2种蜜蜂和3种胡蜂蜂毒前肥大细胞脱粒肽原基因的克隆及同源性分析

从中华蜜蜂、意大利蜜蜂、大胡蜂、墨胸胡蜂和亚非马蜂5种雌成蜂毒腺中快速抽提总RNA，用RT-PCR方法分别扩增各得到大小约为350bp的cDNA片段，进一步将这5个片段克隆入pGEMT-easy载体，进行测序和序列分析。结果表明：所扩增得到的5个片段长度均为341bp，均包含一个完整的开放阅读框和3′端未编码区的188bp核苷酸序列，证实为5种蜂的蜂毒前肥大细胞脱粒肽原的cDNA。经序列比较，意大利蜜蜂、中华蜜蜂、大胡蜂、墨胸胡蜂和亚非马蜂前肥大细胞脱粒肽原核苷酸序列彼此间的同源性都为90％以上。中华蜜蜂、大胡蜂、墨胸胡蜂和亚非马蜂与意大利蜜蜂的前肥大细胞脱粒肽原氨基酸序列的同源性分别为96％、100％、94％和98％。尽管大胡蜂和墨胸胡蜂与意大利蜜蜂属于不同的科，但它们的肥大细胞脱粒肽却完全相同，而中华蜜蜂与意大利蜜蜂属于同一个属，它们的肥大细胞脱粒肽却不相同。中华蜜蜂和亚非马蜂肥大细胞脱粒肽第5号位的氨基酸为精氨酸，替代了意大利蜜蜂5号位的半胱氨酸，该位置的半胱氨酸与19号位的半胱氨酸组成意大利蜜蜂肥大细胞脱粒肽分子的一个对蛋白活性起重要作用的二硫键。

Cloning and Characterization Analysis of the Genes Encoding Precursor of Mast Cell Degranulating Peptide From 2 Honeybee and 3 Wasp Species

The precursors of mast cell degranulating peptide (MCDP) genes were amplified by RT-PCR from the total RNA of venom gland of two honeybee species,Apis mellifera ligustica,Apis cerana cerana,and three wasp species,Vespa magnifica,Vespa velutina nigrothorax and Polistes hebraeus,respectively.Their PCR products were ligated into pGEM ((R))T-easy vector and the nucleotide sequences were analyzed.The length of five fragments was the same,it was 341 bp containing an ORF of 153 bp coding the precursor of MCDP and 188 bp 3′ noncoding region.They have more than 90% homologues with each other in nucleotide sequences.The precursors of MCDP of A.cerana cerana,V.magnifica,V.velutina nigrothorax and P.hebraeus shared 96%,100%,94% and 98% homology with A.mellifera ligustica,respectively.The two species of wasps,V.magnifica and V.velutina nigrothorax,contained the same MCDP as A.mellifera ligustica,though they belong to different families with quite different biological properties,while A.cerana cerana contained the different MCDP in their venom as A.mellifera ligustica though they belong to the same genus.The fifth amino acid residue of MCDP in A.cerana cerana and P.hebraeus is arginine,replacing the cysteine,an important disulfide bridges element,in the position as in A.mellifera ligustica.