PCR法扩增盐藻叶绿体atpA基因

目的：克隆杜氏盐藻叶绿体atpA基因片段。方法：把GenBank中近10种藻类的atpA全基因的氨基酸序列进行比较，设计简并引物，利用PCR方法从盐藻叶绿体DNA中扩增atpA基因片段，然后胶回收所扩片段并与T—vector相连，转化大肠杆菌JM109，挑阳性克隆鉴定，并测序，再将序列与所选藻类有关序列比较同源性。结果：从盐藻叶绿体基因组中扩增出约400bp的片段。测序结果显示此片段长405bp，推导的氨基酸序列与莱茵衣藻的同源性为920k，，普通小球藻为88％，，原始绿藻为87％，卵形肾藻为86％，Cyanidioschyzon merolae为85％。结论：本实验中所克隆的序列为杜氏盐藻的叶绿体atpA基因片段。

Amplification of atpA gene fragment from chloroplast of Dunaliella salina by PCR

Aim: To clone atpA gene fragment from the chloroplast of Dunaliella salina. Methods: According to conserved motif of the homologous amino acid sequence of about ten kinds of algae, we designed a pair of degenerate primers and amplified atpA gene fragment from the chloroplast of Dunaliella salina by PCR technique.The resulting PCR products were inserted into pMD-18 T-vector,and then transformed into E.coli JM109. Positive colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequence were performed by BLAST and subsequently compared with GenBank data. Results: The nucleotide sequences were 405 bp which encoded 135 amino acid. The sequence shared high homology with atpA, with identity 92%, 88%, 87%, 86% and 85% to Chlamydomonas reinhardtii, Chlorella vulgaris, Mesostigma viride, Nephroselmis olivacea and Cyanidioschyzon merolae, respectively. Conclusion: It can be concluded that the cloned sequence is atpA gene fragment from the chloroplast of Dunaliella salina.