| Excellent biocontrol effect of Trichoderma on various plant fungal diseases was identified,in order to study the biocontrol mechanism of Trichoderma,transcriptomic database of Trichoderma harzianum ACCC30371 strain was constructed firstly and quality Unigenes were got,after that biocontrol genes from transcriptome were classified according to function of biocontrol;two biocontrol functional genes were selected from database and studied further,eliciting plant response protein gene epll and protease gene ss.The results are as follows:Transcriptome of T.harzianum ACCC30371 strain was constructed after it grown in 8 culture media as follows:1/2PD,minimal medium MM(containing dextrose lOg/L),C starvation medium(derived from MM without dextrose),N starvation medium(derived from MM without ammonium sulphate),and C starvation medium harboring 1%(w/v)powder of phytopathogenic fungi Sclerotinia sclerotiorum,Alternaria alternate,Rhizoctonia solani,Cytospora chysosperma respectively.The transcriptome database has a size of 4Gbp and 96.68%of it has a sequencing error rate not higher than 1%.Data statistics was carried out according to this criterium and 25013 Unigene sequences were obtained.Mean length of them is 1135 nt and 2571 of them are over 3000 nt.16360 Unigenes were annotated to Nr protein database,9875 were annotated to SwissProt database,10266 were annotated to KEGG database,7164 were annotated to COG database,1508 were annotated to GO database along with their protein functional annotations.16723 functional genes were found.402 bio-control genes were classified into four major categories by analyzing transcriptome data of T.harzianum,including 14 genes related to competition,311 genes related to mycoparasitism,76 related to antibiosis and 1 related to eliciting plant response.It reveals that T.harzianum ACCC30371 has integrated biocontrol mechanisms and mycoparasitism is the most important in proportion,antibiosis and induced systemic resistance(ISR)play also important roles.The results possess extremely important significance in further researching bio-control mechanism of Trichoderma and developing and utilizing biocontrol functional genes and biological fungicides.The full-length cDNA and promoter were cloned of gene epll.The cDNA sequence of it has been submitted to GenBank and number is KC876056.Protein of it belongs to Cerato-platanin protein family(Pfam07249).15 similarity proteins of Epll were found on four Trichoderma genomes,which came from T.atroviride,T.virens,T.harzianum,T.asperellum.The conserved domains of them had a length range of 113-120.Epll-Tha which was located on chromosome scaffold 6 of T.harzianum CBS 226.95 had a similarity of 100%with Epl1.The expression of gene epll on transcriptional level under 8 nutrient conditions was studied using RT-qPCR technique and revealed that expression level of epll increased periodically over time when T.harzianum grown in the MM medium.And the amount of expression of epll increased continuously when T.harzianum grown in medium which powder of roots,stems and leaves of Shanxin-poplar was added respectively and even over 32 times control team amount.The amount of expression of epll increased rapidly from T.harzianum grown in C starvation medium and tended to be constant after having reached a certain level and small amount of up-regulated expression of epll was found when T.harzianum grown in N starvation medium.The amount of expression of epll rose when mycelial powder or zymotic supernatant of A.alternata was added in medium,but the degree of change was lower than that of MM at the same time point.Overall,regulated expression of epll appeared when T.harzianum grown in the eight inducing media.The ecombinant vector pGEX-epll and the recombined strain BL21-epll were successfully constructed.SDS-PAGE analysis determined an over-expression protein band,which had a molecular weight of 40.5-kDa similar to the predicted.It laid the foundation for protein production,separation and functional studies.The full-length cDNA of gene ss was cloned successfully.The cDNA sequence of it has been submitted to GenBank and number is KC876057.Protein of it belongs to Peptidases_S8_PCSK9_ProteinaseK_like protein family(Pfam00082).21 similarity proteins of SS were found on four Trichoderma genomes,which came from T.atroviride,T.virens,T.harzianum,T.asperellum.SS1-Tha which was located on chromosome scaffold 6 of T.harzianum CBS 226.95 had a similarity of 100%with SS.Analysis of regulatory-elements-binding-motifs in promoters of these serine protease coding genes on four Trichoderma genomes indicated that serine protease is closely associated with control of Trichoderma spp.The expression of ss on transcriptional level under 8 nutrient conditions was studied using RT-qPCR technique and revealed that the expression level of gene ss changed between 0.7and 3.8 times of control team amount.Overall,expression level of ss changed little when T.harzianum grown in the eight inducing media.The recombinant vector pGEX-ss and the recombined strain BL21-ss were successfully constructed.SDS-PAGE analysis determined an over-expression protein band,which had a molecular weight of 68.7-kDa similar to the predicted.Results of this study provide a theoretical support for the development of biological pesticides from T.harzianum and a practical reference for exploiting proteases in biological pesticides. |
