杜氏盐藻cDNA文库的构建及KCBP基因的克隆

Construction of cDNA library of Dunaliella salina and cloning of kinesin like calmodulin-binding protein gene

目的:构建杜氏盐藻cDNA文库并从该文库中筛选类驱动蛋白钙调素结合蛋白(KCBP)基因cDNA序列。方法:取对数生长期的杜氏盐藻细胞,提取总RNA并分离纯化mRNA,反转录合成双链cDNA,连接到pAP3neo载体上,采用电转化法将重组质粒转化大肠杆菌DH10B。计算平板上单菌落数,测定文库的滴定度和重组率。运用PCR法筛选含有KCBP基因特异片段的质粒。结果:文库滴定度为5.6×106pfu/mL,重组率在90%左右,插入片段长度在0.4～6.0kb,平均长度为1.9kb。利用PCR法从该文库中筛选到了含有新基因KCBP特异片段的单菌落,经测序与cDNA末端快速扩增(RACE)-PCR获得的杜氏盐藻KCBP基因cDNA序列相符,此序列属于kine-sin-14家族。结论:成功构建杜氏盐藻cDNA文库,并从该文库筛选出一个kinesin样cDNA序列。

Aim：To construct the cDNA library of Dunaliella salina and clone a new gene,kinesin like calmodulin-binding protein（KCBP）.Methods：Total RNA was isolated and purified from Dunaliella salina. cDNA was then synthesized from isolated mRNA and ligated into the pAP3neo predigested vector. The recombinant plasmids were transformed into Escherichia coli DH10B by electroporation. Then the titers and recombinant rates were determined by the number of single clones.KCBP was screened from the cDNA plasmid library of Dunaliella salina by PCR.Results：The titer of the cDNA library was 5.6×106 pfu/mL, the recombination rate was about 90%, the sizes of most inserted fragments ranged from 0.4 to 6.0 kb with an average size of 1.9 kb. The screened gene KCBP belonges to kinesin-14 family.Conclusion：KCBP is cloned and screened successfully from the cDNA library of Dunaliella salina constructed in this study.