绿色荧光蛋白基因(GFP)在抗虫转基因植物研究中的应用(英文)

用合成的cry1Ac基因与绿色荧光蛋白基因 (GFP)构成融合蛋白基因 ,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg ,经根癌农杆菌介导转化了烟草。在紫外灯照射下 ,观察到转基因植株叶片中有较强的绿色荧光 ;经抗虫试验、PCR、Southernblot和Westernblot等检测 ,表明该重组植物表达载体能够在转基因植物中有效表达外源基因 ,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统 ,简化了抗虫转基因植物筛选程序 ,有助于快速获得双价抗虫转基因植株。     

AtRGS1-GFP融合蛋白对AtRGS1细胞定位的研究

应用Gateway克隆技术构建了以CaMV35S为启动子,含AtRGS1-GFP融合基因的植物表达载体,并分别用根癌农杆菌介导法和PEG介导法转化拟南芥野生型（C01）悬浮细胞系和幼苗叶片原生质体,利用荧光显微镜观察AtRGS1-GFP融合基因在转化受体系统中的表达与定位。结果显示,在含AtRGS1-GFP融合基因的转化细胞系中,GFP绿色荧光在细胞膜（壁）上特异表达;原生质体瞬时表达系统中,GFP绿色荧光在细胞膜上强烈表达,表明AtRGS1蛋白定位于细胞质膜上。     

绿色荧光蛋白基因（G卯）在抗虫转基因植物研究中的应用

用合成的crylAc基因与绿色荧光蛋白基因(GFP)构成融合蛋白基因,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg,经根癌农杆菌介导转化了烟草。在紫外灯照射下,观察到转基因植株叶片中有较强的绿色荧光;经抗虫试验、PCR、Southern blot和Western blot等检测,表明该重组植物表达载体能够在转基因植物中有效表达外源基因,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统,简化了抗虫转基因植物筛选程序,有助于快速获得双价抗虫转基因植株。     

利用荧光分光光度计定量分析GFP基因的表达水平

以3种表达水分高低不一的绿色组织特异性启动子驱动绿色光蛋白（green fluorescent protein,GFP）基因转化烟草植株,设计了一种利用荧光分光光度计对组织中GFP的表达水平进行定量分析的新方法,利用该方法对获得的102株转基因烟草中不同部位叶片中的GFP表达水平进行了定量分析.其结果与荧光显微镜观察结果高度一致,从而证实利用这种新方法对GFP基因进行定量分析是可行的。     

GFP基因转化香樟胚性愈伤组织的研究

以香樟胚性愈伤组织作为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因(GFP)的遗传转化研究。经农杆菌侵染后的胚性愈伤组织通过共培养、选择培养后获得抗性愈伤组织和体胚,对抗性愈伤组织及体胚的诱导过程进行了GFP荧光检测。结果表明,GFP基因能在抗性愈伤组织和体胚中强烈表达,证明GFP基因能够在香樟遗传转化中得到应用。对抗性愈伤组织的PCR检测初步证实外源GFP基因已整合到香樟胚性愈伤组织的基因组中。     

发根农杆菌介导丹参牻牛儿基牻牛儿基焦磷酸合酶1基因RNA干扰载体的转化

目的：利用发根农杆菌ACCC10060介导丹参牻牛儿基牻牛儿基焦磷酸合酶1基因（SmGGPS1）RNA干扰（RNAi）载体转化丹参叶片,生成SmGGPS1的RNAi转基因毛状根。方法：根据已克隆到的SmGGPS1特异区域设计并合成2段RNAi序列,分别插入RNAi双元载体pK7GWIWG2D（Ⅱ）中,构建2个含卡那霉素（Kan）和绿色荧光蛋白（GFP）双筛选标记的植物表达载体pK7GWIWG2D（Ⅱ）-SmGGPS1-RNAi2和pK7GWIWG2D（Ⅱ）-SmGGPS1-RNAi3;利用带有上述2个RNAi载体的发根农杆菌ACCC10060侵染丹参叶片,诱导生成转基因毛状根;通过Kan抗性筛选和GFP绿色荧光观察统计转化率。结果：分别得到SmGGPS1-RNAi2和SmGGPS1-RNAi3转基因毛状根301根和399根,平均转化率为60.34%。结论：首次建立了发根农杆菌介导的外源基因转化丹参的体系。     

玉米花粉体外萌发方法改进及其对花粉介导转基因的作用

超声波处理花粉介导植物基因转化方法由山西省农业科学院生物技术研究中心发明, 已被国家知识产权局授予发明专利(专利号ZL 99121152.9)。在该专利的基础上, 针对玉米(Zea mays)花粉取样、保存和处理条件等因素对其体外萌发的影响进行深入研究, 提出了改进玉米花粉体外萌发实验的方法。研究结果表明, 在不同时期对开花的玉米进行花粉培养时所需蔗糖溶液的浓度不同; 确定了玉米花粉的保存时间、条件及其对超声波处理后花粉萌发率的影响, 以提高该转化方法中花粉的活力, 并进一步验证了该转基因方法的可靠性; 讨论了玉米花粉体外萌发的操作技巧和各因子的参数, 对提高花粉介导植物基因转化效率有一定的参考价值。     

