高效棉酚降解菌株的筛选鉴定及其差异蛋白质组学研究
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摘要
棉籽饼粕是一种重要的植物蛋白饲料资源,但是其中含有一种对动物有害的抗营养因子—棉酚,从而限制了其在饲料中的应用。目前,现有的一些脱毒方法,如有机溶剂萃取、硫酸亚铁处理、微生物发酵等,对棉籽饼粕的解毒起到了一定的作用。尤其是微生物发酵降解棉酚,不仅可使棉籽饼粕脱毒,同时还可提高棉籽蛋白的营养价值。虽然棉籽饼粕发酵脱毒优点已被公认,但是迄今为止,人们对微生物发酵脱毒的分子机理尚不清楚。本研究以纯棉酚为碳源,从土壤中筛选了2株棉酚降解菌,采用形态学观察及分子生物学方法进行菌种鉴定,通过分析棉酚降解菌的培养条件对其生长特性进行初步探讨,并利用蛋白质组学技术分析棉酚降解菌在不同碳源中的蛋白质表达差异。主要研究结果如下：
1.以醋酸棉酚为唯一碳源,从土壤中筛选棉酚降解菌。从平板上得到2株高效降解棉酚的菌体,分别命名为AN-1、MM-2。从形态上观察及电镜结果显示：AN-1和MM-2均为丝状真菌。通过分子生物学方法对棉酚降解菌进一步鉴定表明：AN-1的18S rDNA序列与黑曲霉(Aspergillus niger)的同源性高达99%以上,据此推测AN-1可能属于A. niger; MM-2的18SrDNA序列与镰刀霉属(Fusarium)的同源性达98%以上,据此推测MM-2可能属于Fusarium sp.。
2.对分离到的棉酚降解菌进行培养特性研究,以获得其最佳生长条件,为进一步研究奠定基础。生长曲线试验结果显示：棉酚降解菌AN-1和MM-2表现出相似生长趋势,即在葡萄糖中生长优于在棉酚中生长,培养60～84 h取种用于研究最好。温度试验结果显示：棉酚降解菌AN-1和MM-2在棉酚及葡萄糖中的生长情况类似,即30℃时菌体生物量达最大值,且在葡萄糖中的生长略好于棉酚。pH试验结果显示：棉酚降解菌AN-1和MM-2在葡萄糖和棉酚中培养时pH值均呈下降趋势,但在葡萄糖中较之棉酚中下降明显。氮源试验结果显示：不同氮源对棉酚降解菌AN-1和MM-2的影响均有相似趋势,即在铵盐及硝酸盐中生长比在酵母膏和尿素中好,且最佳氮源为NaNO3。棉酚耐受性试验结果表明：棉酚降解菌AN-1在5种不同浓度(0.05%,0.1%,0.2%,0.4%,0.8%)的棉酚中生长时具有明显差异,且AN-1对棉酚具有极强的耐受性,确定棉酚最适添加量为0.2%。
3.采用TCA／丙酮沉淀法提取棉酚降解菌AN-1在葡萄糖和棉酚中生长72 h后的菌体蛋白,得到了分辨率较好的蛋白质双向电泳图谱,蛋白质分子量主要集中在25.0～66.2 kDa之间,各pI段均有分布。通过PDQuest软件分析,葡萄糖凝胶图谱中平均检测到478个蛋白点,棉酚凝胶图谱中平均检测到592个蛋白点；以葡萄糖为参考胶,棉酚为碳源的2-DE图谱中平均匹配的蛋白点为293个,未匹配的蛋白点有299个,其中有51个为差异蛋白点。采用MALDI-TOF MS方法对差异蛋白点进行质谱鉴定,获得了47张PMF图谱,鉴定出其中20个蛋白点,包括1个kinesin,2个glycerol-3-phosphate dehydrogenase,1个citrate synthase,1个ubiquitin,1个unnamed protein和14个hypothetical protein。
Cottonseed meal (CSM) is an important plant protein resource. However, only a little of cottonseed meal is used as a protein resource in feed industry because of the existence of free gossypol (FG), which is an anti-nutritional factor and harmful to animals. Recently, several methods have been developed for removing FG from CSM, such as solvent extraction of FG, chemical treatment with ferrous sulfate, microbial fermentation, etc. These methods play an important role in detoxification of CSM. The method of microbial fermentation not only can detoxicate cottonseed meal, but also can improve the nutritional value of cottonseed protein. The advantages of fermentation detoxification of cottonseed meal have been recognized. But so far, the molecular mechanism of fermentation detoxification is not clear. In this study, two new gossypol-degraded strains were isolated from a cotton-planted soil, which could grow on solid medium containing gossypol. The strains were identified by morphological and molecular methods. Meanwhile, the growth characteristics of gossypol-degraded strains were preliminarily investigated by analysing their culture parameters. Additionally, the differential expression proteins of gossypol-degraded strains under different carbon source were analyzed by proteomics. The main research results were as follows:
1. Two strains with high efficiency of gossypol degradation were screened from soil with gossypol as sole carbon source and named AN-1 and MM-2, respectively. The results of the morphological observation and electron microscopy showed that AN-1 and MM-2 were filamentous fungi. And then the two gossypol-degraded strains were identified by molecular biology. The results showed that:the 18S rDNA sequence of AN-1 was found to have 99%similary with those of Aspergillus niger isolates, indicating AN-1 was referred to be A. niger; the 18S rDNA sequence of MM-2 was found to share 98%similary with those of Fusarium isolates; indicating MM-2 was referred to be Fusarium sp.
