禾谷镰孢菌原生质体的制备和再生研究

为建立禾谷镰孢菌（Fusarium gromineanum Schwabe）转化体系，选用禾谷镰孢菌株F128为受体，分别用裂解酶、蜗牛酶、崩溃酶、纤维素酶、几丁质酶消化该菌细胞壁，都可获得一定数量的原生质体。其中产生原生质体效率最高的是崩溃酶，而且以5g／L浓度产生的原生质体数量最多，消化3．5－4．5h即可获得相当数量的原生质体，菌量的加入以每毫升酶解液中加入20mg湿菌性产生的原生质体最多。所制备出的原生质体的再生方式至少有2种。     

棘孢小单孢突变株原生质体制备及再生

以棘孢小单孢突变株（Ｍｉｃｒｏｎｏｚｐｏｒａ ｅｃｈｉｎｏｚｐｏｒａ ＪＩＭ－４０１）为出发菌株,进行原生质体制备及再生条件的研究,结合ＵＶ照射及高能电子流诱变,筛选出ＭＳ－１１６菌株,３０吨发酵罐试验,其Ｃ２ｂ组分达８５．６％。     

河北省丝孢菌的研究：Ⅱ.弯孢属

对弯孢属(Curvularia Boedijn)的分类学作了简短的阐述。报道了河北省弯孢属真菌的10个种,其中4种为国内新记录种,5种为河北省新记录种。国内新记录种是枝孢弯孢(C.brachyspora Boedijn),香茅弯孢(C. cymbopogonis Grove & Skolko),中隔弯孢(C. intermedia Boedijn)和管突弯孢(C. protuberata Nelson & Hodyes);河北省新记录种是须芒草弯孢(C. andropogonis (zimm.)Boedijn),苍白弯孢(c.pallescens Boedijn),棒状弯孢(C. clavata Jain),膝曲弯孢(C. geniculata(Tracy & Earle)Boedijn)和近缘弯孢(C. affinis Boedijn)。文中有种的鉴定特征描述和分种检索表。     

四川省玉米弯孢菌对玉米的致病性研究

为了明确四川省玉米弯孢菌叶斑病(Maize Curvularia leaf spot)病原菌的致病性分化情况,在四川省玉米弯孢菌种类鉴定和生物学特性研究的基础上,选用不同种类或不同来源的27个弯孢菌菌株,在感病品种川单13和抗病品种Mo17进行致病性测定.把病斑大小和产孢量作为弯孢菌致病性鉴定与评价的标准,结果发现不同种类的弯孢菌或同一种类不同来源的菌株对玉米均有致病性,而且各菌株之间存在致病性的分化,可初步划分为强、中等和弱致病力三种类型.     

小诺霉素产生菌棘孢小单孢菌的原生质体诱变育种

通过对菌株产量分布参数的统计学分析，棘抱小单孢菌７４＃被选作原生质体诱变育种的出发菌株．获得小诺霉素含量为８０％的高产突变株的概率以紫外线对原生质体悬液３０秒诱变处理为最高（２２．８％），５株高产突变株的小诺霉素组份与效价平均分别比出发菌株提高３３．３％与５１．２％．初步探讨了甘氨酸对原生质体化的作用机理以及原生质体诱变与再生处理提高小诺霉素产生菌生产能力的机理．     

四川省玉米弯孢叶斑病菌的种类及生物学特性研究

为了明确引致四川省玉米弯孢菌叶斑病（Maize Curvularia leaf spot）的病原菌种类和优势种,以便为病害的防治和抗病育种提供依据,对所获得的78个弯孢菌（Curvularia）菌株采用形态学分类法进行种类鉴定,结果鉴定出6个种或变种,其中属于新月弯孢菌（Curvularia lunata）种类的菌株占种群的61．5％,新月弯孢菌是四川省玉米弯孢菌叶斑病的主要致病菌种类．对属于其中3个种的4个弯孢菌菌株进行生物学特性研究,结果表明,各供试菌株在不同条件下的生长速度都存在一定的差异．     

双孢蘑菇原生质体制备条件的优化

采用L16(45)正交分析方法,对双孢蘑菇原生质体制备工艺进行了优化.试验结果表明,最佳制备条件是取菌龄7 d的液体菌丝体,以2.5%溶壁酶 7%蜗牛酶为组合,0.6 mol/L NaCl作为渗透压稳定剂,在30℃条件下,酶解3 h,原生质体产率为2.34×108个/mL.本研究可为双孢蘑菇原生质体的融合及转基因研究提供理论依据.     

