CODEHOP 法设计引物克隆中华绒螯蟹5-ht1和5-ht2受体基因片段及序列分析

摘要

根据已报道的中华绒螯蟹近缘物种的5－ht1 r和5－ht2 r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－ht1 r和5－ht2 r部分片段并克隆到pMD－19T载体。测序结果表明扩增到的中华绒螯蟹5－ht1 r和5－ht2 r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 ( Con-sensus Degenerate Hybrid Oligonucleotide Primers ) software was used to design degenerate primers to obtain partial cDNA fragments of 5-HT1 and 5-HT2 receptors (5-ht1r and 5-ht2r) in Chinese mitten crab (Eriocheir sinensis).The fragments of 5-ht1 r and 5-ht2 r were amplified by reverse transcription polymerase chain reaction ( RT-PCR) and then cloned into pMD-19T vector.The sequencing results revealed that 5-ht1 r and 5-ht2 r fragments contain 366 and 177 base pairs, which en-code 121 and 58 amino acids respectively .BLAST analysis indicated that the amino acid homology of 5-ht1 r was 98%sim-ilar to Macrobrachium rosenbergii and Procambarus clarkia, and the 5-ht2 r was most similar to Panulirus interruptus and Procambarus clarkia (95%) .