Construction Of Trichoderma Harzianum Induced CDNA Library And Est Sequences And Analysis

Trichoderma harzianum was a biocontrol related fungus widely used as an efficient biological fungicide. In order to acquired biocontrol genes and explore the antifungal mechanism, a unidirectional cDNA library was constructed from mycelium of T. harzianum induced by oligosaccharide.Here, a cDNA library using Trichoderma harzianum mycelium growing on the culture with colloidal chitin at 48h was constructed. Then clones from cDNA library are sequenced from 5'end, and analyzed by bioinfomatics tools to screen biocontrol genes.The titer of original library was 1.06×106 cfu/mL and the recombinant efficiency was 93.4%. The average of insert size was above 700bp. The redundancy of the cDNA library was 3.7% after randomly selected 151 clones was sequenced and annotated. The cDNA library sufficed the criteria on EST analysis with high quality and wide representatives 1632 clones of the cDNA library were randomly selected to be sequenced by single pass sequencing. 1386 EST were achieved after discarding the sequences representing vector, rRNA, mitochondrial DNA and shorter than 100bp. and then was deposited in the GenBank database. The 1386 high-quality sequences were integrated into 915 unigenes, consisted by 463 contigs and 1277 singlets. After BLAST alignments on Internet, 235 novel ESTs were unkonwn, which pieced together and expected to create 142 novel unigenes. Of all known genes, the largest numbers of the clones were associated with biological process by Gene Ontology (GO) analysis, and the proportion of which was the highest. Bio-control related proteins were encoded by 41 genes from the induced cDNA library, and the proportion of bio-control genes was upto 4.5% in total number of genes of this cDNA library, which was higher than 3.7% from Trichoderma harzianum mycelium cDNA library constructed by normal incubation. Based of bio-control mechanism to resist pathogeny fungus, the protein proction expressed by 41 genes were divided into cell wall degrading enzymes, protease, antibiotics, cellular component and protective enzymes after bioinformatics analysis.All the results indicate that a successful gene expression profile of...