三叶青早期结瘤类基因ThENODL1及ThENODL2的克隆及表达分析

Molecular Cloning and Expression Analysis of ThENODL1 and ThENODL2 of Tetrastigma hemsleyanum Diels et Gilg

摘要

近年来的研究显示, 早期结瘤类(early nodulin-like, ENODL)基因在植物地下储藏器官的形成与发育中起到关键作用。课题组前期研究发现, 一些早期结瘤类基因在三叶青块根初始发育期及膨大期高丰度表达。本研究克隆了两个三叶青(Tetrastigma hemsleyanum Diels et Gilg)的早期结瘤类基因ThENODL1与ThENODL2的cDNA, 初步的生物信息学分析表明, ThENODL1的开放阅读框长度为528 bp, 编码175个氨基酸, ThENODL2的开放阅码框长度为651 bp, 编码217个氨基酸, ThENODL1与ThENODL2都具有水稻早期结瘤类基因(OsENODL1_like)保守域, 并具备早期结瘤类基因的基本分子结构特征。相对荧光定量分析显示, ThENODL1的cDNA表达量在块根中显著高于其他器官且在块根的初始发育阶段及膨大期显著提高, ThENODL2的cDNA在块根中的表达量最低且在块根发育的不同时期无显著差异。大肠杆菌原核表达体系的验证结果显示, 所克隆的ThENODL1与ThENODL2的cDNA均可表达出与预期大小相符的蛋白质。本研究为进一步明确ThENODL基因在块根发育中的功能提供了基本分子生物学参考依据。

Abstract

Recent studies have found that early nodulin-like (ENODL) genes play a vital role in the formation and development of underground storage organs in plants. Our previous study found that some ENODL genes were expressed abundantly during the root formation and tuber expansion stages of Tetrastigma hemsleya-num. In this study, two ENODL genes ThENODL1 and ThENODL2 from T. hemsleyanum were cloned. Preliminary bioinformatics analysis showed that the open reading frame (ORF) for ThENODL1 is 528 bp, enco-ding 175 amino acids, and the ORF for ThENODL2 is 651 bp, encoding 217 amino acids. Both ThENODL1 and ThENODL2 have the conserved domain of rice ENODL gene (OsENODL1_like), and possess the basic molecular structure characteristics of ENODL gene. Relative fluorescence quantitation PCR revealed that the expression level of ThENODL1 was significantly higher in tuberous roots than in other organs, and signifi-cantly increased in the initial development stage and expansion stage of root tuber, while the expression level of ThENODL2 was the lowest in tuberous roots and there was no significant difference in expression profiles at different development stages of root tuber. Furthermore, the proteins encoded by cloned cDNAs of both ThENODL1 and ThENODL2 were expressed with the expected size using E. coli prokaryotic expression sys-tem. This study lays a basic molecular biology foundation for further ascertaining the functions of ThENODL genes in tuber development.

关键词

三叶青 / 早期结瘤类基因 / 块根发育 / 原核表达

Key words

T. hemsleyanum / early nodulin-like (ENODL) gene / root tuber development / prokaryotic expression