Cloning And Function Of BmICE-912 Gene In Silkworm, Bombyx Mori

Silkworm not only has the potential to became a good model organism,but also has been the ideal material to study apoptosis, it is dependent on the uniqueness of the silkworm, strain diversity, genetic variation of the polymorphism. Apoptosis is a necessary process for silkworm to complete metamorphosis. Analysis of apoptosis in silkworm is an important way to understand mechanism of metamorphosis. Caspase family plays an important role in apoptosis, but the research is still in its infancy. In this study, according to the Bmlce gene (GenBank accession number:AY885228), the special primers were designed, a new novel isoform of ICE named BmICE-912 is obtained from silkworm, analysis the function and information of the new novel isoform. At the same time, we analysis the relationship between the four novel isoforms. The results are as follows:1 Gene cloning and sequence analysis of BmICE-912 geneThe open reading frame(ORF) of the new novel isoform contain 912 base pairs, encoding 303 amino acids. Sequence analysis of BmICE-912 showed that it contains 8 exons and 7 introns. Protein of BmICE-912 gene contains the specific active site sequences QACRG and classic large and small subunits which is unique in caspase family member. The two homologous genes produce four novel isoforms of ICE in silkworm by two splicing ways.2 BmICE-912 protein has enzyme activity of caspase-1The activity of BmICE-912 protein of prokaryotic expression was studied by caspase-1, caspase-3, caspase-9 activity Assay Kit. Determination of each sample using UV spectrophotometric, Analysis the A-value data in 405 nm, the results may indicate that the activity of the target protein cleave the colorimetric substrate Ac-YYAD-pNA under saturated substrate concentrations in caspase-1. That is means that BmICE-912 protein has enzyme activity of caspase-1.3 BmICE-912 can inhibit apoptosis in silkworm cells and affect the growth of wing of silkworm We use lagged plasmid 1180hrsl000A4-BmICE-912-sv40 to transfection BmE cell in certain concentration, then BmE-SWU1 cells dealled with UVB, after 24 hours, The flow cytometry assay examination show that the apoptosis number of tansfection cells is 3.16%, but the control is 35.74%. These changes suggest that BmICE-912 can inhibit apoptosis in silkworm cells. In addition to, using RANi technology, by injection of siRNA (BmICE-912) into silkworm body, we found that the reduction of BmICE-912 gene expression, affecting the growth of wing of silkworm and resulting smaller wings in RNAi silkworm.