马尾松α-蒎烯合成酶基因cDNA全长克隆及序列分析

Cloning and Sequence Analysis of α-Pinene Synthase Gene from Pinus massoniana

[目的]分离克隆马尾松α-蒎烯合成酶基因cDNA全长。[方法]根据其他松科植物α-蒎烯合成酶基因保守区域设计引物,扩增出基因的部分片段,再结合RACE技术分别扩增出基因3’端和5’端序列,通过序列拼接获得cDNA全长,结合生物信息学软件分析该基因编码蛋白的特性。[结果]马尾松α-蒎烯合成酶基因cDNA全长为2 103 bp,编码区1 980 bp,编码629个氨基酸,含有1个N端结构域、1个金属结合结构域和1个天冬氨酸富集基序(DDMYD)。[结论]该方法成功克隆了马尾松α-蒎烯合成酶基因cDNA全长序列,具有单萜烯合成酶基因的典型特征,序列提交至GenBank,获得登录号KF547035。

[Objective] To clone the full-length of α-pinene synthase gene from Pinus massoniana.[Method] According to high conservative amino acids of reported α-pinene synthase gene from pinaceaes,a pair of degenerate primers was designed and partial fragment was obtained by PCR amplification.Based on this known sequence,some new primers were designed,two terminals gene sequence were cloned by 3＇RACE and 5＇ RACE respectively.After splicing the fragments of sequence,analyzed the structures and function of the coded protein by bioinformatics softwares.[Result] The full-length cDNA of α-pinene synthase gene consists of 2 103 bp,contains a 1 890 bp open reading frame （ORF） encoding 629 amino acid proteins,including N-terminal transit peptide,metal-binging domain and DDXXD motif.[Conclusion] The full-length cDNA of α-pinene synthase gene was cloned,which has the typical characteristics of monoterpene synthase gene.The sequence has been submitted to GeneBank,accessin No.KF547035.