薄荷Eβf合成酶基因(Tspa11)的克隆、序列分析与表达特性

Cloning,Sequencing and Expression Characteristic Analysis of Eβf Synthetase Gene from Mentha haplocalyx

为克隆薄荷Eβf合成酶基因(Tspa11)、分析蛋白质结构特性与理化性质,确定其在薄荷不同组织的表达量差异,本试验以薄荷为材料,采用RT-PCR技术扩增获得Tspa11基因cDNA序列,采用生物信息学软件分析编码蛋白质的结构特性,MEGA7.0软件邻接法构建氨基酸同源性系统进化树;采用实时定量PCR技术分析Tspa11在薄荷不同组织(种子、根、茎、叶、花)的差异表达情况。结果表明,得到Tspa11基因序列长度为1929 bp,最大开放阅读框为71~1723 bp,共编码550个氨基酸,分子量为63.74 ku,理论等电点(pI)5.21,偏酸性;表明Eβf合成酶是一种稳定的疏水性蛋白。NCBI保守结构域在线分析表明,Eβf合成酶基因编码的氨基酸具有Isoprenoid_Biosyn_C1 Superfamily的保守结构域(E-value:1.32e-03),蛋白氨基端位于膜内;氨基酸同源性分析,夏枯草Eβf合成酶基因与薄荷Tspa11亲缘关系最近,构成了一个小分支,同源性高达90%;夏枯草Tspa11基因在同条件下种子的基因表达量极显著地高于其他组织(P<0.01),根部表达量最低。

The objectives of this study were to clone the Tspa11 gene of Mentha haplocalyx,to analyze the structure,physical and chemical properties of the protein,and to determine the differences of expression in different tissues and different developmental stages of the worker bees.Tissues of M.haplocalyx were used as materials.The cDNA sequence of odorant receptor gene Tspall was obtained by RT-PCR.The structural properties of encoded protein were analyzed by the bioinformatics software,and the phylogenetic tree of Tspa11 based on amino acid was constructed by MMEGA7.0 neighbor-joining method.The different expression levels of Tspa11 gene in different tissues(seeds,roots,stems,leaves,and flowers) were detected by quantitative real-time PCR at different developmental stages.The results showed that the length of Tspa11 gene was 1 929 bp,encoding a putative protein of 550 amino acids,with an estimated molecular weight 63.74 ku,the theoretical pI 5.21,indicating it was a acidic and stable hydrophobic protein.The N-terminal was intracellularly located(a conserved Isoprenoid_Biosyn_C1 superfamily domain);the amino acid homology analysis showed that the amino acid sequence of Eβf synthetase was consistent with that of Prunella vulgaris’s gene with the similarity of as high as 90%.The expression of Tspa11 in seeds was significantly higher than that in other tissues at the same stage(P<0.01),and the expression level in root was lowest.