CDNA Isolation And Protein Expression Of OBP3 In Six Insects And Identification Of Proteins Associated With Olfaction In Sitobion Avenae

Olfaction plays a crucial role in host detecting and locating of both pests and their natural enemies. And EβF is one of the significant research objects for finally clarifying molecular mechanism of olfactory signal transmission in aphids and their natural enemies, as it is important in chemical communication among trophic relations of host plant, aphids and natural enemies. OBP3s are highly conserved among aphids, which indicates that this protein plays a very critical role in the process of ahpids'olfaction, what more, ApisOBP3 showed its specific affinity to EβF. So, it is believed that OBP3 involves in insect EβF perception as the EβF binding protein. Study on insect EβF perception aids in deeper insight into behaviors of insect and answering why and how the aphids are repelled but the natural enemies are attracted by EβF, and also supply theorical principle for refining'Push and Pull'strategy based on EβF for integrated pest management (IPM).We treated six species with different responses to EβF as the experimental subjects, they are,Sitobion avenae, Brevicoryne brassicae, Drepanosiphum platanoidis, Harmonia axyridis, Episyrphus balteatus, Eristalis tenax. Eight cDNAs coding for OBP3 were isolated from them; and further recombinant proteins were expressed after construction of expression vectors; olfactory response investigation on Drepanosiphum platanoidis was performed by four arms olfacometer; proteomic investigation focusing on heads of Sitobiom avenae was carried out which based on 2D electroporesis technique. The main results and our conclusions are as follow:1st. we contributed the evidences at molecular level to prove that EβF complicatedly involves in chemical communication among trophic relations of host plants, aphids and natural enemies.OBP3s were identified in all experimental subjects by molecular biological methods. And what we obtained here showed evidences at molecular level for that EβF being alarm pheromone for Sitobion avenae but kairomone for natural enemies, such as adult of Harmonia axyridis and of female Episyrphus balteatus. Whereas, OBP3s were also identified in other experimental subjects whose response to EβF were unknown or not significant, which implied that EβF probably have more physiological functions other than known; OBP3 was identified in Brevicoryne brassicae which is insensitive to EβF indicated that they can detect EβF but not consider it as alarm pheromone anymore. This consist with previous thesis that EβF from Brevicoryne brassicae is utilized to distinguish host plant and its conspecifics; we demonstrated that Drepanosiphum platanoidis has intraspecific alarm pheromone with no EβF; Drepanosiphum platanoidus is repelled by allogenetic EβF, which could be relevant with host plant detective and effective avoidance of being hunted; EβF perception by male Episyrphus balteatus and Eristalis ttenax which could help them to locate host plant (nectar source) by detecting EβF from aphids settled on the host plants or some plants which could release EβF; male Episyrphus balteatus could also increase chances to meet their mates through detecting EβF. 2nd Drepanosiphum platanoidis express two OBP3s named as DplaOBP3-1 and DplaOBP3-2.Their amino acid sequences share highly identity with only 5 residue substitutions. This showes more evidence for the opinion that insect odorant binding protein family was derived from tandem gene replication and gene mutation based on"birth-and-death evolution model".3rd obp genes become divergent before insects differentiating from orders under insecta. We deduced this through the mechanisms proposed by the birth-and-death model. The experimental subjects are from three orders such as Hemiptera ( Sitobio avenae, Brevicoryne brassicae, Drepanosiphum platanoidis), Coleptera (Harmonia axyridis), Hymenoptera (Episyrphus balteatus, Eristalis tenax ), and their OBP3s share high identities among different species.4th sexual dimorphic OBP3s are expressed by Episyrphus balteatus Male and femal Episyrphus blateatus express different OBP3, the difference between two sequences is 1 amino acid.5th 10 proteins related to olfaction or gustation were identified in Sitobion avenae based on 2D electrophoresiss They were named as Save Tol-1,Save Tol-2,Save Tol-3,Save Tol-4,Saor49a,Saor5,Saor5′,Saor7,Saveobp2b,Sagr28b.