苹果山梨醇转运子cDNA的克隆及其序列分析

Cloning and Sequence Analysis of cDNA of Sorbitol Transporters from Apple Leaf

[目的]为进一步研究糖运输蛋白的功能、作用机理奠定基础。[方法]以嘎啦苹果叶片总RNA为模板,根据报道的山梨醇转运子的保守区设计引物,对山梨醇转运子cDNA片段的PCR扩增,PCR产物的克隆和测序和序列分析。[结果]经RT-PCR获得一条长度为581bp的片段,回收并进行测序,该片断编码181个氨基酸。应用B lastn和B lastx软件,通过与GenBank蛋白数据库比对分析发现其蛋白序列与MdSOT4、MdSOT6、MDSOT1 3种苹果(Malusx domestica)同源性分别为95%、92%、92%;酸樱桃(Prunus cerasus)78%;大豆(Glycinemax)77%;应用SMART软件分析,含有4个AgrB结构域,为一种跨膜蛋白结构。[结论]克隆得到的片段确定为苹果山梨醇转运子基因。

[ Objective] The study was to lay the base for the further investigation on the function and acting mechanism of sugar transporters. [ Method] With the total RNA of Malus pumila Mill cv. Royal Gala Leaf as templet, and the primer was designed according to the conserved region of sorbitol transporters that had been reported ,then the cDNA fragments of sorbitol transporters were amplified by PCR, and then PCR productions was cloned, sequenced and its sequence was analyzed. [ Result] Through RT-PCR, a fragment with length of 581bp was obtained, retrieved and sequenced and it encoded 181 amino acids. When Blastn and Blastx softwares were uses to the comparative analysis with GeneBand protein database, it was found that the homologies of its protein sequence with 3 apples （Malus x domestica） such as MdSO.T4,MdSOT6, MDSOT1 were 95% ,92% and 92% resp. , that with amarelle （Prunus cerasus） was 78% and that with soybean （Glycine max） was 77%. Analysis with SMART software showed that the fragment was a＇structure of transmembrane protein with 4 AgrB domains. [ Conclusion] The fragment obtained by clone was indeed the gene of sorbitol transporters of apple.