山药PAL基因全长cDNA序列的克隆、表达与分析

CLONING,EXPRESSION AND ANALYSIS OF A FULL-LENGTH cDNA SEQUENCE ENCODING PAL IN YAM

为了阐明苯丙氨酸解氨酶（PAL,EC4.3.1.5）基因在山药地下块茎酚类物质积累过程中的分子机理,本研究运用普通PCR、RACE和TAIL-PCR技术从大薯（Dioscorea alataL.）’Zixiao’地下块茎中克隆到同一个PAL基因的3个片段,即长度分别为1 145bp、1 151bp和424bp的中间片段、3’端和5’端的cDNA序列;将3段cDNA序列拼接后获得大小为2 376bp的PAL基因全长cDNA序列,该序列含有一个1986bp的最大开放阅读框（ORF）,一个28bp的5’端非翻译区,一个362bp含25 nt的Poly（A＋）尾的3’端非翻译区,最大ORF可推测编码一个包括PAL酶活性中心的特征序列（GTITASGDLVPLSYIA）在内的661个氨基酸的多肽,分子量为71.974kDa,等电点6.310;大薯地下块茎的PAL基因分别在核苷酸与氨基酸水平上和GenBank中所选已知其他物种的同源性为73%~77%和76%~82%,说明PAL基因在系统进化上相对保守;RT-PCR分析表明该基因仅在地下块茎中表达,而且表达丰度在收获前逐渐降低。

To elucidate molecular mechanisms in the effects of phenylalanine ammonia-lyase （PAL, EC 4.3.1.5） gene on phenols accumulation in the underground tuber of yam, in the present study, the eDNA sequences of middle segment, 3＇-and 5＇-ends in the same PAL gene were isolated from the underground tuber of Dioscorea alata L. ev. ＇Zixiao＇ by traditional PCR, RACE and TAIL-PCR with the designed primers, which were 1 145bp, 1 151bp and 424bp in sizes, respectively. The three eDNA segments were combined and constituted a full-length 2376-bp eDNA sequence of PAL gene, which contains a 1986-bp largest open reading frame （ORF）, a 28-bp 5＇ noncoding region, and a 362-bp 3＇ noneoding region with a 25-nt poly（A＋ ）. Its largest ORF putatively encodes a polypeptide of 661 amino acids containing the typical sequence of PAL enzyme activity centre region （GTITASGDLVPLSYIA）. Both the calculated molecular mass and the theoretical pI of the deduced polypeptide are respectively 71.974kDa and 6. 310. PAL gene in yam underground tuber shares the similarities of 73 % -77 % and 76 % - 82 % at the levels of nucleotides and amino acids, respectively, to those of the selected other plant species reported in GenBank Database, which indicated that PAL gene was relatively conserved in systematic evolution. Analysis of the homologous gene expression by RT-PCR showed that it was expressed only in yam underground tuber and its expression abundance was gradually reduced before the harvesting.