基于转录组测序的青风藤内青藤碱合成途径分析  

Analysis of sinomenine synthesis pathway based on transcriptome sequencing in Sinomenii Caulis

作　　者：曾茜垚 乔克威 李雨嫣 周喜新[1] 杨华[1,2,3] 熊兴耀 ZENG Xi-yao;QIAO Ke-wei;LI Yu-yan;ZHOU Xi-xin;YANG Hua;XIONG Xing-yao(College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha 410128,China;Hunan Zhengqin Pharmaceutical Group Co.,Ltd.,Huaihua 410000,China;Horticulture and Landscape College of Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学生物科学技术学院,湖南长沙410128 [2]湖南正清制药集团股份有限公司,湖南怀化410000 [3]湖南农业大学园艺学院,湖南长沙410128

出　　处：《中草药》2019年第22期5537-5544,共8页Chinese Traditional and Herbal Drugs

基　　金：湖南省科技重大专项(2015SK1001);湖南省科技计划项目(2015RS4059);长沙市技术及产品开发重点项目(kq1801015)

摘　　要：目的探究控制青藤碱含量的表达基因、青藤碱合成途径中的控制位点以及表达路径。方法利用HPLC法对6个种群共49株青风藤的根和茎分别进行了青藤碱含量的测定,选定青藤碱含量差异倍数大的2个种群,即陕西宝鸡与贵州遵义,分别选取其中最具代表性的青风藤植株,并取其根和茎进行转录组测序,命名为HR/LR、HS/LS。结果将clean reads进行拼接得到355201个转录本,其中包括275491个Unigene。在HR/LR和HS/LS中差异基因分别有23562和37143个。GO分析显示这些差异基因功能明显富集在天冬氨酸型肽链内切酶活性与天冬氨酸肽酶活性上,推测这些差异基因可能编码这2种酶。KEGG富集结果表明HR与LR以及HS与LS 2组中的差异基因共同参与糖类代谢、蛋白质与细胞膜结合、维生素C合成。qRT-PCR验证了异喹啉生物碱合成途径上游关键基因表达情况,发现与青藤碱的积累呈正相关。结论本研究初步了解了造成青藤碱含量差异的分子机制,为深入了解青藤碱积累规律与合成途径提供了参考。Objective In order to explore the expression of sinomenine content control genes,synthetic control sites and expression pathway.Methods In this study,high performance liquid chromatography(HPLC)was used to determine the content of sinomenine in the roots and stems of 49 Sinomenii Caulis in six populations.Two populations with large multiple differences in sinomenine content were selected,namely Shanxi Baoji and Guizhou Zunyi.The most representative of them were selected,and their roots and stems were taken for transcriptome sequencing and named as HR/LR and HS/LS.Results Sequencing results showed that 355201 transcripts were obtained by splicing clean reads,including 275491 Unigene transcripts.There were 23562 and 37143 differentially expressed genes in HR/LR and HS/LS,respectively.GO database analysis showed that the functions of these differentially expressed genes were significantly enriched in aspartic-type endopeptidase activity and aspartic-type peptidase activity,it is speculated that these two enzymes might be encoded.The results of KEGG enrichment explained that the differentially expressed genes were involved in carbohydrate metabolism,protein binding to cell membrane and vitamin C synthesis.The results of qRT-PCR verified the expression of upstream key genes of the isoquinoline alkaloid synthesis pathway and found that it was positively correlated with the accumulation of sinomenine.Conclusion This study provided a preliminary understanding of the molecular mechanism that caused the difference in sinomenine content,and provided a reference for further understanding of the accumulation rules and synthesis pathways of sinomenine.

关 键 词：青风藤 青藤碱 高效液相色谱法 转录组测序 QRT-PCR 

分 类 号：R282.12[医药卫生—中药学]