Construction Of The CRE/loxP Recombination Activation System For Analysising The Function Of Stress Tolerance Genes And Promoters

Abiotic stress seriously affect the normal growth and development of the plant. It is a effective way that use the stress inducible pomoters to cultivate new resistant crops in transgenic plants. Clone a new stress inducible promoters and make the inducible promoter successfully into transgenic plants, regulating expression of the resistance gene is the development direction of resistance gene engineer, how to evaluate the function of the stress inducible promoters and stress resistance genes is the key. Currently, the data derived from the functional identification and expressional regulation of the anti-sress genes and promoters when they are in stress shorter or developmental special stage. In view of this, establishing a CRE/loxP recombination activation system which is suitable for analysis of anti-stress gene and promoter will be great significance for researching mechanism of the anti-stress genes and promoters.The CRE/loxP-based activation system vectors contained stress inducible promoter (AtRD29A or AtHsp18.2) and structrue gene (TvNHX1 or AtNHX1) were constructed and transformed into Arabidopsis thaliana. The expression of structure gene and the GFP reporter gene were co-activated with the same expression patterns owing on the interaction mechamism of the CRE/loxP recombination system and the GAL4-VP16/UAS transactivation system. A CRE/loxP activation system line which could be used to analyses the function of promoter and structure gene rapidly and effectively by visual assessment GFP and morphologically changes in transgenic plants were eatablished. The main results were summarized as follows:The stress inducible RD29A promoter from Arabidopsis thaliana were cloned, and the RD29A promoter, TvNHX1 and AtNHX1 genes from Tripolium vulgare and Arabidopsis thaliana were introduced into the CRE/loxP recombination system plasmids. Plant expression vector of pGII-TvNHX1-HSCREN₂, pGII-AtNHX1-HSCREN₂, pGII-RD29ACREN₂ and pGII-TvNHX1-RD29ACREN₂ were constructed. Each of the plant expression vectors and the helper plasmid pSoup were co-transformed into Agrobacterium tumefaciens (C₅₈C₁) using a freeze-thaw method.Mixture two Agrobacterium strains which harboring different vectors, pGII-TvNHX1-HSCREN2 and pI-GFP, pGII-AtNHX1-HSCREN2 and pI-GFP, pGII-RD29ACREN2 and pI-GFP, pGII-TvNHX1-RD29ACREN2 and pI-GFP were co-transformed into Arabidopsis via Agrobacterium tumefaciens-mediated floral dip. 103 resistant plants survived by herbicide (8 mg/L PPT) and antibiotic (10 mg/L HYG) screening. PCR analysis indicated that 28 of the transgenic plants were co-transformants.GFP fluorescence signal in leaves of co-transformants were detectable under 250 mM NaCl stress. The results showed success of the co-transformtion, and AtRD29A and AtHsp18.2 promoter were inducible by NaCl, and the CRE/loxP–based recombination system lines were activated.Some of PPT-resistant plants grew normally under 250 mM NaCl and, GFP were detectable in which of the leaves .It is concluded that this CRE/loxP-based activation system line can provide a useful tool to univeil functions of the salt tolerance gene and promoters.