2. The isolated gossypol-degraded strains were cultured in different media to establish the optimum incubation conditions. The results of growth curvres of AN-1 and MM-2 both showed that medium containing glucose was better than gossypol and the best period used to be studied was 60-84 h incubation. The maximal biomass of both strains cultured by glucose and gossypol was at 30℃and growth in glucose was better than in gossypol. Cultured by glucose and gossypol respectively, the pH mutative curves of strains AN-1 and MM-2 were both downtrend, and glucose was more obviously than gossypol. Effects of various nitrogen sources on growth of gossypol-degraded strains showed similar trends. The results indicated that both AN-1 and MM-2 cultured by ammonium and nitrate were superior to that by yeast extract and urea, and the best nitrogen source was sodium nitrate. The tolerance of strain AN-1 cultured on solid media was analyzed under five different concentrations of gossypol (0.05%,0.1%,0.2%, 0.4%and 0.8%). The results showed that the gossypol-degraded strain AN-1 had strong tolerance of gossypol, and the optimal dosage of gossypol was 0.2%.
3. The mycelial proteins of gossypol-degraded strain AN-1 cultured in different carbon sources for 72 h were extracted by TCA/acetone method. Some satisfied 2-DE maps were obtained. The protein spots concentrated in the range of 25.0-66.2 kDa and spreaded in different gradient of pI. The 2-DE gels analyzed by PDQuest software showed that average 478 spots in glucose gel,592 spots in gossypol gels. Glucose as the master gel, average 293 spots were matched in gossypol gel and 51 protein spots were differently expressed. Among the fifty-one differential protein spots,47 PMF maps were obtained by MALDI-TOF MS. The PMF maps were searched in Mascot database. Twenty proteins were preliminarily identified, including 1 kinesin,2 glycerol-3-phosphate dehydrogenases,1 citrate synthase,1 ubiquitin,1 unnamed protein and 14 hypothetical proteins。
1.以醋酸棉酚为唯一碳源,从土壤中筛选棉酚降解菌。从平板上得到2株高效降解棉酚的菌体,分别命名为AN-1、MM-2。从形态上观察及电镜结果显示：AN-1和MM-2均为丝状真菌。通过分子生物学方法对棉酚降解菌进一步鉴定表明：AN-1的18S rDNA序列与黑曲霉(Aspergillus niger)的同源性高达99%以上,据此推测AN-1可能属于A. niger; MM-2的18SrDNA序列与镰刀霉属(Fusarium)的同源性达98%以上,据此推测MM-2可能属于Fusarium sp.。
2.对分离到的棉酚降解菌进行培养特性研究,以获得其最佳生长条件,为进一步研究奠定基础。生长曲线试验结果显示：棉酚降解菌AN-1和MM-2表现出相似生长趋势,即在葡萄糖中生长优于在棉酚中生长,培养60～84 h取种用于研究最好。温度试验结果显示：棉酚降解菌AN-1和MM-2在棉酚及葡萄糖中的生长情况类似,即30℃时菌体生物量达最大值,且在葡萄糖中的生长略好于棉酚。pH试验结果显示：棉酚降解菌AN-1和MM-2在葡萄糖和棉酚中培养时pH值均呈下降趋势,但在葡萄糖中较之棉酚中下降明显。氮源试验结果显示：不同氮源对棉酚降解菌AN-1和MM-2的影响均有相似趋势,即在铵盐及硝酸盐中生长比在酵母膏和尿素中好,且最佳氮源为NaNO3。棉酚耐受性试验结果表明：棉酚降解菌AN-1在5种不同浓度(0.05%,0.1%,0.2%,0.4%,0.8%)的棉酚中生长时具有明显差异,且AN-1对棉酚具有极强的耐受性,确定棉酚最适添加量为0.2%。
3.采用TCA／丙酮沉淀法提取棉酚降解菌AN-1在葡萄糖和棉酚中生长72 h后的菌体蛋白,得到了分辨率较好的蛋白质双向电泳图谱,蛋白质分子量主要集中在25.0～66.2 kDa之间,各pI段均有分布。通过PDQuest软件分析,葡萄糖凝胶图谱中平均检测到478个蛋白点,棉酚凝胶图谱中平均检测到592个蛋白点；以葡萄糖为参考胶,棉酚为碳源的2-DE图谱中平均匹配的蛋白点为293个,未匹配的蛋白点有299个,其中有51个为差异蛋白点。采用MALDI-TOF MS方法对差异蛋白点进行质谱鉴定,获得了47张PMF图谱,鉴定出其中20个蛋白点,包括1个kinesin,2个glycerol-3-phosphate dehydrogenase,1个citrate synthase,1个ubiquitin,1个unnamed protein和14个hypothetical protein。
