CAJ | 학술논문

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350 bp的cDNA片段,进一步将这5个片段克隆入pGEM ((R))T-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341 bp,均包含一个完整的开放阅读框和3′端未编码区的188 bp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.
종중화밀봉、의대리밀봉、대호봉、묵흉호봉화아비마봉5충자성봉독선중쾌속추제총RNA,용RT-PCR방법분별확증각득도대소약위350 bp적cDNA편단,진일보장저5개편단극륭입pGEM ((R))T-easy재체,진행측서화서렬분석.결과표명:소확증득도적5개편단장도균위341 bp,균포함일개완정적개방열독광화3′단미편마구적188 bp핵감산서렬,증실위5충봉적봉독전비대세포탈립태원적cDNA.경서렬비교,의대리밀봉、중화밀봉、대호봉、묵흉호봉화아비마봉전비대세포탈립태원핵감산서렬피차간적동원성도위90%이상.중화밀봉、대호봉、묵흉호봉화아비마봉여의대리밀봉적전비대세포탈립태원안기산서렬적동원성분별위96%、100%、94%화98%.진관대호봉화묵흉호봉여의대리밀봉속우불동적과,단타문적비대세포탈립태각완전상동,이중화밀봉여의대리밀봉속우동일개속,타문적비대세포탈립태각불상동.중화밀봉화아비마봉비대세포탈립태제5호위적안기산위정안산,체대료의대리밀봉5호위적반광안산,해위치적반광안산여19호위적반광안산조성의대리밀봉비대세포탈립태분자적일개대단백활성기중요작용적이류건.
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.