腐皮镰孢菌壳聚糖酶CSN1的基因克隆、表达及其对致病性影响的研究
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摘要
壳寡糖(Chitooligosaccharides)是由氨基葡萄糖以β-1,4-糖苷键缩合而成的低聚物,聚合度一般为2—20,具有较低的分子量,呈水溶性,易于被细胞吸收,具有多种生物学活性,广泛应用于医药、保健、日化、农业、生防等领域,有着广阔的市场前景,是近年来研究和开发的热点之一
壳寡糖主要由壳聚糖(Chitosan)降解产生,而壳聚糖是由自然界中储量最为丰富的含氮类有机化合物甲壳素(Chitin)脱乙酰基形成的。目前壳寡糖的制备方法主要有壳聚糖化学降解法和酶解法。化学法包括浓酸降解法和过氧化氢降解法,但两种方法都较难得到低聚合度的壳寡糖,而且反应条件剧烈,容易引发环境问题。酶解法具有反应条件温和,壳寡糖得率高,不造成环境污染等优点。壳寡糖的酶法制备,又分为复合酶解法和专一性酶解法。复合酶解法是混合利用多种水解酶类,包括纤维素酶,蛋白酶,脂肪酶等,共同降解壳聚糖产生不同聚合度的壳寡糖。而专一性酶解法是利用壳聚糖的专一性水解酶—壳聚糖酶(Chitosanase)水解壳聚糖制备壳寡糖的过程。从壳寡糖的转化率、产物分子量分布范围、酶解产品的质量稳定性和酶解过程的可重复性来讲,利用专一性酶解法制备壳寡糖最具有前景和发展优势。壳寡糖市场前景的广阔促使了壳聚糖酶研究的活跃,目前已在多种微生物中分离到了壳聚糖酶,并对其酶学性质、水解机制进行了研究,其中以内切型壳聚糖酶最适合于壳寡糖的生产。
酿酒酵母是非常理想的真核生物表达系统,具有原核细菌所没有的真核生物蛋白翻译后修饰加工系统和安全型基因工程受体系统,并能将外源基因表达产物分泌至培养基中,已广泛用于外源基因的表达。酿酒酵母同时也是广泛用于工业生产的经济微生物,具有培养条件简单、生长迅速,便于大规模、高密度发酵,公认的生物安全菌(GRAS),高抗逆性等优良工业生产特性。
腐皮镰孢菌(Fusarium solani)在世界范围内分布广泛,可在土壤中长期进行腐生生活,同时也可寄生危害寄主植物。由于其在农业生产上的严重危害,腐皮镰孢菌受到了广泛关注Hadwiger (1980,1981)在研究F. solani与豌豆(Pisum sativum)的相互作用时发现,从F. solani细胞壁上释放出的壳寡糖类能够启动豌豆的防御机制,诱导豌豆细胞植保素(Phytoalexin)的产生,从而提高其抗病性,抑制F. solani的侵染,并通过免疫化学的方法检测到壳寡糖从F. solani细胞壁上释放出来,进入植物细胞中。Prapagdee(2007)研究发现低浓度的外源性壳聚糖类就能够抑制F solani的生长,保护豌豆免于病原真菌引发的猝死综合症(Sudden death syndrome)。鉴于壳聚糖和壳寡糖在病原真菌与植物相互作用中扮演着重要的角色,Shimosaka (1993)曾推测F. solani壳聚糖酶可能与真菌细胞壁的降解或致病性有关,但关于真菌壳聚糖酶生理功能的研究极少,至今尚没有实验性的证据被报道。
本实验室前期研究中利用平板透明圈法结合薄层层析法(TLC),筛选到-株能分泌表达壳聚糖酶的腐皮镰孢菌F. solani 0114。对粗酶液的研究表明该菌所产壳聚糖酶的水解产物中不含单糖,水解产物的平均分子量为1.3 kDa,壳寡糖比例较高。本论文的主要研究工作包括：
1. F. solani壳聚糖酶基因的克隆及酶学性质研究
