玉米大斑病菌非核糖体肽合成酶NPS6 基因ATMT 敲除载体的构建

载体构建是利用农杆菌介导建立真菌遗传转化体系的基础。以双元载体pPZP100为骨架,通过限制性内切酶酶切、连接以及去磷酸化将标记基因NptⅡ及StNPS6基因侧翼序列导入目标载体pPZP100中,构建玉米大斑病菌非核糖体肽合成酶6基因敲除载体,并采用冻融法将pPZP100NptⅡNPS6转入农杆菌菌株AGL-1。     

玉米弯孢叶斑病菌NADPH氧化酶生物信息学分析和ATMT敲除载体的构建

从JGI数据库玉米弯孢叶斑病菌(Curvularia lunata)基因组中获得2个编码NADPH氧化酶基因ClNox1和ClNox2。采用生物信息学方法对2个基因编码的蛋白质进行性质、结构和功能等方面的预测。结果表明,ClNox1和ClNox2的分子量分别为6.35和6.41 KD,等电点PI为8.87和8.93,不稳定指数为44.50和39.45;ClNox1是亲水性膜蛋白,包含6个明显的跨膜结构域;ClNox2也是膜蛋白,包含8个明显的跨膜结构域。在亚细胞水平上,ClNox1定位于线粒体上,而ClNox2定位于细胞质膜上;在氨基酸序列方面,ClNox1和ClNox2都含有多个苏氨酸、丝氨酸和酪氨酸激酶磷酸化位点;在功能方面,ClNox1和ClNox2都包含NADPH氧化酶特征结构域,并且与Cochliobolus heterostrophus的NADPH氧化酶基因具有较高的同源性。根据ClNox1和ClNox2序列设计特异性引物扩增目的片段,与标记基因NPTII和HphGFP分别连入骨架载体pPZP100,构建完成ClNox1和ClNox2基因的敲除载体pPZP100NPTIIClNox1和pPZP100HphGFPClNox2,为根癌农杆菌介导的遗传转化提供基础材料。     

根癌农杆菌介导柱花草炭疽菌遗传转化体系的优化

选取含有氯嘧磺隆抗性标记的ILV1基因和报告基因GFP二元载体的农杆菌AGL-1,采用单因子和双因子方法研究根癌农杆菌AGL-1介导柱花草炭疽菌CH008转化过程中各主要因素对转化率的影响,优化构建ATMT转化柱花草胶孢炭疽菌体系条件,使转化效率达到300 ～400个转化子/106个炭疽孢子,即根癌农杆菌AGL-1浓度OD600=0.8,炭疽菌分生孢子浓度为1x106个/mL,AS浓度为100 μmol/L,诱导时间为6h,共培养温度和时间分别为25 ℃和4d.经PCR检测都有GFP片段,并能够表达绿色荧光蛋白.这为进一步开展炭疽菌对柱花草的致病机理研究提供了依据,优化转化体系有利于后续突变体库的扩大、筛选突变体和致病功能基因的研究.     

以bar基因为筛选标记转基因大豆的获得及鉴定

在已优化的大豆农杆菌介导转化体系基础上,以发芽1～2 d的成熟大豆子叶节为外植体,以bar基因作为筛选标记基因,采用农杆菌介导法将胰蛋白酶抑制剂基因(Sporamin)和几丁质酶基因(Chitinase KDEL)转入大豆品种YC-2中.T0代得到生根苗115株,其中采用除草剂叶片涂抹法和目的基因PCR检测法鉴定出阳性...     

花生CpTI基因转化——花粉介导法

利用花粉作为外源基因的载体将抗虫基因(CpTI基因)转入花生中。以花生仲恺01号为受体,以PCB302-3质粒外源基因为供体。在花生开花期,收集花粉与质粒DNA混合并附加超声波处理,然后以人工授粉的方法将外源基因导入受体中。利用花粉作为载体介导外源基因转化,避免了传统的基因枪法和农杆菌转化所要求的组织培养技术,转化方法简单、易操作,具有很强的实用性。     

农杆菌介导向玉米茎尖导入HAL1基因的初步研究

选用玉米优良自交系郑58的茎尖为受体,研究以玉米茎尖为受体进行农杆菌转化体系的可行性。用农杆菌介导法将耐盐碱基因HAL1转入玉米中,获得70株转化苗,经过300 mg/L的除草剂(Basta)筛选,共获得9株转基因植株。进一步进行 PCR检测,其中6株表现阳性,转化率达8.57%。初步证明外源基因已经整合到玉米基因组中,以玉米茎尖作为受体孢子体的转化系统可用于基因转化。     

