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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Gen Bank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c DNA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c DNA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(Gen Bank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c DNA,初步预测其参与了三叶青块根的发育过程。
Objective To clone the expansin gene of Tetrastigma hemsleyanum and analyze the expression of Tetrastigma hemsleyanum and discuss its function. Methods A pair of degenerate primers was designed based on the conserved regions of the expansin genes registered in GenBank. RT-PCR was carried out using c DNA of Rhizoma Cyanophyta as template. Combined with RACE technique, Long c DNA sequence, named Th-exp. Semi-quantitative RT-PCR and quantitative PCR were used to analyze the expression of Th-exp gene in different organs. Results A total of 782 bp of the extended family protein gene was cloned. It contained a 630 bp open reading frame and encoded 209 amino acids (GenBank accession number KP693606). The protein encoded by Th-exp has the typical structure of exfoliated protein, the domain containing 8 cysteines at the N-terminus, the 4 conserved tryptophan domains at the C-terminus and histidine domain in the middle. Blast analysis showed that Th-exp is similar to the expanse gene in grapevine and cleomeria. Semi-quantitative RT-PCR and real-time PCR analysis showed that Th-exp was expressed in leaves, stems, common fine roots and spherical tuberous roots, but the expression in the tuberous roots was stronger than that in other organs. Conclusion The full-length c-DNA of Thy-Exp Expansion family protein was cloned and predicted to be involved in the root development of Clover.