| Insect innate immunity is divided into cellular immunity and humoral immunity.The cellular immunity refers to the defense responses of various hemocytes,including cytophagy,nodulation and encapsulation;the humoral immunity involved in the induction of antimicrobial peptides,activation of prophenoloxidase(PPO)and melanization.Serine protease inhibitors(Serpin),is a kind of protein,which inhibits serine protease in the hemolymph,used to adjust the balance of the inner environment of insect.Beta-1,3-glucan recognition protein(?GRP),as a kind of pattern recognition protein to identify cell wall components of the fungus,plays an essential role in the insect immune system.In the present study,we cloned Ostrinia furnacalis Serpin2 gene using RT-PCR and RACE amplification technology.The full length cDNA of the Serpin2 is 1447 bp,including a 1137 bp open reading frame(ORF),a 163 bp 5-untranslated region(5' UTR),and a 147 bp 3-untranslated region(3' UTR)with a poly(A)signal.ORF is beginning from the 164th nucleotide and ending at the 1300th nucleotide,and encodes a 378 amino acid residues.The Serpin2 was predicted with a molecular weight of 41.71 kDa and a pI of 5.29.Bioinformatics analysis showed that:Of-Serpin2 has no transmembrane domain structure,neither N-terminal signal peptide nor glycosylation sites,but contains 39 phosphorylation sites uniformly distributed in the polypeptide chain.BlastP analysis results showed that the amino acid sequence of the cloned Of-Serpin2 was highly similar to those of Lonomia obliqua Serpin2,Spodoptera exigua Serpin2,Bombyx mandarina Serpin2,Bombyx mori Serpin2,Danaus plexippus Serpin2.Three-dimensional structure indicated that Of-Serpin2 has 8 alpha helixes and 3 beta sheets.The expression levels of immune-related genes,such as Serpin1,PAP,PPO,as well as the preliminary effects on the cellular immune ability were analyzed after RNA interference in the 4th instar larvae of Ostrinia furnacalis,The DEPC water and the green fluorescent protein(GFP)treatment group were applied as the negative control,dsRNA of ?GRP3 and GFP were prepared for the injection of 4-instar O.furnacalis larvae,respectively.Real-time PCR was used to detect the transcription level of the related immune genes after RNAi,and the PO activity and numbers of various hemocytes were analyzed after RNAi to explore the function of ?GRP3 in the process of immune recognition and defence of O.furnacalis larvae.The results showed that ?GRP3 expression decreased significantly in 72 h after RNAi,indicating that RNAi could effectively down-regulate the expression of ?GRP3 in Asian corn borer larvae.Meanwhile,the expression levels of the immune-related genes,such as prophenoloxidase activation protease(PAP),prophenoloxidase(PPO),was also significantly down-regulated when compared with the negative control group of GFP dsRNA injection;The PO activity was significantly decrease in 72 h post injection(HPI)after ?GRP3-dsRNA injection in RNAi,suggesting Of-?GRP3 are regulating the PO activity,which play an important role in the PPO cascade.The analysis of the numbers of various hemocytes showed that the number of oenocytoid,plasma hemocytes and prohemocyte began to decline at 72 HPI after RNAi of ?GRP3 gene,However,the number of granulocyte hemocytes increased significantly,and the number of plasma hemocytes increased again after 96 HPI.Compared with the control group,the total number of hemocytes of RNAi group did not change obviously,which indicated that the hemocytes could be self-adjusted in insect hemocoel when affected by foreign invaders to maintain normal physiological activities.Following RNA interference,the injection of Escherichia coli was conducted,The results showed that the median lethal time(LT50)of E.coli-infected wild-type larvae was 3.0 days,which was significantly longer than that of E.coli-infected Of-?GRP3-RNAi larvae(1.0 day)(P<0.01)These data suggest that?GRP3 is required for resistance against infection by bacterial pathogens. |
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