根癌农杆菌介导的地衣型真菌Cladonia metacorallifera的转化

以潮霉素抗性和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)作为筛选标记,利用地衣型真菌Cladonia metacorallifera的菌丝,成功实现了根癌农杆菌介导的遗传转化,PCR检测证明转化子中存在潮霉素抗性基因,共聚焦显微镜检测到转化子菌丝能够产生绿色荧光,证明EGFP能够在trp C启动子控制下在地衣型真菌中表达。     

里氏木霉分泌型表达载体的构建及绿色荧光蛋白的表达

【目的】构建里氏木霉分泌型表达载体,通过表达绿色荧光蛋白论证载体的可行性并初步观察绿色荧光蛋白在里氏木霉中的分泌过程。【方法】应用PCR及分子克隆技术将里氏木霉(Trichoderma reesei)纤维二糖水解酶(CBH1)的启动子及CBH1自身信号肽、终止子和潮霉素筛选基因依次插入骨架质粒pUC19中,构建出T.reesei表达载体Ppth15。将增强型绿色荧光蛋白(eGFP)基因装载入Ppth15中,获得eGFP表达载体Ppth15-eGFP。再将Ppth15-eGFP转化进T.reesei原生质体,通过潮霉素抗性筛选、基因组PCR检测等方法鉴定,获得阳性重组转化子。【结果】用PDA培养基培养阳性转化子2-3 d后,可在菌丝顶端、隔膜及培养基中清晰地观察到大量绿色荧光。【结论】表达载体构建成功且能够用于eGFP的表达,实验为进一步研究T.reesei表达其他基因提供了有效工具,同时为T.reesei胞外蛋白分泌的研究提供了参考。     

里氏木霉绿色荧光蛋白表达载体的构建及功能鉴定

为了后续研究里氏木霉(Trichoderma reesei)纤维素酶基因的表达与调控,利用overlap PCR及分子克隆技术构建了含有Col E1原核复制起始位点、氨苄青霉素抗性、里氏木霉的丙酮酸脱羧酶启动子、丙酮酸脱羧酶终止子、潮霉素B抗性的筛选标记并能表达增强型绿色荧光蛋白(Zs Green)的表达载体p LXT-Zs Green。将该载体转化里氏木霉QM9414原生质细胞,使用潮霉素B筛选平板得到阳性转化子,随后使用荧光显微镜在488 nm激发光下观察菌丝,并随机挑取4个转化菌株进行Western-blot验证。结果显示,里氏木霉菌丝体可发出明亮的绿色荧光,而且Western-blot验证了该载体能够在里氏木霉中有效地表达增强型绿色荧光蛋白。上述研究表明,载体p LXT-Zs Green在里氏木霉中能够稳定高效地表达外源基因,为研究里氏木霉的基因表达调控奠定了实验基础。     

绿色荧光蛋白在枯草芽孢杆菌WB600中的表达

根据绿色荧光蛋白(green fluorescent protein,GFP)的特性,将GFP作为目的基因,与穿梭载体p HY300PLK结合,构建一个芽孢杆菌WB600的蛋白表达体系,为在枯草芽孢杆菌中表达其他蛋白提供了一定的技术和理论支持。本文结合p HY300PLK的克隆位点,设计gfp基因的引物,PCR扩增后回收的gfp基因片段进行双酶切,与相同双酶切的pHY300PLK连接获得pHY300PLK-GFP,通过电转化将pHY300PLK-GFP转化至芽孢杆菌WB600中并检测。获得转化子WB1,检测到转化子在荧光显微镜下显示绿色荧光,且后续测序检测表明重组质粒构建成功。     

