Cloning, Expression And Function Analysis Of Chitinase Gene From Mycoparasitic Fungus

Chitinases produced by mycoparasites are directly involved in the parasitic process. Chintinase is thought to be an enzyme related to fungi disease resistance, because it can degrade the cell wall of pathogenic fungal hosts, suppress mycelium growth, spore germination and germ tube elongation. Except in bio-control against plant diseases, mycoparasitic chitinase is widely used in bio-conversion of chitin, where chitin can be partially hydrolyzed by chitinase to generate chitooligosaccharide. Chitooligosaccharide is a kind of bio-activator with broad application in agriculture, medicine, chemical, food industry and environmental fields. Transgenic crops encoding mycoparasitic chintinase gene have high disease resistance with long persistence and broad spectrum and their pest resistance could also be enhanced. So far, studies in chitinase and its genes are mainly focusing on Trichoderma spp., but less on other mycoparasites. Cloning chintinase genes from other mycoparasites will lead us to understanding its enzymatic properties and activities to suppress pathogens, which is important for researches in bio-control and breeding disease-resistant crops at the molecular level.Trichothecium roseum and Talaromyces flavus are two important mycoparasites. In this work, three chitinase genes were completely and one was partially sequenced under using RT-PCR, RACE-PCR and TAIL-PCR technology. These three chitinases with complete sequences were successfully expressed in Pichia pastoris and their properties were well studied in this work. One of the chintinases from T. roseum was transformed into tobacco and its effect on disease resistance was analyzed.1 Degenerate primers were designed according to conserved regions in homologous sequences of chitinase genes. Chitinase gene trchi1 (accession number in GenBank GU361768 for mRNA, and GU361767 for DNA, respectively) was cloned from T. roseum, by using RT-PCR, RACE-PCR and TAIL-PCR technology. trchi1 belonged to Family 18 glycoside hydrolases, its ORF was 1278 bp and it encoded 425 amino acids containing a signal peptide with 20 amino acids. Yeast expression vector with trchi1 was transformed into Pichia pastoris to produce secretory protein. A protein, Trchi1, its molecular weight was 43.97 kDa, with optimal temperature was 50℃and optimal pH 6.5. Trchi1 also was stable at the temperature below 45℃and pH ranging from 6-8. Its activity was significantly suppressed by metal ions such as Hg²⁺, Cu²⁺, Ag⁺, Zn²⁺ and Fe²⁺. Its optimal substrate was colloidal chitin. It could strongly inhibit mycelium growth of Cryphonectria parasitica, and could obviously suppress spore germination of Colletotrichum nicotianae and Alternaria alternate, it had significant effect on spore germination of Curvularia lunata, namely promoting germination at low concentration, but inhibiting at high concentration.2 Two chitinase genes, tfchi1 and tfchi2, were cloned from T. flavus ACCC3096. tfchi1 (accession number in GenBank GU361770 for mRNA, and GU361769 for DNA, respectively) was 1194 bp and encoded 397 amino acids which had no signal peptide. The chitinase encoded by tfchi1 was a member of Family 18 glycoside hydrolases. A protein, Tfchi1, having chitinase activity was expressed in Pichia pastoris. Its MW was 42.53 kDa, optimal temperature at 35℃and optimal pH at 5.5. Tfchi1 was stable at the temperature below 30℃and ranging from 6-8. It was significantly suppressed by Hg²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and Ag⁺. Its optimal substrate was colloidal chitin. It had strongest inhibition to mycelium growth of Cryphonectria parasitica and Cytospora chrysosperma, but had weaker inhibition to that of Alternaria alternate and Fusarium graminearum. The higher chitinase concentration there is, the stronger inhibition is observed. It significantly influenced spore germination of Curvularia lunata and the germ tube hardly extended after the spore germination.The gene, tfchi2 (accession number in GenBank was HQ896839) included an ORF with 1191 bp and encoded 396 amino acid which had no signal peptide. The chitinase encoded by tfchi2 was also involved in Family 18 glycoside hydrolases. A protein, Tfchi2, with chitinase activity was detected. Its MW was 44.06 kDa, with optimal temperature at 70℃and optimal pH at 6. Tfchi2 was stable at the temperature below 55℃and pH ranging from 6-9. It could be activated by Ba²⁺, K⁺, completely inactived by Fe²⁺, Hg²⁺, Mg²⁺ and significantly reduced its activity by Cu²⁺, Zn²⁺, Ag⁺ and NH4. The protein had a specific activity to degrade colloidal chitin. It inhibited mycelium growth of fungal pathogen, as well as spore germination and germ tube extending. Therein, strong inhibition to mycelium growth of Cryphonectria parasitica and Cytospora chrysosperma was observed. It partially inhibited spore germination of Colletotrichum gloeosporioedes at lower concentration, while completely inhibited it at high concentration. But results also showed that spore germination of Alternaria alternate and Curvularia lunata was facilitated at low recombinant chitinase concentration but inhibited at high concentration. High concentration of recombinant chitinase also inhibited germ tube elongation after spore germinating.3 One partial sequences of chitinase gene, tfchi3 were cloned from T. flavus ACCC30404. cDNA of the cloned sequences had 865 bp with TAA as termination codon and encoded 267 amino acid which was predicted as a member of Family 18 glycoside hydrolases.4 Plant expression vector with gene trchi1 was constructed and successfully transformed into tobacco using Leaf Disc via Agrobacterium rhizogines. The Southern blot and protein expression analysis result indicated that trchi1 was integrated into tobacco genome, and was expressed effectively in tobacco. It was observed that the resistance of tobacco with gene trchi1 against Alternaria alternate and colletotrichum gloeosporioedes was obviously enhanced.