| The Asian corn borer(Ostrinia furnacalis Guenee)is a major borer pest causing serious damage to crops in China.It is an important research direction to use more environmentally friendly and safer biological control methods to control O.furnacalis at present.In this paper,the full-length cDNA sequence of the EDA-like cell death trigger(Eiger),a ligand member of the tumor necrosis factor superfamily of O.furnacalis,was cloned by cDNA end rapid amplification(RACE).The amino acid sequences encoded by Eiger gene were analyzed by bioinformatics.The expression of Eiger gene in different tissues and different developmental stages of O.furnacalis was investigated.The expression of Eiger gene in O.furnacalis larvae was successfully down-regulated using RNA interference(RNAi)technology.The changes of mRNA expression of IMD,Toll and JNK signal pathway related genes,Of-Hemocytin,Of-Myosin,Of-TEP2,Of-Rab5 and Of-ARP genes and six antimicrobial peptides(AMPs)such as Of-Lebocin,Of-Defensin,Of-Attacin,Of-Cecropin,Of-Gloverin and Of-Moricin after RNAi were further detected,and the changes of phenoloxidase(PO)activity after RNAi were detected.The function of Eiger gene of O.furnacalis in the process of resisting bacterial infection was explored by injecting Pseudomonas aeruginosa(Pa.)and Micrococcus luteus(Ml.),respectively.The effect of Eiger RNAi on phagocytosis of hemocytes was also studied.The main research results are as follows:(1)The full-length cDNA sequences of Of-Eiger gene were cloned by RACE method.The full-length of Of-Eiger gene is 1533 bp,including an open reading frame(ORF)of 1464 bp.The start codon is ATG and the stop codon is TAA,which encodes a total of 487 amino acids,including a 5’non-coding region(5’UTR)of 25 bp and a 3’ non-coding region(3’UTR)of 44 bp(GenBank accession number:MN833226).The calculated molecular mass of Of-Eiger is about 54.75 kDa and the predicted isoelectric point is about 5.34.The protein encoded by Of-Eiger has a glycosylation site at the position 298 amino acid.There are 25 serine(Ser)phosphorylation sites,13 threonine(Thr)phosphorylation sites and 7 tyrosine(Tyr)phosphorylation sites,and a total of 45 phosphorylation sites are distributed in the whole polypeptide chain.Of-Eiger protein is a hydrophilic protein.Amino acids 1-52 are intramembrane proteins,53-75 amino acids are located on the membrane,76-487 amino acids are extramembrane proteins,and there is a transmembrane domain.The results of three-dimensional structure prediction show that it has one α helix and thirteen β sheets.The BlastP results showed that the amino acid sequence of Of-Eiger was closely related to Helicoverpa zea Eiger,Helicoverpa armigera Eiger,Trichoplusia ni Eiger-like isoform X1,Melitaea cinxia Eiger,Spodoptera litura Eiger and Manduca sexta Eiger.(2)The expression levels of Of-Eiger in different tissues of the 4th instar of O.furnacalis larvae were detected by quantitative real time PCR(qPCR).The results showed that the expression of Of-Eiger was the highest in hemocytes,followed by the fat body and head,and the lowest in midgut.Meanwhile,the expression levels of Of-Eiger in different developmental stages of O.furnacalis were also detected.The results showed that the expression level of Of-Eiger increased with the increasement of day-age.During the whole development statges of O.furnacalis,the expression of Of-Eiger in 3-day-old pupae was the highest,and the expression of Of-Eiger in male adults was higher than that in female adults(P<0.05).(3)Of-Eiger gene expression of the 4th instar larvae was successfully down-regulated by RNAi.After Of-Eiger RNAi,the qPCR was used to detect the expression levels of Of-Eiger and immune-related genes prophenoloxidase 2(Of-PPO2),Nitric oxide(Of-NOS1 and Of NOS2),and Of-Lysozyme,and IMD signaling pathway-related immune genes,such as Of IMD,Of-Relish and Of-FADD,Toll signaling pathway-related immune genes Of-MyD88 and Of-Dorsal,MAPK signaling pathway-related immune genes Of-JNK,Of-P38 and Of-ERK,and five cellular immunity-related genes Of-Jemocytin,Of-Myosin,Of-TEP2,Of-Rab5 and Of-ARP;and six antimicrobial peptides,such as Of-Lebocin,Of-Defensin,Of-Attacin,Of-Cecropin,Of-Gloverin and Of-Moricin.The results showed that the expression of Of-Eiger could be inhibited successfully at 60,72,and 84 h after injection of dsEiger,and the interference efficiency was the highest at 72 h post injection,with the expression decreased by(85.72±4.28)%;In the successful RNAi period of(60-84 h),the expression of Of-PPO2 was significantly up-regulated at 60,72 and 84 h,which was 2.42±0.52 times,4.55±1.67 times and 1.30±0.17 times of those in the control group,respectively.The expression of Of-NOS1 gene was significantly down-regulated at 60 and 72 h.The expressions of Of-NOS2 and