| Mythimna separata(Lepidptera: Noctuidae)harms mainly three major food crops-maize,wheat and rice.It is a critical area of concern in pest control.M.separata is a typical long-distance migration pest,and its larvae have a population density-dependent behavior,which further affect the differentiation of the migratory behavior.Olfactory plays a vital role in the density-dependent behavioral activities of M.separata.Odorant binding proteins(OBPs)are important olfactory-related proteins that take participate in the recognition of chemical information substances in the environment through the sense of smell in insects.Therefore,the research on the odorant binding protein of M.separata has certain theoretical significance for elucidating the olfactory mechanism of insects,and it may provide basic data for the molecular mechanism of olfactory responding to population density regulation and population migration,and further may provide the theory data for control of specific agricultural pests.Based on the transcriptome data of 5th instar larvae of M.separata,12 unigenes sequences of odorant binding proteins(OBPs)in M.separata larvae were identified.Using real-time fluorescent quantitative PCR(RT-q PCR)technology,the OBPs expression patterns of two life types(solitary phase: 1 larva/jar;gregarious phase: 10larvae/jar)were established.According to the expression patterns of OBPs,OBP6 and OBP11 were selected as candidate genes,and the full-length c DNA sequences of candidate genes were cloned and analyzed using RACE-PCR and bioinformatics.Spatiotemporal expression patterns of OBP6 and OBP11 genes were established.Using plant-mediated insect RNAi approach,the effects of OBP6 and OBP11 genes on the foraging selection behavior of M.separata were investigated.1.The transcriptome database of 5th instar larvae in two life-types was constucted.High-throughput transcriptome sequencing and construction of c DNA libraries of 5th instar larvae in two life-type were performed by RNA-seq technology.The base correctness rate(Q20)of all samples was higher than 98%,and the GC content was between 51.29% and 53.04%.The sequencing results were reliable,and58 094 unigenes were obtained.Similarity searches and comparisons in Nr,Swissprot,KOG and Kegg,and a total of 30 184 unigenes were annotated.2.The identification of OBPs and their expression patterns of M.separata larvae in two life-types was established.1)Through screening and verification of the transcriptome database,12 genes related to odorant binding proteins were obtained and named OBP1-OBP12,respectively.The expression levels of OBPs were detected by RT-q PCR.The results showed that 12 OBP genes were expressed in the head and epidermis of the larva.The m RNA levels of OBP1,OBP3,OBP4,OBP6,OBP7,OBP8,OBP9 and OBP11 in the head were significantly higher than those in the epidermis(P<0.05),and the m RNA levels of OBP2,OBP5,OBP10 and OBP12 in the epidermis were significantly higher than those in the head(P<0.05).The m RNA levels of OBP1,OBP3,OBP4,OBP6,OBP7,OBP8,OBP9 and OBP11 in the head of solitary larvae were significantly higher than gregarious ones(P<0.05),and the m RNA levels of OBP2,OBP5,OBP10 and OBP12 in the head were no significant difference between solitary larvae and gregarious ones;the m RNA levels of OBP3,OBP5,OBP8,OBP10 and OBP12 in the epidermis of solitary larvae were significantly higher than gregarious ones,and the m RNA levels of OBP2 in the epidermis of gregarious larvae was significantly higher than solitary larvae(P <0.05),and there was no significant difference in the m RNA levels of OBP1,OBP4,OBP6,OBP7,OBP9,and OBP11 between solitary larvae and gregarious larvae.2)The construction of a phylogenetic tree of based on OBPs.Phylogenetic tree construction of 42 OBPs of M.separata and other 12 Lepidoptera species was carried out by using MEGA7.0 by employing Neighbor-joining.The results showed that M.separata OBPs were same as those in the other Lepidoptera insects,they were appeared in various branches and there was no clustering of OBPs.This finding indicated that 13 species may have common ancestors.3.The cloning and bioinformatics analysis of full-length c DNA sequences of OBP6 and OBP11 genes.By RACE-PCR technology,the full-length c DNA sequences of OBP6 and OBP11 genes were cloned.The 750-bp 5’-RACE and 600-bp 3’-RACE fragments of OBP6 gene are spliced with the OBP6 unigene sequence and obtained a full-length c DNA sequence of 1 028 bp.The start codon ATG is located positions211-213,the stop codon TAA is located positions 924-926,and the open reading frame is 714 bp.It encoded a total number of 237 amino acid peptide chain.The 1-19 amino acids in the peptide chain(MNNKVFVLVFLTYMSLAAAS)are the signal peptide region,and the 5-23 amino acids are the transmembrane region.The 600-bp5’-RACE and 600-bp 3’-RACE of OBP11 are spliced with OBP11 unigene sequence and obtained a full-length c DNA sequence of 912 bp.The start codon ATG is located positions 50-52,the stop codon TAA is located positions 466-468,and the open reading frame is 417 bp.It encoded a total number of 138 amino acid peptide chain.The 1-17 amino acids in the peptide chain(MKSFVVFCLVLVLVVGVYAN)are the signal peptide region,and