杜氏盐藻硝酸盐还原酶cDNA片段的克隆与分析

Cloning and analysis of nitrate reductase cDNA fragment of Dunaliella salina

目的 :获得杜氏盐藻硝酸盐还原酶cDNA片段。方法 :根据Dunaliellatertiolecta、衣藻、团藻等生物硝酸盐还原酶 (nitratereductase ,NR)高度保守序列EGWWFKP、WNVMGMM设计一对简并引物 ,以含硝酸盐培养基培养的的盐藻cDNA为模板 ,进行PCR扩增 ,产物克隆至T载体后转化大肠杆菌JM1 0 9,经筛选后测序 ,将测序结果推导成氨基酸序列 ,并与Dunaliellatertiolecta、团藻、衣藻、小球藻等进行同源性分析。结果 :在盐藻中所获得的cDNA片段 ,核苷酸长度为 381bp ,编码 1 2 7个氨基酸。推导的氨基酸序列与下列物种的硝酸盐还原酶进行同源性比较 :Dunaliellatertiolecta为 88.2 %同源 ,团藻为 78% ,衣藻为 6 7% ,小球藻为 5 9%。结论 :所克隆的序列为盐藻硝酸盐还原酶cDNA片段。

Aim: To obtain nitrate reductase cDNA fragment from Dunaliella salina. Method: A pair of degenerate primers were designed according to the homologous amino acid sequences conserved motifs of EGWWFKP, WNVMGMM and used to amplify nitrate reductase cDNA fragment from Dunaliella salina by PCR technique. The resulting PCR products were inserted into T-vector then transformed into JM109. Four colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences were performed by BLAST and subsequently compared with GenBank data. Result: Three of four nucleotide sequences were obtained, of which contain 381bp coding 127 amino acid. The sequences shared high homology with assimilatory nitrate reductase, with identity 88.2% to Dunaliella tertiolecta, 78% to Volvox carteri, 67% to Chlamydomonas reinhardtii, and 59% to Chlorella vulgaris, respectively. Conclusion: The cloned sequence is nitrate reductase cDNA fragment from Dunaliella salina.