银耳芽孢完整细胞高效转化体系的建立

银耳（Tremella fuciformis）属于高等担子菌,其担孢子芽殖产生酵母状分生孢子称为银耳芽孢．银耳芽孢单核,能像酵母那样快速生长且容易培养,具备优良外源基因表达宿主的特点．本研究以gpd—Gl启动子分别与绿色荧光蛋白基因gfp和潮霉素抗性基因hph连接构建表达载体pG1g—gfp和pG1q—hph;设置潮霉素浓度梯度在3种培养基中对银耳芽孢的敏感性进行测定,结果表明银耳芽孢在不同的培养基上对潮霉素的敏感性不同,在MA培养基上其最低敏感浓度为5μg／mL;采用电击法把pG1q—hph质粒转化进银耳芽孢完整细胞,假定转化子经MA筛选培养基筛选,结果表明银耳芽孢完整细胞电击转化的最佳参数为：STM电击缓冲液、银耳芽孢浓度1．0×10^8个／mL,电击体积200μL,表达质粒6μg,电击电压2．0kV／cm,电击后采用MB液体培养基静置预培养48h,转化率达277个／μg DNA．采用最佳电击参数把质粒pG1g—gfP和pG1g—hph按1：1共转化银耳芽孢,转化子经过筛选培养基筛选、PCR鉴定及Southern杂交验证,结果表明受鉴定的8个转化子中有3个整合了gfp基因,其共转化率为37．5％．这3个gfp基因转化子的芽孢在激光荧光显微镜下可观察到发出强烈的荧光,表明了外源gfp基因能够在银耳芽孢中获得高效率的表达．     

板栗疫病菌绿色荧光与红色荧光蛋白共定位载体的构建

低毒病毒-板栗疫病菌组合是研究病毒与宿主相互作用的一个优秀的模式系统.我们构建了含绿色荧光蛋白基因gfp的载体pCPXHY2GFP与含红色荧光蛋白基因rfp的载体pCPXG418RFP,并用于转化野生型菌株EP155,获得了以潮霉素为筛选标记、表达绿色荧光蛋白的转化株pCPXHY2GFP/EP155和以G418为筛选标记、表达红色荧光蛋白的转化株pCPXG418RFP/EP155.将载体pCPXG418RFP转化pCPXHY2GFP/EP155,获得的转化株能观察到绿色荧光蛋白与红色荧光蛋白共定位的现象.板栗疫病菌绿色荧光与红色荧光共定位载体pCPXHY2GFP与pCPXG418RFP的构建,为深入研究病毒与宿主相互作用的分子机制提供了强有力的研究材料.     

一株水稻促生长内生真菌的绿色荧光蛋白基因标记与示踪

将绿色荧光蛋白基因(gfp)转入到碱蓬内生真菌JP4-1中并检测菌株在水稻幼苗中的定殖情况。采用PEG-Ca Cl2介导的原生质体转化方法将携带gfp基因的p CT74质粒与菌株基因组整合获得转化子,用转化子侵染水稻幼苗,荧光显微镜下示踪JP4-1菌株及其侵染特性。转化子经连续传代6次仍能发出绿色荧光且荧光强度良好,能够稳定遗传;经PCR验证gfp基因已成功转入JP4-1菌株和水稻幼苗植株内并表达。转化可获得稳定表达GFP的JP4-1转化子,JP4-1菌株可定殖于水稻幼苗的根、茎、叶,定殖位置为细胞间隙,其促生作用与野生型菌株无明显差别。     

螺旋藻耐盐相关基因启动子区功能分析

文章利用绿色荧光蛋白基因作为报告基因,研究2个螺旋藻耐盐相关基因启动子区域的功能。通过启动子预测软件预测螺旋藻耐盐相关基因5′端非翻译区的启动子结构,用Primer3.0程序在线设计引物,以pMD18-T载体和pUC18载体克隆螺旋藻启动子序列、gfp和卡那霉素抗性基因,将螺旋藻启动子-GFP基因-卡那霉素抗性基因(pro-gfp-kanr)三联DNA片段克隆至pKW1188载体,并将该重组质粒pKW1188::pro::gfp::kanr转化至受体菌集胞藻6803,激光共聚焦显微镜观察不同盐浓度培养条件下、不同时间段集胞藻表达GFP的情况。结果显示,通过不同盐浓度和不同时间的诱导,2个螺旋藻启动子在0.4~0.6 mol/L NaCl条件下,培养6~8 h表达的绿色荧光蛋白最多。文章成功构建了以绿色荧光蛋白为报告基因、卡那霉素抗性基因为选择标记、集胞藻6803作为外源基因表达受体,进行螺旋藻耐盐相关基因功能研究的平台;另外,从螺旋藻启动子能被盐诱导大量表达GFP的结果看,与启动子相关的螺旋藻基因很可能与螺旋藻的耐盐性相关。     

