中药材2024，Vol.47Issue(7)：1612-1618,7.DOI:10.13863/j.issn1001-4454.2024.07.002
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
摘要
Abstract
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 2 137 bp,acoding region length of 1 674 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 5.93×104 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 Pseudostellaria heterophylla.关键词
太子参/萜类化合物/HMGR/克隆/异源表达Key words
Pseudostellaria heterophylla(Miq.)Pax ex Pax et Hoffm./Terpenoids/HMGR/Clone/Heterologous expression分类
中医学引用本文复制引用
林思妮,许雨琼,陈观水..太子参萜类合成关键酶HMGR基因的克隆及异源表达分析[J].中药材,2024,47(7):1612-1618,7.基金项目
福建省自然科学基金项目(2022J01598) （2022J01598）
福建省教育厅中青年教师科研项目(JT180138) （JT180138）
