根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T—DNA插入突变体的体系,在镰孢菌孢子浓度10^6／mL、农杆菌OD600=0．15—0．20、乙酰丁香酮浓度为200μmol／mL的条件下共培养36h转化率最高,可达60—120个／10^6个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。     

根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T-DNA插入突变体的体系,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15-0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化率最高,可达60-120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。     

根癌农杆菌介导的球毛壳菌遗传转化及T-DNA插入突变体的获得

根癌农杆菌介导的遗传转化系统是植物基因工程常用方法,目前已将这一转化系统应用到酵母、丝状真菌以及人类细胞的转化。利用这一转化系统,成功地实现了丝状真菌球毛壳菌（Chaetomium globosum）的遗传转化,转化率约为60～180个转化子/10.7个孢子 。通过对转化子的PCR检测和Southern 杂交分析表明,TDNA已整合进毛壳菌基因组中,而且在所检测的转化子中都是以单拷贝的形式整合,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具。     

农杆菌介导的小孢拟盘多毛孢遗传转化及突变体筛选

本研究通过农杆菌EHA105介导的方法,以含潮霉素抗性基因和GFP基因的双元载体pCAMBgfp为转化载体,对小孢拟盘多毛孢Pestalotiopsis microspora KFRD-2菌株进行遗传转化。基于潮霉素及GFP荧光抗性进行转化子的初步筛选,随后,进一步对转化子的菌落特征、菌丝生长速率、产孢量、荧光稳定性及致病力进行验证。结果获得阳性转化子100余株,转化效率达200个转化子/10⁶个孢子。大部分转化子与野生型菌株无明显形态及致病力差异。同时,获得了14株菌丝形态、产孢量或致病力与野生型存在明显差异的突变株,可用于小孢拟盘多毛孢关键致病基因的挖掘验证及致病机理等研究。     

根癌农杆菌介导的水稻遗传转化

就根癌农杆菌介导水稻遗传转化的研究历程和影响农杆菌转化水稻的几个关键因素以及这一问题的前景作了评述和展望     

根癌农杆菌介导的大豆遗传转化

农杆菌介导法是大豆遗传转化的重要方法之一 ,许多实验室应用该方法得到了转基因大豆 ,但目前使用该方法进行转化的效率还比较低 ,尚需深入研究。农杆菌菌株、大豆基因型、组织培养条件、T-DNA的转移效率和转化后的筛选模式都会影响大豆转化的效率。概述了近年来根癌农杆菌介导的大豆遗传转化的一些重要成果 ,以及转化过程中大豆的易感性与农杆菌的转化能力、乙酰丁香酮促进vir基因活化、转化的受体系统和巯基混合物减轻受体材料的褐化、提高T DNA的转移效率等几个重要因素的研究进展 ,并介绍了转化中常用的几个筛选标记基因 (nptⅡ、hpt、bar基因和突变的ahas基因 )及通过共转化法去除标记基因的方法 ,同时对今后研究的重点进行了讨论.     

根癌农杆菌介导丝状真菌遗传转化的研究进展

根癌农杆菌介导的丝状真菌遗传转化是近年建立起来的一种新方法,该方法和以往的真菌转化体系相比具有转化方法简单、材料易得、效率高以及转化子中T-DNA单拷贝插入比例高等特点。就根癌农杆菌转化的丝状真菌种类、转化的具体过程以及影响转化效率的因素等方面进行了综述,并展望了该方法的应用前景。     

根癌农杆菌介导的香蕉遗传转化研究进展

香蕉的栽培品种绝大多数是三倍体, 常规育种难度大, 工作周期长, 难以采用传统的育种方法进行遗传改良, 因此转基因技术成为改良香蕉种质的有效方法。根癌农杆菌介导的香蕉的遗传转化从20世纪90年代起取得的了较大的进步, 但仍然存在着很多问题。文章分析了影响农杆菌介导香蕉转化的几个重要环节, 并对其研究现状、存在的问题及应用前景作简要概述。     

根癌农杆菌介导真菌遗传转化的研究进展

根癌农杆菌介导的真菌遗传转化是近年来发展的一种新方法 ,与其它方法相比 ,该方法具有操作简便、转化效率高和易得到稳定转化子等特点。目前 ,在根癌农杆菌介导下已实现了多个属种真菌的遗传转化 ,显示出良好的应用前景。综述了根癌农杆菌介导真菌遗传转化的转化机理和T DNA在真菌细胞中的存在方式等方面的研究结果 ,并展望这一方法的应用前景。     

根癌农杆菌介导的草莓遗传转化研究进展

随着植物离体再生技术的日益成熟和多种外源目的基因的分离与克隆,转基因技术以目的性强、周期短等优点成为草莓品种改良的重要途径。在草莓上采用的遗传转化方法主要是农杆菌介导法。概述了农杆菌介导法的转化机理以及基因型、农杆菌菌株、侵染时间和菌液浓度、共培养时间、酚类物质等对草莓遗传转化的影响,并从草莓果实贮藏保鲜、抗除草剂、抗病毒、抗虫、抗真菌、转基因疫苗等方面对近年来草莓的转基因研究进展进行了总结。     

