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．     

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.     

不同抗性玉米自交系对玉米弯孢菌侵染应答反应差异蛋白分析

采用Mg／NP-40及PEG预分离技术,在去除叶片高丰度表达蛋白主要是Rubisco的基础上,研究了鲁原92（高抗）,78599-1（抗）,E28（感）和黄早4（感）4个玉米自交系苗期叶片对玉米弯孢菌（Curvularia lunata）强致病株系CX-3侵染应答反应差异蛋白质图谱．接种病菌后抗、感自交系蛋白质点总数均明显增加;抗性自交系表达丰度上调二倍的蛋白点数多于感病自交系．对可能与抗、感病性明显相关的8个差异蛋白点进行了质谱鉴定,发现玉米萌发素类似蛋白GLP和翻译起始因子elF-5A与玉米对玉米弯孢菌叶斑病的抗性关系密切．     

寄主抗性胁迫下玉米弯孢叶斑病菌致病力相关蛋白与基因表达的分析

采用RT-PCR方法,研究了玉米弯孢菌的弱致病性分化型WS18在抗性玉米自交系M017,沈135,785991上分别继代接种后所得的各个世代菌株中Sod,Scdl,Brnl三种基因表达量的变化．Sod基因变化比较灵敏,继代前期有剧烈变化,而后恢复与原始菌株相当;而Scdl与Brnl基因表达量在继代前后均未发生显著性变化;这与基因所编码蛋白的表达量变化趋势有所差异．因此,在寄主一定程度的选择压力下病菌致病力相关蛋白的变化可能在mRNA水平并没有发生同步改变．     

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。     

农杆菌介导的玉米茎尖遗传转化及优化

通过用农杆菌介导法,以玉米自交系B73的种子培养成的黄化苗的茎尖作为转化的受体材料.将具有玉米C4-PEPC基因的植物表达载体p TCK303-P-ZF导入玉米自交系B73,根据载体上所带有的抗性基因,以潮霉素筛选获得抗性植株,进而为玉米遗传转化的相关研究打下坚实的基础.     

稗弯孢菌（Curvularis lunata）原生质体的制备

研究了不同菌龄、酶浓度、渗透压稳定剂、缓冲液pH值以及酶解温度和时间等因素对稗弯孢菌(Curvularia lunata)原生质体形成的影响。将培养18h的菌丝,以0.7mol/L KCl作为渗透压稳定剂,30℃下,经过2％溶壁酶和4％纤维素酶混合酶液(pH5.8)酶解4h,原生质体在静止条件下最大释放量达到 1.7×10~6/mL,再生率为0.76％。     

根癌农杆菌介导的异角状拟盘多毛孢菌转化系统研究

以携带潮霉素B磷酸转移酶抗性基因(hph)的pBHt1作为转化载体,根癌农杆菌AGL-1作为转化介体,实施转化异角状拟盘多毛孢菌。研究发现,异角状拟盘多毛孢菌的最优转化体系为:异角状拟盘多毛孢菌分生孢子悬浮液浓度为1×106个/mL,根癌农杆菌OD600为0.3,共培养时间48 h,共培养温度为25℃,诱导培养基中乙酰丁香酮(AS)浓度为200μmol/L,选择培养基添加250μg/mL潮霉素B、250μg/mL头孢噻肟钠。1×106个异角状拟盘多毛孢菌分生孢子可以产生200～300个转化子,随机挑选10个转化子进行PCR检测,均能扩增出预期条带,且在不含潮霉素B的PDA培养基平板上转化子连续培养5代后,hph基因仍能稳定遗传,这表明T-DNA已经插入到异角状拟盘多毛孢菌的基因组中。此次建立的异角状拟盘多毛孢菌的转化体系可为该病菌的功能基因研究和寄主与病原菌的互作研究提供有效的工具。     

16个黄瓜品种对棒孢叶斑病的抗病性鉴定

为了确定不同黄瓜品种对棒孢叶斑病菌的抗性,采用田间调查结合人工接种孢子悬浮液的方法测定了16个黄瓜品种抗病性.结果 表明:黄瓜品种田间抗病性程度均低于人工接种的抗病性;博美70,博美80-16,津优308和中荷10为高抗品种;中荷15,中蔬C56,中荷16,中农12,津优35和中荷102为抗病品种.而且同一品种针对培养...     

