杜氏盐藻硝酸盐还原酶5’端cDNA片段的快速扩增  

Rapid amplification of 5' cDNA fragment of Dunaliella salina nitrate reductase

作　　者：谢华[1] 许培荣[1] 姜国忠[1] 吕玉民[1] 郭玉忠[1] 薛乐勋[1] 

机构地区：[1]郑州大学细胞生物学研究室,郑州450052

出　　处：《郑州大学学报（医学版）》2004年第1期20-22,共3页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家高技术研究发展 ( 863 )计划 2 0 0 2AA62 80 5 0 ;国家自然科学基金资助项目 3 0 2 70 0 3 1

摘　　要：目的 :根据已扩增的杜氏盐藻硝酸盐还原酶cDNA的部分序列 ,利用 5’RACE的方法扩增其 5’上游未知片段。方法 :根据盐藻硝酸盐还原酶cDNA已知的部分序列 ,设计一条磷酸化反转录引物、2对特异性反向PCR引物。提取盐藻细胞mRNA ,利用 5’RACE的方法反转录成cDNA ,然后利用PCR方法扩增 5’上游序列 ,产物与T载体连接后转化至大肠杆菌JM1 0 9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 :在盐藻中所获得的cDNA片段 ,核苷酸长度为 4 31bp ,编码 1 4 3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 83% ,衣藻为 6 8% ,团藻为 6 6 % ,小球藻为 6 4 %。结论Aim: To obtain nitrate reductase cDNA fragment from Dunaliella salina. Method: mRNA from Dunaliella salina was isolated and reverse-transcribed into single-stranded cDNA by means of rapid amplification of 5' cDNA ends (RACE-PCR), followed by full-length PCR amplification. The resulting PCR products were inserted into T-vector then transformed into JM109. Three 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 nucleotide sequences were obtained, of which contain 431bp coding 143 amino acid. The sequences share high homology with assimilatory nitrate reductase, with identity 83% to Dunaliella tertiolecta, 66% to Volvox carteri, 68% to Chlamydomonas reinhardtii, and 64% to Chlorella vulgaris, respectively. Conclusion: The cloned sequence is nitrate reductase cDNA fragment from Dunaliella salina.

关 键 词：杜氏盐藻 硝酸盐还原酶 CDNA RACE 

分 类 号：Q781[生物学—分子生物学]