Expression And Analysis Of Binding Characteristics Of Sex Pheromone Gland-specific Chemosensory Protein AipsCSP2 In Agrotis Ipsilon(Hufnagel)

In the long-term evolution process,insects have developed a complex chemical information sensing mechanisms.Highly developed chemical sensing system plays an important role in the connection between insects and environmental chemical information.Chemosensory proteins(CSPs)can recognize host plant volatiles and pheromones,which play a very important role in insect chemosensory system.But the molecular mechanism of their chemosensory is not clear at present.Agrotis ipsilon(Hufnagel)belongs to Noctuidae of Lepidoptera.In recent years,it has caused more and more serious damage in China,mainly harming corn,cotton,sorghum,tobacco and vegetable seedlings.The functional analysis of the chemosensory protein is helpful to explore the molecular mechanism of chemosensory,and also to provide a theoretical basis for the use of pheromones to regulate the population density and occurrence range of the A.ipsilon.What's more,it can provide us with new methods,such as interfering with the identification of pheromones,so as to achieve effective control of it.In this paper,AipsCSP2 gene was cloned by RT-PCR.The length of the open reading frame of this gene was 360 bp,encoding 119 amino acids.There are four conserved cysteines in the amino acid sequence,which conform to the structural characteristics of typical chemosensory proteins.The expression levels in different tissues of male and female adult were studied by qRT-PCR.The results showed that it was highly expressed in the sex pheromone gland of females and the accessory glands of males,but not in other parts.The spatiotemporal expression profile showed that the expression level of this protein was the highest in the sex pheromone gland on the day of female insect emergence,and the expression level was significantly different from that in the days after emergence.The expression vector was successfully constructed and induced by prokaryotic expression technology.SDS-PAGE analysis showed that the recombinant protein was mainly expressed in the inclusion body.The AipsCSP2 recombinant protein was purified by cation exchange chromatography column,the concentration was 0.48mg/mL.We select 1-NPN as probe,the binding ability of AipsCSP2 protein to the sex pheromone components of A.ipsilon and other Lepidoptera insects,as well as the volatiles of the main host plants was determined by fluorescence competitive binding test.The results showed that AipsCSP2 protein had a very strong binding capacity to Z11-16:Ald,the precursor of Z11-16:Ac,Ki=1.48 ?M,indicating that they participated in the synthesis process of sex pheromone.In addition,the alcohol and aldehyde pheromones of other lepidopteran insects,such as tetradecanol,Z9-16:Ald,Z7-16:Ald,also have extremely strong binding capacity to AipsCSP2 protein.The binding ability of Z7-12:Ac and Z8-12:Ac was moderate.AipsCSP2 protein was weakly bound or not bound to the volatiles of the main host plants of A.ipsilon.It was speculated that AipsCSP2 protein was mainly involved in the synthesis and recognition of sex pheromone of A.ipsilon and played an important role in reproductive activities.