用绿色荧光蛋白和洋葱表皮细胞检测拟南芥rd29A基因启动子活性的方法

将rd29A基因的启动子与绿色荧光蛋白基因(GFP)融合在一起,构建成植物表达载体,并以CaMV35S启动子驱动的GFP基因的植物表达载体为对照,用基因枪介导法转化置于4种类型培养基上的洋葱表皮细胞.对其进行不同温度下的培养,16 h后观察GFP基因瞬时表达水平的结果表明,rd29A启动子对高盐和脱水逆境的响应较温度显著,特别是在含PEG6000的培养基上,细胞无破损,绿色荧光强烈,适合于GFP的瞬时表达.而高盐由于易导致细胞出现离子毒害,不宜作为GFP瞬时表达的培养基.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

绿色荧光蛋白cDNA在腺病毒重组载体转染中的应用

绿色荧光蛋白（green fluorescent protein, GFP）基因是目前发现的唯一能在细胞内表达,且不需要其他外源底物参与的全新报告基因.将GFP cDNA与腺病毒载体pAdE1CMV重组,以lipofectin转染293细胞（一种人胚肾细胞）,观察其在真核细胞内的表达情况,为转基因技术提供了新的监测方法.     

β-Glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment

 Gene constructs containing the β-glucuronidase (GUS) gene or green fluorescent protein (GFP) gene under the control of pollen-specific promoter Zm13-260 from maize were introduced by particle bombardment into de-exined pollen of Nicotiana tabacum. The de-exined pollen exhibited transient expression of the GUS or GFP gene as indicated by histochemical and fluorescent assay, respectively. The frequency of de-exined pollen transformation with the GUS or GFP gene was approximately 6 and 3 times higher, respectively, than that of pollen with intact walls, indicating that pollen deprived of the exine barrier responded better to foreign gene transfer than did the original. Cytological observation of GUS-expressing pollen grains showed that introduced gold particles were visible in the cytoplasm and vegetative nucleus as well as in the generative nucleus. GFP-expressing pollen tubes were observed in the style even after pollination. Received: 28 October 1997 / Revision accepted: 13 April 1998     

Pollen vegetative cell-specific expression of Bra r 1: useful tool for observation of the vegetative nucleus and identification of transgenic pollen by nuclear-targeted GFP

Bra r 1 encodes a novel Ca²⁺-binding protein specifically expressed in pollen and is localized in cytoplasm of pollen and pollen tubes. In this study, we demonstrated the expression of green fluorescent protein (GFP) with a nuclear localization signal under the control of Bra r 1 promoter in tobacco pollen. A fluorescent signal was detected in the vegetative nucleus (VN) but not in generative and sperm cell nuclei, indicating pollen vegetative cell-specific expression of Bra r 1. The fluorescent signal in elongating pollen tubes was stronger than that in mature pollen, indicating that the expression of Bra r 1 was more activated during pollen tube growth. This result suggests that Bra r 1 protein might be necessary for pollen tube growth. The pattern of green fluorescence in the VN revealed that VN chromatin is dispersed during the mid-bicellular pollen stage and condensed at the mature stage. This suggests that the level of chromatin condensation might be linked with gene expression in pollen vegetative cells. We also found that the expression of GFP and its targeting of the VN have no detrimental effect on pollen maturation and pollen tube growth. Expression of GFP in pollen thus makes rapid non-destructive monitoring of transgenic pollen and pollen tubes possible. The GFP which moved into the VN was found to be a convenient tool for observation of the VN and could be useful as a selectable marker of transgenic pollen for the analysis of pollen-specific genes. Received: 6 December 2000 / Revision accepted: 20 March 2001     

绿色荧光蛋白在草鱼肾细胞中的表达

应用阳离子脂质体介导法,将含绿色荧光蛋白（GFP）基因的质粒pEGFP-N1转染到培养成单层的草鱼肾细胞（CIK）中,通过荧光倒置显微镜和特异性RT-PCR方法检测GFP的表达.在荧光倒置显微镜下可见CIK细胞的胞质和胞核均呈现绿色荧光,且细胞核的绿色荧光强度强于细胞质.转染细胞中的转录产物经RT-PCR扩增后,凝胶电泳鉴定出与GFP基因片段分子量大小一致的条带,经测序证明其为GFP基因序列.结果表明,GFP基因可以在草鱼CIK细胞内高效率成功表达,为构建以GFP为报告基因的真核重组质粒及研究草鱼出血病DNA疫苗奠定了重要的基础.     

