小球藻Δ12脂肪酸去饱和酶基因的克隆与序列分析  

Cloning and sequence analysis of Δ12 fatty acid desaturase of Chlorella vulgaris

作　　者：迟晓元[1,2] 路延笃[1,3] 王明清[1,4] 卞曙光[1,5] 杨庆利[1,2] 秦松[1] 

机构地区：[1]中国科学院海洋研究所,山东青岛266071 [2]山东省花生研究所,山东青岛266100 [3]中国科学院烟台海岸带可持续发展研究所,山东烟台264003 [4]中国海洋大学,山东青岛266071 [5]南京农业大学,江苏南京210095

出　　处：《海洋科学》2009年第8期11-20,共10页Marine Sciences

基　　金：国家自然科学基金项目(30670165);中国科学院知识创新工程项目(KSCX2-YW-G-002;KZCX2-YW-209)

摘　　要：利用已报道的Δ12脂肪酸去饱和酶基因序列的保守区域,设计简并引物,通过RT-PCR的方法获得了小球藻NJ-7的内质网型Δ12脂肪酸去饱和酶基因(CvFAD2)的部分序列,然后采用RACE的方法分别克隆到5′片段和3′片段,拼接后设计特异引物扩增到全长cDNA。该基因全长为2 032 bp,ORF为1 158 bp,编码385个氨基酸,分子质量约为44 ku。根据已经得到的CvFAD2序列推导成氨基酸序列与一些已知物种的FAD2氨基酸序列相比较,同源性分别为普通小球藻(Chlorella vulgaris)75%,莱茵衣藻(Chlamydomonas reinhardtii)57%,石榴(Punica granatum)57%,麻疯树(Jatropha curcas)52%。系统发育分析表明,南极小球藻CvFAD2基因与真核微藻(衣藻和普通小球藻)的FAD2基因聚在一起,介于真菌与高等植物之间,并且真核微藻FAD2基因与高等植物的同源性更高,推测其在进化上与高等植物的亲源关系更近。A pair of degenerate primers was designed to amplify specific DNA fragment of FAD2 using cDNA of Chlorella vulgaris from the South Pole according to the homologous sequences. The middle fragment of interested cDNA was obtained by RT-PCR. Then the full length of the cDNA was isolated by 5′ RACE and 3′ RACE. The clone contains 2 032 bp nucleotides with an open reading frame （ORF） of 1 158 bp comprising 385 amino acid residues with the predicted molecular mass of 44 ku. The sequence shares a high homologue with the following FAD2 ： C. vulgaris 75%, Chlamydomonas reinhardtii 57% , Punica granatum 57%, and Jatropha curcas 52% . Phylogenetic analysis shows that the microsomal △12 desaturase genes from C. reinhardtii and C. vulgaris are clustered together with genes from fungi and higher plants while they are separated from those of marine cyanobacteria, diatoms, rhodophytes and prasinophyceae. Moreover, the microsomal △12 desaturases of eukaryotic microalgae and higher plants may arise through gene duplication events independently from a common ancestor.

关 键 词：南极小球藻 脂肪酸去饱和酶 基因克隆 序列分析 

分 类 号：Q73[生物学—分子生物学]