The Study On The High-productiont Trehalose Strain By Using Protoplast Electrofusion Technique

As2.1416 produce trehalose highly ,As.l887 with STA gene could degrade starch.In order to construct the highly producing trehalose strain which has the ability of saccharificition.the research introduce STA gene into As2.1416 by means of protoplast electrofusion technique.Factors of protoplast formation,regeneration and parameters of eletrofusion were study in the research.Looking forward to finding the regulation and optimum parameters,the research will offer some data for microorganism breeding by using protoplast electrofusio and contribute to clarify the mechanism of fusion.The result of the research as follows:1 Influencing factors of formation and regeneration of protoplast1) From plenty of trials,the conformation is:the optimum concentration of snailase 0.3%;the time of snailase treatment 20min;the temperature of snailase treatment 30-37 t;the pH of snailase treatment 6~7.2) Contrast the connection of different usages of B-mercaptoethanol to the formation and regeneration of protoplast.The final scheme is the blend dissolution liquid of snailase and B-mercaptoethanol.3) The amount of protoplast is fewer than synchronization culture's,which go against the fusion.So the synchronization culture is necessary.The experiment adopt the method that the fungus activated 24h is put into the refrigeratory 4V, 2h.4) Contrast the effect of the different osmotic stabilizer of5) Confirm the parameter of 0.6% 32min and 60C 45min.2 The choice of fusion parameter1) The results of the experiment show the two fungus have different characteristic of string.The peak value of ideal cell string of As2.1416 is on the 200v/cm,the As.1887 is onthe 250v/cm.So the electric field intensity is 250v/cm in the experiment.2) The protoplast has different velocity in the electric field,the adjustment of protoplast density is necessary to have the maximal ratio of ideal string.The density is 105.3) The stillness time of protoplast is confirmed.The high ratio of ideal string is on 60second.4) After the adjustment of electric density.the ratio of ideal string improve from 45% to 55%,50% to 59% separatly.The conclusion is that the adjustment has evident effect on the ratio of ideal string.5) The impulse intensity is 5.5v/cm,the livability of protoplast is 71 % 61 %.6) The impulse time is 30second, the livability of protoplast is 77% 70%.7) The impulse number is 5, the livability of protoplast is 72% 69%. From the fusants.a strain could produces trehalose and degrades starch.