玉米弯孢叶斑病菌ATMT转化条件优化及转GFP单孢的获得

为了明确玉米弯孢叶斑病菌的侵入机理,对农杆菌介导法（ATMT）转化弯孢叶斑病菌的条件进行了探究。以农杆菌AGL-1为侵染菌系、pCAMDGFP作为转化载体。结果表明,玉米弯孢叶斑病菌的ATMT最佳转化条件为：分生孢子萌发12 h后,与农杆菌在25℃黑暗条件下共培养48 h,共培养介质为微孔滤膜。转化效率可达到100～1...     

绣球菌原生质体制备及再生条件的初步研究

为进一步改善绣球菌栽培特性、提高栽培产量,对绣球菌原生质体制备与再生条件进行了初步探索.以绣球菌原生质体再生量为指标,对等渗剂、酶反应温度、pH和反应时间等影响因素进行了优化.结果表明,采用2%的溶壁酶溶解绣球菌菌丝细胞壁,制备原生质体的最佳条件是,以0.6 mol/L蔗糖为等渗剂,在30℃、pH值6.0环境下反应3 h.在此条件下,原生质体的再生量为11 850个/mL.     

谷氨酸产生菌原生质体的制备和再生

6株谷氨酸产生菌以甘氨酸、青霉素和溶菌酶不同组合处理,对其原生质体形成进行了比较,在最佳条件下,原生质体形成率可达95～100％。B9-15 菌株细胞形态特殊,表面呈凹陷状,仅在含1.5～2.0％甘氨酸的高渗培养液中就能形成原生质体,在加有壁制剂的丁二酸钠高渗培养基上,所试菌株的原生质体再生率从10.5％～61.8％,差异性较大。     

Proteomic comparison of four maize inbred lines with different levels of resistance to Curvularia lunata(Wakker)Boed infection

Protein profiles of leaves in four maize inbred lines with different disease resistance to pathogen Curvularia lunata(Wakker)Boed were studied by two-dimensional electrophoresis(2-DE)and mass spectrometry.Proteins were extracted from the forth leaf of maize seedlings 24 h after fungal inoculation,and fractionated by polyethylene glycol to precipitate the most abundant leaf protein,Rubisco,before gel separation.Protein profiles from 2-DE showed that total numbers of protein spots were increased in all four inbred lines inoculated with C.lunata CX-3 strain compared with the control.The numbers of changed protein spots in abundance were higher in resistant inbred lines than in susceptible ones,which implied that resistant inbred lines were more sensitive than susceptible ones to pathogen infection.Among proteins identified by MALDI-TOF MS,germin-like protein GLP and translation initiation factor eIF-5A were supposed to play important roles in maize resistance against C.lunata infection.     

Proteomic comparison of four maize inbred lines with different levels of resistance to Curvularia lunata （Wakker） Boed infection

Protein profiles of leaves in four maize inbred lines with different disease resistance to pathogen Curvularia lunata(Wakker)Boed were studied by two-dimensional electrophoresis(2-DE)and mass spectrometry.Proteins were extracted from the forth leaf of maize seedlings 24 h after fungal inoculation,and fractionated by polyethylene glycol to precipitate the most abundant leaf protein,Rubisco,before gel separation.Protein profiles from 2-DE showed that total numbers of protein spots were increased in all four inbred lines inoculated with C.lunata CX-3 strain compared with the control.The numbers of changed protein spots in abundance were higher in resistant inbred lines than in susceptible ones,which implied that resistant inbred lines were more sensitive than susceptible ones to pathogen infection.Among proteins identified by MALDI-TOF MS,germin-like protein GLP and translation initiation factor eIF-5A were supposed to play important roles in maize resistance against C.lunata infection.     

Analysis of the Protein Electrophoresis Patterns of Curvularia Lunata in Maize and Immunological Detection of the Pathogenic Culture

Twelve isolates of Curvularia lunata and six related species from maize or other host plants are examined for the analysis of polyacrylamide gel electrophoresis (SDS-PAGE) patterns of their soluble proteins,Differences exist significantly among different species and the same species.There is a special protein band for Curvularia at relative mobility value 0.177(Rf 0.177) and a special protein band for C.lunata at Rf 0.225,Two kinds of rpimary antibodies to C.lunata are used to indirect ELISA and Avidin-Biotion-peroxidase Complex(ABC) immunoassay to evaluate their detecting sensitivity and specificity,The sensitivity of ABC immuno- assay is higher than indirect ELISA.APAbs to C.lunata is special to C.lunata.     

