摘要
采用逆转录聚合酶链反应(RT-PCR)及cDNA末端快速扩增(RACE)技术克隆中华绒螯蟹(Eriocheirsinensis)的Hsp70基因并进行序列分析.以中华绒螯蟹卵组织cDNA为模板,克隆测序后拼接得到一条长2 429 bp的cDNA序列,序列分析表明其覆盖了完整编码区,编码649个氨基酸.经antheprot分析发现2个Hsp70基因的签名序列:IFDLGGGTFDVSIL,IVLVGGSTRIPKIQK;Dnak特征基序DLGTT-S-V;非细胞器基序:RARFEEL;核定位信号标签:KKDPSESKRALRRL;胞质Hsp70特征基序GPTIEEVD.经BLASTn和BLASTx软件分析,该编码区核苷酸序列与凡纳滨对虾(Litopenaeus vannamei)、斑节对虾(Penaeus monodon)的相似性分别为85.30%和84.89%.根据核苷酸序列所推导出的Hsp70氨基酸序列,其与斑节对虾、罗氏沼虾(Macrobrachium rosenbergii)的相似性分别为93.23%和93.40%.本研究克隆了中华绒螯蟹的Hsp70基因,为进一步深入研究锯缘青蟹的抗逆机理及其遗传改良奠定了基础.
Reverse Transcription-Polymerase Chain Reaction （RT-PCR） and rapid amplification of cDNA ends （RACE） were employed to clone the Hsp70 gene using cDNA of the ovum of Eriocheir sinensis as template. A DNA fragment of 2429 bp was cloned and sequencing analysis indicated that it carried an entire open reading frame and encoded a protein of 649 amino acid residues. By the analyse of antheprot, two signature sequences of HSP70 family-IFDLGGGTFDVSIL and IVLVGGSTRIPKIQK, Dnak sequence-DLGTT-S-V, the non-organellar motif-RARFEEL, bipartite nuclear localization signal-KKDPSESKRALRRL, and C-terminal four amino acids of Hsp70-EEVD were detected in the predicted amino acid sequence. With BLASTn and BLASTx from GenBank, the open reading frame of the Hsp70 gene sequence of Eriocheir sinensis has 85.30% identity with the Hsp70 cDNA sequence of Litopenaeus vannamei, and 84. 89% identity with Penaeus monodon. Under the alignment of Hsp70 amino acids deduced from DNA sequences, there is 93.23% identity between Eriocheir sinensis and Penaeus monodon, and 93.40% identity between Eriocheir sinensi and Macrobrachium rosenbergii. The Hsp70 gene of Eriocheir sinensis was cloned successfully.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2008年第5期516-521,共6页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金资助项目(04300664)
关键词
HSP70基因
CDNA末端快速扩增
逆转录聚合酶链反应
中华绒螯蟹
Hsp70 gene
Reverse Transcription-Polymerase Chain Reaction （RT-PCR）
rapid amplification of cDNA ends （RACE）
Eriocheir sinensis
