几种丝状真菌原生质体的形成与再生

本文报道从黑曲霉、米曲霉、酱油曲霉、康宁木霉、拟青零等工业上有重要用途的7种丝状真菌菌丝制备原生质体,以及对这些真菌原生质再生过程中形态学变化进行观察的结果。实验表明用商品纤维素酶、蜗牛酶、溶菌酶配成的混合酶液,可成功地对上述丝状真菌制备出原生质体。在所有供试菌株中均观察到由原生质体直接长出菌丝,及原生质体先形成酵母状物再长出菌丝这两类再生方式。     

去壁酶与酶解方式对曲霉原生质体释放的影响

研究了纤维素酶、蜗牛酶、溶菌酶以及菌丝体培养方式和酶解方式对黑曲霉和米曲霉菌丝释放原生质体的效应。发现黑曲霉菌丝原生质体制备最佳条件为固体透析培养菌丝体,2%纤维素酶,在平皿中,28℃和80r/min条件下酶解3h;米曲霉原生质体制备最佳条件为2%纤维素酶+1%蜗牛酶+5mmol/L二硫苏糖醇,酶解时间6h,其它条件与黑曲霉的相同。     

γ——亚麻酸生产菌深黄被孢霉原生质体的形成和再生

采用２％的溶壁酶加４％的蜗牛酶,从深黄被孢霉的菌丝中获得了大量的原生质体,同时对菌丝的培养时间,渗透压稳定性,酶解系统和酶解温度等因素了进行了系统的观察,从而获得了制备深黄被孢霉原生质体的最适条件,并对该原生质体在高渗培养基上进行了再生实验,其再生率为３２％。     

γ-亚麻酸生产菌深黄被孢霉原生质作的形成和再生

采用2％的溶壁酶加4％的蜗牛酶,从深黄被孢霉的菌丝中获得了大量的原生质体。同时对菌丝的培养时间、渗透压稳定剂、酶解系统和酶解温度等因素进行了系统的观察,从而获得了制备深黄被孢霉原生质体的最适条件。并对该原生质体在高渗培养基上进行了再生实验,其再生率为32％。     

γ—亚麻酸生产菌深黄被孢霉原生质体的形成和再生

采用2％的溶壁酶加4％的蜗牛酶,从深黄被孢霉的菌丝中获得了大量的原生质体。同时对菌丝的培养时间、渗透压稳定剂、酶解系统和酶解温度等因素进行了系统的观察,从而获得了制备深黄被孢霉原生质体的最适条件。并对该原生质体在高渗培养基上进行了再生实验,其再生率为32％。     

生防木霉菌原生质体的制备及再生研究

ACCC 30150是由本实验室筛选的一株对黄瓜枯萎病、青椒疫病等多种土传病害具有较好防治效果的生防木霉菌.为了建立该生防木霉菌的CaCl2/PEG转化体系,对其原生质体制备及再生条件进行了摸索.结果表明,以0.6 M NaCl作为渗透压稳定剂,用PDA平板上培养36 h的微小菌丝接种液体菌丝培养基,菌龄16 h,用20 mg/ml的纤维素酶和蜗牛酶以1:1的比例混合,按照菌丝重量(g):酶液体积(ml)=1:20的比例,36℃酶解2 h原生质体产量最高.同时,酶解2 h,以CMR1为再生培养基原生质体的再生率最高.     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的:采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法:经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶(0.8%溶菌酶+1.2%蜗牛酶+1.6%纤维素酶)处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果:在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论:采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

米曲霉两株营养缺陷型原生质体的形成和再生的条件实验*

采用1%溶壁酶加1%玛瑙螺酶(褐云玛瑙螺消化液的冷冻干粉)的混合酶,自米曲霉(Aspergillus oryzae)的两株营养缺陷型中获得了大量的原生质体,并比较了渗透压稳定剂、温度、菌丝体的培养基成分等因素对原生质体形成和再生的作用。无机盐类稳定剂(NaCl、KCl)获得了高产量的原生质体,而有机类(蔗糖、甘露醇、山梨醇)做为稳定剂不甚理想。对120和720菌株的原生质体在高渗再生培养基上进行再生试验,再生率分别为52%和65%。     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的：采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法：经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶（0.8%溶菌酶＋1.2%蜗牛酶＋1.6%纤维素酶）处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果：在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论：采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

