【摘要】 根据中国明对虾(Fenneropenaeuschinensis)Hsp70的cDNA序列及日本对虾(Marsupenaeusjaponicus)Hsp70cDNA序列片段设计引物,用RT-PCR方法,从经热休克处理的日本对虾鳃总RNA中克隆到长1992bp的Hsp70cDNA序列,包括长1959bp的完整编码序列(CDS)。根据编码序列推导出相应的652个氨基酸,与其他真核生物Hsp70家族成员进行同源性比较,发现与凡纳滨对虾(Litopenaeusvannamei)、斑节对虾(PinaeusmonodonFabricius)、中国明对虾(Penaeuschinesis)的相似度分别为99.5%、99.4%、99.4%,与日本沼虾(Macrobrachiumnipponense)和罗氏沼虾(M.rosenbergii)的相似度分别为93.7%和92.9%,与虹鳟(Oncorhynchusmykiss)、原鸡(Callusgallus)、家鼠(Musmusculus)和人(Homosapiens)的相似度为89.2%、85.4%、88.3%和88.3%,表现出较高的保守性。分析表明所得到的日本对虾Hsp70是一种Dnak亚家族类型的细胞质Hsp70,拥有完整的N端ATP酶结构域(约45kDa)、底物肽结合结构域(18kDa)及C端结构域(10kDa)。以上结果说明我们所得到的序列是一条包含完整CDS的Hsp70基因序列。更多还原

【Abstract】 In order to amplify the Hsp70 gene of Marsupenaeus japonicus and study the features of the sequence,we designed a pair of primers according to the Hsp70 cDNA sequence of Fenneropenaeus chinensis(GeneBank accession No.AY748350) and the partial sequence of Marsupenaeus japonicus(GeneBank accession No.DQ663761),then used the RT-PCR program to amplify the complete Hsp70 gene.Total RNA was isolated using Trizol Reagent according to the manufactures instructions.The first strand cDNA was synthesized using reverse transcriptase Superscript.The polymerase chain reaction(PCR) was performed by the two-step method and the product was cloned to pMD-18 vector and sequenced.The sequence of cDNA,which was 1992bp in size,analyzed with software DNAstar,has a 1959bp open reading frame(ORF),encoding 652 amino acids with an ATPase domain of 386 amino acids,the substrate peptide binding domain of 60 amino acids and a C-terminus domain of 112 amino acids.The ORF of the Hsp70 gene sequence of Marsupenaeus japonicus has 94.5%,94.2% and 94.2% identity with the Hsp70 cDNA sequence of Litopenaeus vannamei,Penaeus monodon,and Fenneropenaeus chinensis,respectively.Under the alignment of Hsp70 amino acids deduced from DNA sequences,there is 99.5%,99.4% and 99.4% identity with Litopenaeus vannamei,Penaeus monodon,and Fenneropenaeus chinensis,respectively.A complete coding sequence(CDS) of Hsp70 gene was successfully amplified from M.japonicus.更多还原