Molecular Cloning And Functional Analysis Of CSN1 In Fusarium Solani

Chitooligosaccharides are polymers of 0-1,4 linked D-glucosamine residues with the polymerization below 20. Chitooligosaccharides have low molecular weight, high water-solubility and absorbability. Therefor, they have many applications in various fields, for instance, they can strengthen immunity systerm, control the growth of cancer cell. chitooligosaccharids are obtained mainly by chemical or enzymatic hydrolysis of the chitosan chains. Chemical hydrolysis is carried out by two alternative methods:acid hydrolysis with concentrated acids or oxidative degradation with hydrogen peroxide. Both methods have been applied successfully to chitosan degradation, but both show some drawbacks, including the difficulty to obtain low polymerization degree oligosaccharides, and to control the extent of hydrolysis, which frequently results in hydrolysates containing a high rato of monosaccharides. In addition, the harsh reaction conditions required may cause environmental problems. Alternatively to the aggressive chemical hydrolysis, chitosan may also be hydrolyzed in a milder way using enzymes. Enzyme catalyzed chitosan hydrolysis is more specific and allows a greater control of the extent of reaction and, therefor, of the product size. Enzymes that can hydrolyze chitosan include unsepcificity and specificity ones, the former includes cellulase, hemicellulases, pectinase, proteases and lipase, the latter is chitosanase. Chitosanase catalyzed hydrolysis of chitosan has many advantages and has been paid a lot of attentions by reseachers.S. cerevisiae, which can grow rapidly on simple medium to a high-cell density, is the most useful eukaryotic microorganism for heterologous protein production. More importantly, S. cerevisiae is a GRAS strain that causes no harm to plants or humans. The rapid growth, ease of genetic manipulation, and a well-defined genetic system of S. cerevisiae make it vey usefull for applied studies.F. solani is a soilborne filamentous fungus of worldwide distribution that has been recognized for a long time as important plant pathogens. It causes an important economic loss in the agriculture industry. Hadwiger et al (1980,1981) found that chitosan polymers released from F. solani cell walls can inhibit fungal growth and elicit disease resistance responses in pea pod tissue. Prapagdee et al (2007) also found that low concentrations of chitosan can inhibit the F. solani f. sp. glycines growth and protect soybeans from sudden death syndrome (SDS). Therefore, the fungal chitosanase probably is also involved in the plant-pathogen interactions (Shimosaka 1993).In a previouse work, we obtained a chitosanase-producing strain F. solani 0114 using the plate halo secreening and TLC analysis. In this study, we want to clone the chitosanase gene of F. solani 0114 and investigate its characterizations. We also want to study the physiological functions of chitosanase in F. solani growth and pathogenicity. 1. Cloning of the chitosanase gene (csnl) from F. solani and research on biochemical characterizations of CSN1The chitosanase cDNA of F. solani 0114 was amplified by reverse transcription-mediated PCR (RT-PCR), The cDNA sequence, which containing an open reading frame (ORF) encoding 300 amino acid residues, was submitted to GeneBank with the accession number EU263917. The ORF of the cDNA was ligated into pET-15b and expressed in E. coli BL21 (DE3). The recombinant protein was expressed as inclusion bodies. After solubilization, the denatured proteins were purified with the Ni-NTA Purification System. The molecular weight of the purified protein was about 30 kDa. A chitosanase assay showed that the specific activity of the renatured protein was 2.5 U/mg. The purified enzyme functioned between pH 3 and 6 with an optimum at pH 5.6. The enzyme was most active at 50℃. Kinetic analysis results showed that the Km of the enzyme was 0.063 mg/ml,Vmax was 126.58μmol/ml.min. TLC and HPLC results indicated that most of the enzyme hydrolysates were chitooligosaccharides with a polymerization below 10, and no monomers were detected. The chitosanase from F. solani 0114 is an endochitosanase. Because of its special characteristics, this enzyme is very useful in chitooligosaccharides production.2. Expression of csnl in Saccharomyces cerevisiae industrial strainBased on the yeast multiple integration plasmid pYMIKP, a CSN1 expression vector pYMIKP-CHO was constructed. The vector was introduced into S. cerevisiae industrial strain N-27. To direct the recombinant protein into the secretory pathway, the INU1A signal sequence was fused to 5'end of the csn cDNA, because the INU1A signal sequence can