砂梨CONSTANS基因的克隆及原核表达分析

为了探索梨树植物的开花调控机制,本研究以砂梨(Pyrus pyrifolia Nakai)叶片为材料,采用同源序列克隆法,进行了开花调控相关基因CONSTANS的克隆,命名为PyCO,GenBank登录号为KF246572。序列分析表明,该基因包含一个1023 bp的开放阅读框,编码340个氨基酸,推测蛋白质分子量为37.81 kD,等电点为5.95。PyCO蛋白具有典型的植物CO家族的结构特征,含有2个高度保守的B-box及CCT结构域。进化树分析表明,该氨基酸序列与苹果的同源性接近93%,与碧桃、可可树、草莓等其他高等植物的CO类蛋白同源性也在70%以上。原核表达获得了具有较高表达水平的融合蛋白,分子量约为58.5 kD,为进一步探索梨树开花调控机理奠定了基础。

CONSTANS Gene Cloning from Pyrus pyrifolia Nakai and Its Prokaryotic Expression

In order to explore the regulation mechanism of flowering, the flowering gene CONSTANS gene, designated as PyCO (GenBank accession number: KF246752), was isolated from Pyrus pyrifolia Nakai by homologous cloning strategy. Sequence analysis revealed that the full-length cDNA sequence of PyCO is 1023 bp and encodes 340 amino acids, molecular weight and isoelectric point of the protein is 37.81 and 5.95 respectively. The sequence of PyCO that contains B-box and CCT domain has the typical structure characteristics of CO gene in plants. According to the phylogenetic analysis of CO genes, deduced amino acid of PyCO gene demonstrates high homology with MdCO (93%), and more than 70% homologous with that in Prunus persica, Theobroma cacao and Fragaria vesca. In the end, the fusion protein was produced at a high level by prokaryotic expression and then 58.5 kD protein was obtained. This study will provide a foundation for further discuss the flowering mechanism of pear.