| Diaphorina citri is the only known vector for transmission of huanglongbing(HLB).HLB is called citrus cancer.The world citrus industry has caused huge economic losses.At present,the control of D.citri includes agricultural control,chemical control and biological control,but it mainly depends on chemical control.Chitin synthase(CHS)is the last and most critical step in the chitin synthesis pathway,and its deletion can affect the normal development of insects.Therefore,the chitin synthesis pathway is relatively safe as a target for pest control.It is of great theoretical significance to develop new pesticides based on this pathway and apply it to pest control.In this paper,D.citri is used as the research object,and PCR combined with RACE technology is used to amplify the full-length cDNA sequence of the chitin synthase A gene of D.citri,and the sequence characteristics are analyzed.RT-qPCR technology was used to investigate the mortality and molt of the fifth-instar nymphs of D.citri,at different stages of D.citri growth and development at different stages of D,citri growth and development,as well as mRNA expression levels.The main results are as follows:The full-length cDNA of the chitin synthase A gene of D.citri is 4677 bp,the open reading frame(ORF)is 4437 bp in length,encoding 1478 amino acids,the 5 ’end non-coding region(UTR)is 156 bp,and the 3’ end UTR is 84 bp.The start codon is ATG,the stop codon is TAA,and the tail signal(AATAA)is located 10bp after the stop codon.Citrus psyllid chitin synthase A protein has a molecular weight of 169.74KD and an isoelectric point of 6.35.It contains 164 positively charged amino acid residues(R,K)and 175 negatively charged amino acid residues(D,E),no signal peptide.TMHMM analysis showed that the DcCHSA gene has 15 transmembrane domains,including two chitin synthetase tag sequences:EDR(852-854)and QRRRW(889-894);phylogenetic analysis showed that the DcCHSA gene and the same half The CHSA genes of the Aphis citricidus and Aphis glycines of the winged insects are grouped together,and then together with the CHSAs of Laodelphax striatellu,Sogatella furcifera,and Nilaparvata lugens;DcCHSA genes are expressed throughout the developmental period.The expression level of the instar nymph stage was the highest,and the expression level was the lowest in the egg stage,and the expression level of the fifth instar nymph was significantly higher than that of other development stages(P<0.05).The LC50 of diflubenzuron on D.citri fifth instar nymphs was 140.992 mg/L.Treatment with low,medium and high concentrations of DFB treatment for 48 h resulted in death and molting failure of fifth instar nymphs.Mortality and molting failure rates increased with DFB concentration.Keep rising.When the concentration was 500 mg/L,the mortality rate was as high as 83%.Compared with the normal phenotype,the phenotype of failure is a phenotype that is trapped in the old stratum corneum and cannot be completely moulted,and the wingspan fails to phenotype,etc.,and the body color becomes darker and the abdomen becomes green.It is worth mentioning that in the 500mg/L treatment group,a number of nymphs had a special phenotype of molting failure,bulging on the left front side of the head,and green abdomen.RT-qPCR was used to detect the DcCHSA gene expression level of the nymphs after treatment.The results showed that as the concentration of diflubenzuron increased,the DcCHSA gene expression level continued to increase,and the expression in the 500 mg/L treatment group was significantly different from the control group(P<0.05).After synthesizing dsRNA in vitro,the fifth-instar nymphs of D.citri were fed by feeding method for RNA interference.After 48 hours,the mortality of the fifth-instar nymphs in the experimental group was significantly higher than that of the other two control groups,reaching 24%.The molting failure phenotype appeared in nymphs,but not in the control group.Compared with the normal phenotype,it is mainly manifested in the inability to completely get rid of the old stratum corneum and the failure of wingspan or even death.The results of RT-qPCR showed that the expression level of the DcCHSA gene was significantly reduced after feeding dsRNA in the experimental group.Compared with the GFP control group,the expression level was decreased by 38.26%and 44.91%compared with the blank control,and the expression level of DcCHSA gene was significantly different from that of the two control groups(P<0.05).In summary,on the basis of cloning the full length of the chitin synthase A gene from D.citri,and in this paper,by targeting DcCHSA gene,performing DFB treatment and RNA interference experiments,the fifth-instar nymph of D.citri molted.Failure and high lethality indicate that the DcCHSA gene can be used as a potential target for pest control,providing ideas and theoretical basis for the subsequent control of D.citri. |
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