根癌农杆菌介导的巨大口蘑遗传转化体系的构建

以巨大口蘑菌丝为受体材料,利用含有双元质粒plasmid4的根癌农杆菌EHA105介导,首次成功建立了巨大口蘑的遗传转化体系。通过潮霉素抗性筛选、PCR鉴定和绿色荧光蛋白的检测,表明潮霉素抗性基因（Hyg）已经整合到巨大口蘑基因组中,增强型绿色荧光蛋白基因（eGFP）在巨大口蘑菌丝中获得表达,并能够稳定遗传。本研究建立了农杆菌介导的巨大口蘑遗传转化体系,为今后巨大口蘑的基因功能研究奠定了基础。     

利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测

源自噬菌体P1的Cre重组酶可以识别 34bp的靶DNA序列loxP ,进行位点特异性的重组反应。为了简便地检测Cre酶在大肠杆菌中的重组活性 ,分别将cre基因和上下游带有loxP的绿色荧光蛋白基因 (gfp)克隆到具有不同抗性的两种不相容质粒中 ,然后将构建的原核表达载体pET30a Cre和pET2 3b loxGFP电击共转化大肠杆菌BL2 1(DE3) ,利用卡那霉素和氨苄青霉素双抗生素抗性进行筛选。通过直接观察转化子的绿色荧光 ,便可以显示Cre酶的体内重组活性 ,并进一步通过SDS PAGE分析、质粒酶切鉴定进行了验证。结果表明 :以gfp为报告基因、通过两种不相容质粒共转化大肠杆菌可以为研究和改进Cre loxP重组系统提供一种简便直观的检测方法     

灰葡萄孢犬尿氨酸途径关键酶基因的鉴定

【背景】灰葡萄孢是一种重要的植物病原真菌,实验室前期明确了灰葡萄孢犬尿氨酸单加氧酶(kynurenine3-monooxygenase,KMO)基因BcKMO参与调控病菌的生长发育和致病力。犬尿氨酸单加氧酶(KMO)是犬尿氨酸途径的关键酶,但灰葡萄孢是否存在犬尿氨酸途径及其在病菌生长、发育和致病过程中的功能尚未见相关报道。【目的】鉴定灰葡萄孢犬尿氨酸途径中的关键酶基因,确定灰葡萄孢犬尿氨酸途径的存在,为阐明灰葡萄孢生长发育和致病力的分子机理奠定基础。【方法】利用生物信息学方法,对灰葡萄孢犬尿氨酸途径中犬尿氨酸酶(kynureninase,KYN)、吲哚-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)、犬尿氨酸氨基转移酶(kynurenine amino transferase,KAT)的编码基因进行分析;利用Real-time PCR技术,检测灰葡萄孢野生型BC22、BcKMO基因T-DNA插入突变体BCG183、恢复菌株BCG183/BcKMO中犬尿氨酸途径关键酶基因的表达水平;利用真菌犬尿氨酸酶KYN检测试剂盒,测定BcKMO突变体中犬尿氨酸酶(KYN)的含量。【结果】灰葡萄孢中含有2个犬尿氨酸氨基转移酶(KAT)的编码基因、3个吲哚-2,3-双加氧酶(IDO)的编码基因、10个犬尿氨酸氨基转移酶(KAT)的编码基因。灰葡萄孢KYN编码基因、IDO编码基因、KAT编码基因在突变体BCG183中的表达水平显著高于或低于在野生型和恢复菌株。突变体BCG183中犬尿氨酸酶(KYN)的含量显著低于野生型BC22和恢复菌株。【结论】灰葡萄孢中存在犬尿氨酸途径,灰葡萄孢BcKMO基因突变影响KYN、IDO和KAT编码基因的表达以及犬尿氨酸酶(KYN)的含量。     

