牛樟芝鲨烯环氧酶基因的克隆、生物信息学及表达分析

目的克隆牛樟芝三萜合成途径中鲨烯环氧酶基因(AcSE),并进行生物信息学和表达差异分析。方法以牛樟芝菌丝体cDNA为模板,利用快速末端扩增(RACE)进行AcSE序列扩增,利用生物信息学分析AcSE基因的理化性质并预测其蛋白二级结构、三级结构及其功能;同时利用实时荧光定量PCR(q RT-PCR)测定不同培养时间的液体牛樟芝菌丝体及子实体中AcSE基因的表达情况。结果 AcSE的cDNA全长1 446 bp(Gen Bank编号:KT070558),编码481个氨基酸,相对分子质量为53 300,等电点为6.36。结构域分析表明AcSE内含3个跨膜的结构域,无卷曲螺旋结构,且疏水区和亲水区交替存在。AcSE的g DNA全长为1 607 bp,含4个外显子和3个内含子。AcSE在液体培养7 d的牛樟芝菌丝体中表达量最高,为子实体的7.89倍,且随着培养时间的延长逐渐下降。结论首次从牛樟芝中克隆了AcSE基因,为进一步阐明该基因在牛樟芝三萜合成途径中的作用奠定基础。

Cloning, bioinformatics, and expression analysis of squalene epoxidase in Antrodia cinnamomea

Objective To clone the squalene epoxidase gene of Antrodia cinnamomea (AcSE) and analyze the bioinformatics and expression of the gene.Methods AcSE was cloned by rapid-amplification of cDNA ends (RACE) from cDNA of A. cinnamomea. The physical and chemical properties of AcSE protein were analyzed, and its secondary structure, tertiary structure, and function were predicted by using bioinformatics analysis. The expression of AcSE in mycelium and fruit body of A. cinnamomea at different culture time was detected by using quantitative real-time PCR (qRT-PCR).Results The full-length cDNA sequence of AcSE were 1 446 bp (Genbank:KT070558), encoding a 481-amino-acid polypeptide. The molecular weight of AcSE was 53 300 and pI was 6.36. Domain analysis results showed that AcSE had three transmembrane domains without coiled-coil structure, and hydrophobic and hydrophilic regions existed alternately. The gDNA sequence of AcSE was 1 607 bp, contained four exons and three introns. A gene expression analysis by relative qRT-PCR showed that the highest expression level of AcSE was in mycelia incubated for 7 d of A. cinnamomea, and it was 7.89 times than that in fruiting body, and a gradual decline was observed with the extension of the culture time.Conclusion Gene AcSE was firstly cloned from A. cinnamomea, and it would lay a foundation for exploring the mechanism of terpenoid biosynthesis in A. cinnamomea.