文献类型：期刊文献

中文题名：杨树SAMT基因超表达载体的构建及遗传转化

英文题名：Construction of Salicylic Acid Methyltransferase Gene Over-expression Vectors and Genetic Transformation in Poplar(Populus alba × P.glandulosa)

作者：董慧霞[1,2] 理永霞[3,4] 吕全[1,4] 贾秀贞[1,4] 张星耀[1,4]

第一作者：董慧霞

机构：[1]中国林业科学研究院森林生态环境与保护研究所/国家林业局森林保护学重点实验室;[2]河南师范大学生命科学学院;[3]中国林业科学研究院林业新技术研究所;[4]南京林业大学南方现代林业协同创新中心

年份：2016

卷号：24

期号：11

起止页码：1709-1717

中文期刊名：农业生物技术学报

外文期刊名：Journal of Agricultural Biotechnology

收录：CSTPCD;;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；

基金：林业公益性行业科研专项经费(No.201204501);中央级公益性科研院所基本科研业务费专项资金项目(No.CAFYBB2014MB002)

语种：中文

中文关键词：84K杨;水杨酸甲基转移酶基因(SAMT);超表达;载体构建;遗传转化

外文关键词：84K Poplar, Salicylic acid methyltransferase gene （SAMT）, Over-expression, Vector construction,Genetic transformation

分类号：S763.11;Q319.2

摘要：一年生草本植物受到病原菌侵染时,体内的水杨酸甲基转移酶基因(salicylic acid methyltransferase,SAMT)能将植物侵染部位产生的水杨酸(salicylic acid,SA)转化为水杨酸甲酯(methyl salicylate,Me SA),在SA信号转导和植物系统获得抗性(systemic acquired resistance,SAR)中起着重要作用。而多年生木本植物中SAMT基因的功能还有待进一步验证。本研究根据毛果杨(Populus trichocarpa)SAMT基因序列,设计了84K杨SAMT引物,利用Gateway克隆技术,在BP反应酶作用下,使带att B接头的SAMT基因全长开放阅读框与入门载体p DONRTM222进行BP重组反应,将目的基因转入入门载体;转化大肠杆菌(Escherichia coli)感受态细胞DH5α后提取的质粒用MLUⅠ进行酶切,然后在LR酶作用下与超表达载体p MDC32进行LR重组反应,将SAMT转入表达载体,成功构建了SAMT的超表达载体。通过农杆菌(Agrobacterium tumefaciens)介导的遗传转化,获得了SAMT超表达的84K杨转基因植株。为今后研究该基因在杨树抗溃疡病中的功能以及杨树在获得SAR和其他方面的作用提供了基础资料,同时也为研究该基因在其他多年生木本植物中的功能提供参考。
When plants of annual herb are being invaded by a pathogen, the gene of salicylic acid methyltransferase （SAMT）, which converts salicylic acid （SA） produced at the inoculation site into methyl salicylate （MESA）, plays an important role in plant systemic acquired resistance （SAR） and signal transduction of SA. However, the function of SAMT in perennial woody plants needs to be further verified. In this study, a pair of primers was designed according to the complete CDS of Populus trichocarpa SAMT and was used to amplify the SAMT gene of 84K popular by polymerase chain reaction. Under Gateway technology, we performed a BP recombination reaction between the attB-flanked SAMT fragment and an attP-containing donor vector pDONRTM222 with the action of BP clonase enzyme to convert the gene of interest into entry vector. Then the vector were transformed competent Escherichia coli DHSct. Plasmids, which were extracted and digested by MLU I enzyme, and over-expression vector pMDC32 were performed an LR recombination reaction with the action of LR clonase enzyme to transform the SAMT into expression vector. Successfully over-expression vector of SAMT gene were constructed, and SAMT-overexpressing transgenic 84K popular plants were obtained by Agrobacterium-mediated genetic transformation using leaf-disk transformation method. Then transgenic lines were tested and analyzed by RT-PCR and qPCR methods. From the results, leaf- disk transformation is appropriate transformation method, with high conversion efficiency. Eccept one line of the SAMT- overexpressing genetically modified lines is lower, 57 times more than CK. Compared with control, other lines of transgenic 84K popular plants are more than 3 729 times. These results laid the foundation for the future research of the gene function and SAR in poplar and other roles, as well as provided the reference about the function of the gene in other perennial woody plants.