利用RT-PCR方法扩增得到F. solani 0114壳聚糖酶的cDNA序列,序列分析表明该cDNA中包含一个编码300个氨基酸残基的开放读码框(ORF,903bp)。该序列已提交至GeneBank,登录号为EU263917。将此序列N末端与His标签融合并在大肠杆菌DE3中异源表达,通过对该重组酶的镍柱纯化,透析复性,得到了纯化的壳聚糖酶CSN1。SDS聚丙烯酰胺凝胶电泳显示CSN1的分子量约为30 kDa,酶学性质分析表明CSN1的最适温度为50-℃,最适pH值为5.6,以脱乙酰度85%的壳聚糖为底物时Km值为0.063 Ing/ml, Vmax为126.58μmol/ml.min,比酶活为2.5 U／mg。利用薄层层析法(TLC)和高效液相色谱技术(HPLC)对CSN1的酶解产物进行分析,结果表明,水解产物中不含单糖,10糖以下组分占75%以上,为内切型壳聚糖酶。本研究得到的壳聚糖酶CSN1非常适合于壳寡糖的专一性酶解法生产。
2. CSN1在酿酒酵母工业菌株中的表达
将CSN1的cDNA序列与克鲁维酵母的菊粉酶(INU1A)信号肽序列融合,以实验室前期构建的多拷贝整合性酿酒酵母表达载体pYMIKP质粒为骨架,构建得到CSN1表达质粒pYMIKP-CHO,并将该质粒导入酿酒酵母N-27中,摇瓶发酵实验表明,酿酒酵母重组菌株N-27C所产壳聚糖酶粗酶液的体积酶活达到50.2 mU/ml,成功实现了CSN1在酿酒酵母中的分泌表达,TLC法酶解产物分析表明,重组壳聚糖酶的酶解特性与纯酶及F. solani 0114所产壳聚糖酶相一致。酿酒酵母重组菌株的构建对于CSN1的规模化生产具有潜在的应用价值。
3.根瘤农杆菌介导的腐皮镰孢菌转化体系的建立
选择两种转化筛选标记：潮酶素B (Hygromycin B)和草丁磷除草剂(PPT)测定其对腐皮镰孢菌孢子生长的最低抑制浓度,结果显示：400μg／ml潮霉素B或750μg/ml PPT能够完全抑制F. solani 0114孢子的萌发和生长。以质粒pCAMBIA1300为骨架,构建适合腐皮镰孢菌转化的双元载体pCABAR和pCAHPH,将构建好的质粒转入根瘤农杆菌LBA4404,利用根瘤农杆菌介导的转化技术(ATMT)转化腐皮镰孢菌F. solani 0114。根据前期本实验室对ATMT的研究结果,采用400μg／ml AS,6 h预培养时间处理用于转化的根瘤农杆菌,根瘤农杆菌与腐皮镰孢菌孢子的共培养时间为48小时。并对根瘤农杆菌与真菌孢子比进行优化,结果显示根瘤农杆菌与真菌孢子比为1.0／106-ml-1时转化效果较好。测定了两种选择标记：bar和hph对转化效率的影响。结果显示以bar为选择标记时,转化效率为13转化子／106孢子；以hph为选择标记时,转化效率为21转化子／106孢子。与PEG-CalCl2转化法相比(27转化子／107原生质体),ATMT法具有较高的转化效率,而且可以省略繁琐的原生质体制备与再生过程,操作更为简单。ATMT转化体系的建立为下一步的F. solani功能基因研究奠定了基础。
4.腐皮镰孢菌CSN1超表达菌株和表达下调菌株的构建
以质粒pCAMBIA1300为骨架,以勾巢曲霉(Aspergillus nidulans)强组成型启动子gpdA和终止子trpc,构建带有CSN1表达原件的双元载体pCHO。通过ATMT技术将pCHO转入F. solani 0114基因组中,构建超表达CSNl的腐皮镰孢菌。挑取12个阳性克隆进行产酶培养实验,结果表明有5个克隆的壳聚糖酶体积酶活明显增加,最高为出发菌株的2.1倍。
根据CSN1 cDNA序列,对其mRNA二级结构进行软件分析,设计出两个能自我配对形成带有544 bp茎和229 nt环结构的发夹片段。以CSN1 cDNA为模板,PCR扩增得到两个片段,将两个片段反向连接,形成反向重复序列(IR)。以质粒pCAMBIA1300为骨架,构建带有IR转录原件的双元载体pCIR,通过ATMT技术将pCIR转入F. solani 0114基因组中,构建得到CSN1的RNA干扰(RNAi)菌株。利用平板透明圈法挑选到5株CSN1表达下调的菌株,Northernblot分析表明5株RNAi转化子胞内的CSN1 mRNA量与出发菌株相比明显降低。
