Cloning And Function Analysis Of Xylanase Genes Of Valsa Mali Var. Mali

Valsa mali var. mali (Vmm), a destructive ascomycetous pathogen of apple tree, secretes various xylanase, cellulase and pectinase in planta that degrade plant cell walls and facilitate pathogen entry into the host. V. mali causes serious economic losses in China. The lack of understanding of the pathogenic genes and mechanisms of V. mali, which poses difficulties to control the disease. Previous studies have indicated that xylanase produced by V. mali is a key pathogenic factor. To better understand the role of xylanase in the pathogenicity of Vmm, we cloned and identified two endo-β-1,4-xylanase gene, Xylanase I and Xylanase II, analyzed the expression characteristics of two genes, and primarly verified the gene function by gene knockout technology.The full-length cDNA of Xylanase I is 1626 bp harbouring 5’-and 3’-non-encoding regions, as well an open reading frame of 1320 bp that encodes a protein with a calculated molecular mass and isoelectric point of 43.8 kDa and 4.4, respectively. The predicted amino acid sequences show significant homology with a family 10 of glycosyl hydrolases. The three-dimensional structure of this protein was predicted based on the crystal structure of XynAS9, the endoxylanase family 10. It is "bowl" on the spatial structure,149 and 256 glutamic acid residues are identified as the catalytic domain. The full-length cDNA of Xylanase II is 969 bp harbouring 5’-and 3’-non-encoding regions, as well an open reading frame of 681 bp that encodes a protein with a calculated molecular mass and isoelectric point of 23.8 kDa and 5.29, respectively. The predicted amino acid sequences show significant homology with a family 11 of glycosyl hydrolases. The three-dimensional structure of this protein was predicted based on the crystal structure of Xylanase 11C, the endoxylanase family 11. It is "left half grip-like" on the spatial structure, the first 122 and 213 glutamic acid residues are identified as the catalytic domain, moreover,113 and 124 tyrosine amino acid residues are identified as a substrate binding region.Two xylanase genes were constructed into the expression vector pET-32 and obtained recombinant expression plasmid pET-Xylanase. The expression conditions of pET-Xylanase were optimized. Xylanase Ⅰ protein expressed in Escherichia coli reached a maximum level when induction with 0.5 mmol/L isopropyl s-thiogalactopyranoside (IPTG) for 12 h at 30℃. Xylanase Ⅱ protein expressed in E. coli reached a maximum level when induction with 0.1 mmol/L IPTG for 10 h at 15 ℃. High xylanase activity could be deteced in the supernatant of Xylanase Ⅱ. The recombinant protein was confirmed by Western blot analysis using Anti-His mouse monoclonal antibody and the result showed that the induced ～63 kDa and ～42 kDa bands in E. coli were Xylanase protein, respectively.By quantitative RT-PCR, the expression of two xylanase genes was determined in culture media and apple tissues in vitro. The results showed that the expression of Xylanase Ⅰ and Xylanase Ⅱ were induced when the pathogen was grown in minimal media containing apple branch extract and oat spelt xylan as the only carbon sources at a higher temperature (from 25 to 30℃). However, only Xylanase Ⅰ expression was induced when glucose as the only carbon source. In addition, the expressin of Xylanase Ⅰ and Xylanase Ⅱ was significantly increased in apple detached branches and calli throughout the infection process of the pathogen, which suggested that the two Xylanase genes play a key role in the pathogenicity of Vmm.To further verify Xylanase genes function in V. mali, Xylanase Ⅰ was knockout by PGE-mediated protoplast transformation technique. The knockout vectors were constructed though Double-joint PCR. Then, using the PEG-mediated transformation,139 mutants were obtained byhygromycin resistance screening. However, only one mutant of Xylanase Ⅰ knockout was obtained by PCR detection. The biology charecteristics and pathogencity of gene deletion mutant were analyzed. Compared with the wild type LXS080901, there was no significant difference on colony morphology and growth rate of the gene deletion mutant. However, the pathogenicity of the gene deletion mutant was significantly decreased in apple detached branch. Determination of xylanase activity in disease spot that geng deletion mutant of xylanase activity was lower than that of the wild type. All the results suggested thai Xylanase Ⅰ plays an important role in the process of pathogenicity of V. mali.In this study, xylanase, an important pathogenic factor of V. mali, was identified by cloning its genes and analyzing gene expression and function. All the results will lay a theoretical foundation for further understanding the pathogenetic mechanism of V. mali.