简并引物在克隆盐藻热休克蛋白70基因家族中的应用

Application of degenerate primers in cloning hsp70 gene families of Dunaliella salina

目的 :利用简并引物扩增盐藻热休克蛋白 70 (hsp70 )家族。方法 :根据衣藻、矮牵牛花等真核生物hsp70氨基酸高度保守序列DIDLGTT ,DQGNRTTP ,PAYFNDS和ATKDAG设计 2对简并引物 ,以盐藻hsp70cDNA为模板进行巢式PCR扩增 ,产物经T -A克隆转化至JM1 0 9大肠杆菌中 ,经筛选后测序 ,将测序结果推导成氨基酸序列进行同源性分析。结果 :得到 2个分别为 372bp和 35 4bp编码 1 2 6及 1 1 8个氨基酸残基的部分cDNA片段。推导的氨基酸序列与衣藻、矮牵牛花、番茄、果蝇和酵母hsp70和hsp70b比较有高度同源性。结论 :所克隆的序列为盐藻hsp70和hsp70b部分cDNA片段。

Aim: To obtain heat shock protein (hsp) 70 families from Dunaliella salina by using degenerate primers. Method: Two pairs of degenerate primers were designed based on conserved homologous amino acid sequences of DIDLGTT,DQGNRTTP,PAYFNDS and ATKDAG, and were used to amplify cDNA fragment from heat-shock-treated Dunaliella salina by nest PCR technique. The PCR products were inserted into T-vector and then transformed into JM109. Five colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences was performed by BLAST and subsequently compared with GenBank data. Results: Five nucleotide sequences were obtained, and three of them were 372 bp encoding 126 amino acids and the other two were 354 bp encoding 118 amino acids. The sequences shared high homology with hsp70 and hsp70b of Chlamydomonas reinhardtii, Petunia, Pisumsativum, tomato, Drosophila and yeast. Conclusion: The cloned sequences are putatively hsp70 cDNA and hsp70b cDNA fragments from Dunaliella salina and it is a practical method to screen cDNA liberaries by using degenerate primers.