Of-Lysozyme genes were significantly down-regulated at 60 h compared with the control group.The IMD signalling pathway-related immune genes Of-IMD,Of-Relish and Of-FADD were significantly down-regulated.The Toll signaling pathway-related immune genes Of-MyD88 and Of-Dorsal were down-regulated.The MAPK signaling pathway-related immune genes Of-JNK,Of-P38 and Of-ERK also showed a down-regulation trend.All five cellular immunity-related genes showed a down-regulation trend.Of-Lebocin,Of-Attacin,Of-Gloverin and Of-Moricin showed a down-regulation trend,Of-Defensin showed no significant trend and Of Cecropin showed an up-regulation trend.The activity of PO after the successful RNAi of was measured by enzymatic method,and the results showed that Of-Eiger negatively regulated the activity of PO.(4)Pa.and Ml.were injected into the 4th instar Ofurnacalis larvae,respectively,and the expression levels of Of-Eiger,IMD pathway-related immune genes Of-IMD and Of-FADD,Toll pathway-related immune genes Of-MyD88 and Of-Dorsal,the MAPK pathway-related immune genes Of-JNK and Of-P38,the immune-related genes Of-NOS1 and Of-NOS2 and six antimicrobial peptides Of-Lebocin,Of-Defensin,Of-Attacin,Of-Cecropin,Of-Gloverin and Of-Moricin were also analyzed.The results showed that the expression of Of-Eiger was significantly up-regulated,and the expression of reached the peak at 24 h after the injection of Pa.and Ml.,with 1.95±0.22 and 4.66±1.04 times,respectively.The immune-related genes Of-NOS1 and Of-NOS2,and the genes related to the IMD,Toll and MAPK signalling pathways and six antimicrobial peptides Of-Lebocin,Of-Defensin,Of-Attacin,Of-Cecropin,Of-Gloverin and Of-Moricin were significantly altered after injection of the two bacteria.It suggested that Of-Eiger and the related immune genes mentioned above are involved in the innate immunity of O.furnacalis against Pa.and Ml.infection.(5)In order to further verify the function of Of-Eiger,Pa.and Ml.were injected after interfering with Of-Eiger expression,respectively.The expression levels of the following genes were examined respectively,IMD signalling pathway-related immune gene Of-Relish,Toll signalling pathway-related immune gene Of MyD88;MAPK signalling pathway-related immune gene Of-JNK,immune-related genes Of-NOSl and Of-PPO2 and six antimicrobial peptides Of-Lebocin,Of Defensin,Of-Attacin,Of-Cecropin,Of-Gloverin and Of-Moricin.The results showed that successful interference with Of-Eiger followed by Pa.injection resulted in a significant down-regulation of Of-Relish at 12 h by(77.15 ± 1.76)%of the control group,Of MyD88 at 1,4 and 12 h by(74.08±6.10)%,(72.45±3.83)%and(47.85 ±13.51)%,Of-JNK was significantly up-regulated at12 h by 1.45 ± 0.16 times of the control group,respectively,Of-PPO2 was significantly up-regulated at 4 and 24 h by 23.26 ± 1.57 and 3.50 ± 0.42 times,respectively,and Of-NOS1 was significantly down-regulated at 12-24 h,by(2 6.22±6.05)%and(22.39± 5.12)%,respectively,and the expression of all six antimicrobial peptides was significantly down-regulated by Pa.injection after interference.Of-Eiger interference followed by injection of Ml.,Of-Relish was significantly down-regulated at 1,4 and 24h by(85.15±1.29)%,(35.11±4.72)%and(64.80±5.68)%,respectively,and Of-MyD88 was significantly down-regulated at 1,4 and 12 h by(71.09±5.04)%,(77.79± 3.66)%and(48.00±3.84)%,Of-JNK was significantly down-regulated at 24 and 48 h by(79.08±3.00)%and(79.41±2.14)%,respectively,and Of-PP02 was significantly down-regulated at 4,12,24 and 48 h by(80.94±1.68)%,(37.01±6.74)%,(97.24±0.22)%and(76.39±5.53)%;Of-NOSl was significantly downregulated at 1,24 and 48 h,with the most significant downregulation at 24 h,by(75.87±2.45)%compared to the control,and post-interference injections of Ml.There was no significant trend in Of-Lebocin,Of-Gloverin and Of-Defensin Of-Attacin and Of-Cecropin were significantly up-regulated,and Of-Moricin were significantly down-regulated.All these results demonstrated that Of-Eiger may be involved in the innate immunity of Ofurnacalis against bacterial injections through the synergistic action of the IMD,Toll and MAPK pathways.(6)To verify the role of Of-Eiger in cellular immunity,Of-Eiger and five cellular pathway-related genes were detected in Ofurnacalis hemolymph after interference with Of-Eiger expression,which was significantly reduced at 48,60,72,84 and 96 h post-injection.The expressions of Of-Hemocytin,Of-Myosin and Of-ARP were significantly down-regulated,and the expressions of Of-TEP2 and Of-Rab5 were significantly up-regulated.The results also tested whether interference with Of-Eiger affected the phagocytosis of hemocytes,and showed that the phagocytosis of hemocytes of O.furnacalis was reduced after interference with Of-Eiger. |
PDF Full Text Request