烟草SKP1基因cDNA的克隆分析及原核表达

Cloning and Analysis of the Full Length cDNA of SKP1 Gene from Nicotiana tabacum var. samsun NN and Its Expression in E. coli

应用DDRT-PCR法获得可被壳寡糖诱导的枯斑三生烟SKP1基因cDNA的3′端序列,通过5′RACE扩增该基因cDNA的5′端,进而扩增此基因cDNA的全长,通过同源性比较分析表明,与本塞姆氏烟草的SKP1基因同源性达81%,其cDNA全长653bp(GenBank登录号:AY702087),其中编码区位于73～540bp,编码一条155个氨基酸的多肽。经预测该多肽的分子量为17527.78Da,等电点为4.57,定位于细胞质,功能结构域分析结果显示在4～105位氨基酸有一明显的Skp1结构域,其E值为1.25e-52,因此推断该基因为枯斑三生烟的SKP1基因。将该基因编码区亚克隆到原核表达载体pET23b(+)上,转化大肠杆菌BL21(DE3),表达出与预测分子量一致的蛋白。为进一步研究该基因的功能奠定了基础。

To investigate the ability of oligochitosan to affect gene transcription, the messenger RNA （mRNA） differential display technique was applied to the identification and isolation of the genes whose transcription were altered in cultured Nicotiana tabacum var. samsun NN plants treated with oligochitosan. Four differential displayed cDNA were subcloned and confirmed by reverse Northern blot analysis. Then the 3＇ end cDNA of gene SKP1 was obtained by sequence, the 5＇end and full-length cDNA of gene SKP1 from Nicotiana tabacum var. samsun NN was cloned using the SMART-RACE technique. BLAST analysis revealed that the sequence of full-length cDNA showed 81% identity to that of Nicotiana benthamiana. Analysis of domains showed there was one predicted Skpl domain between 4 and 105 amino acids whose Evalue is 1.25e-52, therefore, the gene was named SKP1 of Nicotiana tabacum var. samsun NN, and submitted to GenBank （accession number： AY702087）. To express protein of the gene, cDNA of SKP1 from Nicotiana tabacum var. samsun NN was ligated with the expression vector pET23b. SDS-PAGE indicated that one fused protein was expressed efficiently. These results are useful for studying the function of SKP1.