生防细菌CNY-04筛选、鉴定及其对灰霉病菌的防效

【目的】为研究土壤细菌对蔬菜灰霉病的生防价值, 从辽宁、山东等地区的蔬菜种植基地采集土壤样本56份, 分离、筛选出对灰霉病具有稳定拮抗作用的细菌9株。【方法】采用平板对峙培养法进行初筛、复筛, 用抑菌圈法测定其抑菌效果, 并进行离体果实试验验证其对蔬菜灰霉病的防治效果, 通过形态学特征、生理生化特征及16S rRNA基因序列分析研究其分类地位。【结果】细菌CNY-04对蔬菜灰霉病的拮抗能力最强且遗传稳定, 抑菌圈直径达到34 mm; 初步鉴定该菌株为格氏沙雷菌(Serratia grimesii), 尚未见该菌在生防上的报道; CNY-04液体菌剂对离体番茄果实灰霉病的防效为69.23%, 50%多菌灵防效为75.39%, 24 h时接种CNY-04处理的番茄发病率为40.0%, 而48 h时接种处理的发病率为51.1%。【结论】CNY-04是一株较为理想的拮抗菌, 丰富了生防资源。     

番茄灰霉病高效拮抗菌株的鉴定及通过导入β-1,3-葡聚糖酶基因提高其生防效果

从淄博市温室土壤中分离到蜡样芽孢杆菌B-04菌株,对灰霉病菌表现较高的拮抗作用。本研究从质粒pUC1940得到4.1kb的β-1,3-葡聚糖酶基因片断,将该基因与大肠杆菌-芽孢杆菌穿梭质粒pBE2和pHY300PLK连接,获得重组质粒PBE2-glu和pHY300PLK-glu,转入蜡样芽孢杆菌（Bacillus cereus）B-04菌株,获得工程菌株B-04-glu。限制酶切分析、ABP平板、PCR实验证实B-04成功转入β-1,3-葡聚糖酶基因。与野生菌株相比,平板拮抗试验表明工程菌株较原始菌株对番茄灰霉病（Botrytis cinerea）抑菌效果明显增强。     

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Different products for biological control of Botrytis cinerea examined on wounded stem tissue of tomato plants

For the moment the agents that are used against Botrytis cinerea, in glasshouses were tomatoes are cultivated, are from chemical origin. For reducing the use of chemical agents in the future it is important to search for effective biological control agents against the fight of Botrytis cinerae. The following biological products Vital pasta, Vital gel and Elot-Vis were examined in there possibility to control Botrytis cinerea. Elot Vis was tested out in experiments that were carried out in climate chambers were leafs of 3 week old tomato plants were artificially infected with Botrytis cinerea spores. Also the biological products of Vital were first investigated in experiments that were carried out in climate chambers. In stead off leafs of tomato plants it were stem wounds of tomato plants who were treated with the pasta or the gel that was spread over the wounded surface after this has been inoculated with a suspension of conidia of Botrytis cinerae. The results of these first tests that were executed in the climate chambers were the circumstances for Botrytis cinerea were ideal seemed promising. In the next step these products were tested out on large scale in glasshouses. For each plant 5 wounds were created by removing the leafs, these wounds were or first treated with the Biological product and thereafter artificially infected with Botrytis cinerea spores to check out if these products can be used as a preventive agent or the wounds were first inoculated with a suspension of Botrytis cinerea spores and thereafter treated with the product. For the product Elot-Vis a few plants were totally sprayed with an Elot-Vis suspension before leafs were removed and the wounds were inoculated with conidia of Botrytis cinerea to check out if this product was able to activated the induced systemic resistance pathway. The experiments that were executed in glasshouses showed different percentages of succeeded Botrytis cinerea infections. This is probable due to the different weather conditions during both days that the experiments were executed. For the wounds that were treated with pasta it was difficult to distinguish wounds were Botrytis cinerea succeeded to infect the plant, because these wounds frequently didn't show any sign of infection on the surface but when the wounds with the pasta were cut open it was possible to see Botrytis cinerea infections inside the stem.     

