Express Sequence Tag Sequencing And Cloning-Expression Of Chitinase Gene From Chaetomium Cupreum

Chaetomium cupreum Ames is an important biocontrol fungus with effective control ability to some plant pathogenic fungi. The molecular biocontrol mechanisms were studied with the cDNA library construction, expressed sequence tags, bioinformatics analysis and transgenic technology to C. cupreum. It's significant greatly for the research and exploitation of new type microbial-biofungicide of C. cupreum.Two cDNA libraries of C. cupreum were constructed successfully with the mycelia grown on culture using glucose or chitin as carbon source. They were named mycelial cDNA library (abbreviation: C1 cDNA library) and induced mycelial cDNA library (abbreviation: C2 cDNA library), respectively. The titer and recombinant rate of C1 cDNA library was 0.93×106 pfu/mL and 94.2 %; and the average insert size was 1.3 kb. The titer and recombinant rate of C2 cDNA library was 1.02×106 pfu/mL and 95.3 %, the average length of cDNA inserts was 1.2 kb.ESTs sequencing and bioinformatics analysis were done to randomly selected cDNA clones. 3069 ESTs of high quality in C1 cDNA library were acquired which were assembled into 1471 unigenes including 392 contigs and 1079 singlets. Among them, 874 unigenes exhibited strong similarity to proteins in non-redundancy public databases representing known genes, the others were new genes with unknown function. The results of GO ontology showed that known genes with biological process function (61.75%) accounted for most represented level, then cellular component (4.60%) and molecular function (33.65%).1221 ESTs from C2 cDNA library were acquired which were assembled into 828 unigenes including 176 contigs and 652 singlets. 468 unigenes represented known genes; the others were new genes with unknown function. Genes with biological process function had the largest scale accounting for 63.31%, genes with molecular function and cellular component function accounted for 33.77% and 2.92%. The ESTs of two cDNA libraries of C. cupreum have been deposited in the GenBank database with following accession Nos. DV544375-DV548659. The analysis of genes different expression was done to C1 and C2 cDNA library. Limited inter-library overlap of gene expression (333) was seen and 1138 unigenes were library C1 specific and 495 unigenes were library C2 specific containing 11 unigenes with biocontrol function.The analysis of metabolic pathways enabled the identification of 70 metabolism including 67 in C1 and 63 in C2 cDNA library. The results demomstrated that C. cupreum may be induced metabolism of gluconeogenesis to maintain fast cell growth rate in response to the competition provided by the plant fungal pathogen.Twenty genes and 23 genes related to biocontrol were obtained in C1 and C2 cDNA library. The analysis revealed that the biocontrol function of C. cupreum was complicated which was involved in many mechanisms including the degradation of cell wall, hydrolyzation of proteases, antagonism and so on.The biocontrol genes in two cDNA libraries were different. The special genes related to biocontrol in C2 cDNA library were PTR2 protein (DV547977),β-N-acetylglucosaminidase (DV545166),α-glucosidase (DV548040) and ABC transporter (DV546007).The full-length cDNA, encoding 46kDa endochitinase (Chi46,EU142805) of C. cupreum was successfully cloned, and transformed into Saccharomyces cerevisiae H158 using the constructed vector of pYES2-Chi46. Optimal culture condition was investigated to transformant. Ferment optimization of transformant showed that high level of chitinase activity was induced to 1.96U in the medium containing 2.5 %β-galactose, 0.4 % chitinase, pH6.