几种绿僵菌的核相及脉冲电泳（PFGE）核型方法研究-微生物专业论文.docxVIP

贵州人学2005届硕士研究生学位论文中文摘要 贵州人学2005届硕士研究生学位论文 中文摘要 本研究采用细胞核荧光染色技术、原生质体细胞脱壁技术和脉冲电 泳技术首次比较系统的对5种绿僵菌(Metarhizium．spp)各生长阶段的 核相进行系统观察分析，并进行了脉冲电泳核型分析中完整染色体DNA 制备方法和电泳方法的研究。 核荧光染色发现，011108菌株和两种来自于虫草无性型的戴氏绿僵 菌、金龟子绿僵菌大孢变种的分生孢子多为单核，少有双核，各生长阶 段没有明显的核数变化：金龟予绿僵菌小孢变种的分生孢子几乎全为双 核：双型孢绿僵菌培养性状及细胞核数发生了一定变化，出发菌株的大、 小予6i子几乎都具有细胞核，大孢子的核数为2～4个，转接20多代后的 菌株，大孢子多为单核，小孢子很少具有细胞核。 本研究证明了原生质体法是制备绿僵菌完整染色体DNA样品最可 靠的方法，并建立了适合脉冲电泳核型分析的供试绿僵菌原生质体制备 体系，解决了供试菌株完整染色体DNA样品制备的问题。即以1 5～ 20mg／ml的溶壁酶为细胞壁破壁酶，O．7mol／LNaC!，pH5．8为渗压稳定剂， 30℃处理1．5～2h。 以8株酿酒酵母商品菌株和自制marker为对比得到比较清晰的电 泳核型图谱，具有10～1 3条染色体DNA带；对来自于虫草无性型的一 株金龟子绿僵菌大孢变种的电泳核型结果表明，该菌株至少有5条染色 体，最大的染色体分子量为5．7Mb±。 关键词：绿僵菌；荧光染色：核相；原生质体：PFGE：核型 几种绿僵菌的核相及脉冲电泳(PFGE)核型方法研究Abstract 几种绿僵菌的核相及脉冲电泳(PFGE)核型方法研究 Abstract Each growth stage nuclear phase，preparation methods of intact chromosome DNA and electrophoretic methods in PFGE(Pulsed Field Gel Electrophoresis) analysis ofMetarhizium spp．were studied with nuclear fluorescence staining technique，protoplast technique and PFGE in the thesis． With fluorescence staining，conidia ofM．taii，M anisopliae vat：m巧or and 01 I 108 strain were discovered having majority monokaryou and few diplokaryon、no clear nuclear number transformation in each growth stage．Conidia ofM．anisopliae Var,anisopliae have whole diplokaryon nearly．However,conidia M biformisporae have particular transformation of cultural characteristics and nuclear numbeL The big condiospore and small condiospore ofprimitive strain almost have nuclear and 2 to 4 nuclear in the former．After more 20 generation，the big condiospom just have monokaryou and small condiospore have few nuclear． Protoplast methods WaS proved being credible preparation methods of intact chromosome DNA．Protoplast preparation system suited PFGE analysis of offering M．spp was built and being resolved the problem ofpreparation ofintact chromosome DNA．It is the optimum condition to preparing protoplast that mycelia are digested selecting 0．7mol／LNaCl，pH5．8 as stablizer in 15～20mg／ml lywallzyme at 30 4C for