菊叶薯蓣DcPMK基因克隆及互作蛋白筛选  

Gene Cloning and Interaction Proteins Screening of DcPMK in Dioscorea composite

作　　者：王宏鹏[1] 李一丹 汪耀 谭晓宇 陈成彬[1] 张力鹏[1] WANG Hongpeng;LI Yidan;WANG Yao;TAN Xiaoyu;CHEN Chengbin;ZHANG Lipeng(College of life science,Nankai University,Tianjin 300071)

机构地区：[1]南开大学生命科学学院,天津300071

出　　处：《植物研究》2022年第5期855-865,共11页Bulletin of Botanical Research

摘　　要：以菊叶薯蓣为研究材料,采用RT-PCR技术克隆获得DcPMK基因,进行生物信息学、组织特异性和诱导表达分析,并构建DcPMK的诱饵载体以筛选拟南芥酵母文库中互作蛋白,为深入研究菊叶薯蓣萜类物质的合成积累提供一定理论基础。结果表明:DcPMK基因开放阅读框大小为1536 bp(GenBank登录号MZ171241),编码511个氨基酸。蛋白质序列比对分析发现DcPMK与近源种基因序列一致性达88.85%,具有1个保守的ATP结合位点Gly-X-Gly-XX-Ala。N-J进化树显示DcPMK与海枣(Phoenix dactylifera)等单子叶植物的遗传距离较近。HPLC结果显示菊叶薯蓣皂素主要在根茎中积累,qRT-PCR结果表明DcPMK基因在各组织中均有表达,在老茎中表达量最高,根茎中表达量最低;水杨酸诱导后皂素含量的变化与DcPMK表达量的变化趋势吻合:随着叶片中皂素含量提高,DcPMK上调表达。与阴性对照相比,DcPMK基因没有自激活性和细胞毒性,并筛选到27个互作明显的拟南芥基因,如生长发育相关基因AtKCR1(AT1G67730)、AtRPS9M(AT3G49080)、AtASY4(AT2G33793),非生物与生物逆境相关基因AtVDAC2(AT5G67500)、AtVDAC3(AT5G15090)、AtRH8(AT4G00660)及花色素苷积累相关基因AtPHR2(AT2G47590)等。以上结果说明DcPMK能够参与菊叶薯蓣的萜类物质合成,并通过蛋白质-蛋白质相互作用的方式广泛参与其生长发育和胁迫响应等代谢途径。To explore the synthesis path of terpenoid in Dioscorea composite and to provide theoretical reference for studying,D.composite was used as material,DcPMK gene was cloned by RT-PCR and the bioinformatic analysis,tissue-specific expression analysis and expression analysis after salicylic acid(SA)treatment were carried out respectively,and the bait vector of DcPMK was constructed to screen interacting proteins from Arabidopsis yeast library.The results showed that the open reading frame(ORF)of DcPMK was 1536 bp(GenBank accession No.MZ171241),which encoded 511 amino acids.By multiple sequence alignment,the results showed that similarity between DcPMK and PMK amino acid sequence of other plants was as high as88.85%,DcPMK obtained 1 ATP binding site Gly-X-Gly-XX-Ala.The result of phylogenetic tree showed that DcPMK was closely related to monocotyledon such as Phonenix dactylifera.HPLC analysis showed that the content of diosgenin was the highest in rhizome,qRT-PCR analysis indicated that DcPMK was constitutively expressed among flower,leaf,stem,rhizome and root,with the highest levels in old stem and the lowest in rhizome.After SA treatment,the changes of diosgenin content were in parallel with the expression levels of DcPMK,the diosgenin content was improved as DcPMK up-regulated expression in leaf.Yeast two-hybrid results showed that DcPMK was no autoactivation and cell toxicity,then 27 significant interactional genes were screened,which were growth and development-related genes AtKCR1(AT1G67730),AtRPS9M(AT3G49080),AtASY4(AT2G33793),abiotic and biotic stress-related genes AtVDAC2(AT5G67500),AtVDAC3(AT5G15090),AtRH8(AT4G00660),anthocyanin accumulation-related gene AtPHR2(AT2G47590).The results illustrated that DcPMK could participate in the synthesis of terpenoids in D.composite,and was widely involved ingrowth and stress response pathways through protein-protein interaction.

关 键 词：菊叶薯蓣 PMK 生物信息学 表达分析 互作蛋白 

分 类 号：S567.9[农业科学—中草药栽培]