the 2-19 amino acids are the transmembrane region.OBP6 and OBP11 both belong to small molecular proteins with relative molecular weights of 26.299 k Da and 14.978 k Darespectively.The tertiary structure of OBP6 and OBP11 predicted showed they are mainly composed of α-helix and β-sheet,and theα-helix is connected by disulfide bonds for supporting the tertiary structure of the protein.The identification of the conserved domains of OBP6 and OBP11 revealed that both proteins belong to the odorant binding protein family.The homologous sequence alignment showed that the sequence similarity between OBP6 and Athetis dissimilis OBP was 65.69%,and the sequence similarity between OBP11 and Agrotis ipsilon OBP5 was 73.17%.Therefore,we determined that the cloned OBP6 and OBP11 gene sequences of M.separata are complete gene sequences.4.The expression patterns of OBP6 and OBP11 genes.(1)The expression patterns of OBP6 and OBP11 genes in the larval mouthparts and antennae.The m RNA levels of OBP6 and OBP11 genes in the mandible,maxilla,labrum,labium,hypopharynx and antennae of 5th instar M.separata larvae were detected by RT-q PCR.The experimental results showed that OBP6 and OBP11 genes are all expressed in various tissues and the m RNA levels are different.The m RNA levels of OBP6 in the labrum were significantly higher than those in the other tissues(P <0.05),the antennae was the second,and the m RNA level of hypopharynx was the lowest;the m RNA level of OBP11 in the mandible was significantly higher than those in other tissues(P <0.05),and the labium was the second,and the m RNA level of hypopharynx was the lowest.(2)The expression of OBP6 and OBP11 genes in M.separata larvae of two life types.RT-q PCR was used to detect the m RNA levels of OBP6 and OBP11 of 4th,5th and 6th instars larvae in two life types.The results showed that the m RNA levels of OBP6 in solitary larvae at 4th,5th,and 6th instars were significantly higher than those in gregarious ones(P <0.05).The m RNA levels of OBP11 gene in solitary larvae at4 th,5th,and 6th instars were significantly higher than those in gregarious ones(P<0.05).OBP11 gene m RNA level decreased with the increase of larvae.5.The function of OBP6 and OBP11 genes were preliminarily verified by the host plant-mediated insect RNAi approach(1)Establishment of corn-mediated M.separata RNAi system.The target fragments for interference were determined according to the OBP6 and OBP11 gene sequences,and specific primers with Bam HI and Kpn I restriction enzyme sites were designed at the 5 ’end,and the fragments were cloned by PCR.Using p TRV2 as the RNA expression vector,426 bp OBP6 and 413 bp OBP11 c DNA interference fragments were ligated into p TRV2 after digestion with Bam HI and Kpn I,and obtained p TRV2-OBP6 and p TRV2-OBP11 recombinant plasmids.p TRV1 Agrobacterium tumefaciens strain GV3101 and various A.tumefaciens strain GV310 carrying different recombinant plasmids(p TRV2-OBP6,p TRV2-OBP11,and p TRV2-GFP(negative control))were mixed in a 1:1 ration as agroinfiltration liquid.The germinated maize seeds in agroinfiltration liquid were transformed by vacuum to make maize express OBP6-ds RNA,OBP11-ds RNA and GFP-ds RNA.The m RNA levels of OBP6 and OBP11 genes and the foraging selection behavior of M.separate after feeding the maize seedlings expressing OBP6-ds RNA,or OBP11-ds RNA,or GFP-ds RNA were investigated.The results showed that the m RNA levels of OBP6 and OBP11 are not significantly different between two control groups,while the m RNA levels of OBP6 and OBP11 in 4th and 5th instars larvae of the experimental group are significant down-regulation,which indicates that maize mediated RNAi have silencing system is effective.2)The role of OBP6 and OBP11 genes on the foraging selection behavior in M.separata larvae were investigated by Y-type olfactory instrument.This investigation recorded the selection behavior of maize and air odorant source.Three groups were set up including blank group,negative control group and experimental group,10 larvae of 5th instar in each group,each larva repeated selection 10 times.The results showed that blank group(selection rate of 61.8%)and negative control group(GFP-ds RNA)(selection rate of 64.8%)had significant selection behaviors on maize odorant(P<0.05).The experimental group(OBP6-ds RNA)(selection rate of 55.6%)and experimental group(OBP11-ds RNA)(selection rate of 55.9%)had no significant selection behavior to maize odorant(P> 0.05).In summary,this study screened M.separata odorant binding protein genes(OBPs)based on the high-throughput sequencing data of 5th instar larvae from two life types,and established their expression patterns and phylogenetic relationships.Using OBP6 and OBP11 as candidate genes,cloned their full-length c DNA sequence and analyzed their spatiotemporal expression patterns.Using host plant-mediated insect RNAi approach,the roles of OBP6 and OBP11 on their foraging selection behavior was investigated.According to these findings,we speculated that OBP6 and OBP11 are involved in the odorant-selective behavior of maize.Their roles maybe response to the density-dependent behavior of M.separata. |
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