CODEHOP法设计引物克隆中华绒螯蟹5-ht_1和5-ht_2受体基因片段及序列分析

CODEHOP PCR primers for cloning 5-ht_1 and 5-ht_2 receptor fragments in Eriocheir sinensis

根据已报道的中华绒螯蟹近缘物种的5-ht1r和5-ht2r氨基酸序列保守区,利用CODEHOP设计了两对简并引物,采用RT-PCR法扩增得到了中华绒螯蟹5-ht1r和5-ht2r部分片段并克隆到p MD-19T载体。测序结果表明扩增到的中华绒螯蟹5-ht1r和5-ht2r片段长分别为366 bp和177 bp,编码121和58个氨基酸。经BLASTp分析显示,中华绒螯蟹5-ht1r与罗氏沼虾(Macrobrachium rosenbergii)和克氏原螯虾(Procambarus clarkii)的5-ht1r同源性高达98%,而中华绒螯蟹5-HT2R与断沟龙虾(Panulirus interruptus)和克氏原螯虾(Procambarus clarkii)5-ht2r同源性高达95%。

According to the conserved amino acid sequences of known 5-Hydroxytryptamine receptors,CODEHOP（ Consensus Degenerate Hybrid Oligonucleotide Primers） software was used to design degenerate primers to obtain partial c DNA fragments of 5-HT1 and 5-HT2receptors（ 5-ht1 r and 5-ht2r） in Chinese mitten crab（ Eriocheir sinensis）. The fragments of5-ht1 r and 5-ht2 r were amplified by reverse transcription polymerase chain reaction（ RT-PCR） and then cloned into p MD-19 T vector. The sequencing results revealed that 5-ht1 r and 5-ht2 r fragments contain 366 and 177 base pairs,which encode 121 and 58 amino acids respectively. BLAST analysis indicated that the amino acid homology of 5-ht1 r was 98% similar to Macrobrachium rosenbergii and Procambarus clarkia,and the 5-ht2 r was most similar to Panulirus interruptus and Procambarus clarkia（ 95%）.