拟南芥防御素基因PDF1.2启动子与GUS重组载体的构建与转化

PDF1.2基因在植物的防御系统中扮演着重要的角色,其编码产生的植物防御素参与了植物对真菌入侵、病毒感染、不良环境等逆境胁迫的防御反应。试验利用从拟南芥中通过PCR扩增和克隆的该基因启动子构建GUS报告基因的表达载体,通过根癌农杆菌浸渍转化法将表达报告基因转入拟南芥中,筛选获得了转化植株。获得以下主要结果:(1)成功克隆了拟南芥PDF1.2基因的启动子并将其与带GUS标记基因的载体进行了融合,得到了重组的融合载体;(2)成功的将带GUS标记基因的拟南芥PDF1.2基因启动子融合载体转入到拟南芥植株中并利用其自带抗性标记筛选到了抗性植株;(3)利用GUS染色技术检测了转基因植株中PDF1.2基因启动子的表达情况。     

Mineirao柱花草MSRA基因的克隆及其植物表达载体的构建

根据抗病柱花草中的1个AFLP特异片段SG2950-1设计引物,利用RACE技术从Mineirao柱花草中克隆到一个MSRA基因:经测序比对,该序列与细胞色素氧化酶系基因有较高的相似性.构建了植物表达载体,通过冻融法将该载体质粒转入根癌农杆菌LBA4404,为进行该基因的功能分析奠定了基础.     

无机焦磷酸化酶基因的克隆与植物表达载体的构建

提取面包酵母基因组DNA,用无机焦磷酸化酶(PPase)基因特异引物通过PCR扩增出864bp的片段,并将该片段克隆至pMD18-T上。序列分析结果表明,该克隆序列与GeneBank上的PPase基因序列同源性达到100%。进一步将叶肉细胞特异性启动子rbcS和PPase基因克隆至植物表达载体pCBI上,构建了由rbcS启动子调控的PPase基因植物表达载体pCBIPR。通过冻融法将重组质粒导入根癌农杆菌EHA105中,为农杆菌介导的PPase基因对植物的遗传转化奠定基础。     

Bt基因表达载体的构建及对茶树遗传转化的研究

用限制性内切酶HindⅢ和BglⅡ从pGA4 71质粒中切取Bt基因并转入载体pCAMBIA2 30 1中 ,构建后的质粒含Bt基因、intron -GUS基因和NPTⅡ基因。将构建后的质粒转化大肠杆菌 ,通过三亲交配法分别导入农杆菌菌株LBA4 4 0 4、EHA10 5、pRi15834中。以茶树叶片、愈伤组织为转化的受体材料 ,用农杆菌介导法将其转入茶树中 ,获得了GUS瞬间表达。以潮霉素作为愈伤组织筛选的选择性试剂 ,效果明显优于卡那霉素 ,适宜浓度为 2 0 μ/ml;以卡那霉素作为茶树叶片材料的筛选试剂 ,50 μ/ml为宜。     

玉米圆斑病拮抗菌芽孢杆菌21的鉴定及抑菌研究

利用形态特征、培养性状、生理生化特征及16Sr DNA序列分析,对获得的具有生防潜力的芽孢杆菌21进行鉴定,确定该菌株为枯草芽孢杆菌(Bacillus subtilis spp.)。利用显微观察法,研究枯草芽孢杆菌21对玉米圆斑病菌的抑制作用,结果显示,该菌株不同浓度发酵液处理后玉米圆斑病菌孢子萌发率出现下降趋势,萌发率最低为0;经发酵液处理后的玉米圆斑病菌芽管产生畸形,在发酵液浓度为90%时,芽管畸形最严重;处理后的玉米圆斑病菌菌丝出现畸形,菌丝中的内容物有外渗趋势。     

顶孢霉菌代谢产物对玉米圆斑病菌生长和发育的影响

利用菌丝生长速率法和孢子萌发法研究顶孢霉代谢产物对玉米圆斑病菌生长的抑制能力。结果表明,顶孢霉发酵液对玉米圆斑病菌分生孢子萌发和菌丝生长均有抑制作用。菌丝生长速率测定表明,含有顶孢霉菌代谢产物的培养基（D+P）圆斑病菌菌丝的生长速率低于含有玉米圆斑病菌代谢产物的培养基（Y+P）和空白对照PDA的生长速率,菌丝生长速率分别为65.0、86.5、87.5 mm/d;以Y+P和PDA为对照,D+P对玉米圆斑病菌在第1天和第2天时抑制率最高,均达58%以上;在1 mL PDA体系中,顶孢霉发酵液添加量为0.5 mL时,抑制率可达100%,不同浓度处理间及其与对照处理间顶孢霉发酵液抑制率差异显著（P=0.05）;同一处理不同培养天数顶孢霉发酵液抑制率也有显著差异（P=0.05）。孢子萌发实验表明,经顶孢霉发酵液处理后的玉米圆斑孢子萌发率降低,在发酵液浓度为0.9 mL/mL时,萌发率仅为24%,抑制率可达66.67%, EC50约为68 mL/100 mL,且孢子经发酵液处理后产生畸形,不能正常发育。     

Effect of a continuous hot water treatment of potato tubers on seed-borne fungal pathogens

Summary The viability of five pathogens was decreased by treatment with hot water when tested in vitro.Polyscytalum pustulans was most sensitive andRhizoctonia solani least sensitive. Potato tubers were exposed to 55°C for 5 min in a commercial continuous hot water treatment plant using naturally contaminated seed tubers and tubers which had been inoculated by dipping in comminuted cultures. The frequency of eyes colonised byP. pustulans, Helminthosporium solani, andR. solani was reduced to virtually zero and the effect persisted on tubers subsequently stored at 4°C and at 15°C for up to 16 wk. Results withColletotrichum coccodes were inconclusive. Treatment suppressedPenicillium spp. which, however, rapidly recolonised the eyes during storage, leading to higher contamination levels in the treated than in the untreated tubers. With tubers inoculated withPhoma foveata, good control was achieved when the incubation period before treatment was 10 d but not when the period was 42 d.     

