农杆菌介导的番茄遗传转化的研究进展

番茄(Lycopersicon esculentum)传转化体系对其功能基因的研究和基因工程育种有重要影响,对农杆菌(Agrobacterium tumefaciens)介导的番茄遗传转化的研究进展进行了综述,主要包括影响番茄遗传转化效率的几个因素,如番茄的基因型、外植体状态、预培养和侵染过程、分化培养基中的激素和抗生...     

利用gfp报告基因优化农杆菌介导的水稻遗传转化

为提高农杆菌介导的水稻遗传转化效率,以晚粳97为转化材料,绿色荧光蛋白gfp基因为报告基因,采用正交试验L9(33)对影响农杆菌介导水稻的遗传转化因子进行优化。通过观察愈伤组织荧光表达情况,分析菌液浓度、共培养温度与共培养时间对农杆菌转化水稻的影响。结果表明,在OD660值为0.1、共培养21℃～23℃黑暗条件下,农杆菌与水稻愈伤共培养72 h,最有利于水稻的遗传转化,该条件下晚粳97愈伤组织荧光表达率达到70.9%。     

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。     

农杆菌介导的棉花枯萎病菌转化体系的优化

【背景】海岛棉相对陆地棉更易感枯萎病,一旦发生很难根治,使得枯萎病逐渐成为威胁新疆海岛棉产业发展的主要病害,但其致病机理目前还不是十分明确。【目的】揭示棉花枯萎病菌的遗传变异和致病机理,同时获得带有绿色荧光蛋白(Green Fluorescent Protein,GFP)标记的棉花枯萎病菌转化子用于观察其侵染海岛棉的途径。【方法】采用农杆菌介导的遗传转化(Agrobacterium tumefaciens-Mediated Transformation,ATMT)方法,对棉花枯萎病菌7号生理小种st89进行了遗传转化并对转化条件进行优化。【结果】农杆菌介导的遗传转化法转化棉花枯萎病菌的最佳条件为:150 mg/L的潮霉素浓度能完全抑制棉花枯萎病菌的生长,浓度为200 mg/L的头孢噻肟钠能完全抑制农杆菌LBA4404生长,农杆菌起始浓度OD₆₀₀为0.2,农杆菌预培养时间为8 h,棉花枯萎病菌分生孢子浓度为10⁵个/mL,枯萎病菌孢子悬液和农杆菌LBA4404比例为1:1,乙酰丁香酮浓度为200μmol/mL,共培养时间为4 d,转化后培养温...     

农杆菌介导的粘帚菌遗传转化北大核心CSCD

【目的】建立分离自西藏林芝的能够产生一系列具有抗肿瘤活性的二酮哌嗪类化合物(epipolythiodioxopiperazine,ETP)的一株冬虫夏草定殖真菌——粘帚菌(Gliocladium sp.)6.102的遗传转化体系。【方法】利用农杆菌介导的方式建立了粘帚菌的遗传转化体系;用正交试验研究了影响转化效率的因素,包括共培养所用农杆菌与真菌孢子的比例、共培养的时间、pH值和乙酰丁香酮的浓度。【结果】成功建立了农杆菌介导的粘帚菌的遗传转化体系,得到了转化的最佳条件,其转化效率为50-100个转化子/106真菌孢子。通过农杆菌介导的方式分别将潮霉素B磷酸转移酶基因(hph)和绿色荧光蛋白基因(egfp)转入粘帚菌中,实现了表达并且可以稳定存在。【结论】首次建立了农杆菌介导的粘帚菌遗传转化体系,为研究ETP化合物的生物合成及其调控机制奠定了基础。     

农杆菌介导的玉米遗传转化

Several maize inbreds were transformed with Agrobacterium tumefaciens EHA101 (pGIH). Transgenic maize plants were obtained. Frequency of transformation of maize inbred Suyu No. 1 can reach 8.1%. Results of PCR and Southern blot analysis proved that T-DNA was stably integrated into the genome of maize. Staining with X-gluc confirmed the expression of GUS gene in maize cells. The band amplified by inverse PCR showed that the copy number of transgene in three transformants was single. After long term of subculture, some hygromycin resistant calli lost their regeneration ability. Although Southern blot probed the integration of gusA gene in their genome, GUS activity cannot be detected in those calli. Southern blot analysis of HpaII digest DNA showed that transgenic gusA gene was highly methylated.     

