缺刻缘绿藻碳酸酐酶(CA)基因的序列克隆及特征分析  

Molecular cloning and characterization of carbonic anhydrase( CA) genes from Myrmecia incisa Reisigl H4301

作　　者：梅守华 毕燕会[1] 周志刚[1] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2015年第5期658-668,共11页Journal of Fisheries of China

基　　金：国家海洋局可再生能源专项基金(SHME2011SW02);国家自然科学基金(31172389);上海高校海洋科学一流学科建设项目

摘　　要：从缺刻缘绿藻的转录组数据库中搜索到5条编码该藻碳酸酐酶(carbonic anhydrase,CA)的contig序列,据此序列设计基因特异性引物,利用c DNA末端快速扩增(rapid amplification of c DNA ends,RACE)技术,克隆得到c DNA全长序列,分别命名为M iαCA1、M iαCA2、M iβCA1、M iβCA2和M iγCA,开放阅读框(ORF)长分别为963、1 089、1 041、738和687 bp,相应编码由320、362、346、245和228个氨基酸组成的蛋白。这些蛋白均富含疏水性氨基酸,分别占氨基酸总量的41.25%、45.31%、43.35%、42.45%和43.42%。基于缺刻缘绿藻和其他物种CA的蛋白序列所构建的Neighbor-Joining系统演化树显示,这些CA很明显地被聚类成α-、β-和γ-CA等3支。缺刻缘绿藻的2个α-CA都存在与Zn2+结合的3个His残基,2个β-CA也具有与Zn2+结合的2个Cys残基和1个His残基,但MiγCA中的Zn2+结合位点分别为Arg、His和Asn,不同于报道中的3个His。MiαCA1与莱茵衣藻的CAH3亲缘关系较近,因具有2个信号肽,它可能位于叶绿体的类囊体腔中并发挥作用。MiαCA2与莱茵衣藻的CAH1亲缘关系较近,因具有1个信号肽,可能在细胞的周质空间起着与CAH1类似的功能。M iβCA1和M iβCA2与莱茵衣藻的CAH7和CAH8亲缘关系更近,可能在细胞质中参与CO2和HCO-3之间的转化。MiγCA则与高等植物的γ-CA聚在一起,可能位于线粒体内发挥作用。由此推测,自缺刻缘绿藻所克隆的5个CA基因应在细胞的不同部位协同作用,通过参与CO2和HCO-3之间的转化以调节p H并实现CO2在细胞内转运的目的。Based upon five contigs coding for carbonic anhydrase（ CA） from a pyrosequencing transcriptome of Myrmecia incisa,gene-specific primers w ere designed for CA gene cloning from this green microalga.Five CA genes,named MiαCA1.MiαCA2,MiβCA1,MiβCA2 and MiγCA were cloned by using the technique of rapid amplification of cDNA ends. The open reading frames of the cloned full-length cDNAs w ere 963 bp,1 089 bp,1 041 bp,738 bp and 687 bp in length,encoding putative proteins composed of 320,362,346,245 and 228 amino acids,respectively. The encoded CA proteins w ere rich in hydrophobic amino acids w hich accounted for 41. 25%,45. 31%,43. 35%,42. 45% and 43. 42% of total amino acids,respectively. Neighbor-joining（ NJ） phylogenetic tree inferred from the putative proteins of CA genes indicated that these cloned CA genes w ere divided into three clades： MiαCA1and M iαCA2 in α-CA clade,M iβCA1 and M iβCA2 in β-CA one and M iγCA in γ-CA one. MiαCA1and M iαCA2 are characterized by 3conserved His residues,M iβCA1 and M iβCA2 are by 2 conserved Cys and 1 conserved His residues w hereas M iγCA is found to use Arg,His and Asn instead of the reported 3 residues of His to bind Zn2 ＋to form their catalysis centre. MiαCA1clustered w ith CAH3 from Chlamydomonas reinhardtii in the NJ phylogenetic tree,had a tw o-part leader sequence,suggesting that it might be localized and associated with PSII in the thylakoid lumen like CAH3. M iαCA2,close to CAH1 after protein alignment,had a signal peptide which could lead the mature M iαCA2 to the periplasmic space resembling CAH1 in C. reinhardtii. It was predicted that this CA could function as CAH1 to transfer HCO-3into CO2 for photosynthesis. Two β-CAs,M iβCA1and MiβCA2,w ere clustered w ith CAH7 and CAH 8 from C. reinhardtii,w hich might be involved in interconversion betw een HCO-3and CO2 in the cytoplasm of M. incisa. M iγCA,grouped w ith γ-CA from higher plants,was expected to be associated w ith the complex I of mitochondrial electron transport chain to wo

关 键 词：缺刻缘绿藻 莱茵衣藻 碳酸酐酶 克隆 CO2 

分 类 号：Q785[生物学—分子生物学] S917.3[农业科学—水产科学]