米曲霉两株营养缺陷型原生质体的形成和再生的条件实验

采用1%溶壁酶加1%玛瑙螺酶(褐云玛瑙螺消化液的冷冻干粉)的混合酶,自米曲霉(Aspergillus oryzae)的两株营养缺陷型中获得了大量的原生质体,并比较了渗透压稳定剂、温度、菌丝体的培养基成分等因素对原生质体形成和再生的作用。无机盐类稳定剂(NaCl、KCl)获得了高产量的原生质体,而有机类(蔗糖、甘露醇、山梨醇)做为稳定剂不甚理想。对120和720菌株的原生质体在高渗再生培养基上进行再生试验,再生率分别为52%和65%。     

丝状真菌AL18的原生质体制备和再生条件的优化

目的:为了建立起产苝醌类光敏剂丝状真菌AL18原生质体制备和再生体系。方法:采用单因素实验法研究了预处理方式、渗透压稳定剂和酶解条件对原生质体制备率和再生率的影响。结果:原生质体制备及再生的最佳条件是用0.3%的β-巯基乙醇预处理15 min,酶解液以0.6 mol/L的MgSO4·7H2O作为渗透压稳定剂,0.02 mol/L的磷酸盐缓冲液pH值为5.8,纤维素酶:蜗牛酶=2:3,酶的总浓度为15mg/mL,36℃酶解2h;以0.6mol/L的蔗糖作为再生培养基的渗透压稳定剂。结论:原生质体的制备率和再生率可分别达到1.42×107/mL和3.2%。     

灭蚊真菌——大链壶菌原生质体形成和再生的研究

采用1％纤维素酶与1％真菌脱壁酶混合液作脱壁酶,0.6mol/L山梨醇为渗透压稳定剂,从摇床培养的12—14小时菌龄的大链壶菌(Lagenidium giganteum)菌丝体获得原生质体。酶解3—5小时后,产量可达1.4—2.0×10~6/mL。并在双层培养基上初步实现了原生质体再生。     

Development of a sample preparation method for fungal proteomics

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.     

酿酒酵母W5及休哈塔假丝酵母20335原生质体制备条件的确定

确定了酿酒酵母W5及休哈塔假丝酵母20335原生质体制备的最佳条件。选取不同脱壁预处理时间及不同酶解时间,对酿酒酵母W5、休哈塔假丝酵母20335进行原生质体制备和再生,比较制备率和再生率。确定脱壁预处理30 min后,以终浓度2%的蜗牛酶,30℃、100 r/min酶解处理15 min为双亲株原生质体制备的最佳条件。利用原生质体融合的方法,以酿酒酵母W5和休哈塔假丝酵母20335为亲本株,构建可以利用木糖生产生物乙醇的新型酿酒酵母融合株,该前期工作为W5、20335原生质体融合工作奠定了重要的基础,对于将木质纤维素原料转化为生物乙醇的研究具有极其重要的意义。     

籼稻原生质体培养和植株再生

用籼稻IR52、IR8和IR45的幼花序和幼胚愈伤组织在LS培养基建立了稳定的悬浮培养物。悬浮系的建立经历三个阶段:褐变期,长根期,成熟期。建立了适合籼稻原生质体生长的Y8培养基,其植板率显著高于KPR和PCM培养基。悬浮细胞系间差异明显,只有部份系可以提供有分裂能力的原生质体或具看护活性。以上三个品种的原生质体均分裂良好,但只有IR52和IR8分化出苗,其中IR52分化率1.25%,得再生植株50余株,移至田间生长结实正常。     

