大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析(英文)

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

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 1 300 bp,包含一个长为 1 191 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 oxysporumf. 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。

A full-length cDNA encoding PAL was cloned from leaves of plantain （Musa ABB cv. Dongguandajiao） by RT-PCR and RACE. The cDNA was 1 300 bp in length and contained a complete ORF of 1 191 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 43 KD expressed in Escherichia coli BL21 （DE3） was the same as deduced molecular weight and showed phenylalanine ammonia-lyase activity. The transcript ofM-PA L 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.