农杆菌介导的获取疏绵状嗜热丝孢菌插入突变体体系的建立

[目的]建立疏绵状嗜热丝孢菌的稳定遗传转化体系并获得插入突变体.[方法]利用农杆菌介导的方法建立疏绵状嗜热丝孢菌的遗传转化体系 ;分别通过Southern杂交、克隆转移DNA(T-DNA)侧翼序列来确定T-DNA在疏绵状嗜热丝孢菌基因组中的拷贝数和插入位点.[结果]成功建立了可靠的疏绵状嗜热丝孢菌的遗传转化体系.共培养过程中使用萌发孢子是成功建立疏绵状嗜热丝孢菌遗传转化体系的必要条件.疏绵状嗜热丝孢菌萌发的孢子与农杆菌在28℃共培养48h时,转化效率最高.乙酰丁香酮(AS)在农杆菌预培养及疏绵状嗜热丝孢菌萌发的孢子与农杆菌的共培养阶段都是必需的,且在共培养阶段当AS浓度为500 μM时转化效率最高.Southern杂交验证表明,79.2％的转化子为T-DNA单拷贝插入,且通过热不对称PCR (TAIL-PCR)分析得出T-DNA在该菌基因组中的插入位点是随机的.通过该转化系统筛选到部分表型突变体.[结论]我们首次报道了利用ATMT技术成功转化嗜热真菌-疏绵状嗜热丝孢菌,证明了该方法是一种简单有效的获得插入突变体的方法,并为该嗜热真菌进行基因定位提供了工具.     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum

We transformed haploid mycelium of Hebeloma cylindrosporum via Agrobacterium tumefaciens and optimised the procedure to develop a new tool for insertional mutagenesis in this fungus. Southern blot analysis of 83 randomly selected transformants showed that they all contained plasmid inserts. Each of them showed a unique hybridisation pattern, suggesting that integration was random in the fungal genome. Sixty percent of transformants obtained in the presence of bacteria pre-treated with acetosyringone integrated a single transferred DNA copy. Thermal asymmetric interlaced polymerase chain reaction allowed us to recover the left border and the right border junctions in 85% and 15% of transformants analysed, respectively. Results show that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in H. cylindrosporum.     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris

Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C.?militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of 30-600 transformants per 1×10(5) conidia. Acetosyringone (AS) supplement in C. militaris ATMT was not necessary during either precultivation or cocultivation. The transformation procedure was optimised based on the ratios between donor A. tumefaciens and recipient conidia, and pH value of cocultivation media. The integration of the hyg gene into C. militaris genome was determined by PCR and Southern blot analysis, suggesting that 67-88% resulting transformants in cultivation conditions with or without AS were inserted by T-DNA and 55-80% were single-copy. Special mutants with altered phenotypes and growth potentials were characterised. The efficient TAIL-PCR approach was established for identifying T-DNA flanking sequences from C. militaris mutants. The successful construction of the mutant library indicated the usefulness of this approach for functional genetic analysis in this important fungus.     

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。     

根癌农杆菌介导的黄瓜转基因研究进展

黄瓜Cucumis sativus是世界性的重要蔬菜作物。农杆菌介导的转基因技术是研究植物基因功能及品种改良的重要手段。为进一步加快黄瓜的转基因研究和育种进程,文中针对农杆菌介导的黄瓜遗传转化方法,从黄瓜再生能力的影响因素、遗传转化条件和过程中各类添加物质等方面,阐述了根癌农杆菌介导的黄瓜转基因研究进展及存在的问题,并对提高黄瓜遗传转化效率和安全筛选标记的应用等前景进行了展望,以期为黄瓜抗逆育种和果实品质改良等研究提供参考。     

Agrobacterium tumefaciens-mediated transformation of Mentha spicata and Mentha arvensis

The stable integration of GUS and NPTII genes in Mentha arvensis and M. spicata has been achieved by Agrobacterium tumefaciens-mediated gene transfer. Transformation assays were performed by cocultivating plant leaf disks with either GV2260/GI or EHA105/MOG Agrobacterium strains. Transgenic plants were selected on medium containing 150 mg l⁻¹ kanamycin. Transgene presence and structure was studied by the use of PCR analysis and Southern blot hybridization. Transgene expression was evaluated by RT-PCR and transgene product activity by a histoenzymatic GUS assay. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil

Dunaliella bardawil, a unicellular microalga, grows in relatively high concentrations of salt and has so far been refractory to Agrobacterium-mediated transformation. An inverse relationship between salt concentration and hygromycin resistance was observed. Co-cultivation at 0.2?M NaCl allowed growth of both D. bardawil and A. tumefaciens. Lowering salt concentrations also enabled the use of lower concentrations of hygromycin, the selection agent. Cells resistant to 100?mg?l^?1 hygromycin were selected and growth of Agrobacterium was completely eliminated in these cells using cefotaxime/potassium clavulanate. The concentration of sodium chloride was gradually increased to 1.0?M with simultaneous reduction of hygromycin concentration for better growth of D. bardawil. Agrobacterium was unable to survive in the growth medium used for Dunaliella. Expression of β-glucuronidase (uidA), green fluorescent protein (GFP) and hygromycin phosphotransferase (hpt) in the hygromycin-resistant culture was detected using X-gluc as substrate and Western blotting using GFP antibodies and RT-PCR respectively. Cells growing in 1.0?M NaCl (in the absence of hygromycin) retained their ability to grow in hygromycin even after 18 months of cultivation. These cells expressed GFP and PCR for hpt gene was positive. The stability of the integrated transgene and resistance to hygromycin in three different transformation events were ascertained periodically. Southern blotting of DNA extracted from hygromycin resistant cells (HRC) that were 15–18 months old established the presence of the integrated transgene in the DNA of D. bardawil. Results of the present study substantiate A. tumefaciens-mediated transformation of the unicellular marine alga D. bardawil. Agrobacterium tumefaciens-mediated transgene integration along with the massive outdoor cultivation methods used for D. bardawil may allow the commercial synthesis of secondary metabolites and heterologous proteins.