钩藤管家基因筛选及生物碱合成相关基因的表达分析  

House-keeping Genes Screening and Expression Patterns Analysis of Genes Involved in Alkaloid Biosynthesis in Uncaria rhynchophylla

作　　者：穆德添 万凌云 章瑶 韦树根[2] 陆英[1] 付金娥[2] 田艺 潘丽梅[2] 唐其[1] MU De-tian;WAN Ling-yun;ZHANG Yao;WEI Shu-gen;LU Ying;FU Jin-e;TIAN Yi;PAN Li-mei;TANG Qi(College of Horticulture,Hunan Agricultural University,Changsha 410128;Guangxi Botanical Garden of Medicinal Plants,Nanning 510023)

机构地区：[1]湖南农业大学园艺学院,长沙410128 [2]广西壮族自治区药用植物园,南宁510023

出　　处：《生物技术通报》2023年第2期126-138,共13页Biotechnology Bulletin

基　　金：湖南省教育厅重点项目(21A0138);广西自然科学基金(2019GXNSFBA185026);湖南省自然科学基金项目(2022JJ30305);国家自然科学基金项目(81903752);湖南农业大学园艺学科开放课题(2021YYXK002);广西壮族自治区药用植物园基金项目(桂药基202013)。

摘　　要：为研究钩藤不同部位中最适管家基因与钩藤生物碱上游合成途径中关键酶基因的表达模式。以钩藤根、茎钩、叶片、蒴果为实验材料,通过RT-qPCR技术、GeNorm、NormFinder、Bestkeeper软件及△Ct程序、RefFinder在线网站分析了12个候选管家基因(18S、SAM、TUA、TUB、EF-1β、EF-1α、RNA L13、GAPDH、Actin6、PAL、CYP、cdc73)表达稳定性。结果表明,最适管家基因为SAM。再以SAM为管家基因,基于“基因组+转录组+代谢组”共表达分析筛选出钩藤生物碱上游合成途径中的15个重点候选相关基因(G8H、8-HGO、IS、CYP76A26、7-DLGT、7-DLH、LAMT、SLS、AS、AnPRT、IGPS、TSA、TSB、TDC、STR)进行表达分析,结果显示这些基因的表达量与含量趋势较为一致,很可能参与钩藤中TIAs的合成。This work aims to study the expression pattern of the most suitable house-keeping genes in different parts of Uncaria rhynchophylla and the key enzymes genes in the upstream synthesis pathway of U.rhynchophylla indole alkaloids.The root,stem hook,leaf and capulse from U.rhynchophylla were used as experimental materials.The expression stabilities of 12 candidate house-keeping genes(18S,SAM,TUA,TUB,EF-1β,EF-1α,RNA L13,GAPDH,Actin6,PAL,CYP,and cdc73)were evaluated by RT-qPCR,GeNorm,NormFinder,Bestkeeper,△Ct,and RefFinder.The results showed that SAM can be used as the most suitable house-keeping genes for gene expression analysis in U.rhynchophylla.Having SAM as house-keeping gene,15 key enzyme genes involved in alkaloid biosynthesis upstream pathway,i.e.,G8H,8-HGO,IS,CYP76A26,7-DLGT,7-DLH,LAMT,SLS,AS,AnPRT,IGPS,TSA,TSB,TDC,and STR were screened based on the co-expression analysis of“genome+transcriptome+metabolome”,and their expression levels were analyzed.The results showed that the expressions of these genes were consistent with the content trend,indicating that the genes are likely involved in the synthesis of terpenoid indole alkaloids.

关 键 词：钩藤 实时荧光定量PCR 管家基因 吲哚萜类生物碱 关键酶基因 表达 

分 类 号：S567.19[农业科学—中草药栽培]