| In our past research we have found that both the mating and oviposition of yellow peach moth(Conogethes punctiferalis)were enhanced by dim red light in scotophase.Moreover,the sensitivity of male antennae to pheromones was enhanced by Electroantennography(EAG)tests.In this study,we used prokaryotic expression and fluorescence competition binding experiments to further explore the effect of weak red light in the dark period on male antennal odorant binding protein genes of the yellow peach moth.The main research contents and results are as follows:1.The full-length transcriptome of C.punctiferalis.We obtained 39.77 Gb clean data of the full-length transcriptome of C.punctiferalis.61550 non-redundant transcript sequences and 45846 complete ORF sequences were obtained,and 51274 unigenes have been annotated in the data.17306 unigenes have annotated in the GO database totally,among them,the majority are annotated as participating in cell and cell composition,catalytic activity,metabolic processes and cellular processes.There are 18142 items annotated in the COG database,of which post-translational modification,protein turnover,general function prediction only,chaperones,carbohydrate transport and metabolism account for more;26603 items are annotated in the KEGG database,which are related to metabolic pathways.36628 in KOG,40674 in Pfam,32978 in Swiss-Prot,50239 in eggNOG and 34190 in the Nr.2.Antenna transcriptome of C.punctiferalis.Compared with the control,the expression of many genes changed in the dark period under the dim red light condition.Among the differential genes of males,7 genes related to odorant binding protein were up-regulated.Through qPCR verification,the seven odorant binding protein genes of males were also up-regulated,among which CpunGOBP2,CpunOBP2,and CpunPBP5 were significantly different.3.Gene cloning and prokaryotic expression.In this study,we cloned the full-length sequence of the three genes CpunGOBP2,CpunOBP2,CpunPBP5 successfully,which lengths are 486 bp,456 bp and 507 bp respectively and the base encodes 161 amino acids,151 amino acids,and 168 amino acids,respectively.The predicted molecular weights are 18.38 kDa(CpunGOBP2),16.55 kDa(CpunOBP2),19.17 kDa(CpunPBP5),and the molecular weights of the three are about 15 kDa after removing the tags.Using pET-32 a as the prokaryotic expression vector,the expression vector of three genes(pET-32a/CpunGOBP2,pET-32a/CpunOBP2,pET32a/CpunPBP5)was successfully constructed using homologous recombination technology.After the expression vector transferred into E.coli was induced by ITPG and sonicated,CpunOBP2 and CpunPBP5 successfully obtained soluble protein,while CpunGOBP2 did not gain soluble protein.4.Fluorescence competition binding experiment.The obtained soluble protein was purified by a Ni-NTA affinity column,and using recombinant bovine enterokinase for tags removal of the purified protein,then purified and concentrated the protein for fluorescence competition binding experiment.The experimental results show that both CpunOBP2 and CpunPBP5 can bind to two sex pheromones,(Z)-10-hexadecenal and(E)-10-hexadecenal,CpunOBP2 with binding constants of 6.86±2.50 μM and8.48±4.06 μM,respectively;CpunPBP5 with a binding constant of 1.47±0.62 μM and4.17±1.68 μM.The research results of this paper show that the dim red light treatment during scotophase of C.punctiferalis male adults can promote the up-regulation of the odorant binding protein gene in the male antennae.Moreover,after the quantitative and functional verification showed that the up-regulated expression of CpunPBP5 and CpunOBP2 can bind to the sex pheromone of female C.punctiferalis. |
