4种胡蜂前溶血肽原基因的克隆与序列比较  

Cloning and Comparative Analysis of the Venom Prepromelittin Genes from Four Wasp Species

作　　者：施婉君[1] 张素方[1] 张传溪[1] 程家安[1] 

机构地区：[1]浙江大学应用昆虫学研究所,杭州310029

出　　处：《Acta Genetica Sinica》2003年第6期555-559,共5页

基　　金：国家自然科学基金项目 (编号 :3 0 2 710 0 8)资助~~

摘　　要：从雌性亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂毒腺中抽提总RNA ,通过RT PCR方法扩增得到 4种胡蜂蜂毒前溶血肽原的cDNA ,再将扩增产物克隆到pGEM Teasyvector上。测序结果表明 :扩增得到的片段长度均为2 13bp ,系蜂毒前溶血肽原编码区的cDNA。经序列比较 ,4种胡蜂蜂毒前溶血肽原之间的氨基酸序列同源性都超过95 %。亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂各自与意大利蜜蜂蜂毒前溶血肽原氨基酸序列的同源性分别为95 8%、10 0 %、97 2 %、97 2 %。结果表明 :蜂毒前溶血肽原一级结构序列具有很高的保守性 ,尽管胡蜂和蜜蜂属于膜翅目不同的总科 。The total RNAs were isolated from the venom glands of female Polistes hebraeus,Vespula maculifrons,Vespa velutina nigrithorax and Vespa magnifica ,respectively.Using the ReverAid TM First Strand cDNA Synthesis Kit,the first cDNAs were synthesized from the total RNA templates.PCR was carried out for 30 cycles (40s at 94℃,40s at 52℃ and 1 min at 72℃),with a final extension of 10 min at 72℃.RT PCR products were examined by electrophoresis in 1 5% agarose gels with ethidium bromide staining.After purification using QIAquick PCR Purification Kit,the PCR products were ligated into pGEM T easy vector.Positive clones were identified by double digestion of Xho Ⅰ and Hin dⅢ and PCR amplification and then were sequenced.The sequencing results showed that the amplified cDNAs containing the open reading frames of pre promelittin,and their lengths were all 213 bp.The ORFs were potential to encode polypeptides of 70 amino acid residues with predicted molecular weight of 7 7 kDa,including a signal peptide of 21 residues and a promelittin of 49 residues.Comparative analysis showed that the pre promelittins from different wasp species shared more than 93% identities in nucleotide sequences and more than 95% identities in amino acid sequences with each other,respectively.The sequences of pre promelittins of Polistes hebraeus,Vespula maculifrons,Vespa velutina nigrithorax and Vespa magnifica share 94%,100%,94%,93% homologies in nucleotide sequences,and 95 8%,100%,97 2% and 97 2% identities in amino acid sequences with those of the European honey bee, Apis mellifera ,respectively.There are 67 common amino acid residues among the pre promelittins of the four wasps and two honey bee species, Apis mellifera and Apis cerana. Phylogenetic relationships based on the pre promelittin nucleotide sequences were also examined using the software DNAStar5 01.The result showed that a closer relationship lied in between Polistes hebraeus,Vespa velutina nigrithorax and Vesp

关 键 词：胡蜂 蜂毒 前溶血肽原 序列比较 

分 类 号：Q78[生物学—分子生物学]