| Monochamus alternatus Hope, is a serious pest management concern in pine stands. It transmits pine wilt disease and causes huge economic losses. Because of M.alternatus behavior is hidden, it is difficult to control M.alternatus by chemical control which causes serious pollution to the environment. Biological control and physical control were not effective. It is necessary to find an environmental and efficient measure to control M.alternatus. Specialized sensilla in the chemosensory organs sensitive olfactory system seem to be particularly important in insects for reproduction(locating mates and oviposition sites) and nutrition(detecting food sources). Terpenes volatiles were attractive to Monochamus alternatus Hope, but the mechanism of the insects how to perceive the volatiles is unclear. Odorant-binding proteins(OBPs) play an important role in the olfactory recognition. But the funcation of the OBPs in olfactory system remain unclear. Here, we coloned 9 Malt OBPs genes and analysised expression pattern of the MaltOBPs genes and measured the bingding properties of MaltOBPs with pine volatiles, and checked the reaction of Monochamus alternatus Hope to odorants. All of these establish a foundation about the function of Malt OBPs in Monochamus alternatus Hope olfactory system. The main results are as follows: 1 Sequence analysis and cloning of OBPs about M.alternatusNine OBPs genes were cloned, MaltOBP6, MaltOBP7, MaltOBP12, MaltOBP13, MaltOBP14, Malt OBP15, MaltOBP16, MaltOBP18, Malt OBP20 and all of them coded 143,128,143,136,166,133,142,124,131 amino acids respectively. The IDs in GenBank are KF977559, KF977560, KF977565, KF977566, KF977567, KF977568, KF977569, KF977571 and KF977573 separately. The blast of MaltOBP6, MaltOBP13, Malt OBP14, MaltOBP15, MaltOBP16 contained six conserved cysteine sites which belonged to Classic OBPs and MaltOBP7, MaltOBP12, MaltOBP18, MaltOBP20 showed four conserved cysteine sites which belonged to Minus-C OBPs. 2 Spatial and temporal expression patterns of MaltOBPs genesThe results of q-PCR showed that nine MaltOBP genes, including MaltOBP6, MaltOBP7, MaltOBP12, Malt OBP13, MaltOBP14, Malt OBP15, MaltOBP16, MaltOBP18, MaltOBP20 by using q-PCR were mainly expressed in the antenna and the last abdominal of M.alternatus. The expression quantity in male and female adults were consistent with the different stages, and there were no regular. While the expression of MaltOBPs genes in antenna were different from sex, age and mating status. 3 Expressed in prokaryotic, purified and analysized of the fluorescent binding properties of MaltOBP13In our research, we got the recombinant plasmids pET30a-MaltOBP13 successfully, and exoressed in E.coli BL21(DE3). PET30a-MaltOBP13 mainly expressed in inclusion bodies, we obtained purified Malt OBP13 through ni sepharose high performanc column and recombinant bovine enterokinase digested fusion protein.In acidic(pH=5.0) and neutral(pH=7.4) environment, we used fluorescence competitive binding assay and 1-NPN as a probe to test the binding characteristics of MaltOBP13 to 20 kinds of pine volatiles respectively. It suggested that the binding affinity of the Malt OBP13 in acidic environment were better than in neutral environment, and showed better binding affinities with almost all of the pine volatiles. 4 Olfactometer bioassay of M.alternatus to odorantsThe behavioral responses of M.alternatus to 7 pine volatiles which had higher binding affinities with MaltOBP13 in neutral(pH=7.4) environment were tested. Combined with the life cycle of M.alternatus adults, the insects were devided into before sexual maturity and after, male and female to measure the choice behavior of the pine volatiles by Y-tube olfactometer, there were no significant difference in our research. |
