| The corn worm（Dioryctria abietella）is an important pest that harms conifers.Due to its small size,borehole hazard and hidden life history,it is difficult to control.In recent years,the cones of Pinus koraiensis have been seriously harmed in Northeast China,and the cone damage rate is usually as high as 90%to 100%,causing huge economic losses.The chemosensory system plays a crucial role in the host location,finding food and mates,and laying eggs.Therefore,in-depth research on the functions of genes related to olfactory communication and sex pheromone biosynthesis in D.abietella will help to master the insect olfactory sense.Identify molecular mechanisms of sex pheromone communication and provide a rationale for biological control for controlling outbreaks of this pest.In this study,using the HiSeq XTen sequencing platform to sequence the second-generation transcriptomes of female moth antennae,male moth antennae and female sex pheromone glands,respectively.We also identify a series of olfactory-related genes and sex pheromone biosynthesis,transportation and degradation genes.The expression profiling of odorant-binding protein,chemosensory protein and other genes was analyzed by real-time quantitative PCR（RT-qPCR）method,and the key genes were screened for cloning,prokaryotic expression and protein function analysis.Finally,the electroantennography（EAG）and behavior of male and female adults were tested in order to find the plant volatiles that could attract or repel D.abietella.The results of this study are as follows:1.Based on the antennae transcriptome of D.abietella,we identified 132 olfactory related genes,including 15 odorant-binding proteins,18 chemosensory proteins,65 odorant receptors,5 sensory neuron membrane proteins,24 ionotropic receptors and 5 gustatory receptors.Using RT-qPCR technology to examine the tissue expression profiles of 6 DabiOBPs and 15DabiCSPs.We found that the majority of odorant-binding proteins and chemosensory proteins were specifically expressed in the antennae（except for DabiCSP8 and DabiCSP10）,indicating that these genes are likely involved in during the process of recognizing odor molecules by the antennae.In addition,the expression levels of some genes were significantly different between male and female antennae.The expression level of DabiCSP14 in female antennae was significantly higher than that in male antennae,and the expression levels of DabiPBP1,DabiOBP4 and DabiCSP6 in male antennae were significantly higher than that in female antennae,demonstrating the existence of odor perception-related gene expression and functional differences between different sexes.2.Based on the transcriptome of the female sex pheromone glands（PGs）of D.abietella,identified 139 type Ⅰ sex pheromone biosynthesis pathway related genes and 71chemoreception genes.The genes involved in the sex pheromone biosynthesis pathway include:12 acetyl-CoA carboxylases,19 fatty acid synthases,12 desaturases,47β-oxidases,7 fatty acyl-CoA reductases,10 acetyltransferases,16 very long-chain fatty acid elongases,7 short-chain dehydrogenases,8 fatty acid transport proteins and 1 acetyl-CoA-binding protein.The genes involved in sex pheromone recognition and degradation mainly include:8 odorant-binding proteins,1 chemosensory protein,4 odorant receptors,5 ionotropic receptors and 53odorant degradation genes.Tissue-specific expression results showed that four desaturases（DabiDES4-7）and one fatty acyl-CoA reductase（DabiFAR6）were highly expressed in female sex PGs,indicating that they play a crucial role in sex pheromone synthesis.In addition,we also found that most odorant-binding proteins were highly expressed in female antennae（except DabiOBP4 was specifically expressed in sex PGs）,suggesting that they may play different physiological functions.3.The recombinant proteins DabiGOBP2 and DabiCSP2 were successfully obtained by prokaryotic expression system,and RT-qPCR results showed that DabiGOBP2 was mainly expressed in the antennae of female adults,while DabiCSP2 was widely present in various tissues and developmental stages.Western blot experiments further confirmed that the expression of these proteins in the antennae was consistent with the results at the mRNA transcription level.Fluorescence competition binding assay showed that DabiGOBP2 exhibited stronger binding ability than DabiCSP2,and the two proteins both showed ligand specificity for terpenoids.Among them,DabiGOBP2 has the ability to bind to 10 odorant molecules such asα-pinene,β-pinene,ocimene,myrcene,α-terpinene,decanal,dodecanal,isobutyl benzoate,2-ethylhexyl acetate,3-methylheptane.It has strong binding ability to myrcene（8.19±0.05μM）,dodecanal（16.14±1.67μM）,isobutyl benzoate（18.80±6.05μM）,andα-pinene（21.57±0.41μM）.However,the binding spectrum of DabiCSP2 to the tested host plant volatiles is very narrow,and it only has the ability to bind to four odor molecules:α-pinene,β-pinene,myrcene,and decanal.And it has strong binding ability toβ-pinene（20.66±1.70μM）and decanal（21.96±5.56μM）.4.The EAG test of 6 kinds of host plant volatiles with strong binding ability to recombinant proteins DabiGOBP2 and DabiCSP2 showed thatα-pinene and myrcene could induce higher EAG response values in female antennae,while male antennae had a higher EAG response value with myrcene,decanal and isobutyl benzoate.The Y-tube behavior assay showed that different concentrations ofα-pinene,β-pinene,decanal and dodecanal had significant attracting effects on females,and only 10-1 and 1 mol·L-1 ofα-pinene and 10-2mol·L-1 isobutyl benzoate showed strong attraction to males,and other volatile substances had strong repelling to males.In summary,this paper excavated a large number of olfactory-related genes and sex pheromone biosynthesis related genes of D.abietella through transcriptome sequencing,and clarified the binding characteristics of key proteins DabiGOBP2 and DabiCSP2 with 26 kinds of host plant volatiles,and preliminarily screened some host plant volatiles with attracting or repelling effects on the D.abietella adults.The research results provide a molecular basis for elucidating the molecular mechanisms of its olfactory recognition and sex pheromone communication,and lay a theoretical basis for the further development of environmentally friendly insecticides. |
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