Expression And Function Of The Chitinase MDCht9 In Musca Domestica

Objective:The phylogenetic tree of MDCht9 gene was constructed,The purified recombinant protein was obtained by prokaryotic expression system,and its enzymatic activity and stability were determined.The temporal and spatial expression patterns and functions of MDCht9 in the development of housefly were studied.Methods:Neighbor-Joining tree to study the classification of MDCht9 gene and its evolutionary relationship with Drosophila melanogaster,Anopheles gambiae and Culex quinquefasciatus chitinase.Analysis of physicochemical properties of MDCht9gene and encoded protein,signal peptides,and secondary structure of MDCht9 gene and its encoded protein,and to predict protein function;Primers were designed based on their cDNA sequences and amplified by PCR.The recombinant plasmid was constructed with pET28a?+?as vector and transformed into E.coli.The expressed product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The purified protein was identified by mass spectrometry.Chitinase activity assay:Recombinant protein stability and enzyme activity were determined using4MU-?GlcNAc?₃ oligomer as a substrate.Results:Neighbor-Joining tree indicates that the MDCht9 gene has the highest homology with the MDCht9.MDCht9 gene has a full-length cDNA of 1401bp and encodes 466 amino acids with a theoretical molecular weight of 50827.2 Da and an isoelectric point of 6.97.It is a hydrophilic protein.The amino acids 1-22 of the protein encoded by the MDCht9 gene are signal peptides,and the cleavage site is between the 22nd and 23rd amino acids.The functional domain of MDCht9 protein is located between amino acids 24-357 and is the catalytic domain of the 18 family chitin glycosyl hydrolase.The sequence between the amino acids 413-466 is the chitin binding domain.The secondary and tertiary structures of MDCht9 protein are shown in three types:predominantly random coils,followed by alpha-helical and beta-sheet.Construction of MDCht9recombinant expression plasmid with correct gene sequence and expression of recombinant protein in E.coli BL21?DE3?.The recombinant protein purified by affinity chromatography was identified by mass spectrometry and was consistent with the MDCht9 sequence,suggesting that the MDCht9 recombinant protein was successfully obtained.Enzymatic activity indicated that different concentrations of MDCht 9 recombinant protein had chitinase activity and showed a dose-effect relationship.MDCht9 has an optimum pH of 9,and has the best stability at pH 9;the optimum temperature is 45°C,and the heat stability is best at 40°C;Urea and metal ions have an inhibitory effect on MDCht9 enzyme activity,and Tris enhances its enzyme activity.Real-time PCR for detection of relative expression of MDCht9 gene in the different developmental stages of Musca domestica,with the egg stage as a reference.The relative expression levels of mRNA were ranked as 3rd instar larvae,2nd instar larvae,1st instar,male,egg,female,and pupa.The expression level in the flood season was no difference between the first day and the second day.The amount of expression from the most to the least is the first day,the fourth day,and the third day.In the different tissues of the 3rd instar larvae of the housefly,the epidermis was used as a reference,and the relative expression levels of mRNA were salivary gland,trachea,intestinal tract,fat body,malpighian tube and epidermis.Western blot was used to detect the expression of MDCht9 protein in different developmental stages of housefly.The expression of protein was from 3rd instar larvae,1st instar larvae and2nd instar larvae.The protein expression was consistent with the expression of nucleic acid level in males,and the protein level was consistent with the mRNA level.It is expressed in the main larval stage,and it is speculated that this gene may be involved in the molting of the larval stage.3.RNA interference with MDCht9 gene,the results showed that the best silencing effect was achieved at 24h after injection.Compared with the negative control group,the survival rate of Musca domestica larvae decreased by 23%,the emergence rate decreased by 31.8%,and the deformity rate reached 5%.Conclusion:The expression of MDCht9 in the growth and development of housefly was mainly in the larval stage,and the expression in the egg stage and the pupa stage was lower.The expression of salivary gland was the highest in the 3rd instar larvae of the housefly.The injection of dsMDCht9 effectively silenced the target gene compared with the negative control.In the group,the larval mortality increased and the emergence rate decreased,and the worm body could not be drilled from the clam shell,and the wing veins were incompletely deformed.