| Ostrinia furnacalis Guenee,Asian corn borer,is a sort of worldwide pest that harms crops,which mainly damages corn,sorghum,millet and other crops.The biological control of the O.furnacalis through bio-control bacteria is an important research direction at present.In this paper,the full-length cDNA sequence of the matrix metalloproteinase 14 was obtained by the method of Rapid Application of cDNA End(RACE)in O.furnacalis larvae,and analyze the amino acid sequence of MMP14 was analyzed bioinformaticly.The expression of MMP14 gene in different tissues and different developmental stages of O.furnacalis was studied.Using RNA interference(RNAi)technology,the down-regulated expression of MMP14 gene in O.furnacalis larvae was successfully achieved,and the mRNA expression profiles of four antimicrobial peptides,such as Attacin,Cecropin,Glover in and Moricin,after RNAi were further detected.Meanwhile the activities of phenoloxidase(PO)and Lysozyme after RNAi were also detected.Two kinds of biocontrol bacteria,Pseudomonas aeruginosa(Pa)and Micrococcus luteus(Ml)were injected respectively for the functional analysis of O.furnacalis MMP14(Of-MMP14)gene in the defense of the infection of Pa and Ml The main results are as follows:(1)The full-length cDNA sequence of Of MMP14 gene was cloned by RACE method.The total length of MMP14 gene is 2565 bp,including an open reading frame(ORF)of 1611 bp,a start codon of ATG and a stop codon of TAG,encoding a total of 536 amino acids,including a 178 bp 5 ’non-coding region(5’UTR)and a 776 bp 3 ’non-coding region(3’UTR)(GenBank accession number:MN833228).Bioinformatics analysis showed that the calculated molecular mass of Of-MMP14 gene was about 59.77 kDa;Isoelectric point is 8.83;There is no transmembrane domain;There are three glycosylation sites,located at the 66th,92nd and 137th amino acids.There were 27 phosphorylation sites of serine(Ser),20 phosphorylation sites of threonine(Thr)and 10 phosphorylation sites of tyrosine(Tyr),and the above 57 phosphorylation sites were distributed in the whole polypeptide chain.MMP14 protein was hydrophilic protein.The predicted three-dimensional structure results showed that there are 11 α helices and 13 βfolds.The amino acid sequence of Of-MMP14 was analyzed by BlastP,the results show that:The amino acid sequence of Of-MMP14 had high homology with Spodoptera frugiperda MMP14 isoform X5、Zerene cesonia MMP14 isoform X10、Trichoplusia ni MMP14 isoform X3、Helicoverpa armigera MMP 14 isoform X2、Vanessa tameamea MMP 14 isoform X6 and Bicyclus anynana MMP14 isoform X7.(2)The expression levels of Of-MMP14 in different tissues of the 4th instar of O.furnacalis were detected by using quantitative real time PCR(qPCR).The results indicated that the expression of Of-MMP14 was the highest in the midgut,and low expression in integument and fat body,and the lowest expression in blood cells.The expression levels of Of-MMP14 in the different developmental stages of the O.furnacalis were also tested.The results showed that the expression level of Of-MMP14 increased with the increasement of age.During the whole development process,the expression level of 1-day-old pupae was the highest,and the expression level of Of-MMP14 in male adult was higher than that in female adult.(3)The gene expression of MMP 14 in the 4th instar larvae of the O.furnacalis was successfully down-regulated by RNAi.The expression levels of MMP14,immune-related gene PPO2,Lysozyme and four kinds of antimicrobial peptides,such as Attacin,Cecropin,Gloverin and Moricin were detected by qPCR after RNAi.The result indicated that the expression of Of-MMP 14 could be successfully down-regulated at 84 h,96 h and 108 h after dsMMP14 injection,and the highest RNAi efficiency was(52.95±11.89)%at 84 h;The expression of PPO2 was also significantly decreased during the period of MMP14 RNAi(84 h to 108 h),which was(65.42±2.78)%,(61.77±3.71)%and(70.76±1.94)%of the control group,respectively;The expression of Lysozyme increased significantly,which was 4.35±0.23 fold,10.47±0.10 fold and 3.56±0.49 fold to the control group;The expression levels of Attacin,Gloverin and Moricin were up-regulated,while the expression levels of Cecropin were significantly down-regulated(P<0.05).Enzymatic methods were used to determine the phenoloxidase(PO)activity and lysozyme activity of O.furnacalis larvae after MMP14 RNA interference.The results showed that the PO activity decreased during the effective period of interference(84 h-108 h)comparing with the control group,while the lysozyme activity increased.(4)The expression levels of Of-MMP14、Of-PPO2 and Of-Lysozyme genes were detected by qPCR in the 4th instar O.furnacalis larvae by injecting P.aeruginosa and M luteus.The results showed that the expression of Of-MMP14 in O.furnacalis larvae was significantly up-regulated post the injection of two kinds of bacteria,respectively,and the expression of Of-MMP14 reached the maximum at 72 h after the injection of P.aeruginosa,which was 4.51±0.35 fold to PBS control group;The highest expression was 4.29±0.21 fold to the control group at 12 h post injection of M.luteus.The expression of Of-PPO2 and Of-Lysozyme also increased significantly after injection.The results showed that PO activity and lysozyme activity increased significantly after injection of the two kinds of bacteria.These results indicated that Of-MMP14 and Of-PPO2 were involved in the innate immune response of the O.furnacalis against P.aeruginosa and M luteus invasion.(5)To further verify the function of MMP14 gene,P.aeruginosa and M.luteus.were injected respectively after MMP14 RNAi,the expression levels of two immune-related genes,such as Of-PPO2 and Of-Lysozyme,and four kinds of antimicrobial peptides were detected.The results showed that the expression of Of-PPO2 decreased significantly at 24 h and 72 h,which was(57.50 ± 0.66)%of the control group,when the expression of Of-MMP14 was down-regulated by Of-MMP14 RNAi before the injection of P.aeruginosa;The expression of Of-Lysozyme increased significantly at 24 h and 72 h.The expression of Of-PPO2 decreased significantly at 72 h,which was(66.67 ± 9.25)%of the control group,When the gene expression of Of-MMP14 was down-regulated by Of-MMP14 RNAi before the injection of M.luteus.The expression of Of-Lysozyme increased significantly at 24 h and 48 h,which were 1.90 ± 0.20 and 1.36 ± 0.44 fold to the control group,respectively.Two kinds of bacteria were injected after Of-MMP14 RNAi,and the expression of Of-Attacin、Of-Gloverin and Of-Moricin increased significantly,the expression of Of-Cecropin was significantly down-regulated.Meanwhile,the results showed that PO activity was down-regulated and lysozyme activity was up-regulated post the injection of two kinds of bacteria after Of-MMP14 RNAi.All together,our results suggested that MMP 14 was involved in the innate immune response of the O.furnacalis against bacterial infection.This study provides an ideal bridge for exploring the interaction between insect growth and development and innate immunity,and lays a foundation for further understanding the function of matrix metalloproteinases. |
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