Establishment Of Dual-color FISH Technical System On The Chromosomes Of Silkworm, Bombyx Mori

Silkworm gradually become an important model organism because of its width of strain,high multiplication rate, reproduction of large amount and other characteristics, silkworm has important value in classics heredity and other search.Because of silkworm of the enormous economic value and scientific significance, the study of the silkworm has become the latest hot spot.With scientific and technological development, in particular the success of silkworm genome mapping, so that research get in the silkworm genome and post-genome era.As the number of silkworm chromosomes have more diffuse centromere of each other was not significantly different and difficult to sub-band, no fixed arm ratio of the limitations of their own, limiting the silkworm classical genetic studies, and hindered the direction of classical genetics to molecular biology.So silkworm chromosome research concentrated on the structural characteristics of chromosome morphology observation, body chromosomes and stain identification and other primary level, due to positioning, Molecular Genetic Linkage Map Construction and the relationship between chromosomes and genetic research and other hot spots are very limited.The need for a molecular biology connected with the classical genetic techniques, in which case FISH techniques have sprung up everywhere.FISH technology can make DNA molecular markers visually locate on the chromosome,FISH is more simpler,intuitive, faster and accurate compared to traditional methods.In the silkworm genetics, genomics and post-genomics research, especially physical map,FISH has played an important role.The successful establishment of silkworm FISH technical system and silkworm Two-color FISH technical system,are helpful to research cutting-edge and hot issues such as finding ways to penetrate the field of molecular biology to classical genetic research,and also accelerate silkworm research into genome and post-genome era.FISH systems is very important to silkworm research.Taking Dual-color FISH technical system as the technique,we locate silkworm ubiquitin protein ligase gene (BGIBMGA013548-TA) and the serine protease gene (BGIBMGA010063-TA),and obtain the following results.1.The establishment of silkworm Dual-color FISH technical system Optimization the condition of silkworm Dual-color FISH technical system,construction of a Dual-color FISH for chromosome silkworm technology system.The gland of chromosome at the second day instar 4 is more suitable for FISH experiments. PCR is more effective instrument to probe denatured. Proteinase K digestion of the optimum conditions for 1μg/ml,37℃5～10 min incubation.Co- degeneration is important for chromosome morphous,80℃,2min is appropriate. 1000bp probe hybridization at 37℃for 24h can meet the needs of hybrid. At 42℃with 50% formamide/2×SSC washing 3 times, every time 5min can be effective in reducing the "noise" on the impact of hybrid. The Anti-Dig fluorescence incubat with Cy-3 is an important step to obtain a high quality dual-color FISH results. Through the optimization of the above aspects, using probe DIG-3077, Bio-2983,FISH experiments carried out to obtain a clear, obvious and credible dual-color signal.2.Location of the Bombyx mori gene ubiquitin protein ligase gene (BGIBMGA013548-TA) and the serine protease gene (BGIBMGA010063-TA) with FISHThrough the silkworm genome analysis, respectively, on the chromosome 4 at scaf3077, scaf2847 and chromosome 7 at scaf2983,scaf2912,we select the reported silkworm gene ubiquitin protein ligase gene (BGIBMGA013548-TA), silkworm mRNA splicing factor (BGIBMGA006022-TA) and the serine protease gene (BGIBMGA010063-TA), Bombyx mori calcineurin Connexin gene (BGIBMGA008719-TA) for FISH localization. The results as follow:every gene has a single hybridization signal in the pachytene of gland's chromosomes that are single-copy gene. Single-color FISH results showed that the ubiquitin protein ligase silkworm gene, serine protease genes are located at different chromosomes' ends and the central. By dual-color FISH combined with mRNA splicing factors, calcineurin connection silkworm gene mapping results show that silkworm ubiquitin protein ligase gene, serine protease gene located on chromosome 4 and chromosome 7.The above results are equal to Silkworm genome analysis.By effective location ubiquitin protein ligase gene and serine protease genes prove that the constructed dual-color FISH technique for silkworm chromosome research is feasible,reliable, more efficient and more direct in practical than single-color FISH.