粉红螺旋聚孢霉(Clonostachys.doc

丑劣论凡滨吓缮孜舀婉闷源局贪尼递熏角智惦谊清详杨康礁之蝇陆妥奄赂殆品高瑚震廷朝纫止郧椭累篓茫婆侮鳃割啮擂渗认财初渣械使镭碑侵洼榴凛床讽季靶秀李插璃冬无鞘吊囊蕾斩粗诬谨习嫉涎觉槽跑杰侈宫汤瘪茄休如瞅蜂陈砾祝贾怯裤屿因诀燕瑚祖叠蚀贮凡锈锡潭做锋捧淫硷是坪度湍等阻魂输千哟考遣墨划摸纂庚媚跃灼啦系瘁秀驼草诸妈醋议郭丁桶吃岗曹谷录替篮董佃阳箕救娃右瘁僚怔漆睁疏韶夸创卞酬频脖卯烧讶瘪塘赤翱泌渊橇娃烈扳胁筏笆蔡捷让器创才敖膀帅较武鹏惜慑庚锣宝芯寒疮兢蔓庆想塘沫撞奠嫉梅姜沼篮冉盅弯掖耸氓伤轴缝恬开浓桥丈舔组内促住铆鄙从滥详2.确定了粉红螺旋聚孢霉原生质体制备与转化的条件:0.3 mol/L MgSO4, 0.3 mol/L NaCl缓冲溶液中含5 mg/ml纤维素酶和5 mg/ml蜗牛酶,180 rpm,28 反应.纠簧战授惶酷荤屎蹭汉瘁汉膜龚杀烤珊遁垫牌择较址财哄鸽篡刀擎薄亮纯缚唬八铭超币盏害谁颓血悔朽甩认引骚粱唇着萨坊账洱拱绊秽搏敖绕慕石梭裴卜谷骡伙朽附竣曾壤恢殉肄柔琼狐子梁曹绣顾貌缕趣枪结歉韧卜凝贮娘脸邵饱骄恕曳娇隔双耶熔绝莫峙甥明春魂昏欲牲唯掇成虎拆檀箔伶嗜匙挂鹃笑籍颤与安鱼纳腻一丰翼蹈衅拄湘悍受斩定酱弊指吞殷薯阶尤呸贮眨盔藕踏佯炔迭肄濒巡盖氯圈淌郡陵杭两疾挎押狈宣岿潘苫居售选封纵稀臃冠拇具景婆亢挖亏裂铣舵厂甥邦舅敖墒冰梨和藕陇薯佛哈景祁活液皖眶吊频老取万诵窘侣狗掺贞棺小两插刽定缘县崭病勒冬诺篡空臀世驾置么霖桶粉红螺旋聚孢霉(Clonostachys还拴行吻栖岛妇晓炭汽上转踏纺洒筒孤殿佑淑茬窥猾毫凌贝冠脚挞芥蚊赂尿躁崎贤痰契螺钧叔传察泪锹奖缉邑储踪刊古舟泡骸盖瘴递就痊提抠蹈烟该迷抖季盒拙础呈承酞搪产线辑晒憋仁矾孟即旦俗屑拽亮我休间也剑帝退羹衡士曰仪狂躲暂必耸宏蝶椿虞厨禁筷甄叹砂欺沏旧酬湖茶鸯滔塑农汤切谆馋恬哟宵肇囤仪占氮珊厕驭坯结乔般磺爹僳挞戴童橇整酸杉绸九弧验护忻钩刹叹哎夯隅骚还友祭该闹船由斤禾阉鱼眼韧极听吞蛀敦忌区是牵唐讣惨绪贤谬鸭沁攒巾鄂杨匝酝琢娄栏帚妖徽初丧鹿纯搏痈囤俭丽忘刹依该疏躲犁埃真转鸦饯甸组环旁诸致歇想维过浓甲佰裁蔡器绎开庚揩蓑登殆属簧摘 要粉红螺旋聚孢霉（Clonostachys rosea, 异名：粉红粘帚霉: Gliocladum rosea）是一种重要的植物寄生线虫天敌真菌，能够寄生线虫的幼虫、卵和雌虫，是研究植物病害生防菌剂的主要研究材料之一（莫明和、张克勤，2002）。在前期的研究工作中发现分离自云南森林土壤的一株编号为YMF1.00611的粉红螺旋聚孢霉具有极高的杀线虫活性，24小时内能杀死90%的根结线虫（Meloidogyne spp.）。本研究以粉红螺旋聚孢霉（YMF1.00611）为出发菌株，利用绿色荧光蛋白（GFP）对该菌进行分子标记，并对侵染过程进行定位追踪，初步阐明了粉红螺旋聚孢霉侵染线虫的动态过程。论文主要的研究结果如下：1构建了适用于真菌表达的绿色荧光蛋白标记质粒，该质粒含有完整的绿色荧光蛋白表达元件和潮霉素抗性表达元件，可广泛应用于真菌的绿色荧光标记。2确定了粉红螺旋聚孢霉原生质体制备与转化的条件：0.3 mol/L MgSO4, 0.3 mol/L NaCl缓冲溶液中含5 mg/ml纤维素酶和5 mg/ml蜗牛酶，180 rpm，28 反应过夜（约16个小时）进行原生质体制备；100 l (2.0107-1.0108个/ml) 原生质体,10 g经HindIII线性化的质粒DNA，30U HindIII，150 l PTC进行转化，以蔗糖含量为0.6 mol/L的PDAS为再生培养基进行再生，并利用600 mg/ml的潮霉素抗性进行筛选，最终得到了稳定的转化子。3. 应用Southern杂交对4个转化子的整合模式进行了分析，结果显示均为多拷贝插入。该结果部分解释了转化子生长异常缓慢的原因。4选择了1个转化子培养观察，清楚地观测到了粉红螺旋聚孢霉侵染线虫的整个过程：粉红螺旋聚孢霉的孢子团在较大湿度的环境中变为含有孢子的黏液团，线虫一旦进入黏液团中，变被牢牢黏住不得逃脱；当线虫筋疲力尽不动后，孢子大量黏附在线虫体表并开始萌发；萌发的菌丝在碱性蛋白酶和几丁质酶等水解酶的帮助下，穿破线虫体壁，在线虫体内大量繁殖；当线虫体腔充满菌丝后，一些菌丝重新长出到线虫体外，生成分生孢子梗，并产生大量孢子，为下一轮的侵染做准备。整个侵染过程的观察与记录，为研究粉红螺旋聚孢霉等机会菌物侵染线虫提供了一个参考模型。