大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析

Cloning, Characterization and Expression of a Phenylalanine Ammonialyase Gene (M-PAL) from Plantain (Musa ABB cv. Dongguandajiao)

摘要:

为了研究苯丙氨酸解氨酶基因与大蕉(Musa ABB cv. Dongguandajiao)抗枯萎病的关系，利用RT-PCR和RACE技术克隆了大蕉苯丙氨酸解氨酶基因全长cDNA。此cDNA长1300 bp，包含一个长为1191 bp，编码397个氨基酸的完整开放阅读框（ORF），推导的氨基酸序列与水稻PAL基因氨基酸序列同源性达89%，将此基因命名为M-PAL. Southern 杂交结果表明大蕉中存在一个包含4-5个PAL基因的基因家族，将此基因克隆到大肠杆菌表达载体pET32(a+)中，表达的蛋白质分子量大小与推导的相一致，并且表达的蛋白质表现出PAL酶活性。对接种香蕉枯萎病菌4号生理小种(Fusarium oxysporum f.sp. cubense (FOC) race 4 )后大蕉叶片中M-PAL基因的转录谱进行研究表明，在接种枯萎病菌后M-PAL基因在叶片中的转录水平提高，因此推测M-PAL基因的表达可能与香蕉枯萎病抗性相关。

Abstract:

A full-length cDNA encoding PAL was cloned from leaves of plantain (Musa ABB cv. Dongguandajiao) by RT-PCR and RACE. The cDNA was 1300 bp in length and contained a complete ORF of 1191 bp which encodes a protein of 397 amino acid residues. Its deduced amino acid sequence had 89% identity with rice PAL and then this gene was hence designated as M-PAL. Southern hybridization analysis demonstrated that a small family of four to five PAL genes exists in plantain banana. The complete coding sequence of M-PAL was cloned into an expression vector pET32 (a+) and a protein about 43KD expressed in Escherichia coli BL21 (DE3) was the same as deduced molecular weight and showed phenylalanine ammonia-lyase activity. The transcript of M-PAL gene in the leaves was increased when the plantlets of plantain were inoculated with Fusarium oxysporum f.sp. cubense, indicating that the expression of M-PAL may be related to resistance of banana Fusarium wilt.