薇甘菊水杨酸羧甲基转移酶基因的分离鉴定及表达分析

Cloning,Identification and Expression of Salicylic Acid Carboxyl Methyltransferase Gene from Mikania micrantha

为研究水杨酸羧甲基转移酶基因在植物防御系统中的作用,采用RACE法从薇甘菊(Mikania micrantha)cDNA文库中克隆到薇甘菊水杨酸羧甲基转移酶基因SAMT全长cDNA,并进行外源表达以及水杨酸诱导模式分析。结果表明,该基因cDNA全长1299 bp,其中编码区长1089 bp,编码362个氨基酸,Blast显示该基因编码的氨基酸序列与仙女扇(Clarkia breweri)的相似性为74%,证实其主要功能是将水杨酸(salicylic acid,SA)甲基化成水杨酸甲酯(methyl salicylate,MeSA),由此将该基因命名为MmSAMT,GenBank登录号为FJ869889。将MmSAMT编码序列克隆至pET-32a(+)载体,转化Rosetta-gami(DE3)中表达,SDS-PAGE显示单体分子量在40 kD左右,与预测结果一致。Western blot显示在20℃、0.05 mmol/L IPTG和180 r min-1下诱导6 h,可获得较多的可溶性蛋白质。对喷施100μmol/L SA薇甘菊叶片中MmSAMT的转录谱进行研究,该基因的诱导受到SA的激活,48 h的表达水平达最高,暗示MmSAMT可能通过催化合成MeSA引发系统获得性抗性,提高抗性防御的警戒等级。

In order to study the role of salicylic acid carboxyl methyltransferase （SAMT） in plant defense system, a full-length cDNA of Mikania micrantha SAMT was cloned from the SMART cDNA library by RACE, then the exogenous expression and induction pattern by SA were studied. The results showed that the full-length cDNA was 1299 bp, with a 1089 bp-length coding sequence （CDS） encoding a protein of 362 amino acids. Blast result showed 74% similarity with the amino acids of Clarkia breweri CbSAMT, confirming that Mikania micrantha SAMT may play a role in methylating SA into MESA. Therefore this gene was named as MmSAMT and its GenBank accession number was FJ869889. Subsequently the CDS of MmSAMT was integrated into pET-32a（＋）, then the recombinant plasmid was transformed into Rosetta-gami （DE3） for exogenous expression. A specific monomer about 40 kD could be detected by SDS-PAGE, which was consistent with prediction. Western blot showed that the efficient solubility could be obtained cultured under 20℃, 0.05 mmol/L IPTG,180 r min^-1 for 6 hours. The transcription profile of MmSAMT in M. micrantha leaves sprayed 100μmol/L SA revealed that it could be activated by SA and reached a peak after 48 hours. It implied that MmSAMT may participate in the SA signaling pathway via MESA, and induce the systemic acquired resistance （SAR） as a key signal and enhance the plant defense system.