目的 克隆三叶青Tetrastigma hemsleyanum的扩展蛋白家族基因,对其进行器官表达分析,并初步探讨其功能。方法 根据GenBank中登录的扩展蛋白基因保守区域序列设计1对简并引物,以三叶青球形块根cDNA为模板进行RT-PCR扩增,再结合RACE技术,获得三叶青扩展蛋白基因全长cDNA序列,命名为Th-exp。采用半定量RT-PCR和荧光定量PCR分析Th-exp基因在不同器官中的表达情况。结果 克隆获得扩展蛋白家族基因,共782 bp,其中包含630 bp开放阅读框,编码209个氨基酸(GenBank登陆号KP693606)。Th-exp编码的蛋白质具有典型的扩展蛋白结构特征,N端含有8个半胱氨酸的结构域,C端含有4个保守的色氨酸结构域,中间含有组氨酸功能域。Blast分析显示,Th-exp与葡萄、醉蝶花中扩展蛋白基因具有相似性。半定量RT-PCR和荧光定量PCR分析表明,Th-exp在三叶青叶、茎、普通细根和球形块根中均有表达,但球形块根中表达强于其他器官。结论 克隆得到三叶青扩展蛋白家族基因Th-exp全长cDNA,初步预测其参与了三叶青块根的发育过程。

Objective To clone the sequence and characterize gene expression of an expansin gene from Tetrastigma hemsleyanum, and to predict its probable function.Methods According to the conservative region sequences of expansin gene in GenBank database, one pair of degenerate primers were designed for RT-PCR.The conservative region fragment was first amplified by RT-PCR from cDNA template of T.hemsleyanum.The full-length sequence of expansin gene cDNA(named as Th-exp) was extended by RACE(rapid-amplification of cDNA ends).Th-exp expression levels in different organs were conducted by semi-quantitative RT-PCR and fluorescence quantitative PCR.Results The full-length cDNA sequences of Th-exp gene were obtained in 782 bp.Th-exp gene containsed a 630 bp open reading frame, which encoded a protein of 209 amino acids(GenBank accession number KP693606).The protein encoded by Th-exp habours a typical expansin structure, including eight cysteine domains in N terminal region, four conserved tryptophan domain in C terminal region, and histidine function domain in intermediate region.Blast alignment showed that Th-exp was similar to expansin genes of Vitis vinifera and Tarenaya hassleriana.Semi-quantitative RT-PCR and fluorescence quantitative PCR indicated that Th-exp could express in the leaves, stems, ordinary fine roots, and calabash-shaped roots, while the expression level in calabash-shaped roots was higher than those in other organs.Conclusion The expansin gene(Th-exp) is cloned from T.hemsleyanum, and it could be involved in the development of root tubers in T.hemsleyanum.

浙江省药用植物种质改良与质量控制技术重点实验室开放项目(201302)