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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。
The full-length c DNA of phytoene dehydrogenase (PDS) was cloned by RT-PCR and RACE from the plant of Andrographis paniculata, and a complete open reading frame of 1 752 bp was predicted (the sequence has been registered with Gen Bank, accession number KP982892), encoding 584 amino acids. Sequence homology analysis revealed that the amino acid sequence of PDS encoded by andrographis paniculata has high homology with PDS of other plants such as sesame, patchouli and skullcap, and the protein contains a coenzyme domain of amino acid dehydrogenase, NAD (H ) -binding domain. The expression patterns of PDS gene in roots, stems, leaves and flowers of Andrographis paniculata were all analyzed by semi-quantitative RT-PCR. The expression level was leaf> flower> stem> root. The VIGS vector p TRV2-PDS was constructed by the method of virus-induced gene silencing (VIGS), and the VIGS vector p TRV2-PDS was constructed. The Agrobacterium tumefaciens was used to infect Apoderma lucidum leaves. The plant phenotypes were observed. The results showed slight yellowing of the leaves and significant downregulation of genes, and the function of Ap PDS in vivo was identified. This study firstly cloned and identified the PDS gene of Andrographis paniculata, which laid the foundation for the further study of the function of new genes in Andrographis, especially the andrographolide biosynthesis genes.