5.腐皮镰孢菌壳聚糖酶生理功能研究
利用实时荧光定量RT-PCR技术(qRT-PCR)检测到F. solani 0114在液体培养时,CSN1主要在集中在菌体生长的延滞期表达,同时检测到CSN1在F.solani 0114对豌豆不同组织的侵染过程中也有明显表达。表明壳聚糖酶可能不参与菌体的生长过程,而与其致病性有关。对CSN1超表达菌株和RNAi菌株的生长状态,致病能力进行了测定。结果显示,与野生型菌株相比,CSN1超表达菌株在平板和液体培养时的生长都受到了明显的抑制,而RNAi菌株的生长较野生型无明显变化。对三种菌进行豆荚侵染测试,结果显示：与野生型菌株相比,RNAi干涉菌株的侵染能力有明显提高(>50%)；而CSN1超表达菌株的侵染能力明显下降(<40%)。同时,豆苗侵染测试结果也显示RNAi菌株的毒性最高,野生型其次,CSN1超表达菌株的毒性最低,表明CSN1对F. solani的致病性有抑制作用。虽然CSN1对F. solani致病性的抑制机理有待于进一步的研究,但关于该酶的生理功能是首次报导,对于真菌壳聚糖酶的研究及腐皮镰孢菌致病性的研究都具有借鉴意义。
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 CSN1
The 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 strain
Based 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. solani
Based 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. solani
A 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. solani
Using 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.
壳寡糖主要由壳聚糖(Chitosan)降解产生,而壳聚糖是由自然界中储量最为丰富的含氮类有机化合物甲壳素(Chitin)脱乙酰基形成的。目前壳寡糖的制备方法主要有壳聚糖化学降解法和酶解法。化学法包括浓酸降解法和过氧化氢降解法,但两种方法都较难得到低聚合度的壳寡糖,而且反应条件剧烈,容易引发环境问题。酶解法具有反应条件温和,壳寡糖得率高,不造成环境污染等优点。壳寡糖的酶法制备,又分为复合酶解法和专一性酶解法。复合酶解法是混合利用多种水解酶类,包括纤维素酶,蛋白酶,脂肪酶等,共同降解壳聚糖产生不同聚合度的壳寡糖。而专一性酶解法是利用壳聚糖的专一性水解酶—壳聚糖酶(Chitosanase)水解壳聚糖制备壳寡糖的过程。从壳寡糖的转化率、产物分子量分布范围、酶解产品的质量稳定性和酶解过程的可重复性来讲,利用专一性酶解法制备壳寡糖最具有前景和发展优势。壳寡糖市场前景的广阔促使了壳聚糖酶研究的活跃,目前已在多种微生物中分离到了壳聚糖酶,并对其酶学性质、水解机制进行了研究,其中以内切型壳聚糖酶最适合于壳寡糖的生产。
酿酒酵母是非常理想的真核生物表达系统,具有原核细菌所没有的真核生物蛋白翻译后修饰加工系统和安全型基因工程受体系统,并能将外源基因表达产物分泌至培养基中,已广泛用于外源基因的表达。酿酒酵母同时也是广泛用于工业生产的经济微生物,具有培养条件简单、生长迅速,便于大规模、高密度发酵,公认的生物安全菌(GRAS),高抗逆性等优良工业生产特性。