cause highly efficient secretion of large proteins in S. cerevisiae. In S. cerevisiae N-27. Chitosanase assay results showed that CSN1 was successfully secreted from S. cerevisiae transformant, and the yield of chitosanase reached 50.2 mU/ml. The transformant has better application prospects for the large-scale production of CSN1 than F. solani 0114.3. Agrobacterium tumefaciens-mediated transformation (ATMT) of F. solaniBased on the plasmid pCAMBIA1300, we constructed binery vectors fit for F. solani transformation, and the F. solani was successfully transformed using ATMT. The hygromycin resistent gene hph and herbicide resistance gene bar were used as selection markers. The transformation efficiency was 13 transformants/106 spores when using bar as selection marker, and for hph, the efficiency was 21 transformants/106 spores. Compared to PEG-CalCl2 transformation method in F. solani, whose transformation efficiency was 27 transformants/107 protoplasts, ATMT has higher transformation efficiency. It allows fungal conidia to be used as the starting material, provides a easier way for fungal transformation.4 Construction of CSNl-overexpression strain and CSN1-silenced strain of F. solaniA transformation vector carried csn1, which was under the control of the A. nidulans gpdA promoter and A. nidulans trpC terminator was constructed and denoted as pCHO. It was derived from the s binary vector pCAMBIA 1300. A. tumefaciens LBA 4404 containing vector pCHO was used for transformation of F. solani 0114 conidia and 12 resistant colonies were obtained. Enzyme production results suggested that 5 of the 12 colonies had a significant increase in chitosanase production (～2.1-fold than control). A silencing vector containing an inverted repeat (IR) sequence was constructed. It is derived from the binary vector pCAMBIA 1300 as well. The IR sequence was obtained by ligating, in the opposite orientation, two PCR fragments corresponding to CSN1 cDNA. The pCIR construct was expected to produce a self-complimentary transcript forming a hairpin RNA with a 544 bp stem and a 229 nt loop. The construct was introduced into the F. solani 0114 genome by A. tumefaciens-mediated transformation. Plate halo results showed that five of the transformants had a significant reduction in chitosanase activity Northern blot analysis was carried out to determine the expression levels of Csnl in the silenced transformants. We found that all the mutants showed a strong decrease of the endogenous Csnl mRNA compared with the wild-type strain. Therefore, it was confirmed that the reduction of chitosanase activity was due to the Csnl silencing.5. Functional analysis of CSN1 in F. solaniUsing real-time quantitative RT-PCR, the obvious expression of CSN1 was found when F. solani was applied to pea pod, leaf and seed root separately. Meanwhile, CSN1 expression levels at different developmental stages were also determined. We found that CSN1 was mostly expressed in the staling phase when F. solani 0114 was cultured in CZ liquid medium. These results suggested that the F. solani chitosanase probably is involved in the plant-pathogen interactions, and may be not essential to fungal growth. Further, growth rate assays were performed to determine the effect of chitosanas expression on fungal growth. The CSN1-silenced strain exhibited no distinguishable change in both radial and submerged growth. However, the csn1-overexpression strain showed an obvious growth inhibition compared with the wild-type strain, indicating that CSN1 overexpression had an adverse effect on fungi mycelial growth. To determine the role of chitosanase expression in fungal virulence, both pea pod and seedling infection assays were performed. The silenced transformant showed an increase of virulence (150%) over the wild type strain (100%). Meanwhile, the Csnl-overexpression strain exhibited a reduction in virulence (～60%). In the second assay, seedlings infected with the silenced transformant CKD3 showed a significant increase, and seedlings infected with the csnl-overexpression strain had a little reduction in disease severity, in comparison to seedlings infected with the wild type strain. These data are consistent in that the silenced transformant has the highest, wild type has the medium, and the csn1-overexpression strain has the weakest virulence. Although the mechanism remains unclear, our findings did suggest that F. solani chitosanase has a negative effect on fungal pathogenicity. This finding is important in the regard that chitosanases are found in many plant pathogenic fungi. Further studies and findings for the role of chitosanase in virulence and the host-pathogen function would likely have broad application.