Clone And Functional Analyses Of MaBBI In Musa Acuminata

BBI(Bowman-Birk type protease inhibitor)is a plant protease inhibitor and rich cysteine,which mainly inhibits serine protease including trypsase,chymase,elastase and so on.In this experiment,we cloned 4 MaBBI genes(MaBBI1,MaBBI2,MaBBI3 and MaBBI4)from banana(Musa acuminata L.cv.Brazilian).Preliminarily studied the functions of 4 MaBBI genes mentioned above in response to stresses through bioinformatics,gene expression,yeast complementary,Arabidopsis thaliana transformation,subcellular localization and so on.The major results were shown below:1)The bioinformatics analysis showed that MaBBI1,MaBBI2,MaBBI3 and MaBBI4 both contains a BBI domain,each BBI domain includes 2 protease binding sites.In addition,MaBBI1 contains a Bowman-Birk leg,while MaBBI2,MaBBI3 and MaBBI4 not.2)The gene expression analysis displayed that the expression of MaBBI1,MaBBI2 and MaBBI3 are highest in roots of seedling,while MaBBI4 in leaf sheaths.The expression of the genes mentioned above were influenced by different plant hormones and environmental stresses.It’s worth noting that MaBBI1,MaBBI2,MaBBI3 and MaBBI4 both were induced by JA in roots.What’s more,MaBBI1,MaBBI2 and MaBBI4 were all induced by MV in leaves,and MaBBI1,MaBBI2 and MaBBI3 both answer the CdCl2 stress.These suggest that the MaBBI mentioned above may as defense genes involving in the answer of abiotic and biotic stresses in Musa acuminata.3)GST-MaBBI1,GST-MaBBI2,GST-MaBBI3 and GST-MaBBI4 fusion proteins were all insoluble after prokaryotic expression.We obtained the soluble fusion proteins through denaturing and renaturing the inclusion bodies.However,the soluble fusion proteins renatured do not show obvious antimicrobial effect to Fusarium oxysporum f.sp.cubense race 4 in vitro.4)Yeast complementation test shown that MaBBI1,MaBBI2,MaBBI3 andMaBBI4 were all oxidation resistance,leaving the oxidation sensitive type wine yeast strains Δyap1 and Δskn7 recover the oxidation resistance in varying degrees.At the same time,overexpression the MaBBI mentioned above in arabidopsis thaliana also shown stronger oxidation resistance than wild type.5)Subcellular localization displayed that MaBBI1,MaBBI2,MaBBI3 and MaBBI4 both widely distributed in cytoplasm.Besides,MaBBI3 and MaBBI4 may more concentrately distributed in nucleus or certain organelle.