Comparative proteomic analysis of the response in resistant and susceptible maize inbred lines to infection by Curvularia lunata

Proteins differentially expressed from maize leaves in response to the infection by Curvularia lunata strain CX-3 were identified through a high-resolution two-dimensional gel electrophoresis (2-DE) method. Two inbred lines, 78599-1 and E28, were used, respectively, as resistant and susceptible lines to CX-3 infection. Proteins were extracted from the fourth leaves of six- or seven-leaf stage plants sampled at 24, 36, 48, 60, and 72 h after inoculation with CX-3. Twenty-seven differentially expressed protein spots resolved on the 2-DE gels were identified by MALDI-TOF MS/MS. The results showed that these proteins are associated with photosynthesis, respiration,oxidative and drought stress tolerance as well as signal transduction in maize. Among stress-related proteins, the 22 kDa drought-inducible protein, putative glutathione peroxidase (GPX), and translation initiation factor (eIF-5A) were up-regulated in the resistant inbred line and were implicated in host defense response to C. lunata infection. It suggests that drought-inducible and oxidation stress-related proteins might directly contribute to maize resistance to C. lunata.     

大球盖菇原生质体制备及紫外诱变

对大球盖菇（Stropharia rugoso-annulata）原生质体制备的条件进行了研究,并确定了最佳条件组合:选用15 g/L的纤维素酶+15 g/L的蜗牛酶的混合酶来酶解菌丝,0.6 mol/L的甘露醇作为渗透压稳定剂,菌龄为3d,酶解温度30℃,酶解时间2.5h,在此条件下制得的原生质体产量最高达1.70×...     

一株内生真菌的原生质体制备及再生过程研究

通过对酶系组成和酶解条件的研究,得出了制备禾本科植物内生真菌Neotyphodiumsp.原生质体的最佳条件:鲜菌丝由10.3%蔗糖溶液预处理,酶系组成为1.5%崩溃酶、1.0%蜗牛酶、1.0%纤维素酶和2.0%溶菌酶,酶解温度32℃,pH 6.8,酶解5 h,原生质体得率达7.3×103个/mg鲜菌丝.另外,液体再生涂布平板法结合钙离子作用可使原生质体再生率达4.72%,为不含钙离子处理组的2.5倍.     

霍山石斛原生质体制备条件初探

本实验采用机械法和酶解法对霍山石斛原生质体的分离条件进行研究。结果表明,机械法制备霍山石斛原生质体,得率很低。正交试验法优选出的酶解法最佳组合条件为:纤维素酶1.5% 果胶酶1.5% 离析酶1.0%的酶液组合(溶于0.6mol/L的甘露醇),pH值5.4,28℃温育18 h。     

出芽短梗霉的原生质体形成及再生研究

采用多因素实验正交优选法对出芽短梗霉（Ａｕｒｅｏｂａｓｉｄｉｕｍｐｕｌｕｌａｎｓ）的原生质体制备和再生条件进行了研究．发现在用ＹＥＰＤ作生长培养基,菌龄２４ｈ,复合酶处理（含０．２５％（ｗ）蜗牛酶＋０．２５％（ｗ）纤维素酶）,０．６ｍｏｌ／ＬＫＮＯ３作渗透压稳定剂的情况下,原生质体制备率最高,酶解１ｈ,制备率为１００％,再生率为０．３５％．如用ＰＤＡ为再生基础培养基,在添加５０ｍｍｏｌ／ＬＣａＣｌ２,１．０％（ｗ）ＰＥＧ（ＭＷ６０００）等再生促进因子的情况下,再生率可提高到１．２３％     

产辅酶Q10的类球红细菌原生质体制备和再生研究

 辅酶Q₁₀(CoQ₁₀)是人类细胞自身产生的内源性辅酶因子,是一类天然抗氧化剂,目前已广泛应用于医药、食品、化妆品等诸多领域.基因组改组技术的核心内容为原生质体融合,为了提高基因组改组的效率,提高辅酶Q₁₀的产量,本研究对产辅酶Q₁₀的类球红细菌原生质体制备及再生条件进行了优化,通过生长曲线、正交试验等确定了原生质体制备及再生的适宜条件：溶菌酶浓度1mg/mL、作用温度37℃、作用时间1h以及再生培养基蔗糖浓度10%.在此条件下,类球红细菌原生质体形成率可达到96.1%,再生率可达到28.8%.这为进一步对该菌的基因组改组研究奠定了基础.