天麻二羧酸-三羧酸盐载体蛋白基因的克隆及功能初探  

Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata

作　　者：赵建豪 陈虞超 华中一 刘天睿 赵玉洋 黄璐琦 袁媛[2] ZHAO Jian-hao;CHEN Yu-chao;HUA Zhong-yi;LIU Tian-rui;ZHAO Yu-yang;HUANG Lu-qi;YUAN Yuan(School of Pharmacy,Jiangsu University,Zhenjiang 212013,China;State Key Laboratry Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Institute of Traditional Chinese Medicine Health Industry,China Academy of Chinese Medical Sciences,Nanchang 330115,China)

机构地区：[1]江苏大学药学院,江苏镇江212013 [2]中国中医科学院中药资源中心道地药材国家重点实验室培育基地,北京100700 [3]中国中医科学院中医药健康产业研究所,江西南昌330115

出　　处：《中国中药杂志》2023年第12期3140-3148,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金重大项目(81891013,81891010);中国中医科学院科技创新工程重大攻关项目(CI2021A041,CI2021B014);国家科技性基础资源调查项目(2018FY100800);中央本级重大增减支项目(2060302)。

摘　　要：基于天麻转录组数据,设计特异性引物,克隆天麻二羧酸-三羧酸载体蛋白编码基因GeDTC,并利用ExPASy、ClustalW、MEGA等软件对其进行生物信息学分析,利用马铃薯遗传转化体系,获得阳性转基因植株和微型薯,并对微型薯块茎的大小、质量、有机酸和淀粉含量等农艺性状进行了检测和分析,对GeDTC基因功能进行初步研究。结果显示,GeDTC开放阅读框(open reading frame)全长981 bp,编码326个氨基酸残基,相对分子质量为35.01 kDa。预测GeDTC蛋白的理论等电点为9.83,不稳定系数为27.88,亲水性平均指数为0.104,属于稳定的亲水性蛋白。GeDTC蛋白存在1个跨膜结构,无信号肽,定位于线粒体内膜。系统进化树显示,GeDTC与其他种类植物DTC蛋白之间具有较高的同源性,其中与铁皮石斛DcDTC(XP_020675804.1)同源性最高,为85.89%。运用双酶切方法构建GeDTC过表达载体pCambia1300-35Spro-GeDTC,并通过农杆菌介导转化法获得了马铃薯转基因植株,移栽收获的转基因株系微型薯和野生型株系相比体积更小,质量更轻,有机酸含量低,淀粉含量无显著差异。初步推测天麻GeDTC基因可能为三羧酸的外排通道并参与调控块茎的发育,为深入阐明天麻块茎形成的分子机制奠定了基础。The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G.elata.Bioinformatics analysis on GeDTC gene was carried out by using ExPASY,ClustalW,MEGA,etc.Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system.Agronomic characters,such as size,weight,organic acid content,and starch content,of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated.The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded,with a relative molecular weight of 35.01 kDa.It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83,the instability coefficient was 27.88,and the average index of hydrophilicity was O.104,which was indicative of a stable hydrophilic protein.GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria.The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species,among which GeDTC had the highest homology with DcDTC(XP_020675804.1)in Dendrobium candidum,reaching 85.89%.GeDTC overexpression vector pCambial300-35Spro-GeDTC was constructed by double digests,and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation.Compared with the wild-type plants,transgenic potato minituber harvested by transplanting had smaller size,lighter weight,lower organic acid content,and no significant difference in starch content.It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development,which lays a foundation for further elucidating the molecular mechanism of G.elata tuber development.

关 键 词：天麻 DTC基因 遗传转化 块茎发育 有机酸 

分 类 号：S567.239[农业科学—中草药栽培]