钩藤不同部位总RNA提取及UrSTR基因的克隆与表达分析  

Extraction of Total RNA and Cloning and Expression Analysis of UrSTR Gene from Uncaria rhynchophylla

作　　者：穆德添 万凌云 韦树根[2] 吴长桥 潘丽梅[2] 柳亦松[3] 田艺 付金娥[2] 唐其[1] MU De-Tian;WAN Ling-Yun;WEI Shu-Gen;WU Chang-Qiao;PAN Li-Mei;LIU Yi-Song;TIAN Yi;FU Jin-E;TANG Qi(College of Horticulture,Hunan Agricultural University,Changsha 410128,China;Guangxi Botanical Garden of Medicinal Plants,Nanning 530023,China;College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China)

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 [3]湖南农业大学动物医学院,长沙410128

出　　处：《农业生物技术学报》2022年第9期1737-1746,共10页Journal of Agricultural Biotechnology

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

摘　　要：钩藤(Uncaria rhynchophylla)为我国常用大宗中药材之一,主治头痛眩晕、阿尔茨海默和高血压等症,其有效成分主要为萜类吲哚生物碱(terpenoid indole alkaloids,TIAs)。异胡豆苷合酶(strictosidine synthase,STR)是TIAs生物合成途径中的关键酶,目前在钩藤中还未见报道。为了寻找参与钩藤TIAs生物合成的STR基因,本研究采用LC-MS法测定钩藤的根、叶片、茎钩、蒴果等不同部位中钩藤碱和异钩藤碱的含量,比较筛选4种总RNA提取方法。全长克隆了1条可能参与钩藤TIAs生物合成的候选基因UrSTR,并进行了生物信息学分析,利用qPCR分析了UrSTR在钩藤不同部位中的表达模式。结果表明:(1)通过LC-MS法测定,钩藤碱与异钩藤碱在钩藤蒴果中含量最高,根中含量最低。(2)改良CTAB法为钩藤不同部位总RNA的最佳提取方法。(3)UrSTR基因全长为1122 bp,编码一条374个氨基酸的肽链,具有跨膜螺旋结构,理论分子量为41.446 kD,理论等电点为7.549,在进化分析中发现,其与阿拉比卡咖啡(Coffea arabica)和中果咖啡(C.canephora)的STR基因聚为一类,亲缘关系较近。(4)q PCR结果显示,UrSTR基因主要在钩藤蒴果中表达,根中的表达量最低。通过分析发现,UrSTR基因的表达量与钩藤碱/异钩藤碱含量的趋势一致,提示UrSTR基因可能是钩藤STR候选基因之一,参与了TIAs的生物合成。本研究为UrSTR基因的功能验证和钩藤生物碱合成途径的解析提供了参考。Uncaria rhynchophylla is one of commonly used staple Chinese medicinal materials in China,mainly used for headache,Alzheimer’s disease and hypertension.Its active ingredients are mainly terpenoid indole alkaloids(TIAs).Strictosidine synthase(STR)is a key enzyme in the biosynthesis pathway of terpenoid indole alkaloids.So far,it has not been reported in U.rhynchophylla.In order to search for STR genes involved in the TIAs biosynthesis of U.Rhynchophylla.LC-MS method was used to measure the content of rhyncholphylline and isorhyncophylline in root,leaf,stem hook and capsule of U.rhynchophylla.Total RNA from different parts of U.rhynchophylla were compared by 4 extraction methods.UrSTR gene,which may be involved in the biosynthesis of rhyncholphylline and isorhyncophylline,was cloned and bioinformatics analysis was carried out.The expression patterns of UrSTR in different parts of U.rhynchophylla were detected by qPCR.The results were as follows:(1)The contents of rhyncholphylline and isorhyncophylline were the highest in capsules and the lowest in roots through LC-MS method.(2)The improved CTAB method was the best extraction method for different parts of U.rhynchophylla.(3)The total length of UrSTR was 1122 bp,encoding a peptide chain of 374 amino acids with a transmembrane helical structure.The theoretical molecular weight of UrSTR was 41.446 kD,and the theoretical isoelectric point was 7.549.The evolution of UrSTR gene was clustered with Coffea arabica and C.canephora,showing a close genetic relationship.(4)The results of qPCR showed that UrSTR gene was mainly expressed in capsule and the lowest expression was found in root.The analysis showed that the expression of UrSTR gene was consistent with the trend of the content of rhyncholphylline/isorhyncophylline,indicating that UrSTR gene may be one of the candidate genes for U.rhynchophylla involved in the biosynthesis of TIAs.This study provides reference for the functional verification of UrSTR gene and the analysis of U.rhynchophylla alkaloid synthesis pathway.

关 键 词：钩藤 改良CTAB法 单萜吲哚生物碱 异胡豆苷合酶 

分 类 号：S529[农业科学—作物学]