Cottonseed meal (CSM) is an important plant protein resource. However, only a little of cottonseed meal is used as a protein resource in feed industry because of the existence of free gossypol (FG), which is an anti-nutritional factor and harmful to animals. Recently, several methods have been developed for removing FG from CSM, such as solvent extraction of FG, chemical treatment with ferrous sulfate, microbial fermentation, etc. These methods play an important role in detoxification of CSM. The method of microbial fermentation not only can detoxicate cottonseed meal, but also can improve the nutritional value of cottonseed protein. The advantages of fermentation detoxification of cottonseed meal have been recognized. But so far, the molecular mechanism of fermentation detoxification is not clear. In this study, two new gossypol-degraded strains were isolated from a cotton-planted soil, which could grow on solid medium containing gossypol. The strains were identified by morphological and molecular methods. Meanwhile, the growth characteristics of gossypol-degraded strains were preliminarily investigated by analysing their culture parameters. Additionally, the differential expression proteins of gossypol-degraded strains under different carbon source were analyzed by proteomics. The main research results were as follows:
1. Two strains with high efficiency of gossypol degradation were screened from soil with gossypol as sole carbon source and named AN-1 and MM-2, respectively. The results of the morphological observation and electron microscopy showed that AN-1 and MM-2 were filamentous fungi. And then the two gossypol-degraded strains were identified by molecular biology. The results showed that:the 18S rDNA sequence of AN-1 was found to have 99%similary with those of Aspergillus niger isolates, indicating AN-1 was referred to be A. niger; the 18S rDNA sequence of MM-2 was found to share 98%similary with those of Fusarium isolates; indicating MM-2 was referred to be Fusarium sp.
2. The isolated gossypol-degraded strains were cultured in different media to establish the optimum incubation conditions. The results of growth curvres of AN-1 and MM-2 both showed that medium containing glucose was better than gossypol and the best period used to be studied was 60-84 h incubation. The maximal biomass of both strains cultured by glucose and gossypol was at 30℃and growth in glucose was better than in gossypol. Cultured by glucose and gossypol respectively, the pH mutative curves of strains AN-1 and MM-2 were both downtrend, and glucose was more obviously than gossypol. Effects of various nitrogen sources on growth of gossypol-degraded strains showed similar trends. The results indicated that both AN-1 and MM-2 cultured by ammonium and nitrate were superior to that by yeast extract and urea, and the best nitrogen source was sodium nitrate. The tolerance of strain AN-1 cultured on solid media was analyzed under five different concentrations of gossypol (0.05%,0.1%,0.2%, 0.4%and 0.8%). The results showed that the gossypol-degraded strain AN-1 had strong tolerance of gossypol, and the optimal dosage of gossypol was 0.2%.
3. The mycelial proteins of gossypol-degraded strain AN-1 cultured in different carbon sources for 72 h were extracted by TCA/acetone method. Some satisfied 2-DE maps were obtained. The protein spots concentrated in the range of 25.0-66.2 kDa and spreaded in different gradient of pI. The 2-DE gels analyzed by PDQuest software showed that average 478 spots in glucose gel,592 spots in gossypol gels. Glucose as the master gel, average 293 spots were matched in gossypol gel and 51 protein spots were differently expressed. Among the fifty-one differential protein spots,47 PMF maps were obtained by MALDI-TOF MS. The PMF maps were searched in Mascot database. Twenty proteins were preliminarily identified, including 1 kinesin,2 glycerol-3-phosphate dehydrogenases,1 citrate synthase,1 ubiquitin,1 unnamed protein and 14 hypothetical proteins。
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