穿心莲八氢番茄红素脱氢酶ApPDS的基因克隆及功能鉴定

Cloning and functional characterization of phytoene desaturase in Andrographis paniculata

采用RT-PCR和RACE技术从穿心莲植株中克隆得到了八氢番茄红素脱氢酶(PDS)的全长c DNA,预测有一个1 752bp的完整开放阅读框(序列已登录Gen Bank,登录号KP982892),编码584个氨基酸。序列同源性分析,穿心莲PDS编码的氨基酸序列与芝麻、广藿香、黄芩等其他植物的PDS有很高的同源性,该蛋白包含一个共有的氨基酸脱氢酶的辅酶结构域NAD(H)-binding domain。利用半定量RT-PCR技术进行组织表达模式分析,发现PDS基因在穿心莲的根、茎、叶、花中均有表达,表达量为叶﹥花﹥茎﹥根。用病毒诱导基因沉默(VIGS)的方法在穿心功莲体内鉴定Ap PDS的功能,构建VIGS载体p TRV2-PDS,经农杆菌浸染穿心莲叶片,植物表型观察、定量RT-PCR检测Ap PDS基因的表达,结果观察到叶片轻微变黄和明显的基因下调,鉴定了Ap PDS在体内的功能。该研究首次克隆并鉴定了穿心莲PDS基因,为进一步研究穿心莲中新基因尤其是穿心莲内酯类二萜生物合成基因的功能奠定了基础。

A full-length cDNA of phytoene desaturase （PDS） gene fi＇om Andrographis paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp （ GeneBank ： KP982892 ） , encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD（H）-binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves. Virus induced gene silencing （VIGS） was per- formed to characterize the functional of ApPDS in planta. Significant photobleaching was not observed in infiltrated leaves, while the PDS gene has been down-regulated significantly at the yellowish area. To the best of our knowledge, this represents the first report of PDS gene cloning and functional characterization from A. paniculata, which lays the foundation for further investigation of new genes, especially that correlative to andrographolide biosynthetic pathway.