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拟南芥抗致病疫霉相关基因ACS9的功能验证及其启动子活性研究

标题: 拟南芥抗致病疫霉相关基因ACS9的功能验证及其启动子活性研究
英文标题: Functional Confirmation and Promoter Activity of Phytophthora Infestans-defense Related ACS9 Gene in Arabidopsis
作者: 潘永刚
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: 拟南芥,致病疫霉,ACS9,启动子,瞬时表达
英文关键词: Arabidopsis thaliana,Phytophthora infestans,ACS9,promoter,transient expression
论文级别: 硕士
学位: 学位论文
导师: 刘颖超%董金皋
专业: 植物病理学
提交时间: 2009
摘要: 乙烯作为一种植物激素,对植物种子萌发、生长发育、衰老、果实成熟、性别决定等都有着重要的影响,同时参与抵抗逆境胁迫如机械伤害、冷害、渍水、病原菌入侵等以及参与植物细胞的程序性死亡.ACC合酶是乙烯合成途径中的限速酶,本实验室在前期研究中利用致病疫霉接种不同生态型拟南芥,初步确定ACC合酶中的ACS9基因为抗致病疫霉相关基因.为了深入研究ACC合酶参与抗致病疫霉的机制和信号传导途径,本试验利用反义RNA和RNAi技术对ACS9基因功能进行了初步研究.克隆了ACS9基因上游不同长度的调控序列,并构建了GUS表达载体,研究了该基因的表达特性.结果如下: (1)构建了ACS9基因反义RNA和RNAi载体pCAMBIA1300-ACS9A和pCAMBIA1300-ACS9i.将pCAMBIA1300-ACS9A和pCAMBIA1300-ACS9i载体经农杆菌介导转化拟南芥.通过潮霉素筛选,得到了18株反义RNA转化子和13株RNAi转化子.经PCR鉴定,外源基因已成功整合到了转基因植株的基因组中.RT-PCR结果表明,转基因植株中ACS9基因的表达均受到不同程度的抑制,确定所得转基因植株均为ACS9基因的(反义RNA/RNAi)突变体. (2)经RT-PCR分析发现,突变体病害防御反应中乙烯信号转导途径的下游基因PDF1.2的表达受到了抑制;对突变体进行暗培养,"三重反应"消失;突变体接种致病疫霉后表现感病症状,确定ACS9基因参与了调控拟南芥中乙烯介导的病害防御反应. (3)用PLACE和PLANTCARE软件分析ACS9基因的启动子,发现该序列具有启动子的基本元件TATA-box和CAAT-box,此外还发现了光应答元件G-Box、GATA和刺激诱导元件MBS和LTR等多个顺式作用元件. (4)成功构建了带有GUS报告基因的含有ACS9基因不同长度调控序列的植物表达载体,发现不同长度的调控序列均具有启动子活性;GUS瞬时表达定量检测与组织化学染色结果表明长度为953 bp的调控序列启动子活性最强,推测ACS9基因上游953 bp~1 232 bp之间可能存在负调控元件.
英文摘要: 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.