Cloning And Spatio-temporal Expression Of The Odorant Binding Protein CmonOBP1 And CmonOBP2 Genes From Cryptolaemus Montrouzieri(Coleoptera: Coccinellidae)

Cryptolaemus montrouzieri(Mulsant, 1983)is native to Australia and has been introduced to worldwide to show a bright prospect in biological control as one kind of effective predatory natural enemies. Identifying volatile odor molecule sensitively and peculiarly plays an important role in C.montrouzieri survival and reproduction. The research on OBPs of C.moutrouzieri has provided new ideals of enriching the existing ways of OBPs and preventing mealybug. With the rapid development of molecular techniques such as enzyme engineering, second-generation sequencing and genomics, there are more and more researches on micro-level studies of C.moutrouzieri like m RNA, transcriptome, gene expression and so on. The transcriptome of C.moutrouzieri has laid the foundation of the studies on C.moutrouzieri OBPs which have not been reported yet.1. Cloning and sequence analyzing of Cmon OBP1 and Cmon OBP2 genes from C.montrouzieriThe odorant binding protein gene Cmon OBP1(Gen Bank number: KU170686) and Cmon OBP2(Gen Bank number: KU170685) from C.moutrouzieri were cloned by technology of RT-PCR and RACE. About Cmon OBP1, the full length was 922 bp, containing a 5'UTR of 40 bp and a 3'UTR of 462 bp. ORF was 420 bp which encoded a polypeptide of 139 amino acids with an estimated molecular weight of 15.516 k Da and p I of 6.57. Besides, the sequence had an AATAAA tailing signal. The predicted N-terminal hydrophobic region containing 20 amino residues displayed the characteristic features of a signal peptide, and no transmembrance domain was predicted. Four conservative cysteine residues classified Cmon OBP1 as Minus-C OBP. 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 OBP1 was much identitier to Batocera horsfieldi.About Cmon OBP2, the full length was 620 bp, containing a 5'UTR of 60 bp and a 3'UTR of 116 bp. ORF was 444 bp which encoded a polypeptide of 147 amino acids with an estimated molecular weight of 16.444 k Da and p I of 4.78. Besides, the sequence had an AATAAA tailing signal. The predicted N-terminal hydrophobic region containing 16 amino residues displayed the characteristic features of a signal peptide, and no transmembrance domain was predicted. Cmon OBP2 contained six conservative cysteine residues which classified Cmon OBP2 as classic OBP. The amino acid sequence had three potential phosphorylation sites. Compared with other insect Classic OBPs, Cmon OBP2 was much identitier to the Coleoptera.2. Spatio-temporal expression of Cmon OBP1 and Cmon OBP2 genes from C.montrouzieriBy q RT-PCR and RT-PCR methods, we determined the expression pattern of Cmon OBP1 and Cmon OBP2 in different developmental stages, various adult tissues, other preys and different larval food regimes. Spatio-temporal expression result showed that Cmon OBP1 had the highest expression level in male heads and wings. Whether larval diet changed either poor or rich, the expression level wouldn't be influenced markedly, while when the food changed from Planococcus citri to Megoura japonica, the expression level decreased significantly. Meanwhile, Cmon OBP2 didn't show significant differences between groups in different developmental stages, different larval food regimes or different preys. While it showed markedly significant differences between groups in different adult tissues and it showed the highest expression level in adult heads and wings.The result suggested that Cmon OBP1 gene not only identified common odor molecule but also played an important role in the process of male C.montrouzieri feeling related pheromone, while Cmon OBP2 gene mainly identified common odor molecule, thus indicating a distinct sensitivity in olfactory.