巴戟天MoTPS基因及其启动子克隆与分析

Cloning and Analysis of MoTPS and Its Promoter in Morinda officinalis How

为探究巴戟天中MoTPS基因的序列特征及其在生长素IAA处理下的表达模式。本研究利用RT-PCR获得3个巴戟天MoTPS基因的全长c DNA序列,分别命名为MoTPS1、MoTPS2和MoTPS3。基因序列分析显示,基因全长分别为2841、2126和2144 bp,开放阅读框(ORF)分别为2481、1668和1943 bp,分别编码826、555和640个氨基酸。序列分析结果显示,3个MoTPS基因编码蛋白均为亲水性的不稳定蛋白,3个基因的氨基酸序列含有萜类特有的天冬氨酸(DDXXD)富集基序及RXR保守基序,并含有萜类ClassⅠ超级家族结构域。同源比对分析显示3个MoTPS基因氨基酸序列与咖啡树匹配的TPS基因同源性均达到75%以上。系统进化树分析表明MoTPS1基因属于TPS-f亚族分支,而MoTPS2和MoTPS3基因同属于TPS-d亚族分支。启动子克隆得到序列长度为739 bp的MoTPS3基因启动子,其含有多种光响应、激素响应顺式元件,如G-box、Sp1、P-box、TGA-element等。荧光定量PCR分析表明3个MoTPS基因在巴戟天根、茎、叶中稳定表达且具有组织特异性;经生长素IAA处理后,3个MoTPS基因在根中的相对表达量均显著上调。因此研究推测,3个MoTPS基因参与巴戟天根中萜类次生代谢物合成过程且生长素IAA对MoTPS基因的表达调控发挥重要作用。

To reveal the sequence characteristics of MoTPS gene of Morinda officinalis and its expression pattern under auxin(IAA)treatment.The coding sequence of three MoTPS genes was obtained by RT-PCR.They were named separately MoTPS1,MoTPS2 and MoTPS3.Gene sequence analysis showed that the full-length sequence of three MoTPS genes were separately 2841,2126 and 2144 bp,and contained separately 2481,1668 and 1943 bp open reading frame(ORF),encoding separately 826,555 and 640 amino acids.The sequence analysis showed that proteins encoded by three MoTPS genes were hydrophilic unstable protein and amino acid sequences of three genes contained terpenoid specific aspartic acid(DDxxD)enrichment motif,RxR conservative motif,and terpenoid classⅠsuperfamily domain.Homology analysis showed that amino acid sequences of three MoTPS genes were similar to TPS gene matched in Coffea arabica,and these plants belonged to Rubiaceae.Phylogenetic tree analysis showed that MoTPS1 gene belonged to TPS-f subfamily,but MoTPS2 and MoTPS3 gene belonged to TPS-d subfamily.Among the three MoTPS genes,MoTPS3 gene was screened for promoter isolationm,the length of 5’end promoter sequence was 739 bp,including a variety of regulatory elements related to light response,stress response and hormone response,such as G-box,Sp1,P-box,TGA-element etc.Fluorescence quantitative PCR analysis showed that the expression of three MoTPS genes were stable and tissue-specific in roots,stems and leaves of M.officinalis.the relative expression levels of three MoTPS genes were significantly up-regulated after IAA treatment.Therefore,this study has speculated that three MoTPS genes are involved in the synthesis of terpenoid secondary metabolites in root of Morinda officinalis and auxin IAA was predicted to play an important role in the regulation of MoTPS gene expression.