Ascosphaera Apis Construct CDNA Library And The Qualitative Research Of Extracellular Enzyme

Bee chalkbrood disease,caused by a pathological fungi,Ascosphaera apis mainly infect the honeybee brood.It is one of the main honey disease with great potential effect on apiculture development in China.The present research on chalkbrood are mainly on biological character of the Ascosphaera apis such as the optimal cultivating conditions,prevalence situation,pathology and diagnosis etc.Their pathogenic mechanism is still not clear today.How to control the disease is under exploration.A cDNA library was constructed using ZAP Express(?)cDNA Synthesis Kit and ZAP Express(?)cDNA Gigapack(?)â…¢Gold Cloning Kit from Stratagene.The titre of the cDNA library was 10⁶ pfu·mL^-1,the titer of the amplification cDNA library was 10⁹ pfu·mL^-1,and the recombination rate is over 90%.Such a work will provide a platform for future ectracellular proteinase research.Peritrophic membrane is a thin layer membrane in midgut.It is consisted of protein,chitin and other carbonhydrate compounds.The fungi Ascosphaera apis infect the honeybee brood by secreting the extracellular enzyme such as proteinase,chitinase, esterase and amylase.In this study,we certificated that the extracellular enzyme including proteinase,esterase and amylase by activity staining and silver staining methods.There are two proteinase bands with molecular weight of 37.0 kDa and 110.0 kDa,three bands of esterase bands with molecular weight of 14.1 kDa,20.1 kDa,29.0 kDa under SDS-PAGE repectively.In the condition of PAGE,there are two proteinase bands with molecular weight of 48.0 kDaå’Œ94.0 kDa,one esterase bands with molecular weight of 35.0 kDa and three bands of amylase with with molecular weight of 90.0 kDa,97.2 kDa and more than 97.2 kDa;we also explore the chitin binding protein in the extracellular compositions using their chitin binding capability for different time under PBS buffer system.The binded protein were eluted by SDS-PAGE loading buffer and 1%Calcofluor(M2R)respectively.The results showed that the binding in PBS with pH 7.4 for 6 days and eluted with SDS-PAGE loading buffer was the best binding and elution parameter.