Biocontrol agents of Botrytis cinerea tested in climate chambers by making artificial infection on tomato leafs

To reduce the use of chemical agents, that are causing damage to the environment, in the fight against Botrytis cinerea, different BCA's were tested for their possibility to control Botrytis cinerea in a biological way. In order to investigate the effectiveness of the different micro organisms and Elot-Vis, experiments were carried out in climate chambers with 5 weeks old tomato plants. Leafs on the plant were inoculated with drops of a suspension that contained spores of Botrytis cinerea. The possible antagonists that were tested in these experiments were Trichoderma harzianum (Trichodex), T. asperellum (Biofungus), T. hamatum (T382), Bacillus subtilis (Serenade and Phytovit) and Pseudomonas aeruginosa (7NSK2 and KMPCH). For all these different micro organisms the direct and the indirect influence on Botrytis cinerea was investigated. In tests where the direct influence of the antagonists was examined, the spores of the moulds or the bacteria were suspended together with spores of Botrytis cinerea and subsequently drops of this suspension were pippeted on the leafs. After a while by ideal circumstances for Botrytis cinerea the infections on the inoculated leafs were counted. For the indirect influence of the antagonists, also leafs of 5 weeks old tomato plants were inoculated with a suspension of Botrytis cinerea spores. The roots of the tomato plants that were used for testing the indirect influence were treated during there growth with a suspension of the antagonist to see if induced systemic resistance pathway (ISR) was activated. For testing the effectiveness of Elot-Vis, tomato plants were sprayed a few times with a solution of this product during their growth. Results of the climate chamber test of the plants that were treated with Elot-Vis, showed a reduction of Botrytis cinerea infections on the inoculated leafs. Biological control agents seem to be not always very effective against Botrytis cinerea. The biological control agents that are containing micro organisms are also depending on the circumstances of the environment for an optimal development. These conditions are not always that optimal to compete with Botrytis cinerea or other micro organisms that are present on the plant.     

农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)介导法对灰葡萄孢(Botrytis cinerea)进行转化,构建T-DNA插入突变体库,为从分子水平上认识灰葡萄孢的致病机制打下基础。【方法】以含有pCAMBIA 1390双元载体的农杆菌对灰葡萄孢进行转化,利用潮霉素进行筛选。对抗性稳定的转化子进行生物学和形态学观察,采用离体番茄叶片进行致病性测定。利用TAIL-PCR技术对突变体中T-DNA的旁侧序列进行克隆。【结果】得到了一些突变体,表现为生长速率减缓、产孢能力下降、致病力减弱等。克隆并分析了其中一个突变体中T-DNA插入的位置和旁侧序列。【结论】本实验建立了农杆菌介导的灰葡萄孢转化体系,构建了T-DNA插入的灰葡萄孢突变体库。用TAIL-PCR进行突变体中T-DNA旁侧序列的分析是可行的。     

Characterization of Bc-hch, the Botrytis cinerea homolog of the Neurospora crassahet-c vegetative incompatibility locus, and its use as a population marker

The Botrytis cinerea homolog (Bc-hch) of Nc-het-c and Pa-hch (vegetative incompatibility loci of Neurospora crassa and Podospora anserina respectively) was cloned and sequenced. The gene structure of Bc-hch is very close to those of Nc-het-c and Pa-hch. A PCR-RFLP approach on a 1171 bp fragment was used to screen polymorphism at this locus among 117 natural isolates of B. cinerea. Restriction patterns by the restriction enzyme HhaI fell into two allelic types. Moreover, haplotypes at the Bc-hch strictly corresponded to the resistance phenotypes to fenhexamid, a novel Botryticide. The use of Bc-hch as a population marker thus reveals a new structuring of B. cinerea natural populations into two groups (I and II). This result was confirmed by genic differentiation tests performed with five other markers on a sample of 132 B. cinerea isolates from the French region of Champagne.