Construction Of Trichoderma Mutants By REMI And Exploitation Of Their Function

Trichoderma spp.was the traditional bio-control fungi to plant diseases.The single-functional Trichoderma spp.cannot meet the needs of clean agriculture production.Therefore,it was very important that multifunctional Trichoderma strains were screened.For obtaining multifunctional Trichoderma strains and producing new agent of Trichoderma spp.,the wild strain of Trichoderma spp.was improved through restriction enzyme mediated DNA integration.We studied the effects of Trichoderma mutants on degradation of crop debris,degradation of soil phosphate-compound and inhibition of weeds besides the function of preventing plant disease.For building Trichoderma spp.resource and screening,REMI was used to construct mutants from T.atroviride strain T23 and T.koningii strain CICC 40144. Totally,201 mutants were obtained.Successive culture,PCR and Southern blot analysis were applied to determine that plasmid pV2 was integrated into Trichoderma spp.gene.Frequency of single copy transformation reached 64.3%,which favored further study such as plasmid rescue.The conditions of mycelium growth and spore germination of mutants of Trichoderma strain T23 has been studied.The mutants could grow in a wide range of pH value(from 2 to 10),but the optimal pH value for mycelium growth was from 4 to 6.The optimal temperature for mycelium growth was 30℃.Sucrose and fructose were the best carbon sources and yeast extracts and peptone were the best nitrogen sources.As a whole,mutants were better than Trichoderma strain T23 in the aspect of using carbon and nitrogen.The spores of mutants could germinate in a wide range of temperature(from 15℃to 40℃),but the optimal temperature for spore germination was from 25℃to 35℃.In incubating for 28 hours,the rate of spore germination reached 100%.The spore could germinate in a wide range of pH value(from 2 to 8),but didn't germinate when pH value was 10. Illumination accelerated spore germination.The spore germination need direct proportion to the RH(relative humidity).As in the air,conidia of TE7 could germinate complete when RH reached 100%.The soluble proteins and esterase isozyme of Trichoderma strain T23 and four mutants were analysed with polyacrylamide gel electrophoresis.The results showed there were differences among mutants and between wide strain and mutants.Some of the bands of protein was lost in four mutants.TE7 has lost five bands of protein.The difference between esterase isozyme was more obvious.The strips of esterase isozyme were less than protein.It confirmed the protein were changed when a plasmid insertion,which established the base of theory for allocation and the clone gene further.For excavating more functions of Trichoderma spp.,11 mutants were screened for improved cellulase activity as compared with their parent strain.Higher cellulase activity of parent strain had,less improvement of mutants.Moreover,one mutant, TK-2R-14,was found with the highest CMC(37.6 IU.mL-1) and FPA(16.9 IU.mL-1) activities among all screened mutants,which were increased by 8.05%and 19.07%, respectively.The optimal condition for solid fermentation of TK-2R-14 was carried out for higher cellulase production.The results based on orthogonal experiment showed that the optimal solid materials components were determined as follows: ratio of rice straw to bran 9:1,the ratio of solid materials to water 1:3 in conjunction with(NH₄)₂SO₄ 1%and Tween-20 0.1%.Moreover the single fermentation factors were also crucial to the mutant for improving cellulase enzyme activity,inoculum volume 5%-7.5%,culture temperature 30℃.culture time 96 h and neutral pH value were best for fermentation.The FPA and carboxyl methyl cellulase activity of TK-2R-14 were 6.097 IU/g and 8.123 IU/g respectively under these optimal conditions.Four phosphate-degradading mutants were screened from T.koningii REMI mutants,named TK-2,TK-38,TK-46 and TK-47.TK-46 of them was the best because it's phosphate- degradading capacity was 363.79μg / mL,increased 41% compared with their parent isolates.Studied the optimal condition of TK-46 for phosphate-degradading,the results showed that the optimal carbon source was maltose,the optimal nitrogen source was ammonium chloride or ammonium sulfate, the optimal pH value was 7.0.The phosphate-degradading capacity to CaHPO₄ was far greater than to AIPO₄.But it didn't dissolve FePO₄.It was discovered that the decreasing pH value of culture mediums was not necessarily to the degradation of phosphate,but the degradation of phosphate could lead to pH value decrease.After screening for barnyardgrass-inhibition of REMI mutants of Trichoderma spp.,eight mutants were obtained.They are TA2,TB3,TB7,TE2,TE5,TH8,TJ6 and TJ9.Their culture filtrates(CFs) have bamyardgrass-inhibition activities,and are safe to rice.In Secondary experiments,the bioassays of the spore suspension,ultrasonic broken spore PBS suspension treatment and CFs without spores are conducting.Thus mutant TB3 was the best one.For analysing barnyardgrass-inhibition component of TB3,some extract experiments and protein precipitation were carried on.The result was that barnyardgrass-inhibition component from TB3 was neither gliotoxin nor viridiol,but protein.The protein could lose activity by acetone precipitation,but preserves activity by ammonium sulphate precipitation.Furthermore,ammonium sulphate of high saturation was applicable for the protein precipitation.We must study which kind of protein was the protein further.In purpose of expounding the phenotype change of Trichoderma mutants,protein profiles of T.koningii CICC 40144 and it's mutant TK-2R-1 were detected by 2-DE. Eight unique spots were found and four of them were identified their functions by MS-TOF-TOF which were connected with growth and metabolization of Trichoderma spp.Furthermore,the frank fragment of TB3 and TK-2R-1,TK-2R-14,TK-2R-15 genomics DNA and restriction integration site characterization by REMI was separated and analyzed by plasmid rescue.But the sequence of function gene had not been found in Blast database.Therefore they were unknown amino acid sequences possibly.Searching ORF in ORF Finder,enough big ORF of code protein wasn't found among these sequences.The effect of these sequences would been studying further.