腐皮镰孢菌(Fusarium solani)在世界范围内分布广泛,可在土壤中长期进行腐生生活,同时也可寄生危害寄主植物。由于其在农业生产上的严重危害,腐皮镰孢菌受到了广泛关注Hadwiger (1980,1981)在研究F. solani与豌豆(Pisum sativum)的相互作用时发现,从F. solani细胞壁上释放出的壳寡糖类能够启动豌豆的防御机制,诱导豌豆细胞植保素(Phytoalexin)的产生,从而提高其抗病性,抑制F. solani的侵染,并通过免疫化学的方法检测到壳寡糖从F. solani细胞壁上释放出来,进入植物细胞中。Prapagdee(2007)研究发现低浓度的外源性壳聚糖类就能够抑制F solani的生长,保护豌豆免于病原真菌引发的猝死综合症(Sudden death syndrome)。鉴于壳聚糖和壳寡糖在病原真菌与植物相互作用中扮演着重要的角色,Shimosaka (1993)曾推测F. solani壳聚糖酶可能与真菌细胞壁的降解或致病性有关,但关于真菌壳聚糖酶生理功能的研究极少,至今尚没有实验性的证据被报道。
本实验室前期研究中利用平板透明圈法结合薄层层析法(TLC),筛选到-株能分泌表达壳聚糖酶的腐皮镰孢菌F. solani 0114。对粗酶液的研究表明该菌所产壳聚糖酶的水解产物中不含单糖,水解产物的平均分子量为1.3 kDa,壳寡糖比例较高。本论文的主要研究工作包括：
1. F. solani壳聚糖酶基因的克隆及酶学性质研究
利用RT-PCR方法扩增得到F. solani 0114壳聚糖酶的cDNA序列,序列分析表明该cDNA中包含一个编码300个氨基酸残基的开放读码框(ORF,903bp)。该序列已提交至GeneBank,登录号为EU263917。将此序列N末端与His标签融合并在大肠杆菌DE3中异源表达,通过对该重组酶的镍柱纯化,透析复性,得到了纯化的壳聚糖酶CSN1。SDS聚丙烯酰胺凝胶电泳显示CSN1的分子量约为30 kDa,酶学性质分析表明CSN1的最适温度为50-℃,最适pH值为5.6,以脱乙酰度85%的壳聚糖为底物时Km值为0.063 Ing/ml, Vmax为126.58μmol/ml.min,比酶活为2.5 U／mg。利用薄层层析法(TLC)和高效液相色谱技术(HPLC)对CSN1的酶解产物进行分析,结果表明,水解产物中不含单糖,10糖以下组分占75%以上,为内切型壳聚糖酶。本研究得到的壳聚糖酶CSN1非常适合于壳寡糖的专一性酶解法生产。
2. CSN1在酿酒酵母工业菌株中的表达
将CSN1的cDNA序列与克鲁维酵母的菊粉酶(INU1A)信号肽序列融合,以实验室前期构建的多拷贝整合性酿酒酵母表达载体pYMIKP质粒为骨架,构建得到CSN1表达质粒pYMIKP-CHO,并将该质粒导入酿酒酵母N-27中,摇瓶发酵实验表明,酿酒酵母重组菌株N-27C所产壳聚糖酶粗酶液的体积酶活达到50.2 mU/ml,成功实现了CSN1在酿酒酵母中的分泌表达,TLC法酶解产物分析表明,重组壳聚糖酶的酶解特性与纯酶及F. solani 0114所产壳聚糖酶相一致。酿酒酵母重组菌株的构建对于CSN1的规模化生产具有潜在的应用价值。
3.根瘤农杆菌介导的腐皮镰孢菌转化体系的建立
选择两种转化筛选标记：潮酶素B (Hygromycin B)和草丁磷除草剂(PPT)测定其对腐皮镰孢菌孢子生长的最低抑制浓度,结果显示：400μg／ml潮霉素B或750μg/ml PPT能够完全抑制F. solani 0114孢子的萌发和生长。以质粒pCAMBIA1300为骨架,构建适合腐皮镰孢菌转化的双元载体pCABAR和pCAHPH,将构建好的质粒转入根瘤农杆菌LBA4404,利用根瘤农杆菌介导的转化技术(ATMT)转化腐皮镰孢菌F. solani 0114。根据前期本实验室对ATMT的研究结果,采用400μg／ml AS,6 h预培养时间处理用于转化的根瘤农杆菌,根瘤农杆菌与腐皮镰孢菌孢子的共培养时间为48小时。并对根瘤农杆菌与真菌孢子比进行优化,结果显示根瘤农杆菌与真菌孢子比为1.0／106-ml-1时转化效果较好。测定了两种选择标记：bar和hph对转化效率的影响。结果显示以bar为选择标记时,转化效率为13转化子／106孢子；以hph为选择标记时,转化效率为21转化子／106孢子。与PEG-CalCl2转化法相比(27转化子／107原生质体),ATMT法具有较高的转化效率,而且可以省略繁琐的原生质体制备与再生过程,操作更为简单。ATMT转化体系的建立为下一步的F. solani功能基因研究奠定了基础。
