霍山石斛β-1,4-木糖基转移酶(IRX9)基因克隆及生物信息学分析

Cloning and bioinformatics analysis of β-1,4-xylosyltransferase IRX9 gene from Dendrobium huoshanense

目的克隆霍山石斛β-1,4-木糖基转移酶(IRX9)基因,并进行生物信息学、密码子偏性分析。方法根据霍山石斛转录组数据获得的IRX9基因序列设计特异性引物,通过RT-PCR技术得到IRX9基因cDNA的全长序列,并进行生物信息学分析。结果霍山石斛IRX9基因全长1 553 bp,包含一个长度为1 047 bp的开放阅读框,编码348个氨基酸,理论相对分子质量为39 350,等电点为7.26,为不含信号肽的亲水性稳定蛋白,定位于质膜,具有多个磷酸化位点和糖基化位点。系统进化树表明,该序列与同科植物铁皮石斛的同源性最高,且具有GT-A超家族的保守区域"DXD"。密码子偏性分析表明,该基因偏好使用以A/T结尾的密码子,具有27个偏性密码子,烟草为该基因最适合的外源表达宿主。结论成功克隆IRX9基因,为从分子水平调控霍山石斛的生长发育和改善药材产量和品质提供理论基础。

Objective To clone IRX9 gene from Dendrobium huoshanense and perform the bioinformatics and codon bias analysis.Methods The full-length cDNA sequence was cloned from the IRX9 gene based on transcriptome sequencing data of D.huoshanense generated in our previous study by using RT-PCR and further analyzed by bioinformatic methods.Results The cloned IRX9 gene was 1 533 bp,containing a 1 047 bp open reading frame(ORF)which encoded 348 amino acids.The theoretical molecular weight was 39 350 and its isoelectric point was 7.26.IRX9 protein was a hydrophilic protein and had no signal peptide,which had multiple phosphorylation sites and glycosylation sites and might be located in the plasma membrane.Phylogenetic analysis indicated that sequence of amino acids had the highest homology with D.candidum and the conserved region“DXD”of the GT-A superfamily.Codon bias analysis showed that IRX9 gene prefered to use A/T ending codon,with 27 skewed codons.Nicotiana is the most suitable host for exogenous expression of IRX9 gene.Conclusion The IRX9 gene was cloned from Dendrobium huoshanense successfully and it provided a theoretical basis for regulating the growth and development of D.huoshanense and improving the yield and quality of D.huoshanense.