| Juvenile hormone(JH)is an important substance involved in insect reproductive regulation.As a receptor of JH,Methoprene-tolerant(Met),a member of the BHLH-PAS family,plays an important regulatory role in the functional effect of JH.In this paper,the important vector insect Culex pipiens pallens was used as the research object.The full-length cDNA sequence of the juvenile hormone receptor gene CpMet was cloned and the sequence characteristic structure was analyzed.Real-time fluorescent quantitative PCR technology was used to analyze the expression distribution of CpMet in different tissues and different developmental stages of C.pipiens pallens and the effect of hormone treatment on CpMet gene expression.The effect of CpMet on the reproduction and blood-sucking behavior of C.pipiens pallens and the role of CpMet in the resistance of C.pipiens pallens to pyriproxyfen were studied using RNA interference technology.The results have important scientific significance and application value for further understanding the molecular mechanism of juvenile hormone signaling pathway regulating insect reproduction and the resistance control of bionic juvenile hormone insecticides.The main research results are as follows:The full-length cDNA sequence CpMet C.pipiens pallens juvenile hormone receptor gene was cloned and its sequence and structural characteristics were analyzed.The open reading frame(ORF)was 2,892 bp long and encoded 963 amino acids(AA).The predicted molecular weight(Mw)and isoelectric point(pI)of the protein were 105.10 kDa and 6.44,respectively.The domain analysis showed that CpMet had conserved bHLH(basic Helix-Loop-Helix),PAS(Per-Arnt-Sim,PAS-A,PAS-B)and PAC(PASC terminal)domains.Sequence analysis showed that the amino acid sequences of CpMet were 52.7%and 26.6%consistent With Aedes aegypti and Tribolium castaneum,respectively.Phylogenetic analysis showed that CpMet gene was clustered with other diptera,such as Aedes aegypti,Anopheles sinensis,Anopheles gambiae,etc.The relative expression of CpMet in different developmental stages and tissues of C.pipiens pallens was detected by Quantitative Real-time PCR.The results showed that the expression level of CpMet was significantly higher at egg stage than at other stages,followed by pupae and adult,and was very low at larval stage.The expression level of CpMet was higher in ovary of female adult mosquito,followed by adipose body.The further study found that the CpMet expression of female adult mosquitoes was up-regulated by 1.27 times,4.15 times and 1.26 times after 6 h?12h?24h treatment of Methoprene,indicating that the CpMet was positively regulated by the JH.After treating mosquito pupae with Ecdysterone 20E for 24h,the CpMet gene expression increased sharply,indicating that the CpMet gene expression was induced by 20E.Effects of CpMet on growth,development and reproduction of C.pipiens pallens were studied.RNA interference was performed by micro injection on unfed and unmated female adults of C.pipiens pallens.The results showed that compared with the control group(dsEGFP treatment),the CpMet mRNA expression level in the dsCpMet treatment group was down-regulated by 50.29%and 86.29%h 24 and 48 after interference,respectively.After dsCpMet injection,the survival rate of female mosquitoes was decreased to 74.44%.When injected dsCpMet 1 day later,mosquitoes were allowed to mate and suck blood to observe their oviposition,average spawning number and hatching rate.It was found that the average egg content of female mosquitoes with dsCpMet injection treatment was decreased by 26.58%,and the hatching rate of eggs was decreased to 73.36%.The effect of CpMet on blood-sucking behavior of C.pipiens pallens was studied.The relative expression levels of CpMet were measured between unfed and 3 days after blood feeding.The results showed that there was no significant difference in the expression levels of CpMet.The microinjection Method was used to detect RNA interference in unsucking female adults.The results showed that the response time to blood sucking was significantly increased in the dsCpMet group,and the blood sucking rate and satiety rate were decreased by 34.45%and 19.07%,respectively.The role of CpMet gene in the resistance of C.pipiens pallens to pyriproxyfen was studied The relative expression level of CpMet gene in susceptible and resistant strains was detected by Quantitative Real-time PCR,and it was found that the expression level of CpMet gene in resistant strain was significantly decreased,which was 43.59%of that in sensitive lines.The relative expression levels of CpMet gene in the 4th instar larvae and pupae of were detected after 6,12 and 24h treatment with different concentrations of pyriproxyfen(LC30,LC50 and LC70).The results showed:After treatment with three doses,the mRNA expression level of CpMet in the 4th instar larvae were significantly reduced by 79.67%,88.10%and 92.91%after 6h,and significantly increased after 12h,which was 3.98,4.78 and 4.45 times higher than that in the control group.There was no significant difference between LC30 and LC50 treatment groups after 24h compared with the control group.The expression level of LC70 treatment group was 1.97 times that of control group.Mosquito pupae were treated with three doses,after 6 hours,the expression of CpMet in LC50 treatment group increased,which was 1.35 times that of control group.After 12h,24h,LC30 and LC50 treatment groups,CpMet expression showed a downward trend,which was decreased by 94.73%and 92.93%at 24h,respectively,and CpMet mRNA was almost not expressed.After 6h,12h and 24h,LC70 treatment group had almost no CpMet expression.The sensitivity of CpMet gene to pyriproxyfen was determined by RNA interference.The pupae after RNA interference was soaked in LC30,LC50 and LC70 pyriproxyfen solution,and eclosion was observed.The results showed that there was no significant difference between LC30 and LC50 in the control group and the interference group.The survival rate of LC70 was upregulated to 51.12%. |
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