白木香2个小分子热激蛋白基因的克隆及表达分析  

Cloning and expression analysis of two small heat shock proteins genes from Aquilaria sinensis

作　　者：董先娟 刘晓[2] 张钟秀[1,2] 张乐[1,2] 王娟[1,2] 屠鹏飞[2] 王晓晖[2] 史社坡[2] 

机构地区：[1]北京中医药大学中药学院,北京100029 [2]北京中医药大学中药现代研究中心,北京100029

出　　处：《中草药》2016年第22期4054-4061,共8页Chinese Traditional and Herbal Drugs

基　　金：北京市自然科学基金青年项目(7154216)

摘　　要：目的从白木香Aquilaria sinensis中克隆sHSP1和sHSP2基因,并对它们的生物信息学及表达模式进行分析。方法根据本课题组白木香转录组数据库进行分析获得2条具有小分子热激蛋白(sHSPs)保守结构域的sHSPs基因序列,利用RT-PCR技术克隆sHSP1和sHSP2基因的全长c DNA序列,并对其进行生物信息学分析;利用实时荧光定量PCR检测sHSP1和sHSP2基因在不同组织和高盐及外源ABA、SA、MJ处理下不同时间的表达差异。结果克隆得到的白木香sHSP1和sHSP2基因开放阅读框都为474 bp,编码157个氨基酸。组织表达分析的结果显示,sHSP1和sHSP2基因主要在根中表达,在茎和叶中的表达量较少;白木香愈伤组织在高盐处理下,sHSP1和sHSP2基因分别在36 h和24 h达到最高表达水平,而愈伤组织在外源ABA、MJ处理下,sHSP1和sHSP2基因都在12 h达到最高表达水平,在SA处理下,sHSP1和sHSP2基因分别在12、24 h达到最高表达水平。结论获得了sHSP1和sHSP2 2基因全长c DNA序列,且2个基因在不同组织根、茎、叶中的表达存在差异性,在受到高盐和外源ABA、SA、MJ处理时,在不同时间的愈伤组织中的表达及积累情况也不同,该研究为后续深入研究白木香防御反应奠定了基础。Objective To aim at cloning the open reading frame （ORF） of sHSP1 and sHSP2 genes from Aquilaria sinensis and analyzing the bioinformatics and expression of the two genes. Methods Two unique sequences containing sHSPs domain were discovered in transcriptome dataset ofA. sisnensis. The full-length cDNAs of sHSP 1 and sHSP2 were cloned by RT-PCR strategy with the specific primers. Subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different softwares to analyze the bioinformatics of sHSPs protein. The expression different levels of sHSP 1 and sHSP2 isoforms in different tissues and in responds to salt and ABA, SA, MJ treatment were measured by real-time quantitative PCR. Results The sHSP 1 and sHSP2 cDNA sequence consisted of 474 bp ORF, encoding 157 amino acids. Tissue expression analysis indicated that sHSP1 and sHSP2 were primarily expressed in roots, followed by stems and leaves. Salt treatment experiments indicated that salt treatment caused a rapid increase in sHSPland sHSP2 expression within 36 and 24 h, respectively. Exogenous ABA and MJ treatment experiments indicated that sHSP 1 and sHSP2 genes were induced by exogenous ABA and M J, and all reached the highest expression level at 12 h. Simultaneously, the SA treatment experiments indicated that exogenous SA treatment caused a rapid increase in sHSP1 and sHSP2 expression within 12 and 24 h, respectively. Conclusion The full-length eDNA sequence ofsHSP1 and sHSP2 genes from A. sinensis is obtained, sHSP1 and sHSP2 have the different expression level in different tissues. When subjected to high salt, ABA, SA, and MJ treatment, sHSP1 and sHSP2 show the different expression levels in different time. Cloning and analyzing sHSP1 and sHSP2 genes from A. sinensis will play an important role for further study on plant defense response.

关 键 词：白木香 sHSP1基因 sHSP2基因 表达分析 热激蛋白 生物信息学 

分 类 号：R282.12[医药卫生—中药学]