Functional analysis of chitinase gene Cg10994 in Colletotrichum gloeosporioides

• Main Authors: Yi-Ting Pan, 潘宜庭
• Other Authors: 李敏惠
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/t7d6wz

碩士 === 國立中興大學 === 植物病理學系所 === 107 === The fungal cell wall is composed of β-1,3-glucan, β-1,6-glucan and chitin. It is known that chitinases can hydrolyze the linear polymer chitin and also involved in fungal cell wall degradation and remodeling. The aims of the study are to investigate how many chitinases exist in Colletotrichum gloeosporioides TYC-2, a fungal strain causing many anthracnose, and to characterize the function of chitinase in TYC-2. By searching annotated genome and using conserved domain prediction tools, twenty-one genes encoding chitinases in the genome of TYC-2 were found. The chitinase gene, gene number Cg10994, was selected for gene functional analysis. Plasmids were constructed to generate gene knockout, overexpression and complemented strains for gene functional analysis. Agrobacterium tumefaciens-mediated transformation was used to produce mutants. The mutants of the Cg10994 gene were generated and confirmed by PCR assay or Southern blotting. Cg10994 expression was detected by semi qRT-PCR and the expression level was increased while the fungus was cultured in lower carbon-nitrogen medium. Phenotypic tests showed that deletion of Cg10994 does not affect development, morphology and pathogenicity of TYC-2. In addition, Cg10994 did not be involved in protoplast formation ability and tolerance to osmotic stress and cell wall inhibitors. In the initial analysis of tolerance to thermal and multiple stresses, the ∆Cg10994 strains were found to be less tolerant to thermal stress. These results suggest that Cg10994 gene of TYC-2 might be involved in nutrient deficiencies and thermal stress. Other roles of chitinase gene Cg10994 in C. gloeosporioides TYC-2 remain to be investigated in the future.