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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 annual herbaceous plants are infected by pathogens, salicylic acid methyltransferase (SAMT) in the body can convert salicylic acid (SA) produced by plant infection sites into methyl salicylate (methyl salicylate, Me SA), plays an important role in SA signal transduction and systemic acquired resistance (SAR). The function of SAMT gene in perennial woody plants remains to be further verified. In this study, 84K Yang SAMT primers were designed based on the SAMT gene sequence of Populus trichocarpa. Using the Gateway cloning technique, the full-length open reading frame (ORF) of SAMT with att B linker was ligated with the entry vector p DONRTM222 was used to carry out BP recombination reaction, and the target gene was transferred into the entry vector. The plasmid extracted after transformation of Escherichia coli competent cells DH5α was digested with MLUⅠand then LR recombined with the overexpression vector p MDC32 under the action of LR enzyme The SAMT was transferred into the expression vector and the SAMT overexpression vector was successfully constructed. 84K transgenic plants SAMT overexpressed were obtained by Agrobacterium tumefaciens-mediated genetic transformation. This study will provide the basic information for future study on the function of this gene in poplar anti-ulcer disease and the role of poplar in SAR and other aspects, and also provide reference for studying the function of this gene in other perennial woody plants.