2种类型膜荚黄芪主要药效成分含量及关键酶基因表达量差异研究

目的以2年生2种类型膜荚黄芪Astragalus membranceus(绿茎有毛膜荚黄芪、紫茎有毛膜荚黄芪)根、茎、叶为实验材料,探究其主要药效成分含量及其生物合成途径中关键酶基因表达量的差异,为药材膜荚黄芪资源合理利用及其优良品种选育选育提供科学的理论参考。方法使用HPLC检测膜荚黄芪根、茎、叶中黄芪甲苷与毛蕊异黄酮葡萄糖苷的含量,使用紫外分光光度计检测膜荚黄芪根、茎、叶中总皂苷含量,采用实时荧光定量PCR检测其根中皂苷生物合成过程中关键酶乙酰辅酶A乙酰基转移酶(AATC)、3-羟基-3-甲基戊二酰辅酶A合酶(HMGS)、3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)、异戊二烯焦磷酸异构酶(IDI)、法尼基焦磷酸合酶(FPS)、鲨烯合酶(SS)、鲨烯环氧酶(SE)、环阿尔庭烷合酶(CAS)基因的表达量。结果紫茎膜荚黄芪根中黄芪甲苷含量始终高于绿茎膜荚黄芪;在生殖生长期时,绿茎膜荚黄芪根中毛蕊异黄酮葡萄糖苷含量高于紫茎膜荚黄芪,而枯萎期二者变化浮动较大;绿茎膜荚黄芪根中总皂苷含量普遍高于紫茎膜荚黄芪。AACT、FPS、HMGS基因表达量与绿茎膜荚黄芪总皂苷含量呈显著相关(P0.05),IDI基因表达量与总皂苷含量呈极显著相关(P0.01),说明这4个基因在绿茎膜荚黄芪总皂苷合成中具有重要影响;HMGR基因与紫茎膜荚黄芪总皂苷呈显著相关(P0.05),其他基因与紫茎膜荚黄芪皂苷含量均有相关性,但未达到显著水平。结论探究了2种类型黄芪3种主要药效成分的动态变化和皂苷类化合物关键酶基因的表达,为黄芪皂苷类化合物合成生理生态机制的明晰、膜荚黄芪资源合理利用及优良品种选育提供了科学的理论依据。

Study on content of main active components and expression of key enzyme genes in two types of Astragalus membranaceus

Objective To explore the differences of the main effective components and the expression of key enzyme genes in the biosynthesis pathway of two types of Astragalus membranaceus (A. membranaceus with green stem, A. membranaceus with purple stem) roots, stems, and leaves, so as to provide a scientific theoretical reference for the rational utilization of resources of A. membranaceus and the selection of its excellent varieties. Methods The contents of astragaloside A and genistein glucoside in the roots, stems, and leaves of A. membranaceus were detected by HPLC, the contents of total saponins in the roots, stems and leaves of A. membranaceus were detected by UV spectrophotometer, and the expression of key enzyme genes (AATC, HMGR, CAS, FPS, HMGs, IDI, SE, and SS) in the process of saponin biosynthesis in the roots of A. membranaceus was detected by real-time fluorescence quantitative PCR. Results The content of astragaloside in A. membranaceus with purple stem root was always higher than that in A. membranaceus with green stem. During reproductive growth, the content of isoflavone glucoside in A. membranaceus with green stem root was higher than that in A. membranaceus with purple stem root, but the fluctuation of them was larger in wilting stage. The content of total saponin in A. membranaceus with green stem root was generally higher than that in A. membranaceus with purple stem. AACT, FPS, and HMGs gene expression were significantly correlated with the content of total saponins of A. membranaceus with green stem (P < 0.05), IDI gene expression was significantly correlated with the content of total saponins (P < 0.01), indicating that these four genes had an important influence on the synthesis of total saponins of A. membranaceus with green stem; HMGR gene was significantly correlated with the content of total saponins of A. membranaceus with purple stem (P < 0.05), and other genes were significantly correlated with the content of A. membranaceus with purple stem. The content of saponins was correlated, but did not reach the significant level. Conclusion The dynamic changes of three main active components of two types of A. membranaceus and the expression of key enzyme genes of A. membranaceus saponins were studied, which provided scientific theoretical basis for the clear physiological and ecological mechanism of A. membranaceus saponins synthesis, the rational utilization of A. membranaceus resources and the selection of excellent varieties.