Soil survival and thiabendazole sensitivity ofHelminthosporium solani isolates from Alberta,Canada

Summary Survival of the pathogen causing silver scurf of potato (Helminthosporium solani) in Alberta soils was studied by evaluatingH. solani infection in the progeny ofH. solani-free nuclear seed potato tubers planted in fields where potatoes had never been grown or were grown 1, 2, 3, or 4 years previously. Daughter tubers from all the fields developed silver scurf lesions, andH. solani was isolated from infected tubers. This is the first report of survival ofH. solani in Alberta soils. Soil-borne inoculum appears to have a role in the epidemiology of the disease and in the introduction of the pathogen into silver scurf-free potato seed stock. Of 31 plant species tested, only potato was found to be a host ofH. solani. Most of theH. solani isolates from north central Alberta were more sensitive to thiabendazole than those from southern Alberta, where thiabendazole is much more commonly used.     

玉米对小斑病T小种抗性的遗传模型分析

用广义遗传模型方法,研究了玉米对小斑病T小种的抗性遗传特性.结果表明:玉米对小斑病T小种的核抗性主要受加性和显性效应控制,以显性效应为主.3个病害指标之间有极显著的正相关,相关性主要归因于加性效应,病斑数和病级之间还有显著的显性遗传相关.另外,根据遗传效应预测值对供试亲本作了评价。     

Antifungal textile dyeing with<Emphasis Type="Italic">Mahonia napaulensis D.C.</Emphasis> leaves extract based on its antifungal activity

The antifungal activity of the methanolic extract ofMahonia napaulensis D.C. or Taming (local name) leaves was evaluated with four species of common pathogenic fungi e.g.Colletotrichum capsici (MTCC No. 2071),Leptosphaerulin trifoli (MTCC No. 2328),Alternaria brassicicola (MTCC No. 2102) andHelminthosporium solani (MTCC No. 2075). The present investigation also aims at developing an eco friendly natural antifungal finish from plant extract ofMahonia for textile application. The antifungal textile dyeing was also carried out with aqueous extract of stem and leaves ofMahonia and the dyed fabric was tested against fungal speciesTrichoderma for its antifungal activity in vitro.Mahonia extract showed substantial antifungal activity of 83.33% forLeptosphaerulin trifoli andAlternaria brassicicola by 80 ppm dose in 24 hours and 46% antifungal activity inMahonia dyed pieces in broth againstTrichoderma.     

Fungicide treatment of seed tubers infected with thiabendazole-resistantHelminthosporium solani andPolyscytalum pustulans for controlling silver scurf and skin spot on stored progeny tubers

Summary Seed tubers, cv. Désirée, derived from stocks treated annually with thiabendazole were infected with thiabendazole-resistant strains ofHelminthosporium solani andPolyscytalum pustulans. Samples of seed tubers were either untreated or immersed for 5 min in fungicide suspensions of thiabendazole, imazalil or thiabendazole plus imazalil and planted on four farms in 1988 and 1989. After harvest, tuber samples from each treatment were treated with thiabendazole and stored for 6 months. Applying imazalil or thiabendazole plus imazalil to seed tubers decreased the severity of silver scurf and skin spot on stored progeny tubers. Thiabendazole applied to seed tubers or to progeny tubers after harvest did not affect the severity of either disease, but post-harvest treatment decreased the incidence of black scurf after storage.     

玉米大斑病菌生理小种同工酶的薄层扫描图谱

对属于玉米大斑病菌1、2号生理小种的四个标准菌株的过氧化物同工酶和酯酶同工酶进行PAGE和IEF分析研究．过氧化物同工酶扫描图谱发现1号生理小种在薄板80mm处有明显的馒头状吸收峰(G峰)，而2号小种在薄板80mm处有一微弱吸收(M峰)，紧随其后义在85mm处出现另一微小吸收峰(N峰)，可见玉米大斑病菌1、2号在生理小种的过氧化物同工酶图谱有一定的差异。酯酶同工酶扫描图谱发现，1号生理小种在薄板130m。处有一明显的窄长吸收峰(G峰)，而2号小种在此区域则无明显吸收。如果两性电解质由pH4～6换成pH3.5～10时。2号小种在薄板70mm处有一C峰出现，但1号小种没有出现该峰，可见玉米大斑病菌1、2号生理小种的酯酶同工酶也存在着差异，这将为探讨玉米大斑病菌毒素产生的遗传学和不同小种毒素的毒性成分间的关系奠定基础。