Cloning And Expression Of Odorant Binding Protein From Depressaria Falkovitshi Lvovsky

As a new record species found by the research group in China in 2013,Depressaria falkovitshi Lvovsky is a dominant pest in Chinese Bupleurum chinensis D C.planting area.It mainly feeds on larvae,damaging the seeds,tender leaves,stem tips and buds of B.chinensis.When the pests occur,the damage rate can reach 90%,which seriously affects the yield and quality of B.chinensis.It is difficult to control due to its strong reproductive ability,lack of natural enemies and roll leaf.The study on odorant binding protein of D.falkovitshi can help to understand the olfactory mechanism of insects,and provide guidance to study the relationship between D.falkovitshi and host plants,and may give new ideas for developing new agents such as attractants and repellents.In this study,two OBP genes from D.falkovitshi were cloned using the transcriptome data analysis by the methods of bioinformatics and molecular biology.The expression in vitro was carried out and their expression profiles were determined.The main results are as follows:Combined with adults transcriptome data analysis of D.falkovitshi,and using RT-PCR technology,two OBP genes DfalOBP1(GenBank accession number: MG882075)and DfalOBP3(GenBank accession number: MG882077)were successfully cloned.The open reading frame of gene DfalOBP1 was 420 bp,and it encoded 139 amino acids.The N-terminus contained a signal peptide consisting of 18 amino acids.The molecular weight of the predicted protein was 16.22 kDa.It had typical structural characteristics of OBP,and contained six conserved cysteine residues forming three disulfide bonds,belonging to the Classical OBP.The open reading frame of gene DfalOBP3 was 399 bp,and it encoded 132 amino acids.The N-terminus contained a signal peptide consisting of 16 amino acids.The molecular weight of the predicted protein was 14.97 kDa.It contained four conserved cysteine residues forming two disulfide bonds,belonging to Minus-C OBP.By homologous search(BLAST),the amino acid sequence identity of DfalOBP1 and Danaus plexippus DpleOBP1 and Cnaphalocrocis medinalis CmedGOBP2 was the highest,reaching 82.64%.The amino acid sequence identity of DfalOBP3 and Manduca sexta MsexABP8 was the highest,reaching 83.19%.The two genes obtained were constructed into pET-30a(+)expression vector.and induced by IPTG in E.coli.SDS-PAGE results showed that DfalOBP1 and DfalOBP3 had an obvious band between 15-25 kDa.In this study,EF1,RPL10,RPL32,RPS20 and 28 S rRNA were selected as candidate reference genes,and the expression of candidate reference genes in different developmental stages and different tissues of adults were compared by qRT-PCR.The stability of each candidate reference gene was evaluated by GeNorm,NormFinder and BestKeeper.The results showed that the most stable candidate reference genes were RPS20 and RPL10 in different developmental stages.Among the different tissues of adults,the most stable candidate reference gene was RPS20.So the study suggested that RPS20 could be used as an reference gene in quantitative experiments in different developmental stages and different adult tissues of D.falkovitshi.The gene expression levels of DfalOBP1 and DfalOBP3 in different tissues of male and female adults of D.falkovitshi were detected by qRT-PCR.The results showed that the two genes were mainly expressed in the head of the adult,and the expression level in the female head was significantly higher than that in the male,and little or no expression was observed in other tissues.The results suggested that DfalOBP1 and DfalOBP3 may play an important role in the localization of host plants and the search for spawning ground.