| Myzus persicae(Sulzer)is one of the major tobacco pests in the world,which has the characteristics of short generation cycle,rapid reproduction speed and strong stress resistance.M.persicae can not only harm the stems and leaves of tobacco,but also spread a variety of plant viruses,causing great losses in tobacco production.In nature,M.persicae usually suffers from a variety of adverse environmental factors,such as pathogen infection and parasitic wasps.But it can still survive and multiply well under the influence of various factors,which is inevitably related to the function of immune system.As a key member of innate immune Toll signaling pathway,Toll receptors play an indispensable role in insect growth,development and reproduction in the fight against adverse environment.In order to clarify the role of Toll receptor genes in the immune system of M.persicae,six Toll receptor related genes(MpToll,MpToll3,MpToll6,Mptoll6-1,MpToll7 and MpTollo)were screened by combining the genome and transcriptome database of M.persicae.The real-time polymerase chain reaction(RT-PCR)technology was used for cloning verification,and bioinformatics analysis software was used for sequence analysis.The real-time quantitative polymerase chain reaction(RT-qPCR)was used to analyze the expression patterns of Toll receptor genes in different developmental stages and tissue of M.persicae,and detected the expression changes of six genes after infection with gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli.Finally,RNAi combined with bacterial infection was used to explore the function of Toll receptor genes in the immune response of M.persicae.The main results are as follows:1.Identification and sequence analysis of six Toll receptor genes of M.persicaeRT-PCR was used to clone the open reading frame sequences of six Toll receptor genes:MpToll,MpToll3,MpToll6,MpToll6-1,MpToll7 and MpTollo,the open reading frame lengths were 2952 bp,3234 bp,3789 bp,3882 bp,4008 bp and 3747 bp,respectively.Encoding 983,1077,1262,1293,1335 and 1248 amino acids,respectively.The predicted molecular weights were 113.69,120.29,142.50,146.24,151.83 and 142.14 kDa,and the theoretical isoelectric points were 6.42,6.71,5.54,5.89,6.80 and 6.07,respectively.During the gene coding process,MpToll and MpToll3 were interrupted by three and thirteen introns,respectively,while the other four genes did not contain introns.The proteins encoded by the six Toll receptor genes are all hydrophilic proteins and contain multiple phosphorylation sites,which were easily phosphorylated.The N-terminal of the protein is extracellular and consists of the leueine-rich repeats(LRRs)domain and contains an amino acid residue as a signal peptide.The Mp Toll,Mp Toll6,Mp Toll6-1,Mp Toll7 and Mp Tollo encoded proteins also have a transmembrane structure and a C-terminal TIR(Toll-IL-1R)domain.The results of multiple sequence alignment showed that the extracellular LRRs domain contained multiple characteristic consistency sequences LxxLxLxxNxL,and the intracellular TIR structure contained three motifs that were conserved in human TLRs and other insect Toll proteins.They are the F/YDAxxS motif at the beginning,CLHYRD motif in the middle and FWxxL motif at the end.Phylogenetic analysis of Toll proteins in the evolutionary relationship between M.persicae and other insects revealed that the six Toll proteins were divided into six branches,each of which clustered together with the corresponding Toll proteins of other insects and showed closely related to Acyrthosiphon pisum,Aphis gossypii and Rhopalosiphum maidis.The results of evolutionary selection pressure analysis showed that Toll receptor genes were in a negative selective transition state,eliminating harmful mutations and tending to conserved structure.2.Different developmental stages and tissues expression profiles of six Toll receptor genes of M.persicaeThe expression patterns of Toll receptor genes in different developmental stages and tissues of M.persicae were detected by qPCR.The analysis results show that in the M.persicae just born or molting for 1 h,the expression of MpToll,MpToll3,MpToll6,MpToll6-1,MpToll7 and MpTollo were the highest in the first-instar nymphs or winged adults,and high expression levels were also found in fourth-instar nymphs and wingless adults,but lower in the second-instar nymphs and the third-instar nymphs.In the M.persicae 12 hours after birth or molting,expression patterns of MpToll,MpToll3,and MpToll6-1 were similar to those of just born or molted aphid 1 h,the expression of MpToll6 was the highest in the first-instar nymphs,while there was no significant difference among other stages,and MpToll7 was the most expressed in the wingless adults,and was also highly expressed in the winged adults,and the expression of MpTollo was highest in the first-instar nymphs,but lower in the third-instar nymphs and in winged adults.Furthermore,tissue expression of the six Toll receptor genes were mainly concentrated in the head,embryo and epidermis,but was relatively lower in the gut and hemolymph.3.Expression of six Toll receptor genes of M.persicae under bacterial stressS.aureus and E.coli were used to infect one-day-old wingless adults respectively,and the expression levels of six Toll receptor genes were detected at 6,12 and 24 h after infection.The results showed that MpToll and MpToll6 were strongly sustained at three time point under S.aureus stress,while MpToll and MpToll6 were only slightly induced at 12 and 6 h under E.coli stress.MpToll6-1 and MpTollo were continuously induced expression at three time points under S.aureus,and the response of MpToll6-1 under E.coli stress was stronger,while MpTollo was not induced by E.coli.Under the S.aureus stress,MpToll3 expression was upregulated only at 24 h,while Mp Toll7 expression was upregulated at 6 h and 24 h.Under the E.coli stress,MpToll3 and MpToll7 expression were continuously induced and reached the highest expression levels at 6 h and 12 h.4.Functional response of six Toll receptor Genes of M.persicae to bacterial infection.The MpToll,MpToll3,MpToll6,Mptoll6-1,MpToll7 and MpTollo genes were silenced by RNA interference,respectively.The results showed that after injection of six Toll receptor genes dsRNA in the fourth instar nymphs of M.persicae,respectively,the transcription level of target genes were significantly down-regulated compared with the control group injected with dsGFP.After silencing the target genes,S.aureus or E.coli were used for further infection,and the susceptibility of M.persicae to these two bacteria was analyzed.The results showed that compared with the control group,the mortality under the S.aureus stress were significantly increased by 23.33%,52.22%,39.35%,10.00%,58.78%and 46.56%,and that under the E.coli stress were significantly increased by 30.00%,58.78%,14.98%,10.00%,42.22%and 54.44%,and the average reproduction under the S.aureus and E.coli stress were significantly decreased by 5.23～7.01 and 0.69～4.34,respectively.In conclusion,in this study,combined with RNAi and bacterial infection experiments,the effect of targeted silencing of Toll receptor genes on the resistance of M.persicae,to bacterial infection was evaluated,and the function of Toll receptor genes involved in antibacterial immune response of M.persicae was preliminary clarified. |
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