粗糙脉孢菌产黑色素条件优化及黑色素物理特性研究

优化了粗糙脉孢菌(Neurospora crassa)AS3．1602生产黑色素的条件，结果表明，在葡萄糖0．5g/L,(NH4)2SO4 8g/L,NaCl3 g/L,CaCl2 0.1g/L、酪氨酸取饱和浓度时，有相对高的产量．对所产黑色素进行了分离纯化，并研究了其理化性质．     

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.     

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.     

根癌农杆菌介导的马铃薯遗传转化体系

随着分子生物学研究的深入,植物功能基因组研究以及后续的大规模基因工程都必将依托于高效的植物遗传转化体系,因此植物遗传转化体系越来越成为分子生物学研究和遗传改良的重要技术基础．农杆菌介导转化法被认为是最具发展潜力的植物遗传转化方法．本文介绍了根癌农杆菌介导的马铃薯遗传转化体系．     

原球茎为转化受体的农杆菌介导石斛遗传转化

以携带双元载体pCAMBIA1305.1的农杆菌菌株EHA105转化石斛种子萌发形成的原球茎,通过GUS瞬时表达检测对受体状态、农杆菌活化条件、共培养时间等转化影响因子进行了优化;共培养4 d的原球茎接种到含有30 mg/L潮霉素(Hyg)和500 mg/L头孢噻肟钠的愈伤组织诱导与增殖培养基上以获得潮霉素抗性的愈伤组织,抗性愈伤组织在含Hyg 50 mg/L的再生培养基上再生植株,抗性株经GUS组织化学、PCR和PCR-southem blot检测证明为转化株。     

超声波辅助的发根农杆菌对黄瓜的遗传转化

由于超声波处理可在外植体上产生许多微小伤口,从而提高农杆菌与外植体之间的接触面积,提高农杆菌对外植体的转化频率。用野生型发根农杆菌（Agrobacterium rhizogenes)A4菌株感染黄瓜（Cucumis sativus L.) 无菌苗的子叶和下胚轴,建立了黄瓜的发根遗传转化体系。同时,对超声波处理时间、外植体放置方向、预培养时间、外植体类型、乙酰丁香酮的添加与否等影响因素进行了研究。结果表明,采用外植体在添加0．5mg/L NAA和2mg/L 6-BA的MS培养基上预培养4d,超声波处理2s,共培养3d,在预培养共培养期间子叶上表皮向下可以取得最佳的转化效果,发根诱导率为94．7％。乙酰丁香酮对发根诱导频率影响不明显。冠瘿碱检测表明,发根确定能合成农杆碱。     

农杆菌介导的中国水仙遗传转化体系的建立

利用根癌农杆菌介导法将GUS基因导入中国水仙,组织化学法检测转化后共培养3d的水仙鳞茎盘组织块,GUS基因瞬时表达很好,90％以上的材料呈现大面积的蓝色斑块,取筛选培养20d的材料检测GUS基因的稳定表达,约1％的组织块有蓝色反应。水仙预培养10d后转化,转化前在菌液中加入60μmol/L AS活化2－3h,转化率提高了两倍以上。转化材料在MS＋BA10＋NAA0．2＋Cef300＋Gyg30培养基上培养20d左右分化出小鳞芽,频率约为60％,组织块平均出芽5．7个,最多可达14个,小鳞芽10d后长成小鳞茎。     

根癌农杆菌介导的GFP在洋葱表皮细胞定位研究

采用根癌农杆菌介导的方法,以受控于CaMV35S启动子的携带有GFP报告基因的双元植物表达载体pCAMBIA1300-35S-GFP转化洋葱表皮细胞.荧光显微镜下观察结果显示,GFP基因在经浸染和共培养后的洋葱表皮细胞中得到了表达,绿色荧光分布在细胞核和细胞质中,为进一步研究新基因的亚细胞定位和瞬时表达奠定了基础.