杜氏盐藻叶绿体atpA基因片段的克隆及序列分析

根据莱因衣藻、卵形肾藻、普通小球藻等10种藻类的atpA全基因氨基酸高度保守序列,设计简并引物,利用PCR方法从盐藻叶绿体DNA中扩增出约400bp的片段,将该片段连接到T-vector上进行序列测定。结果表明,核苷酸长度为405bp,编码135个氨基酸。推导的氨基酸序列与莱因衣藻的同源性为92%,普通小球藻88%,Mesostigmaviride87%,卵形肾藻86%,Cyanidioschyzonmerolae85%。以所克隆的DNA片段为探针,与盐藻叶绿体基因组进行SouthernBlot杂交结果有明显的杂交信号。据此可推断本实验中所克隆的序列为杜氏盐藻叶绿体atpA基因片段。该基因序列已被GenBank收录,接受号为AY435096。

Cloning and characterization of atpA gene fragment from the chloroplast of Dunaliella salina

According to conserved motif of the homologous amino acid sequence of ten kinds of algae,a pair of degenerate primers were designed and about 400bp fragment was amplified from the chloroplast of Dunaliella salina by PCR technique. The resulting PCR products were inserted into pMD-18 T-vector and sequenced. The results showed that the nucleotide sequence was 405bp which encodes 135 amino acid,and the sequence shared high homology with atpA,with identity being 92%,88%,87%,86% and 85% to Chlamydomonas reinhardtii,Chlorella vulgaris,Mesostigma viride,Nephroselmis olivacea and Cyanidioschyzon merolae,respectively. The cloned fragment was used as a probe,which would be hybridized with chloroplast DNA of D.salina by Southern Blotting. Southern Blotting result revealed that there were obvious hybridization signals on cpDNA of D.salina. It can be concluded that the cloned sequence is atpA gene fragment from the chloroplast of D.salina. The atpA gene was accepted by GenBank(Accession number:AY435096).