Cloning And Expressing CDNA Of Key Enzyme's Gene Of Amygdalase

Armydase is crude enzymes,mainly containingβ-glucosidase and Hydroxynitrile lyase,and consisting in the seed of apricot,plum,peach,apple and so on,Armydase is correlated with the aromatic of plant and the phenomena of cyanogen,the phenomena of cyanogen is related to the resistance to natural defence of plant,which protect the plant fiom disease and pest.The function of the BGLU combined with HNLs is to degradate cyanogenic glycoside producing hydrogen cyanide,Nowday,it is also an important duty for the researcher to find the genes resources correlating with resistance and quality in the area of crop breeding,more and more researcher will concern them,because of the particularily important function of BGLU and HNLs.The earlier period findings indicated that,tobacco lacks the hydrolytic enzyme that can hydrolisis materials made up of fragrance material,the Armydase from almond(β-glucosidase from almond)can highly hydrolysis tobacco's glycoside, producing fragrance materials.we can clone this kind of materials's gene from the apricot tree and change it over to the tobacco,On the one hand,it can cause the tobacco to express this kind of enzyme which then hydrolisis the tobacco glucoside to release fragrance and enhances the fragrance quality of tobacco;On the other hand, through this enzyme indirect function,it can suppress the growth of fungus in tobacco, when the tobacco receives fungus invades.Prokaryotic experimental system of expression vector pET - 32a(+)and BL21trxB(DE3)strain were employed to express two kinds of tea BGLU(GluⅠand GluⅡ)cDNA gene.The impacts of different induction time and temperature on the expression amount of these two kinds of tea BGLU were studied and the activities of both kinds of BGLU were determined.The expression results revealed that the GluⅠshown the highest proportion of the total protein in the conditions of six hours at 25℃,and then GluⅡshown the highest proportion at six hours at 37℃.Both the resulted products exhibited normal BGLU activity,and both of them can hydrolysis PNPG which is an universal material that BGLU can hydrolysis,and GluⅡ(0.310±0.03 U·mL^-1)expressing product shown much higher BGLU activity than that of GluⅠ(0.234±0.03 U·mL^-1).Both of them can Weakly hydrolisis tobacco glucoside through GC technology,which provide the basis of eukaryotic expression of tea BGLU in tobacco. Through the reaction between the thick enzyme fluid from different organ of apricot tree and the tobacco glucoside,it has been discoveried that the enzyme fluid of almond can hydrolisis tobacco glucoside,and the apricot leaf did not have activeness.This experiment has analyzed the constitution of this kind of commodity enzyme,and has determined that this kind of protein is composed of one kind of proteen called nitrile lytic enzyme protein and other mixed protein.We obtained partial structure information of this kind of protein(amino acid sequence),through the technology of PMF(peptide mass fingerprint).We have designed two pairs of primers such as X1 and X2,X3 and X4 according to these informations and attempted clone the cDNA of this kind of HNLs proteen.