Molecular cloning, sequencing and expression pattern of chaperonin gene in housefly-Musca domesitca

Objective To clone and analyze a novel cDNA, named as Musca domesitca chaperonin containing tailless complex polypeptide η(MD-CCTη)and to detect relative expression levels of MD-CCTη by real time quantitative reverse transcriptase PCR(QRT-PCR).Methods The total RNA was extracted from Musca domesitca larvae of 3rd day;MD-CCTη gene was cloned with PCR and then ligated into vector pMD-19T and transformed into Escherichia coli DH5α competent cells.The relative expression levels of MD-CCTη were detected in different tissues of the larvae of 3rd day and during different developmental stages by QRT-PCR.Results Sequence analysis indicated that the open reading frame was 1 632 bp,encoding a putative protein consisting of 543 amino acids,with a predicted molecular weight of 59.3 kDa and an isoelectric point(pI)of 6.27.The analysis using National Center for Biotechnology Information-Basic Local Alignment Search Tool(NCBI-BLAST)confirmed that the deduced amino acid sequence shared high identity with the reported homologous sequences from other insects(93%).Cluster analysis results showed the genetic relation of Musca domesitca was more close to that of C.capitata and Drosophila(100%).QRT-PCR showed that the expression of MD-CCTη was the highest in the egg,followed by that of in the larvae of 1st day,and lowest in male adult during development;whereas the the expression of MD-CCTη was the highest in the trachea,medium in the salivary glands,and the lowest in the body wall of 3rd day larvae.Conclusion MD-CCTη was cloned and mRNA expression levels of MD-CCTη were different during different development period and in the different tissues of Musca domesitca.