滇重楼甲羟戊酸焦磷酸脱羧酶基因的分子克隆与分析  

Molecular cloning and analysis of pyrophospomevalonate decarboxylase gene from Paris polyphylla var. yunnanensis

作　　者：杨莉 樊小莉[1] 刘琴 王芳[1,2] 刘静 徐智斌[1,2] 冯波 周强[1,2] 王涛 YANG Li;FAN Xiao-li;LIU Qin;WANG Fang;LIU Jing;XU Zhi-bin;FENG Bo;Zhou Qiang;WANG Tao(Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China;University of Chinese Academy of Sciences, Beijing 100049, China)

机构地区：[1]中国科学院成都生物研究所,四川成都610041 [2]中国科学院大学,北京100049

出　　处：《中草药》2019年第6期1435-1441,共7页Chinese Traditional and Herbal Drugs

基　　金：四川省科技计划项目(2017SZ0020)秦巴山区地道中药材多元化种植示范与推广;中国科学院STS区域重点项目(KFJ-STS-QYZD-006)秦巴山区特色高效农业关键技术集成与示范

摘　　要：目的皂苷是滇重楼的主要药效成分,获取滇重楼皂苷合成途径中关键基因(MVD)的cDNA全长序列,并对该序列进行生物信息学及表达量分析。方法通过同源克隆法得到滇重楼MVD cDNA的保守片段,利用RACE技术获得MVD c DNA全长序列,命名为PpMVD,并进行生物信息学分析,通过实时荧光定量(RT-PCR)法检测PpMVD在滇重楼不同组织中的表达情况。结果 PpMVD基因cDNA全长1 583 bp,开放阅读框(ORF)长1 269 bp,编码422个氨基酸,该蛋白质的相对分子质量为46 820,等电点为5.69,不稳定指数为45.40;滇重楼Pp MVD蛋白质二级结构中含有159个α-螺旋,占37.68%;19个β折叠,占4.50%;69个延伸链,占16.35%;175个无规卷曲,占41.47%;该蛋白没有跨膜螺旋区,且不存在信号肽,其全部氨基酸都位于细胞膜表面;该基因包含MVD1-Superfamily家族的保守结构域,属于GHMP激酶蛋白家族成员;RT-PCR结果显示,滇重楼PpMVD基因在种子、叶片、茎和块茎中均有表达,但表达量呈显著差异(P>0.05),由高到低依次为块茎>叶片>种子>茎,其中主要药用组织块茎中表达量最高,为最低表达量(茎)的3.3倍。结论首次克隆了滇重楼Pp MVD cDNA全长序列,并检测了该基因在滇重楼各组织中的表达量,为进一步研究Pp MVD基因在提高皂苷生物合成过程中的影响提供了基础,并对重楼药用资源的可持续利用提供了支持。Objective Saponin is the primary effective component in Paris polyphylla var. yunnanensis. The aims of this study are to obtain the full-length cDNA sequence of a key gene(i.e. MVD) in the saponin synthesis pathway and to analyze its bioinformatics and expression in different tissues. Methods Conserved fragments of MVD cDNA in P. polyphylla var. yunnanensis were obtained by homologous cloning, and its full-length cDNA sequence was acquired by RACE, which was temporarily named Pp MVD.Bioinformatics analysis was then conducted and the expression of PpMVD in different tissues of P. polyphylla var. yunnanensis was detected by real-time fluorescence quantitative PCR. Results The total length of PpMVD cDNA was 1 583 bp, and the length of ORF was 1 269 bp, which encoding 422 amino acids. The molecular weight of this protein was 46 820, isoelectric point was 5.69,and the instability index was 45.40. It contained 159 alpha-helix(accounted for 37.68%), 19 β-pleated sheet(accounted for 4.50%),69 extended chains(accounted for 16.35%), 175 irregular curly(accounted for 41.47%) in the secondary structure of PpMVD protein which had no transmembrane helical region and signal peptide. This gene contained a conserved structural domain of MVD1-Superfamily which belonged to GHMP kinase protein family. The qRT-PCR results showed that the expression of PpMVD gene was significantly different in seed, leaves, stems, and tubers, i.e. tubers > leaf > seed > stem. The highest expression was observed in tuber, which was about 3.3 times of that in stem. Conclusion In this study, we first cloned the full-length PpMVD cDNA sequence, and found that it was highly expressed in tuber, which was the major medicinal organ of P. polyphylla var. yunnanensis. This study laid a foundation for the further study on the effect of PpMVD gene on the saponin biosynthesis, and facilitated the efficient and sustainable utilization of P. polyphylla var. yunnanensis.

关 键 词：滇重楼 Pp MVD 基因克隆 表达分析 RACE RT-PCR 

分 类 号：R282.12[医药卫生—中药学]