| The red turpentine beetle,Dendroctonus valens Le Conte,is a destructive invasive forest pest in China,which is native to North America.D.valens is considered to be a secondary pest in its native range,generally attacks only weakened or dying pines and freshly-cut stumps and logs.However,D.valens has become a primary pest in China and has caused the demise of over 10 million pines,which seriously damaged the forest ecological system.Pheromones play an important role in host location,damage and reproduction of bark beetles.Frontalin is released by female adults of D.valens,which has the dual functions of aggregation pheromone and sex pheromone,plays an important role in the process of mass attack.Pioneer female adults recruit a large number of companions to mass attack on hosts by releasing aggregation pheromone frontalin in the process of attacking,which is conducive to overcome host’s resistance.The synthetic pathway of frontalin has been studied for decades.At present,it is known that frontalin is synthesized de novo via the mevalonate pathway,but it is not clear which genes are involved in the process from geranylgeranyl diphosphate(GGPP)to frontalin.It has been reported that cytochrome P450 genes may be involved in the next steps of frontalin biosynthesis.The screening and functional verification of P450 genes in the frontalin synthesis pathway of D.valens lays a foundation for the study of frontalin biosynthesis pathway,which can provide supports for the prevention and control of the pest as well as references for the study of chemical communication system of other bark beetles.There is no reports on reference genes for D.valens.Combining the transcriptome and referring to the relevant literature of internal reference genes used in other insects,we preliminarily screened out the common reference genes: α-tubulin,actin,GAPDH,tubulin,rpL32,rpS28,rpL39,rpS26,β-actin,rpS23,rpS20 and rpL3812 as candidate genes to test their expression level between stage and tissue for transcriptional level analysis of candidate genes.q RT-RCR analysis showed that rpL39 stably expressed in the guts and heads of adults and larvae,which can be used as reference gene for D.valens.The production of frontalin is gender and tissue specific,that is,it is only produced in the midgut of feeding female adults.The adults of D.valens were treated with feeding and starvation after sex identification,then the heads,guts and antennae were dissected and frozen for transcriptome sequencing.Using genome annotated P450 genes,by transcriptome difference analysis,transcriptome co-expression network analysis,we preliminarily screened eight P450 candidate genes for frontalin biosynthesis.These candidate P450 genes are Dv.864.18,Dv.599.75,Dv.3300.1,Dv.486.3,Dv.6727.1,Dv.513.15,Dv.4209.1,Dv.471.145.Firstly,we verified the gene interference efficiency and interference duration.Compared with the control group,the gene transcription level was significantly decreased by 45.91%,67.67%,63.43%,63.24%,89.88% and 65.89% at 5 d,8 d,11 d,14 d,17 d and 20 d,respectively,the 17 d has the highest interference efficiency.According to biology that callow adults need time to harden wings,then to feed phloem in field,we determined the standard experimental procedure: we let ds RNA injected callow beetles rest on artificial diet for 10 days,then introduce them to bolts to feed for 5 days,beetles were dissected out for volatiles collection for one day,and then dissected for tissues for gene transcription level analysis.Subsequently,the candidate genes were respectively interfered,with Dv.GGPPS as positive control and EGFP as negative control.GC-MS analysis showed that,after interfering with Dv.864.18,Dv.599.75,Dv.3300.1,Dv.486.3,Dv.6727.1,Dv.513.15,Dv.4209.1,Dv.471.145,Dv.GGPPS,the average release amount of frontalin was 73.93 ng/ beetle,43.64ng/ beetle,35.02 ng/ beetle,27.30 ng/ beetle,16.62 ng/ beetle,14.24 ng/ beetle,8.59 ng/ beetle,6.63 ng/ beetle,6.54 ng/ beetle,respectively,with the negative control of 70.11 ng/ beetle.Statistical analysis showed that after interfering of Dv.GGPPS,Dv.513.15,Dv.471.145 and Dv.4029.1,the amount released of frontalin was significantly decreased,when compared with the negative control.q RT-PCR showed that the transcriptional levels of Dv.GGPPS,Dv.513.15,Dv.471.145 and Dv.4029.1 significantly decreased by 84.35%,83.51%,91.60% and 95.29%,respectively.Subsequently,we analyzed the transcriptional level of candidate genes in different tissues,and found that the expression levels of Dv.6727.1,Dv.486.3,Dv.864.18,Dv.4209.1,Dv.475.145 and Dv.GGPPS were significantly higher in gut than other tissues,but the transcriptional levels of Dv.513.15 were higher in head,Dv.599.75 and Dv.3300.1 were higher in fat body.Our results confirmed that the aggregation pheromone frontalin of D.valens was synthesized by mevalonate pathway,and Dv.513.15,Dv.471.145 and Dv.4029.1 were involved in the biosynthesis of frontalin.In order to further determine the specific steps of these three P450 genes in the frontalin biosynthesis pathway,we plan to express the P450 genes in vitro.At present,we have completed the construction of the recombinant plasmids of P450 genes.In summary,with the combination of genome data,using transcriptome difference analysis,transcriptome co-expression network analysis,RNAi,GC-MS,q RT-RCR,we determined successfully that Dv.513.15,Dv.471.145 and Dv.4029.1 were involved in the biosynthesis of frontalin,which constructed solid foundation for further study of the frontalin biosynthesis pathway in future. |
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