月月桂AACT基因的cDNA全长克隆及生物信息学分析

Full length cloning and bioinformatics analysis of Acetyl-CoA C-acetyltransferase gene in Osmanthus fragrans

为了克隆分离月月桂AACT(乙酰辅酶A酰基转移酶)全长基因,并研究其基因特征和蛋白结构,以月月桂花瓣RNA为模板,通过反转录PCR及RACCE技术分离克隆AACT全长。再利用NCBI网站及生物信息学软件对其碱基分布、氨基酸组成、亲疏水性、二级结构、保守区及三级结构进行预测和分析,并对10个物种的AACT氨基酸进行多重比对,对基因进行进化分析。分离克隆出AACT基因的cDNA序列长为1580bp,编码405个氨基酸,该蛋白质相对分子质量为41347.89D,等电点为5.66,其中Ala含量最高为14.53%,平均疏水值为0.215,属Cond-enzymes超家族。氨基酸序列比对中,胡黄连、假马齿苋氨基酸序列同源性最高。OsAACT基因稳定,编码的蛋白为疏水性蛋白,其在进化过程中相对保守。试验获得的基因信息为萜类物质合成途径进一步研究奠定基础。

In order to clone and separate the gene of AACT in Osmanthusfrangtans, and to study its genetic characteristics and protein structure, by taking RNA in O. frangrans flower as template, AACT gene was isolated from flowers of O. frangrans by the method of reverse transcription polymerase chain reaction （RT-PCR） and the rapid amplification of eDNA ends（RACE） technique. NCBI website and bio-informaties sottware were used to predict and analyze base distribution, amino acids composition, hydrophilicity or hydrophobicity, hyper-conservative region, secondary structure, and three-level structure. The results were used to compare with AACT amino acid sequences of other ten species and the related evolution analysis was conducted. The single chain of AACT gene was 1580 bp, code was made up of proteins containing amino acid of 405, and relative molecular mass of protein was 41 347.89 with Aar of 14.53. The isoelectric point was 5.66, and grand average of hydropathicity was 0.215. It belonged to the cond-enzymes superfamily. After many comparisons with other ten species, the amino acid sequences of Picrorohiza and Bacopa monnieri were the highest. The gene of OsAACT was stable and coded albume was hydrophobic protein which was quite conservative in evolution. The information of gene gained in the test paved the way for the further study on synthetic of the terpenoid substances.