杜氏盐藻叶绿体atpA基因片段的克隆及序列分析.docxVIP

维普资讯 维普资讯 2006年9月广 西植物 Guihaia 26 2006年9月 杜氏盐藻叶绿体atpA基因片段的克隆及序列分析 侯桂琴1，2,刘红涛1,潘卫东1,薛乐勋1，2* ,姜东亚3 （1.郑州大学细胞生物学研究室，河南郑州450052； 2.郑州大学第一附属医院，河南 郑州450052； 3.洛阳市新安县第一高级中学，河南洛阳471000 ） 摘 要；根据莱因衣藻、卵形肾藻、普通小球藻等10种藻类的以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，核苜酸长度为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% ,Mesostigma virideS7%，卵形肾藻 86% ,Cyanidioschyzon merolaeSS% o 以所克隆的 DNA 片段为 探针，与盐藻叶绿体基因组进行Southern Blot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以基因片段。该基因序列已被GenBank收录，接受号为AY435096。 关键词：杜氏盐藻；叶绿体；atpM简并引物 中图分类号；Q943.2 文献标识码：A 文章编号：1000-3142（2006）05-0474-05 Cloning and characterization of atpA gene frag-ment from the chloroplast of Dunaliella salina HOU Gui-qin1,2, LIU Hong-tao1, PAN Wei-dong1,XUE Le-xun1,2 * , JIANG Dong-ya3 (1. Laboratory for Cell Biology, Zhengzhou University, Zhengzhou 450052, China； 2. The First Affiliated Hospital,Zhengzhou University, Zhengzhou 450052, Chinaj 3. The First High School of Xinfan County, Luoyang 471000, China )Abstract: According to conserved motif of the homologous amino acid sequence of ten kinds of algae,a pair ofdegenerate primers were designed and about 400bp fragment was amplified from the chloroplast of Dunaliellasalina 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 rein- hardtii , Ch lore Ila vulgaris iMesostigma 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 cpD- NA 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 acc