CDNA Cloning,Expression And Expression Pattern Of CCT Gene In Housefly( Musca Domestica)

Objective: The genes named MD-CCTζ were been screened from the EST sequencing database of Musca domestica and submited to Gen Bank to analyze its molecular characteristics. Then MD-CCTζ genes were cloned,expressed and analyzed its characterization. MD-CCTζ genes expression patterns in different tissues and developmental stages were also investingated. MD-CCTζ genes expression profile of the housefly exposed to different pathogenic microorganisms and heat stress were researched preliminarily. Methods: 1. Sequence analysis and clone,expresssion of MD-CCTζ:The homology analyses of the nucleotides and deduced amino acid sequence of MD-CCTζ were carried out using the BLAST programs at the NCBI. The open reading frame were forecasted. The domain, secondary structure, tertiary structure and subcellular localization of MD-CCTζ were predicted using bioinformatics analysis software. A Phylogenetic tree was constructed using the Neighbour-jonining method with the Molecular Evolutionary Genetics Analysis(MEGA6.0)package.The primer was designed according to its c DNA sequence.The gene coding for MD-CCTζ was amplified by polymerase chain reaction(PCR), and then was inserted into vector PEASY-E1 to construct recombinant plasmid, transformed into E.coli BL21(DE3), induced with IPTG.Recombinant protein was purified by His-Tag protein purification kit. 2.Tissue-soecific and developmental expression patterns of MD-CCTζ.The insect body of different developmental stages were collected from Musca domestica life history( eggs, three instar larvae, pupae,female,male);Anatomy of 3 instar larvae,6kinds of tissues and organs(fat body, salivary glands, midgut, trachea, markov tube and body wall) were choose.Total RNA was extracted for all the tissue samples then reverse transcription c DNA.Using GAPDH gene as reference gene, the expression of MD-CCTζ gene were detected by real-time quantitative PCR(Real Time PCR) technique in differential developmental stages. 3. Function of the MD-CCTζ genes in differences stress.Differences pathogenic microorganisms(Candida albicans, Escherichia coli and Staphylococcus aureus) were injected quantitatively into the third instar larvae of Musca domestica, then the samples were detected to observed different time points(3h, 6h, 12 h, 18 h, 24 h, 36 h, 48h) of MD-CCTζ gene expression levels after induction by real-time quantitative PCR.4.Heat stress third instar larvae of Musca domestica, then the samples were detected to observed different time points(0min, 15 min, 30 min, 1h, 1.5h, 2h) of CCTζ gene expression levels after induction by real-time quantitative PCR.Results: 1.Bioinformatics analysis showed that ORF frame full-length of MD-CCTζ was 1596 bp and MD-CCTζ gene have encoded 531 amino acids. Theoretical molecular weight of MD-CCTζ was 58260.47 Da and isoelectric point of MD-CCTζ was 6.56. MD-CCTζ no signal peptide and transmembrane domain,had two CCT family signature, and functional domain of CCT. The tertiary structure of MD-CCTζ composed of alpha-helix and beta-folding that were connected by irregular. Comparative analysis of the evolutionary tree showed that genetic distance about between housefly MD-CCTζ and Ceratitis capitata CCTζ was closer. MD-CCTζ expression vector successfully constructed, recombinant protein expressed by IPTG inducing,then purif protein were obtained. 2.Spatial and temporal expression patterns of MD-CCTζ gene in housefly indicated that;the expression of MD-CCTζ gene in each developmental stage of the housefly was difference. MD-CCTζ gene expression were highest in eggs(P<0.05), the expression level in order of size was eggs > female > second instar larvae > first instar larvae > pupa> male > third instar larvae.3. Expression levels of MD-CCTζ genes in the salivary glands and the trachea were significantly higher than that of the body wall.The expression levels of MD-CCTζ genes in various tissues in order of size was salivary glands> trachea > Markov tube> midgut > fat body> body wall. 4. MD-CCTζ genes were upregulated in 24 h after Candida albicans,Staphylococcus aureus,E. coli induction. MD-CCTζ gene expression trends have high similarity in three bacterial stress point in time. 5. MD-CCTζ gene of three instar larvae under heat stress decreased expression in 15 min upregulated,expression of 2h were significantly upregulated. Conclusion 1.This study have cloned successfully MD-CCTζ genes of housefly, analyzed the molecular characteristics of MD-CCTζ genes sequence. 2. The expression patterns of MD-CCTζ gene in housefly were revaled initially. The results suggest that MD-CCTζ gene plays an important role in the growth and development of Musca domestica. 3. MD-CCTζ genes expression were significantly up-regulated pathogenic microorganisms or heat stress in the housefly larvae,which suggest that these MD-CCTζ genes have involved in the stress defenses of housefly.