| Carposina sasakii Matsumura is a fruit tree pest of Lepidoptera fruit moth family.Its hosts are mainly more than ten kinds of fruit trees such as apple,hawthorn and peach.It is one of the most serious pests threatening the healthy development of China’s apple industry.Odorant binding protein(OBP),as an important odor transport protein binding to odor molecules in insect olfactory sensing system,plays an important role in insect olfactory perception and other behaviors.In this study,five OBP genes of peach fruit moth were cloned,their molecular characteristics and expression profiles were analyzed,and the functions of three OBP genes were studied by using the techniques of protein expression and purification and protein fluorescence competition,in order to provide a theoretical basis for the development and screening of new attractants of peach fruit moth.The main results are as follows:1.The full-length sequences of five odorant binding protein genes were successfully cloned by RACE technology.Through sequence alignment and phylogenetic tree analysis,it was found that the amino acid sequences of the five odorant binding proteins were highly similar to the odorant binding proteins of other Lepidoptera insects,and could have similar functions.Among them,CsasOBP7,CsasOBP12,CsasOBP15 and CsasOBP21 belong to classic OBP family,and CsasOBP19 belongs to minus-c OBP family.Among the five proteins,CsasOBP7 and CsasOBP19 are closely related and may have similar functions.The functions of the other three CsasOBP may be different from each other.2.Fluorescence quantitative PCR revealed the expression of five CsasOBP genes in egg,larva,pupa and adult stages,as well as the expression of different tissues(antennae,head,thorax,abdomen,legs and wing)during the adults of new emerged,the mating peak of adults and 6h after mating.The expression amount of five CsasOBPs in adult stage was significantly higher than that in different stages of larva stage,and the total expression amount increased in three different stages of adult;At the peak of mating,CsasOBP7,CsasOBP15 and CsasOBP21 were specifically expressed only in male antennae;CsasOBP19 was highly expressed in female wings and male antennae at the peak of mating and 6h after mating;CsasOBP12 was abundantly expressed in males at the peak of mating and 6h after mating,and only in the abdomen of females at the peak of mating.3.The prokaryotic expression vetors CsasOBP/p ET-30 a of CsasOBP12,CsasOBP19 and CsasOBP21 were successfully constructed and the fusion protein expressed in vitro was obtained.4.The binding characteristics of three CsasOBPs to 21 volatiles include sex pheromones and host plant volatiles were determined by fluorescence competitive binding assay.The results showed that CsasOBP12 and phenylethanol(KI=17.14 μM);α-Farnesene(KI=9.2 μM);β-Laurene(KI=15.3 μM);β-Pinene(KI=7.1 μM),cis-7-19c-11-one(KI=4.6 μM),cis-7-eicosyl-11-one(KI=7.7 μM)has strong affinity.CsasOBP19 and cis-7-19c-11-one(KI=2.13 μM),cis-7-eicosyl-11-one(KI=1.7 μM)have strong combination ability.CsasOBP21 and phenylethanol(KI=11.2 μM),hexanal(KI=3.21 μM);α-Farnesene(KI=10.2 μM);β-Laurene(KI=5.1 μM)has strong binding ability and has different degrees of affinity for 17 of the 21 odor ligands,which may be the main protein for the peach fruit borer to recognize the host plant. |
