Cloning, Expression And Function Study Of BmDio, An Aromatic Ring Dioxygenase Gene In The Silkworm, Bombyx Mori

Horizontal gene transfer (HGT), which was also known as gene lateral transfer, refers to gene transmission between different organisms or organelles in the cells. On the contrary, vertical gene transfer refers to genetic materials inherited by their ancestors. Along with deep research of genetics, people constantly reveal the different levels of gene transfer, broaden the concept of gene exchange, and found it the great significance in the field of organism evolution. As an important model for Lepidoptera, silkworm plays an important role in classical genetics research. The research of horizontal gene transfer in silkworm is rare, and the functions of most horizontal transfer genes detected are also not clear. In this study, we selected aromatic ring Dioxygenase genes, a type of horizontal transfer genes in silkworm, as the research subjects and cloned and characterized the function of this gene. The main results were as follows:1. Cloning and sequence analysis of BmDioThere are two copies of Dioxygenase gene in silkworm (Bombyx mori), and the length of the two genes is 819 bp and 885 bp, respectively. The similarity of the amino acid sequences encoded by the two duplicate genes were 62.2%. Specific primers were designed according to the EST and genome sequence of the silkworm, and coding sequence of BmDio was cloend. The results indicated that the CDS of BmDio was 819bp in length and located on the 24th chromosome. There was no intron in BmDio, which encoded a protein with 272 amino acids. The predicted protein molecular weight was 30.8 kD and the isoelectric point is 6.23. 2. Prokaryotic expression of BmDioThe complete CDS of BmDio was subcloned into the pET-28a(+) expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21. After induced by IPTG, a recombinant protein of about 32kD was obtained and the conditions of soluble recombinant protein were 37℃,1 mmol/L IPTG induced 5 hours through optimization.3. Expression pattern of BmDioWe determined the expression profile of BmDio gene at the different developmental stages and in various tissues by RT-PCR analysis. The results revealed that the transcript of BmDio was almost detected in the entire life cycle and all the tissues of the 3rd day of the fifth instar.BmDio protein produced by prokaryotic expression system was purified by affinity chromatography, and then used to produce polyclonal antibody. Protein was extracted from B. mori of the 3rd of 5 instar and Western blot analysis were performed to investigate the expression of BmDio protein. The result is similar to the microarray database analysis, except for that the protein of BmDio was not detected in blood in this study.4. The analysis of the function of BmDioWe utilized the specific color reaction involved in BmDio protein to carry out a preliminary function study. Crude enzyme extract was prepared and appropriate amount of catechol substrate solution was added into supernatant of crude enzyme extract. After incubation at 30℃for 30 minutes, OD375 value was detected with spectrophotometer. The results showed that BmDio had the activity of the typical aromatic ring Dioxygenase.In conclusion, we cloned the BmDio gene in silkworm. BmDio was expressed in the prokaryotic expression system, and pure protein was obtained by purification. We investigated the spatio-temporal expression pattern on mRNA level and protein level, respectively. Finally, we characterized the function of BmDio primarily. The results showed that BmDio was generally expressed in all the periods and tissues and encoded an aromatic ring Dioxygenase with the corresponding activity.