多种被子植物花粉生殖细胞大量分离技术的比较研究

从11种植物的成熟花粉粒中分离出大量生活的生殖细胞。比较了四种分离方法(一步渗透压冲击法、二步渗透压冲击法、低酶法和花粉原生质体释放法)在不同植物中的效果。归纳出三类植物适于采用三种分离方法。研究了影响分离效果的若干重要因素。对5种植物的分离生殖细胞进行了纯化。经鉴定,纯化的细胞群体中80%以上的细胞是生活的。     

原生质体紫外诱变选育竹红菌甲素高产菌株

采用原生质体紫外诱变技术选育竹红菌甲素高产菌株。结果表明:以竹黄菌Shiraia sp.S8为出发菌株,当使用混合酶系(5 mg/mL纤维素酶和10 mg/mL蜗牛酶)在30℃处理菌丝2 h,获得菌丝原生质体3.24×106个/mL。以竹黄菌原生质体在距离15 W紫外灯30 cm处照射诱导,获得诱变菌株C6。其竹红菌甲素产量达到28.1 mg/L,比原始出发菌株提高了53.7%,且遗传稳定,具有较高的医药与工业应用价值。     

Liposome-mediated mycelial transformation of filamentous fungi

Liposome-mediated transformation is common for cells with no cell wall, but has very limited usage in cells with walls, such as bacteria, fungi, and plants. In this study, we developed a procedure to introduce DNA into mycelium of filamentous fungi, Rhizopus nigricans LH 21 and Pleurotus ostreatus TD 300, by liposome-mediation but with no protoplast preparation. The DNA was transformed into R. nigricans via plasmid pEGFP-C1 and into P. ostreatus via 7.2 kb linear DNA. The mycelia were ground in 0.6 M mannitol without any grinding aids or glass powder for 15 min to make mycelial fragments suspension; the suspension was mixed with a mixture of the DNA and Lipofectamine 2000, and placed on ice for 30 min; 100 μL of the transformation solution was plated on potato dextrose agar (PDA) plate and cultivated at 28 °C for transformant screening. The plasmid and the linear DNA were confirmed to be integrated into the host chromosome, proving the success of transformation. The transformation efficiencies were similar to those of electroporation-mediated protoplast transformation (EMPT) of R. nigricans or PEG/CaCl₂-mediated protoplast transformation (PMT) of P. ostreatus, respectively. The results showed that our procedure was effective, fast, and simple transformation method for filamentous fungi.     

Present and potential applications of cellulases in agriculture, biotechnology, and bioenergy

Cellulase (CEL) presently constitutes a major group of industrial enzyme based on its diverse ranges of utilization. Apart from such current and well-established applications—as in cotton processing, paper recycling, detergent formulation, juice extraction, and animal feed additives—their uses in agricultural biotechnology and bioenergy have been exploited. Supplementation of CELs to accelerate decomposition of plant residues in soil results in improved soil fertility. So far, applying CELs/antagonistic cellulolytic fungi to crops has shown to promote plant growth performance, including enhanced seed germination and protective effects. Their actions are believed mainly to trigger plant defense mechanisms and/or to act as biocontrol agents that mediate disease suppression. However, the exact interaction between the enzymes/fungi and plants has not been clearly elucidated. Under mild conditions, removal of plant cell wall polysaccharides by CELs for protoplast preparation results in reduced protoplast damage and increased viability and yields. CELs have recently shown great potential in enzyme aid extraction of bioactive compounds from plant materials before selective extraction through enhancing release of target molecules, especially those associated with the wall matrix. To date, attempts have been made to formulate CEL preparation for cellulosic-based bioethanol production. The high cost of CELs has created a bottleneck, resulting in an uneconomic production process. The utilization of low-cost carbohydrates, strain improvement, and gene manipulations has been alternatively aimed at reducing the cost of CEL production. In this review, we focus on and discuss current knowledge of CELs and their applications in agriculture, biotechnology, and bioenergy.