Cloning And Functional Characterization Of ThChsC Gene Of Trichoderma Harzianum Th-33

Trichoderma harzianum is a widespread and important biocontrol filamentous fungus. Chlamydospore preparation is the most promising future formulation of T. harzianum. Chitin , the major component of the cell wall of the hyphae and spores of filamentous fungi, plays an important role in the maintenance of cell structure and function. Chitin synthase (CS, EC 2.4.1.16) is the key enzyme in the synthesis of chitin. In this research, cloning and silencing of the Chitin synthase gene named ThChsC from T. harzianum were being applied to identify the gene responsible for a detectable phenotype and to characterise the function of the gene. The results were as follows:1. Cloning of chitin synthase gene ThChsC from T. harzianum Th-33The conserved sequences were identified by alignment of the sequences of chitin synthase gene deposited in the nucleotide database of NCBI. The degenerate primers were designed according to the conserved sequences and 372 bp fragment was obtained. It shared 91% similarity with chitin synthase gene (L24910.1) from Sporothrix schenckii. Then, we designed 6 primers based on the 372 bp fragment for cloning of the full length of the ThChsC by chromosome walking and obtained the sequence of 4110bp.2. Bioinformatics analysis of chitin synthase gene ThChsCThe open reading frame (ORF) of ThChsC (HQ419000) spaned 2835 bp was composed of 4 extrons and 3 introns. The ThChsC encoded a protein of 895 amino acids and shared 80%, 81% identity with Gibberella moniliformis chsB (ACY08039.1) and Colletotrichum graminicola chs2 (AAL23718.1) respectively. Phylogenetic analysis indicated that ThChsC belonged to the type III Chitin synthase subfamily. The conserved Chitin_synth₁, Chitin_synth₁N and Chitin_synth₂ domains and three transmembrane domains were found in the predicted amino acids sequence. A conserved DHD domain of glycosyltransferase was found in the tertiary structure of ThChsC predicted.3. Construction of chitin synthase gene ThChsC mutantsThe disruption cassette including of the ptrpC::hph::ttrpC marker flanked by 1417bp and 1124bp of 5'and 3'flanking sequences of the ThChsC, was inserted in to the region between the left and right border repeats of the binary vector pDHt/SK. The resulting plasmids pDHt/ThChsC::hph were subsequently transformed into the genome of T. harzianum by Agrobactirium tumfacience mediated-transformant (ATMT). 162 transformants could grow on the selective medium (hygromycin B,150μg/mL). After five generations on non-selective medium, 116 tranformants still presented resistance to hygromycin B. The results showed that 71.6% of the transformants were genetic stable.4. Screening of chitin synthase gene ThChsC mutantsThe knock-out strain 6-2 was identified by cloning of the 5'flanking sequence, 3'flanking sequence and the deleting sequence of ThChsC. Southern-blotting analysis showed that the T-DNA was integrated into the genome of the mutants and the knock-out strain 6-2 contained 3 copies of T-DNA.5. Phenotype analysis of knock-out strain 6-2Fluorescence analysis demonstrated that the chitin content of the ThChsC knock-out strain 6-2 was fewer than those of the wild-type strain and the cell wall of chlamydospores in the hyphae contained more chitin. In addition, the more septa, fewer branching conidiophores and lots of chlamydospores were observed in the ThChsC knock-out strain 6-2. These results indicated that ThChsC was indispensable for hyphal morphogenesis and sporogenesis in T. harzianum.