植物抗病性信号传导调控基因的克隆与表达检测方法的研究  

Studies on methods in cloning and expression quantification of genes that regulate plant disease resistance signal transduction

作　　者：董宏平[1] 彭建令[1] 余晓江[1] 赵静[1] 王颖[1] 董汉松[1] 

机构地区：[1]南京农业大学农业部病虫监测与治理重点开放实验室

出　　处：《南京农业大学学报》2003年第4期30-35,共6页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金 (3 0 0 80 0 11);教育部博士点基金 (B2 0 0 10 6) ;江苏省农业高技术项目 (BG2 0 0 13 0 7)

摘　　要：建立了从不同植物中克隆抗病性信号传导调控基因及检测其表达的方法 ,并优化了相关条件。用RT PCR和PCR方法从拟南芥、烟草、番茄、中华鳖中克隆了 7个信号传导通路中 18个调控基因的cDNA和DNA序列 ,它们分别是 :抗病基因Pto介导的蛋白激酶级联中的Pti4、Pti5和Pti6 ,细胞编程死亡通路中的Hin1和hsr2 0 3,水杨酸介导的系统性获得抗病性通路中的NPR1,乙烯信号通路中的ETR1、ERS1、CTR1、EIN2和ERF1,茉莉酸信号通路中的COI1,核黄素信号通路中的RFBP和LR ,脱落酸信号通路中的ABF3、ABF4和ABH1,以及调控不同信号通路的通调基因NDR1。进一步研究了这些基因表达的定量测定方法 :用细菌激发子harpinEa处理植物 ,提取RNA作为cDNA第1链合成的模板 ,以在真核生物中广泛同源和高度保守的细胞伸长翻译因子基因EF1α为内参 ,用半定量RT PCR法测定基因mRNA的积累 ,可以比较准确地检测不同基因的相对表达水平。Methods in cloning and expression quantification of plant disease signaling regulatory genes were studied.Application of PCR and RT-PCR techniques resulted in cloning of 18 regulatory genes involved in 7 signal transduction pathways.Genes cloned included Hin1 and hsr203 associated with programmed cell death,NPR1 that regulates salicylic acid-mediated systemic acquired resistance(SAR),ETR1,ERS1,CTR1,EIN2 and ERF1 in ethylene signaling,COI1 involved in jasomonate signaling,Pti4,Pti5 and Pti6 that are components in the resistance gene Pto-mediated protein kinase cascades,RFBP encoding a riboflavin-binding protein and LR encoding lumazine synthase that control riboflavin synthesis,ABF3,ABF4 and ABH1 involved in absisic acid signaling,as well as the NDR1 gene that is required for both gene-for-gene resistance and SAR.Conditions regarding template concentrations were optimized and PCR programs established for the best amplification of the genes.In an attempt to establish methods for determining gene expression levels,expression of the genes in plants treated with the bacterial elicitor harpin Ea were detected by semi-quantitative RT-PCR.The method was proved to feasibly detect relative expression levels of different signaling regulatory genes,based on similar amplification of an inner standard,EF1α, the gene that encodes elongation translation factor and is highly conserved in eukaryotes.

关 键 词：植物 抗病性 信号传导 调控基因 基因克隆 基因表达 检测方法 半定量RT-PCR 

分 类 号：S432.23[农业科学—植物病理学]