4.腐皮镰孢菌CSN1超表达菌株和表达下调菌株的构建
以质粒pCAMBIA1300为骨架,以勾巢曲霉(Aspergillus nidulans)强组成型启动子gpdA和终止子trpc,构建带有CSN1表达原件的双元载体pCHO。通过ATMT技术将pCHO转入F. solani 0114基因组中,构建超表达CSNl的腐皮镰孢菌。挑取12个阳性克隆进行产酶培养实验,结果表明有5个克隆的壳聚糖酶体积酶活明显增加,最高为出发菌株的2.1倍。
根据CSN1 cDNA序列,对其mRNA二级结构进行软件分析,设计出两个能自我配对形成带有544 bp茎和229 nt环结构的发夹片段。以CSN1 cDNA为模板,PCR扩增得到两个片段,将两个片段反向连接,形成反向重复序列(IR)。以质粒pCAMBIA1300为骨架,构建带有IR转录原件的双元载体pCIR,通过ATMT技术将pCIR转入F. solani 0114基因组中,构建得到CSN1的RNA干扰(RNAi)菌株。利用平板透明圈法挑选到5株CSN1表达下调的菌株,Northernblot分析表明5株RNAi转化子胞内的CSN1 mRNA量与出发菌株相比明显降低。
5.腐皮镰孢菌壳聚糖酶生理功能研究
利用实时荧光定量RT-PCR技术(qRT-PCR)检测到F. solani 0114在液体培养时,CSN1主要在集中在菌体生长的延滞期表达,同时检测到CSN1在F.solani 0114对豌豆不同组织的侵染过程中也有明显表达。表明壳聚糖酶可能不参与菌体的生长过程,而与其致病性有关。对CSN1超表达菌株和RNAi菌株的生长状态,致病能力进行了测定。结果显示,与野生型菌株相比,CSN1超表达菌株在平板和液体培养时的生长都受到了明显的抑制,而RNAi菌株的生长较野生型无明显变化。对三种菌进行豆荚侵染测试,结果显示：与野生型菌株相比,RNAi干涉菌株的侵染能力有明显提高(>50%)；而CSN1超表达菌株的侵染能力明显下降(<40%)。同时,豆苗侵染测试结果也显示RNAi菌株的毒性最高,野生型其次,CSN1超表达菌株的毒性最低,表明CSN1对F. solani的致病性有抑制作用。虽然CSN1对F. solani致病性的抑制机理有待于进一步的研究,但关于该酶的生理功能是首次报导,对于真菌壳聚糖酶的研究及腐皮镰孢菌致病性的研究都具有借鉴意义。
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 CSN1
The 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 strain
Based 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. solani
Based 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. solani
A 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. solani
Using 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.
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