子房注射法与农杆菌介导法转化甘蓝型油菜的比较研究

以甘蓝型油菜湘油１３号为试验材料,运用根癌农杆菌介导法和子房注射法成功地将Ｂｔ杀虫蛋白基因导入油菜,在特定的条件下,以子叶柄作为转化受体进行农杆菌介导转化,其转化效率受侵染时间和共培养时间的影响,最佳侵染时间为３－５ｍｉｎ,最佳共培养时间为６０－７２ｈ;子房注射法的最佳注射时间为授粉后第２０－３０ｈ,最适注射部位为子房中部,ＤＮＡ注射量为０．５－１．５μｇ,在最佳转化条件下它们分别获得抗卡那霉素植     

影响农杆菌介导白细胞介素-2基因转化番茄的因素研究

利用农杆菌介导法将白细胞介素-2基因（il-2）导入番茄中,对影响其转化的因素进行了分析。结果表明：农杆菌菌种（EHA105和C58C1）、外植体类型（子叶和下胚轴）、带有不同筛选标记（Kan^r、PPT^r、Hyg^r）的载体质粒几个因素对芽诱导分化及转化均有影响。实验共接种转化2018个子叶和下胚轴外植体,获得了47株抗性再生株,对其进行il-2的PCR扩增检测,有44株呈阳性。PCR-Southern杂交证实PCR结果可靠,显示il-2基因已导入到番茄中。     

插入玉米Ds转座因子的水稻转化群体及其分子分析

转座子标签法是一种利用转座因子插入高等植物基因组中造成基因突变,然后通过分离转座因子插入的旁邻顺序,进而克隆出突变基因的策略。这种策略在高等植物功能基因组学的研究中是十分有用的,为此目的,将玉米的Ｄｓ因子及ｂａｒ基因连接至载体ｐＣＡＭＢＩＡ１３００的Ｔ－ＤＮＡ区域中,构建成重组Ｔｉ质粒ｐＤｓＢａｒ１３００。ｐＤａＢａｒ１３００中Ｔ－ＤＮＡ区域中的潮霉素抗性基因可在转化过程中用作水稻转化植株的选择标     

农杆菌介导Bt基因遗传转化高粱

高粱是全球仅次于小麦、水稻、玉米、大豆和马铃薯等的重要作物之一。以高粱幼穗愈伤组织为转化受体,通过农杆菌介导法和含有抗潮霉素和gus基因的双元载体将杀虫晶体蛋白基因cry1Ab导入高粱品种115、ICS21B和5-27,经Hyg筛选共获得21个独立的转基因株系,52株转基因植株,平均转化率为1.9%。经PCR、Southern杂交和RT-PCR分析表明cry1Ab基因已整合入高粱基因组中并得到正确转录。Bt蛋白Westernblotting分析和ELISA定量测定显示,cry1Ab基因在转基因高粱植株中表达,但不同转基因植株表达量有差异。饲虫试验表明,转基因高粱对大螟(Sesamiainferens)具有一定抗性。     

Agrobacterium tumefaciens Interaction with Suspension-Cultured Tomato Cells

Adherence of Agrobacterium tumefaciens to suspension-cultured tomato cells has been characterized using a quantitative binding assay. Saturable binding of radiolabeled A. tumefaciens to plant cells resulted in 100 to 300 bacteria bound per cell. Specificity of A. tumefaciens binding was also inferred from two additional results: (a) an initial incubation of plant cells with A. tumefaciens reduced subsequent binding of radiolabeled A. tumefaciens by 60% to 75%; (b) tomato cells bound less than three E. coli per cell. Protease treatment of plant cells had no effect on subsequent bacterial binding, but prior treatment of plant cells with pectinolytic enzymes increased binding 2- to 3-fold. Pectin-enriched and neutral polymer-enriched fractions were obtained from tomato cell walls. The soluble pectin-enriched fraction inhibited binding of bacteria to plant cells by 85% to 95%, whereas the neutral polymer fraction only partially inhibited binding. Preliminary characterization of the activity showed it is heat stable, partially inactivated by protease treatment, and substantially inactivated by acid hydrolysis.     

农杆菌介导的单子叶植物转基因研究进展

农杆菌介导法是目前应用最为广泛的植物转基因方法。简单介绍了农杆菌遗传转化的原理,并从农杆菌携带外源基因进入植物的角度对农杆菌转化单子叶植物的关键和相关影响因素进行了论述,同时对近年来利用农杆菌介导法转化的单子叶植物的成功范例做了总结。     

植物转座子及其在番茄功能基因组的应用

转座子作为插入突变原或分子标签被广泛应用于基因的分离和克隆,已成为发现新基因和基因功能分析的有效工具。该文综述了植物转座子及其在基因分离中的研究进展,并讨论其在番茄功能基因遗传资源、功能基因组分离等研究中的应用。     

Transposon tagging of the sulfur gene of tobacco using engineered maize Ac/Ds elements

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.     

口蹄疫抗原决定簇融合基因转化生菜的研究

植物疫苗具有生产简单、成本较低、使用安全方便、易贮存等优点,是目前植物基因工程的一个研究热点。本实验通过农杆菌介导的遗传转化,以O型和A型口蹄疫抗原决定簇融合基因O21-O14-A21-HBcAg转化生菜。通过筛选,获得潮霉素抗性愈伤组织,经胚状体再生得到56棵抗性苗。对部分抗性植株进行PCR和PCR-Southern杂交检测,证实目的基因已经成功整合到生菜基因组中。RT-PCR检测初步表明,O21-O14-A21-HBcAg基因可以在生菜中正常转录表达。     

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.     

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略.