Study On Function Analysis Of PBPs And Preliminary Screening Of Sex Pheromone Biosynthesis Enzyme From Three Stem Borers

The sugarcane bandow(Chilo venosatus Walker)and the red tail white borer(Tryporyza intacta Snellenare)is the main pests of sugarcane in Tropical and subtropical regions.The rice leaffolder(Cnaphalocrocis medinalis Guenee)is a major migratory pest which is harmful to rice and distributes in all rice producing areas of China.At present,chemical pesticides are the main method of pest management,but the abuse and residues of chemical pesticides seriously damage the ecological and soil environment,it is necessary to screen green and efficient environmentally friendly alternatives.The sex pheromone has been proved to be efficient and environmentally safe for the biological control means of pests in the field.At present,there are a few studies on the chemical communication mechanism of C.venosatus,C.medinalis and T.intacta.In this study,CvenPBP1-3,CmedPBP1-3 and TintPBP1-3 proteins were identfied and investigated from the C.venosatus,C.medinalis and T.intacta,respectively.Subsequntly,the binding ability and molecular docking sites of PBPs with sex pheromones were measured in the present study.At the same time,the pheromone glands of three stem borers were subjected to transcriptome analysis,and a large number of sex pheromone synthesis related genes were obtained from the transcriptome datas.Our experimeantal results will provide an important theoretical basis for the comparision of sex pheromone biosynthesis pathway of C.venosatus,C.medinalis and T.intacta,and provide more scientific basis for studying the interaction and co-evolution of insects and host plants.The main results of this study are as follows:(1)The full-length cDNA sequences of CvenPBP1-3,CmedPBP1-3 and TintPBP1-3 genes were amplified by RT-PCR.PBPs were expressed and purified by prokaryotic expression.The binding ability of PBPs to sex pheromone was measured by fluorescence competition assay.The results showed that the binding ability of CvenPBPs to three sex pheromones is strong.The binding ability of CvenPBP1 and CvenPBP2 to Z13-18:AC is very significant,and their Ki value is 1.01 pM and 1.57?M,respectively.The affinity of CvenPBP3 to Z13-18:OH is very significant,its Ki value is 1.87?M.CmedPBPl do not strongly bind to three sex pheromones,CmedPBP2 and CmedPBP3 is able to bind to three sex pheromones.The affinity of CmedPBP2 to Z13-18:OH is very significant,its Ki value is 0.97pM.The affinity of CmedPBP3 to Z13-18:AC is strong also,the Ki value is 1.76?M.TintPBPl and TintPBP3 have better binding ability to trans-hexadecanal,and TintPBP2 has better binding ability to cis-hexadecaldehyde.(2)The sequence identity of CvenPBPs is 50.60%,and the sequence identity of CmedPBPs is 50.20%,while the sequence identity of three TintPBPs is 35.26%,which was relatively low.CvenPBPl and CmedPBP1 sequence identity is as high as 59.39%,CvenPBP2 and CmedPBP2 sequences identity is up to 55.49%,CvenPBP3 and CmedPBP3 sequences identity is 42.18%.The phylogenetic analysis shows that PBPs of Lepidoptera is significantly divided into two groups,among which PBP1 and PBP2 are clustered and PBP3 is single,the results are consistent with PBPs multiple sequence alignment results,and consistent with most studies.(3)Homologous modeling shows that CvenPBPs and CmedPBPs have the typical structure of OBPs which have six a-helix structures.Molecular docking experiments showed that Glu40,Leu43 and Tyr41 may play an important role in the binding of CmedPBP1 to sex pheromones;Seri 11 and Ser56 may play an important role in the binding of CmedPBP2 to sex pheromones;Tyr28 and Leu43 may play an important role in the binding of CmedPBP3 to sex pheromones.Leu43 and Tyr41 may play an important role in the combination of CvenPBP1 to sex pheromone;Ser43 and Ile52 may play an important role in the combination of CvenPBP2 to sex pheromone;Leu24,Tyr28 and Seri may play an important role in the combination of CvenPBP3 to sex pheromone.(4)A total of 74,107,106 enzyme genes related to sex pheromone biosynthesis were obtained from the data of pheromone gland transcripts of C.venosatus,C.medinalis and T.intacta,including 9 major categories:acetyl coenzyme A(ACC),fatty acid synthase(FAS),fatty acyl-CoA reductase(FAR),desaturase(DES),acetyl-CoA oxidase(ACO),alcohol oxidase(AOX),aldehyde dehydrogenase(ADH),acyltransferase(ATF)and carboxylesterase(CEX).(5)The results of evolutionary analysis of ACC,FAS,FAR and DES showed that CvenACC1 is clustered with CmedACC1,CvenFAS1 and CmedFAS1 are clustered into one.CvenFAR1 and CmedFAR1,CvenFAR2 and CmedFAR2,CvenFAR3 and CmedFAR3,CvenFAR4 and CmedFAR4 are clustered,respectively.CvenDES1 and CmedDES1,CvenDES2 and CmedDES2,CvenDES3 and CmedDES3 are clustered,respectively.Lepidoptera insect DESs are divided into four major branches:?9-DES(16C>18C),?9-DES(16C<18C),?11-DES,the desaturase genes of three agricultural pests mostly clustered together.