玉米全生育期黄化材料的光合性能分析

通过对玉米(Zea mays L.)黄化材料和2份正常自交系的光合特性、光响应曲线及荧光动力学参数进行分析,结果表明,黄化材料yglm1的净光合速率(Pn)、气孔导度(Gs)和胞间CO2浓度(Ci)均显著低于正常自交系Mo17和RP128,而蒸腾速率(Tr)却显著高于正常自交系;黄化材料的光补偿点、光饱和点、暗呼吸速率及表观量子效率均高于正常自交系Mo17和RP128,但其净光合速率却低于正常自交系;黄化材料的荧光动力学参数初始荧光(Fo)、最大荧光(Fm)、PSⅡ原初光能转换效率(Fv/Fm)、PSⅡ原初光能捕获效率(Fv'/Fm')、PSⅡ光下实际光化学效率(ΦPSⅡ)、光化学猝灭系数(qP)均显著低于正常自交系Mo17和RP128,非光化学猝灭系数(qN)显著高于正常自交系。相对于正常自交系,黄化材料具有较低的电子传递潜力、原初光能捕获效率和转换效率、光能分配及利用率,这可能是导致其净光合能力降低的根本原因。     

Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny

Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification, i.e., to alter the emission spectra, would increase its utility. Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

转基因大麦中gfp基因的染色体位置及其表达

通过对大麦小孢子进行基因枪轰击获得4株转绿色荧光蛋白基因(gfp)的植株(A、C、D、E),以gfp基因为探针进行荧光原位杂交(FISH)研究转化植株中转基因插入位置和基因表达。4个株系在染色体7L(5HL)的不同位置都有一个插入点,而E株系在染色体5S(7HS)还有第2个插入点。所有的转基因T0代植株都是半合子并在T1、T2代发生分离。D株系GFP未表达,但FISH和PCR分析表明gfp基因已成功插入其染色体。各株系在根尖和花粉中的GFP表达水平不同：C株系在花粉表达强而在根尖表达中等;A株系在花粉中等表达而在根尖表达较淡;E株系则在根尖高表达,花粉中等表达。A和C株系在根尖和花粉的GFP分离都表现单位点特性,而E株系的根尖分离表现重叠作用(15：1)特征,但在花粉中表达GFP的频率低。PCR结果和3个分离株系的根尖表达结果一致。D和E株系的GFP表达不正常可能和加基因插入位置或基因的结构有关。     

腺病毒载体介导的GFP基因在鸭胚成纤维细胞中的表达及RNA干扰

目的：建立在鸭胚成纤维细胞（DEF）中进行RNA干扰（RNAi）的技术平台,为鸭基因组功能的研究提供新的技术手段。方法：以绿色荧光蛋白（GFP）基因为报告基因,脂质体转染化学合成的GFP特异小干扰RNA（GFP-siRNA）,用流式细胞仪测定GFP-siRNA对重组腺病毒（Adv-GFP）介导的GFP基因在DEF中表达的干扰效果。结果：200MOI（感染复数）Adv-GFP介导的GFP基因在DEF中表达效率最高,为31．20％±3．1l％,对DEF的活力无明显影响;GFP-siRNA能有效干扰GFP基因在DEF中的表达,相对抑制率为98．56％。结论：在DEF中进行RNAi是可行的,Adv-GFP是介导外源基因在DEF中表达较为理想的载体;首次建立了在DEF中进行RNAi的技术平台,为鸭基因组的功能等研究提供了新的技术手段。     

Antirrhinum majus microspore maturation and transient transformation in vitro

The male gametophyte of higher plants represents an excellent system to study gene regulation, cell fate determination and cellular differentiation in plants because of its relative simplicity compared to the sporophyte and its accessibility for cytological and molecular analysis. Unicellular plant microspores are single haploid cells, which can be isolated in large amounts at a defined developmental stage. Microspores cultured in vitro in a rich medium develop into mature pollen grains, which are fertile upon pollination in vivo. It is reported here that isolated Antirrhinum majus microspores when cultured in an optimal medium develop to form mature, fertile pollen. Their development closely resembled that of pollen formed in vivo. Isolated microspores were bombarded with Aquorea victoria Green Fluorescent Protein (GFP), Discosoma Red Fluorescent Protein (dsRFP) and beta-glucuronidase (GUS) reporter genes under the control of various promoters and transient expression was observed throughout pollen development in vitro. Bombarded and not bombarded in vitro-matured pollen grains were able to germinate both in vitro and on receptive stigmas and to set seed. The protocol of maturation, transient transformation and germination of Antirrhinum majus pollen in vitro described here provides a valuable tool for basic and applied research.     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。