| Tetranychus cinnabarinus is an important agricultural pest in China and one of the most difficult pests to control in the world.At present,the control methods of T.cinnabarinus is mainly chemical control,but the shortcomings of control are increasingly prominent,which is inconsistent with the development strategy of green pest prevention and control.From the perspective of behavior regulators,regulating the behavior of pest mites through plant-derived natural products to achieve control effects is an important means of green prevention and control.Defining the olfactory mechanism of T.cinnabarinus can provide theoretical support for the development of behavioral modulators.In this study,4 OBP-like genes and 27 NPC2 genes were found from the genome of T.cinnabarinus,and 4 OBP-like genes and 3 NPC2 genes were selected for study.The full-length sequences of 7 genes were obtained by gene cloning,and the recombinant proteins in vitro were obtained by prokaryotic expression technology and and the binding ability of the proteins to plant volatiles was studied by microscale thermophoresis technology(MST).Finally,the effects of corresponding genes on the perception of female adult mites of T.cinnabarinus relative to plant volatiles were explored by RNAi technology and Y-tube experiments.The main results are as follows :1.Gene cloning and bioinformatics analysis of OBP-like and NPC2 genes in T.cinnabarinusThe specific primers of 4 OBP-like and 3 NPC2 genes were designed by Primer 3,and the full-length sequences of TcinOBP-like 1,TcinOBP-like 2,TcinOBP-like 3,TcinOBP-like 4,TcinNPC2-1,TcinNPC2-2 and TcinNPC2-3 genes were obtained by gene cloning using the c DNA of female adults of T.cinnabarinus.The full-length sequences of 7 the genes are 414-642 bp,encoding 137-213 amino acids,and the signal peptide is 16-23 amino acids.Multiple sequence analysis showed that among the 7 genes,4 OBPlike genes had 6 conserved cysteines with TcinNPC2-2 and TcinNPC2-3 genes,while TcinNPC2-1 had 5 conserved cysteines.The results of transmembrane domain analysis showed that all 7 proteins had no transmembrane domain and belonged to the proteins in the membrane.The results of protein secondary structure prediction showed that 4 OBPlike proteins had 6-7 α-helices and 3 NPC2 proteins had 7-8 β-sheets.The results of protein tertiary structure prediction showed that the 4 OBP-like proteins were similar to OBP1 of Varroa destructor,OBP4 of Anopheles gambiae and PBP3 of Epiphyas postvittana;the 3 NPC2 proteins were similar to those of bovine NPC2,Camponotus japonipus NPC2 and Micropltis Mediator NPC2 a.Phylogenetic tree analysis showed that4 OBP-like proteins were clustered with the olfactory proteins of Varroa destructor,Rhipicephalus sanguineus,Dermacentor andersoni,Trichonephila clavata,and Pardosa pseudoannulata,with a bootstrap value of 100%.TcinNPC2-2 and TcinNPC2-3 were clustered with the NPC2 proteins of Anopheles pipiens and V.destructor,and TcinNPC2-1 alone with the NPC2 of V.destructor gather in one branch.The expression levels of 7genes in different mite states were analyzed.The results showed that the relative expression level of TcinOBP-like 1 was the highest in the egg stage and the lowest in the nymph 2 stage.There was a difference between the egg stage with the larva stage and nymph 1 stage,but there was no difference with the nymph 2 stage and the female adult stage.The relative expression level of TcinOBP-like 2 was the highest in the female adult stage and was different from that of other mite stages,while the relative expression level of TcinOBP-like 2 was lower in the larva and nymph 1 stages and there was no significant difference.The relative expression of TcinOBP-like 3 was the highest in the larval stage,and the relative expression of the two mite states was different from that of other mite states.The expression level of TcinOBP-like 4 was the lowest in the female adult stage and was significantly different from other mite stages.The expression level of TcinOBPlike 4 was higher in the larva stage,but there was no difference between the larva stage with the egg stage and the nymph stage.The expression patterns of TcinNPC2-1,TcinNPC2-2 and TcinNPC2-3 were similar,with the highest relative expression in female adult stage and the lowest relative expression in egg stage.2.Prokaryotic expression of OBP-like and NPC2 proteins in T.cinnabarinus6 proteins were successfully expressed by prokaryotic expression system using pCold Ⅱ as vector and Origami B(DE3)as expression strain: TcinOBP-like 2,TcinOBPlike 3,TcinOBP-like 4,TcinNPC2-1,TcinNPC2-2 and TcinNPC2-3.The molecular weights of the 6 recombinant proteins were 22.77 kDa,19.4 kDa,20.53 kDa,15.94 kDa,15.95 kDa and 14.32 kDa,respectively.In addition,TcinOBP-like 1 was successfully expressed using pET-30 a as a vector,and the molecular weight of the recombinant protein was 25.99 kDa.Among the 7 proteins,TcinOBP-like 2 and TcinOBP-like 4 were expressed as inclusion bodies,and the remaining proteins were soluble proteins.Through inclusion body denaturation and renaturation experiments,TcinOBP-like 2 and TcinOBPlike 4 soluble proteins were successfully obtained.3.MST experiment of OBP-like and NPC2 proteins in T.cinnabarinusThe results of dye affinity determination showed that the affinity Kd values of 7recombinant proteins(TcinOBP-like 1,TcinOBP-like 2,TcinOBP-like 3,TcinOBP-like 4,TcinNPC2-1,TcinNPC2-2,TcinNPC2-3)with fluorescent dyes were 91.81 pm,1.55 nm,3.12 nm,3.77 nm,5.07 nm,14.98 nm,5.79 nm.It indicated that the 7 recombinant proteins were successfully labeled with fluorescent dyes and could be used for subsequent experiments.Further experiments on the binding ability of 10 plant volatiles to recombinant proteins showed that TcinOBP-like 2 and TcinOBP-like 4 proteins bound toα-coniferylaldehyde(Kd=84.89 μM,52.06 μM),TcinOBP-like 3 protein bound to nonanal(Kd=36.06 μM),TcinNPC2-2 protein bound to geranyl acetone(Kd=3.32 μM),and TcinNPC2-3 protein bound to farnesol(Kd=9.55 μM).No binding was found between other substances and proteins.4.RNAi and Y-tube behavioral verificationThe dsRNA of TcinOBP-like 2,TcinOBP-like 3,TcinOBP-like 4,TcinNPC2-2 and TcinNPC2-3 were synthesized,and the dsRNA of TcinOBP-like 3,TcinNPC2-2 and TcinNPC2-3 were injected into the female adults of T.cinnabarinus respectively.The dsRNA of TcinOBP-like 2 and TcinOBP-like 4 were co-injected into the female adults of T.cinnabarinus for RNAi experiments.The interference efficiency of TcinOBP-like 2,TcinOBP-like 4,TcinNPC2-2 and TcinNPC2-3 were 68.80%,89.51%,90.81% and 84.98%,respectively,indicating that the gene was successfully interfered.TcinOBP-like3 interference failed.Y-tube behavioral verification was performed after interference.The results showed that after interfering with TcinOBP-like 2 and TcinOBP-like 4,the number of mites that selected DMSO increased compared with the control group;After disturbing TcinNPC2-2,there was no significant difference between the number of mites selected for geranyl acetone and the choice of solvent control;After interfering with TcinNPC2-3,the number of mites that selected farnesol decreased compared with the control group. |
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