巴夫杜氏藻1,5-二磷酸核酮糖羧化酶/加氧酶小亚基基因的克隆和分析

Cloning and characterization of a gene encoding small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Dunaliella parva

1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)是光合作用中的一个关键酶,其小亚基rbcS具有调控羧化反应催化效率和影响该酶对二氧化碳/氧气底物特异性的功能。从巴夫杜氏藻中克隆rbcS基因及其5′-上游序列,并对基因及5′-上游序列进行了分析。根据已知rbcS基因保守核苷酸序列设计引物,分别克隆到1 841 bp的DNA和380 bp的cDNA序列。以此为基础,使用染色体步移(Genome walking)和cDNA末端快速扩增(RACE)技术,获得了799bp的5′端DNA序列和500 bp的3′端cDNA序列。序列分析发现,巴夫杜氏藻rbcS DNA全长为2 031 bp(不包括476 bp 5′-上游序列),cDNA全长包括570 bp开放读码框(GenBank登录号:HQ315783)和294 bp 3′端非翻译区。5′-上游序列区域存在一系列预测的顺式作用元件。该研究旨在为后继的rbcS基因的功能和表达研究、Rubisco的遗传改造奠定基础。同时,rbcS启动子因其可驱动基因高效表达而引起广泛关注,因此获得的rbcS 5′-上游序列经验证和优化后,可用于在嗜盐微藻中驱动转基因的高效表达以及完善微藻转化系统。

Ribulose-l,5-bisphosphate carboxylase/oxygenase（Rubisco）is a key enzyme in photosynthesis,and its small subunit gene rbcS can affect the carboxylation catalytic efficiency and CO2/O2 specificity of the enzyme.We cloned and described the rbcS gene and promoter from Dunaliella parva.Based on the highly conserved nucleotide regions of known rbcS,a pair of specific primers were synthesized to amplify 380 bp cDNA and 1 841 bp DNA sequence in Dunaliella parva.Then the 5′ genomic DNA and 3′ cDNA sequences were cloned by Genome walking and rapid amplification of cDNA ends（RACE）technology.Based on the sequences of the 5′-and 3′-termini,primers were synthesized to obtain the full-length genomic DNA and cDNA.The full-length rbcS cDNA contained 570 bp open reading frame（ORF）（GenBank accession no.HQ315783）,294 bp of 3′ noncoding region.The full-length rbcS genomic DNA was 2 031 bp.In addition,a 476 bp promoter was obtained.Similarity analysis revealed that the highest identity was found between D.parva and Dunaliella salina.The D.parva rbcS also showed wide similarity with other green algae.This study laid foundation for further research on the function analysis and overexpression of rbcS genes,genetic manipulation of Rubisco.In addition,high levels of expression have made the promoter of the rbcS gene an attractive candidate to drive expression of transgene.Therefore the D.parva rbcS promoter after optimization should facilitate the development of transformation system of halotolerant algae and efficient expression of transgene.