凡纳滨对虾(Litopenaeus vannamei)Hsp70基因PCR扩增及序列分析

目的：为获得凡纳滨对虾的热休克蛋白70（Hsp70）基因并分析其基因序列．方法：根据GenBank中斑节对虾（Penaeus monodon）Hsp70基因的cDNA序列，设计引物，对经高盐法提取的凡纳滨对虾（Litopenaeus vannamei）基因组DNA，采用优化的降落PCR（Touch Downeca）程序，扩增凡纳滨对虾Hsp70基因的全长序列．结果：PCR扩增得到一条长1983bp的目的DNA片段，回收纯化该片段并测定其核酸序列．用DNAman软件分析发现，该核酸序列中不含内含子，编码区全长为1959bp；经BLASTn和BLASTx软件分析发现，该编码区核苷酸序列与斑节对虾、罗氏沼虾（Macrobrachium rosenbergii）的Hsp70基因序列的相似性分别为97％和62．2％．根据核苷酸序列所推导出的Hsp70氨基酸序列，其与斑节对虾、罗氏沼虾的相似性分别为99．9％和92．6％．结论：成功地从凡纳滨对虾基因组DNA中直接扩增出Hsp70基因的全长编码区序列。

Amplification and sequence analysis of complete Hsp70 gene of Litopenaeus vannamei

Aim:To amplify the Hsp70 gene of Litopenaeus vannamei and study the features of the sequence. Methods:According to the Hsp70 cDNA sequence of Penaeus monodon accessed by GenBank,designed a pair of primers and used the optimal Touch Down PCR program to amplify the complete Hsp70 gene from genomic DNA of Litopenaeus vannamei. The total genomic DNA was extracted via the protocol of high-salt extraction. Results:A fragment of DNA,which was (1 983) bp in size,was amplified and then sequenced. The sequence,analyzed with software DNAman,has a (1 959) bp open reading frame,encoding 652 amino acids,but no intron. With BLASTn and BLASTx from GenBank,the open reading frame of the Hsp70 gene sequence of Litopenaeus vannamei has 97% identity with the Hsp70 cDNA sequence of Penaeus monodon, and 62.2% identity with Macrobrachium rosenbergii. Under the alignment of Hsp70 amino acids deduced from DNA sequences, there is 99.9% identity between Litopenaeus vannamei and Penaeus monodon, and 92.6% identity between Litopenaeus vannamei and Macrobrachium rosenbergii. Conclusion:Directly and successfully amplified a complete Hsp70 gene from genomic DNA of Litopenaeus vannamei.