象耳豆根结线虫Me-mapk1基因的克隆及RNAi分析

Cloning and RNAi of Me-mapk1 Gene from the Root-knot Nematode Meloidogyne enterolobii

促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是线虫体内信号转导的重要组分,在生长发育过程中具有重要作用。本研究从象耳豆根结线虫中克隆了1个促分裂原活化蛋白激酶基因Me-mapk1c DNA序列,该序列全长1 369 bp,包含1个1 185 bp的完整开放阅读框,推导编码394个氨基酸,蛋白质分子量为45.39 ku。同源性搜索比对结果发现,该基因编码的蛋白序列与南方根结线虫MI-MAPK1具有99%的一致性。通过构建原核表达载体p ET-32a-MAPK,在IPTG的诱导下得到70 ku左右的特异融合蛋白(包括大小约26 ku的标签序列)。利用RNAi技术对象耳豆根结线虫二龄幼虫Me-mapk1基因进行沉默,线虫诱导番茄根结形成的数量显著减少,推测Me-mapk1基因的下调表达有可能影响象耳豆根结线虫二龄幼虫(J2)的生长发育。

Mitogen-activated protein kinases（ MAPKs） are important components in signal transduction pathway involving in the process of nematode growth and development. In this study, a MAPK gene（Me-mapk1） from the root-knot nematode Meloidogyne enterolobii was cloned. The Me-mapk1 c DNA sequence consisted of a 1 185 bp open reading frame（ORF） encoding 394 amino acid residues that were franked by a 27 bp 5′-untranslated region（ UTR）and a 157 bp 3′-UTR. The protein molecular mass was 45.39 ku. Homology analysis showed that the identity was 99% between Me-mapk1 deduced protein ME-MAPK1 and MI-MAPK1 protein of M. incognita. The recombinant protein ME-MAPK1 with a molecular mass of 70 ku which included tag protein sequence of 26 ku was produced by prokaryotic expression. The number of nematode-induced galls on tomato was significantly reduced after knocking-down Me-mapk1 gene by RNA interference, indicating an important role of the gene to nematode development.