杜氏盐藻psaB基因cDNA克隆及系统进化分析

根据真核生物莱茵衣藻(Chlamydomonas reinhardtii)、Chlamydomonas moewusii及Chlorella vulgaris等光系统Ⅰ反应中心蛋白psaB基因的氨基酸高度保守序列,设计一对简并引物,利用TRIzol试剂提取杜氏盐藻(Dunaliella salina)细胞的总RNA,通过RT-PCR,得到的一段长为1.8kb左右的cDNA片段。PCR产物经T-A克隆并测序以及测序结果推导成氨基酸序列进行同源性比较,表明所克隆的1815bp序列为杜氏盐藻光系统Ⅰ反应中心psaB基因的cDNA片段,GenBank收录号为AY820754。根据已经得到的psaB的核苷酸序列推导成氨基酸序列与一些已知物种的psaB氨基酸序列相比较,同源性分别为Chlamydomonas reinhardtii 92%,Chlamydomonas moewusii 91%,Chlorella vulgaris 86%,Mesostigma viride 85%,Phy -scomitrella patenssubsp.Patens 85%,Nephroselmis olivacea 84%。此外,psaB密码子偏爱性分析表明:杜氏盐藻psaB基因第三位密码子A和T的组成分别为35.7%和39.17%,而G和C分别为7.27%和17.85%,即杜氏盐藻psaB基因密码子的组成大多为NNA和NNT。根据psaB基因的特征,作者对绿藻门的50个物种的psaB基因作了进化分析,结果表明:杜氏盐藻与Haematococcaceae中的大多数种类进化地位最为接近,这为更进一步弄清杜氏盐藻的遗传背景提供了理论依据。

Phylogenetic analysis and cloning of psaB gene of Dunaliella salina

One pair of degenerate primers was designed according to conserved motifs of the psaB(A2 subunit of photosystem I) of Chlamydomonas reinhardtii,Chlamydomonas moewusii,Chlorella vulgaris,and a total RNA of Dunaliella salina was extracted with TRIzol reagent.A cDNA fragment,about 1.8 kb in length,from green algal D.salina was obtained through RT-PCR method.The resulting PCR product was cloned into T-vector and screened to determine its sequence.Homologous analysis of the deduced amino acid sequence was performed by BLAST and subsequently compared to GenBank data.The obtained cDNA sequence was 1815 bp long,which encoded 605 amino acids(GenBank accession number:AY820754).The sequence shared high homologue with the following psaB:92%for Chlamydomonas reinhardtii,91% for Chlamydomonas moewusii,86% for Chlorella vulgaris,85% for Mesostigma viride,85% for Physcomitrella patens subsp.Patens and 84% for Nephroselmis olivacea.It can be concluded that the cloned sequence is psaB cDNA fragment from D.salina.In addition,codon bias analysis shows that the content of A and T(35.7% and 39.17%,respectively)is used significantly more frequently in the third position composition than that of G and C(7.27% and 17.85%,respectively),that is to say,the codons of psaB of D.salina are mainly composed of NNA and NNT.At the same time,through phylogenetic analysis of the Chlorophyta with psaB gene,it is shown that D.salina is the nearest to the most species of Haematococcaceae in the phylogenetic relationship,which is contrary to previous opinion that D.salina is the nearest to the most species of Chlamydomonadaceae in the phylogenetic relationship.These studies will help clarify the genetic background of D.salina.