采用RT-PCR和RACE技术,从孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant中成功克隆出气味结合蛋白(Com OBP1)基因的全序列(Genbank登陆号:KU170686)。Com OBP1基因全长922 bp,包括5'端长为40 bp的非编码区域(UTR),及3'端长为462 bp的UTR,开放阅读框ORF长为420 bp,编码139个氨基酸,预测的分子量为15.516 k Da,等电点p I为6.57,存在AATAAA加尾信号。N-末端疏水区包含由20个氨基酸构成的信号肽,无跨膜结构,有4个保守的半胱氨酸,属于Minus-C OBP,也是在孟氏隐唇瓢虫中发现的第一个Minus-C OBP。氨基酸序列中有且仅有一个N-糖基化位点为62 NLSA,并存在2个潜在的磷酸化位点。与其它昆虫的Minus-C OBPs进行同源性比较并构建系统发育树,发现与同为鞘翅目Coleoptera昆虫的同源性较高。利用Real-time PCR、RT-PCR技术对Cmon OBP1基因在孟氏隐唇瓢虫不同发育阶段,不同组织,不同营养条件及不同食性下的表达水平进行了测定,结果显示,Cmon OBP1基因在整个发育阶段均有表达,雄性成虫期具有最高表达量,且多在成虫的头部及翅部表达。当营养条件发生变化时,表达丰度不会发生变化,当猎物由天然猎物柑橘粉蚧Planococcus citri Risso变成碗豆修尾蚜Megoura japonica Matsumura时,表达量会明显下降。该结果表明,孟氏隐唇瓢虫的不同发育阶段、不同组织及猎物种类会影响Cmon OBP1基因的表达,从而进一步影响其嗅觉行为。同时,Cmon OBP1基因可能在雄虫相关的信息素感受过程中发挥着重要作用。
The odorant binding protein gene Cmon OBP1( Genbank number: KU170686) from Cryptolaemus montrouzieri Mulsant was cloned by technology of RT-PCR and RACE. The full length was922 bp,containing a 5' UTR( untranslated region) of 40 bp and a 3' UTR of 462 bp. ORF( Open Reading Frame) was 420 bp which encoded a polypeptide of 139 amino acids with an estimated molecular weight of15. 516 k Da and p I of 6. 57. Besides,the sequence had an AATAAA tailing signal. The predicted Nterminal hydrophobic region containing 20 amino residues displayed the characteristic features of a signal peptide,and no transmembrane domain was predicted. Four conservative cysteine residues classified Cmon OBP1 as the first Minus-C OBP which was found in C. montrouzieri. The amino acid sequence had only one N-glycosylation site 62 NLSA,and two potential phosphorylation sites. Compared with other insect Minus-C OBPs,Cmon OPB1 was much identitier to the other Coleoptera. By Real-time PCR and RT-PCR methods,we determined the expression pattern of Cmon OBP1 in different developmental stages,various adult tissues,different larval food regimes and other food. Spatio-temporal expression result showed that Cmon OBP1 expressed in every stage and the male adult had the highest level. In addition,Cmon OPB1 had a higher expression level in adult head and wing than that of other adult tissues. When larval diet changed either poor or rich,the expression level wouldn' t be influenced markedly,while when the food changed from natural prey Planococcus citri Risso to Megoura japonica Matsumura,the expression level decreased significantly. The result suggested that the expression level of Cmon OBP1 gene was development,tissues and prey type dependent,indicating a distinct sensitivity in olfactory. In addition,Cmon OBP1 gene might play an important role in the process of male adult feeling related pheromone.