| Chitin is a linear biopolymer of N-acetyl-?-D-glucosamine(Glc NAc),covalently linked by ?-(1-4)glycosidic linkages.It is widely distributed in the trachea,integument and the peritrophic matrix of mid-gut as the important structural component.Since chitin metabolism is of vital importance to insect growth and development,the enzymes involved in this process could be potential targets for designing eco-friendly pesticides.Lytic polysaccharide monooxygenases(LPMO)is a new type of oxidase that acts on crystalline polysaccharides,which can cleave glycoside bonds of chitin by an oxidative function.This reaction makes the structures of substrate becoming tractable for further degradation by glycoside hydrolase system.The gene encoding LPMO is widely distributed in the insect genome.The CAZy database classifies this non-microbial-derived LPMO into the 15-family auxiliary activities(AA15).However till now,information about this new family LPMO is still unkown,especially on gene expression profiling and physiological function.In this paper,we focused on AA15 s from Lepidoptera model insect Bombyx mori,Orthoptera model insect Locusta migratoria and agricultural pest Ostrinia furnacalis,provided more basic understanding on how AA15 s function during insect growth and development.The main contents of this thesis include:(1)The AA15 genes of B.mori,L.migratoria and O.furnacalis were obtained by homology analysis and PCR.The AA15 family included three members,namely AA15-I,AA15-II and AA15-III.Phylogenetic analysis showed that BmAA15,OfAA15,and LmAA15 have high sequence identity.They all contained the consrved residues,including the histidine and tyrosine involved with catalysis as well as the aromatic amino acids involved with substrate binding.(2)Real-time PCR results showed that,BmAA15-I,OfAA15-I,LmAA15-I and LmAA15-II had the highest expression level in the epidermis and trachea.BmAA15-I and OfAA15-I were highly expressed in pupa while LmAA15-I and LmAA15-II were significantly up-regulated during locust nymph-adult transition.BmAA15-II and OfAA15-II had the highest expression levels in the testis,and mainly expressed during the pupa-adult metamorphosis.The expression levels of BmAA15-III and LmAA15-III in the mid-gut were higher and they were up-regulated during molting.It is speculated that different AA15 s function at different stages during insect development and they may play important roles in different tissues.(3)To investigate the biological roles of AA15,we performed a RNA interference(RNAi)using the newly molted fifth-instar nymphs of L.migratoria.After the interference,the expression level of each of the AA15 genes was significantly down-regulated,while no significant decrease occurred in the transcript level of the non-target genes.After RNA interference of LmAA15-I,90% of locusts were abnormal,6.7% of fifth instar nymphs died,60% of nymphs failed to shed the old cuticle,and were trapped within the exuviae until death,13.3% nymphs were able to molt to the next stage,but exhibited crumpled wings.Injection of dsLmAA15-II caused an abnormal rate of 20%.After RNA interference of LmAA15-III,100% of nymphs were abnormal.66.7% of the fifth-instar nymphs failed to molt,and died finally.33.3% of nymphs were able to molt to adults,but exhibited abnormal phenotype.It is speculated that LmAA15-I and LmAA15-III may play important roles during the growth and development of locust nymphs.(4)The AA15 domain of Of AA15-I was succesfully heterogeneous expressed in the E.coli cell.It was expressed as soluble form with a fusion Nus-tag at the N-terminus and the yield was 20 mg/L.The recombinant proteins were then purified by metal-chelating affinity chromatography.OfAA15-I could release reduced sugars when using ?-chitin and ?-chitin,but its synergistic activity with chitinase H during the hydrolysis of chitin substrate was not obvious. |
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