Functional Confirmation And Promoter Activity Of Phytophthora Infestans-defense Related ACS9 Gene In Arabidopsis

Ethylene,one of the major phytohomomes,plays a significant role in plant,such as seed germination,floral differentiation,growth,development,decrepitude,sex determination,and fruit setting as well as in various stresses such as wounding,freezing, drought,pathogen infection and so on.1-Aminocyclopropane-1-Carboxylic Acid Synthase(ACC Synthase) is the rate-limiting enzyme of ethylene biosynthesis in higher plants.The different ecotypes of Arabidopsis had been inoculated with Phytophthora infestans early,and ACS9 gene was identified as resistance-related one in Arabidopsis thaliana Columbia ecotype.In order to study the specific function of the ACC synthase genes and search for the resistance mechanisms and signal transduction pathway of P. participating in ACC synthase genes.The efficient plant expression vectors were constructed with the strategy of antiense and RNAi and the mutants were obtained, further more,the preliminary function of those mutants was analyzed.Different length fragments of ACS9 promoter were cloned and fused its 3' terminus with GUS repoter gene.The expression activity and the regulation of ACS9 gene promoter were checked based on this system.The constitutive type plant expressing plasmid vectors pCAMBIA1300-ACS9A and pCAMBIA1300-ACS9i were constructed then the vectors were introduced into Agrobacterium tumefaciens strain GV3101.Both vectors were infected into Arabidopsis thaliana by Floral Dip,and 31 mutants of transgenic plants of Arabidopsis were obtained.The results of PCR analysis indicated that the foreign genes were integrated into the genome of the transgenic plants.The results showed that ACS9 was deferred in transgenic plants.Analysis of downstream genes PDF1.2 in ethylene signal transduction network of defense response of plant diseases showed that the expression was inhibited; Triple response disappeared by cultured in dark;In contrast to wild type,mutants became more easier infected with P.infestans.The promoters of ACS9 were analyzed by the software of PLACE and PLANTCARE.It indicates that the basic core elements of TATA-box and CAAT-box,as well as stress-induced elements such as light-responsive element G-Box,GATA and elicitor-induced element MBS and LTR were existed.Four plant gene expression vectors carrying GUS gene and the ACS9 gene promoter were constructed and introduced into A.strain GV3101.The transient expression of GUS gene was detected by histochemical staining in tobacco leaves and quantitative fluorometric GUS was assaied through A..Those results revealed that the promoter activity of the 953 bp upstream fragment of ACS9 gene was the highest in our study and Cis-elements might be present in the upstream -953 bp to -1 232 bp of ACS9 gene's promoter.