilience gene promoter function carrier system. The main findings are as follows: clone Arabidopsis thaliana RD29A the promoter; RD29A promoter, alkali of Ivana TvNHX1 and the the Arabidopsis AtNHX1 gene introduced into the CRE / loxP recombination system, build regulated by different promoters with different salt tolerance gene the plant expression the the carrier pGII-TvNHX1-HSCREN 2 pGII the-of AtNHX1-HSCREN 2 pGII-RD29ACREN 2 and pGII-TvNHX1-RD29ACREN 2 ; plant expression vector and helper plasmid pSoup by the freeze-thaw method were co-introduced into Agrobacterium the C 58 C 1 . Application of Agrobacterium-mediated floral dip method, through the double strain / double plasmid transformation, the the four the carrier combination pGII-TvNHX1-HSCREN 2 / pI-GFP pGII the-of AtNHX1-HSCREN 2 / pI-GFP pGII-of RD29A CREN 2 / the pI-GFP, and pGII-of TvNHX1-RD29ACREN 2 / pI-GFP into Arabidopsis in. Part of the T0 generation seeds were 8 mg / L PPT and 10 mg / L HYG double resistance screening, 103 resistant plants; the antagonistic genes PPT and HYG and target gene TvNHX1 PCR analysis confirmed that the 28 co-transformed plants . Transformed combinations selected by PCR positive T 1 on behalf of the leaves, 250 mM NaCl stress, GFP was detected in the four vector combinations transgenic plants proved successful co-transformation; and prove the AtRD29A promoter and AtHsp18.2 of promoter is governed NaCl-induced CRE / loxP recombination activating system effectively. Part of the T O generation seeds were 8 mg / L PPT screened 47 transgenic resistant plants, and 250 mM NaCl stress, the normal growth of plants detected different levels of GFP expression. The results showed that the to build CRE / loxP carrier activation system used in the evaluation of salt tolerance gene promoter function is feasible.

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CLC: > Agricultural Sciences > Agriculture ( agronomy ) > Crop Genetics and Breeding and breeding > Biotechnology breeding methods
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