《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(1)》由会员分享，可在线阅读，更多相关《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(1)（57页珍藏版）》请在金锄头文库上搜索。
1、河北农业大学 硕士学位论文 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 姓名：潘永刚 申请学位级别：硕士 专业：植物病理学 指导教师：刘颖超;董金皋 2009-06-11 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
2、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
3、-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-B
4、ox、 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 bp1 232 bp 之间可能存在负调控元件。 关键词：拟南芥；致病疫霉；ACS9；启动子；瞬时表达 Functional confirmation and promoter activity of Phytophthora infestans-defen
5、se related ACS9 gene in Arabidopsis Author：PAN Yong-gang Supervisors：DONG Jin-gao LIU Ying-chao Major：Plant Pathology Abstract Ethylene, one of the major phytohomornes, plays a significant role in plant, such as seed germination, floral differentiation, growth, development, decrepitude, sex determin
6、ation, 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 be
7、en 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. participa
8、ting 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 term
9、inus 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 tumefaci
10、ens 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
11、 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 infect
12、ed 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.
13、 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
14、 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 genes promoter. Keywords: Arabidopsis thaliana, Phytophthora infestans, ACS9, promoter, transient expression 缩
15、写词表 Amp Ampicillin 氨苄青霉素 BSA Bovine serum album 牛血清蛋白 CaMV Cauliflower Mosaic Virus 花椰菜花叶病毒 CTAB Cetyl Trimethy Ammonium Bromide 十六烷基三甲基溴化铵 DEPC Diethylpyrocarbonate 二乙基焦磷酸酯 ddH2O Double Distilled Water 双蒸水 dNTP Deoxynucleotide Triphosphates 脱氧核苷三磷酸 EB Ethidium Bromide 溴化乙锭 EDTA Ethylenediamine Tetr
16、aacetic Acid 乙二胺四乙酸 GUS -glucuronidase -葡萄糖苷酸酶 PCR Polymerase Chain Reaction 聚合酶链式反应 SDS Sodium dodecil sulfate 十二烷基磺酸钠 Tris Tris (Hydroxymethyl) Aminomethane 三（羟甲基）氨基甲烷 TAIR The Arabidopsis Information Resource 拟南芥信息资源 X-Gluc 5-Bromo-4Chloro-3Indolyl-D-Glucuronic acid Cyclohexyla mmonium Salt 5-溴-4-氯-3-吲哚-D-半乳糖酸 独创性声明 本人声明所呈交的学位论文是本人在导师指导下进行的研究工作及取得的研 究成果。据我所知，除了文中特别加以标注和致谢的地方外，论文中不包