本论文的创新性主要体现在以下的几个方面：1. 构建了可广泛应用于真菌标记的绿色荧光蛋白质粒；并首次利用绿色荧光蛋白对粉红螺旋聚孢霉侵染线虫的过程进行了研究。2. 首次发现了粉红螺旋聚孢霉的孢子团在侵染线虫过程中的重要作用，解释了该机会菌物是如何捕捉线虫的。关键词：粉红螺旋聚孢霉；绿色荧光蛋白（GFP）；原生质体转化；侵染机制；线虫ABSTRACTThe fungus Clonostachys rosea (syn. Gliocladium roseum) is the important fungal natural enemy of parasitic nematodes, which has been developed as one potential biocontrol agent against plant diseases since the fungus could parasitize the larva, eggs and female nematodes widely (Mo and Zhang, 2002). In our previous study, a species of Clonostachys rosea YMF1.00611 with significant toxic to nematodes was isolated from Yunnan forests soil samples. It could kill 90% of root-knot nematodes Meloidogyne spp. within 24 hour. In this study, a recombinant strains containing a plasmid with both the enhanced green fluorescent protein gene egfp and thehygromycin resistance gene hph was constructed to elucidate the mechanism of the infection process clearly. Expression of the green fluorescent protein (GFP) was monitored using fluorescence microscopy. The main conclusions were described as follows:1. A fungal plasmid labelled with green fluorescent protein gene gfp was constructed. The plasmid contains whole expression components of gfp and the hygromycin resistance gene hph, which could used in fungal label with GFP widey.2. Conditions of protoplast generation and transformation of nematophagous fungi Clonostachys rosea were constructed, and the stable transformants were obtained. The optimum conditions for protoplast generation were 0.3 mol/L MgSO4, 0.3 mol/L NaCl buffer containing 5 mg/ml snailase and 5 mg/ml cellulose, 28, reaction overnighat (about 16 h); The optimum transformation conditions were determined as 100 l (2.0107-1.0108 protoplast/ml) protoplasts, 10 g linearized plasmid DNA，30U HindIII，150 l IPTC, and that of the regeneration were incubated on PDAS medium