珊瑚菜GlPS1、GlPS2基因克隆及表达分析

Cloning and expression analysis of GlPS1, GlPS2 gene in Glehnia littoralis

目的克隆珊瑚菜Glehnia littoralis补骨脂素合成酶(psoralen synthase,PS)基因全长cDNA序列,并进行生物信息学和表达量分析。方法在前期珊瑚菜转录组测序的基础上,筛选出表达量较高的2条PS基因GlPS1和GlPS2序列。利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法对2个基因cDNA序列3’端进行克隆,DNAMAN拼接后得到基因全长cDNA序列,并对其编码蛋白进行生物信息学分析。利用实时荧光定量PCR(RT-qPCR)方法检测GlPS1和GlPS2基因的组织特异性表达。结果 GlPS1和GlPS2基因cDNA序列全长分别为1 885 bp和1 971 bp,编码495和502个氨基酸残基,蛋白相对分子质量分别为55 740.7和56 363.9,等电点为8.28和6.62,均属于细胞色素P450超家族,含有1个跨膜区,为亲水性蛋白。系统进化分析表明,GlPS1和GlPS2与伞形科欧防风Pastinaca sativa、旱芹Apium graveolens、大阿米芹Ammi majus PS蛋白亲缘关系较近。RT-qPCR结果显示GlPS1基因在根中表达量较高,在叶中表达量较低,而GlPS2基因在花中表达量较高,在根中表达量较低。结论首次获得珊瑚菜GlPS1和GlPS2基因的全长cDNA序列并进行了表达分析,为进一步开展珊瑚菜补骨脂素合成酶基因的功能和遗传调控机制研究奠定基础。

Objective To clone the full-length cDNA sequences of psoralen synthase（PS） genes from Glehnia littoralis so as to perform the bioinformatic and expression pattern analysis. Methods Based on our previous transcriptome sequencing data of G.littoralis, the gene sequences GlPS1 and GlPS2 with high expression level were screened. The 3’cDNA ends of GlPS1 and GlPS2 genes were cloned by the RACE（rapid amplification of cDNA ends） method and the full-length cDNA of genes were assembled by using DNAMAN software. And then encoded GlPS proteins were analyzed by the bioinformatics tools. The issue specific expression of GlPS1 and GlPS2 genes were detected using qPCR. Results The full-length cDNA of GlPS1 gene was 1 885 bp, which encoding a protein of 495 amino acids with a predicted molecular weight of 55 740.7 and isoelectric point of 8.28; The full-length cDNA of GlPS2 gene was 1 971 bp, which encoding a protein of 502 amino acids with a predicted molecular weight of 56 363.9 and isoelectric point of 6.62. GlPS1 and GlPS2 proteins belong to the cytochrome P450 superfamily, which share one transmembrane zone acting as hydrophilic protein. Phylogenetic analysis showed GlPS1 and GlPS2 were genetically closely related to the PS of Pastinaca sativa, Apium graveolens, Ammi majus. Higher expression of GlPS1 gene was observed in roots than leaves. However, GlPS2 gene was expressed at a relatively higher level in flowers than in roots. Conclusion The full-length cDNA of GlPS1 and GlPS2 genes were obtained and the expression patterns were explored in G. littoralis for the first time, which provided a foundation for further studies on gene function and genetic regulatory mechanism of GlPS.