Cloning And Tissue Expression Of CYP6CR1 And ZnoDH From Chinese White Pine Beetle (Dendroctonus Armandi)

The Chinese white pine beetle(Dendroctonus armandi Tsai et Li),which is mainly distributed in the forest of Qinling and Bashan mountain in China,is the main stem borer harmful to Chinese white pine(Pinus armandi Franch),mainly chooses healthy P.armandi which over 30 years,and it has brings huge losses to the ecosystem of Pinus armandi forests in Qinling and Bashan mountain.It has seriously affected the healthy and sustainable development of the ecological environment in Qinling region.This study have shown that exo-brevicomin plays an important role in pheromone synthesis of bark beetle,and it is regulated by juvenile hormones and feeding induction.Therefore,it is of great value and significance to study the expression level of exo-brevicomin pathway related genes in different periods,different tissues and different treatment conditions for revealing the synthesis of D.armandi pheromone.In this study,cloning,RACE and qRT-PCR techniques were used to study the expression of(Z)-6-nonanene-2-alcohol dehydrogenase(Zno DH)and CYP6CR1 genes in different stages,different tissues and different treatments of Dendroctonus armandi.1.By homologous cloning,we obtained the full-length of the amino acid sequence of CYP6CR1 in exo-brevicomin pathway of D.armandi,which is 1769 bp.It was identified as P450 family gene by amino acid sequence similarity analysis;The sequence fragment of Zno DH gene in exo-brevicomin pathway of D.armandi was obtained,which is 566 bp?It was identified as SDR family gene by amino acid sequence similarity analysis.2.CYP6CR1 gene of D.armandi,its molecular weight is about 146.93 k Da,encodes 507 amino acids and its theoretical isoelectric point is 5.00.3.qRT-PCR analysis of CYP6CR1 and Zno DH genes of D.armandi at different developmental stages showed that there were significant differences in the expression of the 2 genes at different developmental stages(p<0.01).The expression of the 2 genes was the highest in pupal,but there was significant difference in the expression of the 2 genes at full emergence adult and invasion stage.4.qRT-PCR analysis of the expression of CYP6CR1 and Zno DH genes in different tissues of D.armandi showed that there were significant differences of the 2 genes in different tissues,male and female adults(p<0.01).The expression of CYP6CR1 in the hindgut was the highest.The expression of CYP6CR1 in fat body of female adult is higher than that of male,and the expression of Zno DH in the foregut and midgut of female adults is higher than that of male,however,in other tissues,the expression of CYP6CR1 and Zno DH was higher than that in female.5.qRT-PCR analysis of the expression of CYP6CR1 and Zno DH genes of D.armandi in different time JH ?-treated showed that the expression of CYP6CR1 and Zno DH were significantly different under juvenile hormone treatment at different times(p<0.05).the expression of 2 genes were increased with the prolongation of JH ?-treated time,and decreased gradually after reaching the maximum expression level.Except the expression of CYP6CR1 gene in male adults was higher than that in female at 36 h,the expression of CYP6CR1 gene in male adults was lower than that in female at each time.6.The expression of CYP6CR1 and Zno DH genes of D.armandi were decreased with the prolongation of feeding time,and gradually increased after reaching the lowest expression level.The expression levels of CYP6CR1 and Zno DH genes of male adults of D.armandi at different feeding time were significantly different(p<0.05),and the expression levels of male adults were higher than that of female adults at different feeding time.