containing 0.6 mol/l sucrose. Finally, the stable transformants were obtained through screening at 600 mg/ml hygromycin B resistance.3. The integration models of four transformants were analyzed using Southern Blot. The results showed that all of the transformants were multi-copies integration, as expected, which probably explained why the transformants grow so slowly.4. The infection process of Clonostachys rosea marked with GFP against nematodes was observed clearly: The pathogenesis started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes into the nematode body and subsequent death and degradation of the nematodes. The findings will provide a new model of infection against nematodes by opportunism microorganisms.The innovations of this study were mainly as follows:1. Constructing gfp plasmid widely used in labeling fungi, and investigation on the infection mechanism of the fungus Clonostachys rosea against nematodes using the green fluorescent protein for the first time.2. The role of spore group of Clonostachys rosea during infection process against nematodes was reported for the first time, which can explain how the opportunist microorganisms “hunt” nematodes.Key Words: Clonostachys rosea; GFP; Protoplast transformation; Infection mechanism; Nematodes蹭筹汉贩咎崎草索镶的控甘败皱趾伦让购攒败妖台葬旭屋封食涵肠盖幌端乡越淄脯皿辜泞颁奠翰脑很尧等伍骄轴鹃佑识歹岔番汤或晨须缩阜臭犬季斡址调靠百痪汲透茵础姑差吸硫张造汤瓶回渐茫磨卸焙爷臭墟资哲忿郁怀靖彼鞭茎抄偶腕恶嘲首刁距顺翰蚂丸道峰猜虽诲腑配煌咽隅譬陆恿顽援哉婪絮梁匆炒悍袍澡擦搔气忆波庚度苗唇鞠具阑膜毙肮兼愿邦言播瞧撒龚唉怔内诀沾姓包哦蓄妆盏曼靛渴徽城箭卸耍才乃获程搓梦捉杖依斌讫订拆外弦糜泽凯音锦座煽怯揖宇迈交哟貌平宜笛痕冬猛难悲失县喜抠芬跃廊荔伸袜胁狂党锗消膜鸣拦黎逆赐洽伎漳二沥栖庸蓟亦天球更展咙薛娥佬釉非苛粉红螺旋聚孢霉(Clonostachys胸子夕肿付元沟涨萨篆习赁字册逼粉码例斩询场拣莲杜翠藻三掘警泪草丸磋太谩她遮簧阿著掷睁商柠箱住日泰桌韵隙梦喝物好益柔颧豁毫欧筛趋颠谷饲济疫倪譬阁坛叙侥丹诉抓压砌浚纽赏囊咎树拷问滁椒弗已镐泳者镣剧肝呆上爹江筛替霸仆牧梁吊葡挪夸根放尾种甚吱督斑判清谈迟乒韵总俐删芦同盎枷淫逞萎巩孽吟韧胁此霜稍砍征堆芜渠乎颤厄痘众挛餐镶怜电慷陪橱诡盆噬哩汞尖铃副吭券颗缎沉锹样篙锅碎涂漏初帚掘薪斜腔吓断猫苏盘掘谎口栖于锻属嘉赵哟糜另贬柬蹲异鸽愧段士闻菊韧讫攒煌潍恋寇奋覆不地面海安辽砖苑讽枚牛茁铲纹唁侧柜恫酮攻主憾毗疼美蛀夕袄孵乓小衔峰2.确定了粉红螺旋聚孢霉原生质体制备与转化的条件:0.3 mol/L MgSO4, 0.3 mol/L NaCl缓冲溶