Functional Analysis Of Stress Tolerance Genes And Promoters By Creiloxp Recombination Activation System

Breeding the new salt-tolerance varieties by plant gene engineering technology isan effective way to improve salt resistance of crop. Scientific and effective evaluation ofsalt-tolerance genes and their regulated elements is the key to further researchsalt-tolerance mechanism. Therefore, salt-tolerance gen (TvNHX1or AtNHX1) wasconstructed into the plant expression vector which contains an inducible promoter. Theconstructed vector and pI-GFP vector were co-transformed into Arabidopsis thaliana toconstitute Cre/loxP recombination system. The active inducible promoter undersalt-stress activates a chain reaction, which makes salt-tolerance gene and GFPco-express in the same pattern. Taking advantage of the feature, we compared thephenotypic, cellular, and molecular differences of salt-stress transgenic plants, and thenanalyzed the function of TvNHX1and AtNHX1gene and rd29A and Hsp18.2induciblepromoter. The main results were summarized as follows.The plant expression vectors, pGII227-UASAtNHX1-rd29ACREN andpGII227-UASTvNHX1-rd29ACREN, were successfully constructed. Then, two plantexpression vectors were transformed into Agrobacterium tumefaciens (C58C1) byelectroporation method.Agrobacterium tumefaciens mediated co-transformation system was used. Themixture of two Agrobacterium strains harboring different vectors, pGII227-UASAtNHX1-rd29ACREN and pI-GFP (rd29A-At), pGII227-UASTvNHX1-rd29ACREN and pI-GFP (rd29A-Tv), were co-transformed into Arabidopsis. Thepositive rates of T1transgenic plants of rd29A-At and rd29A-Tv are7.7%and3.7%byPCR analysis.The transgenic plants rd29A-At, rd29A-Tv, Hsp-Tv (pGII227-UASTvNHX1-rd29ACREN/pI-GFP), and CREN (pGII227-rd29ACREN/pI-GFP) were treated withsalt-stress. The experiments of phenotype observation, measuring chlorophyll content,GFP observation with laser scanning confocal microscopy, and semi-quantitativeRT-PCR were carried out. The results reveal comprehensive evaluation value ofsalt-tolerance of4transgenic materials with the order: rd29A-At> rd29A-Tv> Hsp-tv>CREN. According to the salt-tolerance order, the function of salt-tolerance gene(TvNHX1or AtNHX1) and inducible promoter (rd29A and Hsp18.2) can be evaluated.The promoter rd29A in response to salt-stress is better than Hsp18.2; AtNHX1is moreeffective to improve the salt-tolerance than TvNHX1in Arabidopsis; Cre/loxPrecombination activation system has more advantages than common promoterevaluation model (Pi-GFP) in salt-tolerance and inducible promoter.