Regulation On Expression Of Trichoderma Asperellum Endochitinase 42 Gene And Construction Of Engineering Strain

Trichoderma sp.has great potential in biodegradation and biocontrol.The main purpose of this thesis is to confirm the regulator protein of one of the most important biodegradation protein,ech42;and the way to transport inducer into cell.After that genetic methods would be applied attempted to increase the expression of chitinases including ech42,in order to further exploit potential of Trichoderma sp.biocontrol activity.After confirmation of chitin degradation of T.asperellum,promoter region of ech42 was aquired by Tail PCR and binded with Biotin.Combine intracellular proteins of trichoderma when chitin was only carbon source and promoter sequence with biotin.Collecte specifically binded proteins and analysed with mass spectrometry,cdk5/pho85 protein was the regulator.After changed the expression level of cdk5/pho85,result revealed that when gene expression level of cdk5/pho85 changed to 30 % and 180 % relative to WT,gene expression of ech42 changed to 38 % and 170 %,protein expression level changed to 20 % and 180 %.This result suggested that cdk5/pho85 could change the expression level of Ech42,therefore cdk5/pho85 is one of regulator of ech42 gene.The sequencing result showed that the ABC transporter of T.asperellum was exporter protein abc-b protein.RNAi and expression were applied to abc-b sequence and result revealed that when gene expression level of abc-b changed to 23 % and 161 % compared to WT,the gene expression level of ech42 changed to 12 % and 230 %,protein expression level changed to 0 % and 400 %.When gene expression level of abc-b significantly reduced,trichoderma can not grow when chitin,maltose,cellulose,sucrose were only carbon source respectively but when glucose was only carbon source,trichoderma lost the ability to transport chitobiose;when gene expression level of abc-b increased ocerespression strain grew faster than WT and RNAi except for glucose condition,efficiency of chitobiose transport ability is also higher than WT and RNAi.Enzyme activity of ech42 from abc-b RNAi strain can not be detected when chitobise was only carbon source;enzyme activity of overexpression strain was higher than WT and RNAi.All these results revealed that abc-b transport chitin oligose and chitobiose is inducer of many chitinases including ech42.After double overexpression of cdk5/abc-b,the conditions of fermentation were optimized by single factor experiments and response surfaces to maximize the expression of chitinase.As a result,enzyme activity of cdk5/abc-b double overexpression strain was increased to 11 times as non-optimized WT strain,therefore our goal of increasing the production of specific biocontrol protein was achieved.This thesis confirmed regulator protein cdk5/pho85 to ech42 gene and transport system of chitinase inducer.Gene engineering strain was constructed and certain basic theory of trichoderma biocontrol activity was provided.