The Regulatory Mechanism Of NO-mediated Immunity In Ostrinia Furnacalis Guenée Larvae Infected By Two Biocontrol Bacteria

The Asian corn borer（Ostrinia furnacalis Guenée）is a global pest that mainly harms corn,millet,sorghum and other crops.Biological control of O.furnacalis by bacteria is an important integrated pest management research direction.We cloned the immune-related Nitric oxide synthase 2（NOS2）gene of O.furnacalis larvae by rapid amplification of cDNA ends（RACE）method and analyzed bioinformatically.The O.furnacalis larvae were treated with two bacteria,Pseudomonas aeruginosa（P.a.）and Micrococcus luteus（M.l.）;Meanwhile,calcineurin A（CaNA）and nitric oxide（NO）in larvae were regulated by injection of NO donor NOC,CaNA inhibitor FK506 and NOS inhibitor L-NAME.The functions of NOS and CaNA were further verified by RNA interference（RNAi）,quantitative real-time quantitative PCR（qPCR）,NO donor NOC treatment,CaNA inhibitor FK506 treatment and NOS inhibitor L-NAME treatment in Drosophila S2 cells.The main results are as follows:（1）Reverse transcription PCR（RT-PCR）and RACE were used to clone the open reading frame（ORF）sequence of NOS2 gene from O.furnacalis larvae.Bioinformatics analysis showed that the full length of the open reading frame was 3336 bp,encoding 1111 amino acids.The relative molecular weight was 125.77 kDa.The isoelectric point was 6.62.The whole peptide chain was outside the membrane without transmembrane domain,and no signal peptide and hydrophilic protein were found.There were 97 phosphorylation sites distributed on the whole polypeptide chain.（2）The expression level of Of-NOS2 in various tissues of 4th instar O.furnacalis larvae was detected by qPCR.The results showed that the expression level of Of-NOS2 gene was low in the head and integument.Compared with the head,the expression levels of Of-NOS2 gene in hemocytes,midgut and fat body were 4.27±0.54,3.23±0.87 and 17.15±1.16 folds,respectively.The results showed that the expression level of Of-NOS2 was the highest in fat body.（3）The 4th instar O.furnacalis larvae were injected with P.aeruginosa or M luteus.The expression levels of Of-NOS1,Of-NOS2,Of-CaNA and related antimicrobial peptides（AMPs）including Attacin,Cecropin A,Defensin,Gloverin,Lebocin-4 and Moricin were detected by qPCR.The results showed that the expression levels of related genes were up-regulated at different time points after injection with bacteria.When P.aeruginosa was injected,the expression of Of-NOS2 was significantly increased at 12 h and 48 h post injection.When M.luteus was injected,the expression level of Of-NOS2 was the highest at 24 h post injection（p<0.001）.（4）The content of NO in plasma was significantly up-regulated after injection with NO donor NOC into O.furnacalis larvae（p<0.01）.Compared with the control group,the expression level of Of-CaNA was significantly up-regulated at 12 h and 24 h post injection analyzed by qPCR（p<0.05）.For the injection with CaNA inhibitor FK506,the activity of calcineurin A was inhibited,AMPs were down-regulated on different levels at different time points.However,the injection with NOS inhibitor L-NAME depressed the production Of NO,and the reduction of NO significantly inhibited the expression of Of-CaNA and AMPs（p<0.05）.Taken together,the results indicated that NO was directly or indirectly involved in the innate immune response against bacterial infection.（5）In Drosophila S2 cells,the reduced expression of NOS significantly down-regulated the expression levels of Cecropin A,Defensin,Diptericin and Drosomycin by RNAi（p<0.05）.After knocking down the expression levels of IMD and MyD88,NOS gene was found to be associated with the IMD immune pathways.After the induction of cells by NO donors NOC,the expression levels of CaNA and related AMPs were significantly up-regulated,indicating that NO had certain regulatory effect on CaNA and related AMPs.The addition of CaNA inhibitor FK506 inhibited the expression levels of AMPs;the addition of NOS inhibitor L-NAME inhibited the expression of CaNA gene and AMPs（p<0.05）.In summary,we speculated that NO signal can mediate CaNA directly or indirectly to regulate the antimicrobial peptides expression via IMD pathway.