The Inducement Expression Of The β-mannanase From Trichoderma Reesei RUT-C30 And CDNA Cloning Of β-mannanase

This experiment passing to grope for the carbon source constitutes of the culture medium and using T. Reesei Rut C-30 induced the expression of #-mannanase ( # -1,4-Mannan mannohydrolase EC 3.2.1.78).In this experiment I put the constant carbon source(lactose and locust bean gum) in the foundation culture medium (Mandels nourishment liquid) of T. Reesei Rut C-30,then proceeded the variable carbon source(dragon spruce fiber, com rush pith fiber, wheat straw fiber, wheat straw xylan, corn rush pith xylan, dragon spruce mannan) to single factor,double factor,three factor,four factor and five factor orthogonal experiment.1 determined the activity of P -mannanase using locost bean gum as substract by the 3,5-dinitosalicylic acid method,and observed the growing situation of the gernic At the end I selected the directions for the inducement expression of the #?mannanase from Trichoderma reesei Rut-C30 that contained the dragon spruce fiber, wheat straw xylan, dragon spruce mannan.The total RNA was purified from the germ in the liquid by the guanidine isothiocyantehod method, then the total RNA digested by DNase that had not RNase was used for RT-PCR.I change the magnesium ion dencity in the PCR system in order to optimize the PCR condition.At the end I selected the magnesium ion density as 1.25 mM.The production of RT-PCR was inserted directionally into pGEM?Z(ampr).The pGEM?Z(ampr) was used to transform E coli JM109.I got a positive clone through culling and identificatin.The DNA sequence inserted into pGEM?Z(ampr) was sequenced and blasted with the cDNA sequence of the # -mannanase mature peptide that got from Genbank. The result of the alignment was 100%.