太子参萜类合成关键酶HMGR基因的克隆及异源表达分析  

Cloning and Heterologous Expression Analysis of HMGR Gene,A Key Enzyme Gene for Terpenoid Synthesis of Pseudostellaria heterophylla

作　　者：林思妮 许雨琼 陈观水[1] LIN Si-ni;XU Yu-qiong;CHEN Guan-shui(College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学生命科学学院,福建福州350002

出　　处：《中药材》2024年第7期1612-1618,共7页Journal of Chinese Medicinal Materials

基　　金：福建省自然科学基金项目(2022J01598);福建省教育厅中青年教师科研项目(JT180138)。

摘　　要：目的:克隆太子参类异戊二烯生物合成甲羟戊酸途径的第1个关键限速酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因的全长cDNA,并进行生物信息学分析和异源表达分析。方法:基于其他植物HMGR基因的保守序列设计简并引物,以太子参块根总RNA为模板,利用RT-PCR结合RACE技术获得太子参HMGR基因的全长cDNA,并经生物信息学分析和农杆菌介导转化法进行功能验证。结果:太子参HMGR基因cDNA序列全长2137 bp,编码区长1674 bp,命名为PhHMGR,序列已提交至NCBI,登录号为MZ647966,该基因编码557个氨基酸,理论分子量约为5.93×10^(4) Da,等电点为7.49,不稳定指数为43.45,总平均亲水性为0.128,为不稳定疏水蛋白。根据氨基酸同源性分析,推测PhHMGR氨基酸同其他植物如人参、刺五加、大豆等氨基酸序列相似,包含HMGR蛋白家族的4个保守结合基序,即2个HMG-CoA结合基序、2个NADP(H)结合基序和2个跨膜结构域。PhHMGR基因在模式植物烟草中的异源表达发现,PhHMGR基因在烟草转基因植株的表达可以显著提高转基因植株的总三萜含量。结论:从太子参中成功克隆了太子参HMGR基因的全长cDNA,该基因的异源表达可显著提高转基因植株的总三萜含量,研究结果为进一步解析太子参萜类化合物代谢途径的分子机制奠定了基础。Objective:To clone the full-length cDNA encoding 3-hydroxy-3-methylglutaryl-coenzyme A(HMGR),a key rate-limit-ing enzyme of isoprenoid biosynthesis in mevalonic acid pathway in Pseudostellaria heterophylla and to analyze its bioinformatics and heterologous expression.Methods:The degenerate primers were designed based on conserved sequences of other plant HMGR genes.With the total RNA of Pseudostellaria heterophylla root tuber as template,the full-length cDNA of HMGR gene in Pseudostellaria hetero-phylla was cloned via RT-PCR and RACE techniques.The function was verified by bioinformatics analysis and Agrobacterium-mediated transformation.Results:The cDNA sequence of the HMGR gene of Pseudostellaria heterophylla had a total length of 2137 bp,acoding region length of 1674 bp,named PhHMGR,the sequence had been submitted to NCBI with the login number MZ647966.The gene encoded 557 amino acids.The theoretical molecular weight was about 5.93×10^(4) Da,isoelectric point was 7.49,instability index was 43.45,total average hydrophilicity was 0.128,indicating that it was an unstable hydrophobic protein.According to the analysis of amino acid homology,the amino acid sequences of PhHMGR were similar to those of other plants such as Panax ginseng,Acanthopanax senti-cosus and Glycine max,and contained 4 conserved binding motifs of the HMGR protein family,namely 2 HMG-CoA binding motifs,2 NADP(H)binding motifs and 2 transmembrane domains.The heterologous expression of PhHMGR gene in model plant Nicotiana taba-cum indicated that the expression of PhHMGR gene in transgenic Nicotiana tabacum plants could significantly increase the total triter-pene content in transgenic Nicotiana tabacum plants.Conclusion:The full-length cDNA of the HMGR gene has been successfully cloned from Pseudostellaria heterophylla.Heterologous expression of the gene can significantly increase the total triterpene content in transgenic plants.These results provide a basis for further analysis of the molecular mechanism of terpenoid metabolism pathway of Pseud

关 键 词：太子参 萜类化合物 HMGR 克隆 异源表达 

分 类 号：R282.71[医药卫生—中药学]