Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S . horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S . horneri with analysis of molecular markers. The S . horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that green fluorescent protein (GFP) is an eff ective transgene reporter for S . horneri and that particle bombardment is a suitable method for transformation of S . horneri . Through selection of four diff erent promoters for EGFP and six groups’ bombardment characters, the highest transformation efficiency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and 6 cm distance. This research paves a way for the further research and application of S . horneri .

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii,a high efficient expression vector was constructed.Green fluorescent protein(GFP) was expressed in C.reinhardtii under the control of promoters:RBCS2 and HSP70A-RBCS2.Efficiency of transformation and expression were compared between two transgenic algae:RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ.Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2,and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ.In addition,a threefold increase of GFP in Tran-Ⅱwas induced by heat shock at 40°C.All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C.reinhardtii.

Jellyfish Green Fluorescent Protein(GFP) as a Reporter for Fusarium gramminearum Development on Wheat

The plasmid pGPDGFP under the control ofpgpdA promotor was used together with vector pAN7-1 containing the hygromycin resistance cassette to co-transform protoplasts of HG1, Fusarium graminearum from Hubei Province, China. Twelve out of 14 hygromycin-resistant transformants showed green signal under the UV light and contained one or several copies ofgfp, as indicated by Southern analysis of genomic DNA digested with different restriction enzymes and hybridized to the gfp probe. A single gfp copy transformant （HG1C5） was selected for further evaluation of 80 Chinese wheat cultivars or advanced lines. The results showed different resistance type to F. graminearum were observed. GFP signals observed in the rachis and adjacent spikes of 70 Chinese wheat lines such as Chuanchongzu 104 indicated both type I （host resistance to the initial infection by the fungus） and type II （resistance to the spread of FHB symptoms within an infected spike） were not observed. While other 10 lines showed type II resistance to F. graminearum with GFP signals only in inoculated spikelets. Development of the mycelium can be intuitively observed and the resistance of wheat to F. graminearum can be identified at 7 days post inoculation （dpi） in this way. The results showed no differences were evaluated between the transformed HG1C5 and the non-transgene artificial inoculation by SAS paired chi-square test and McNernar＇s test （P=-0.0625）.

PERFORMANCE AND PERSISTENCE OF GREEN FLUORESCENT PROTEIN (gfp) MARKED AZOTOBACTER CHROOCOCCUM IN STERILIZED AND UNSTERILIZED WHEAT RHIZOSPHERIC SOIL

The persistence and performance (growth promoting potential) of green fluorescent protein (gfp) marked Azotobacter chroococcum strain ABR 4G were studied in sterilized and unsterilized wheat rhizospheric soil. The gfp was integrated via Tn 5 transposition into A. chroococcum chromosome and the resultant gfp marked colonies were identified by green fluorescent emission under UV light. The gfp was stably maintained in A. chroococcum and the gfp insertion had no apparent adverse effect on the growth promoting properties of the marked soil isolate ABR 4G. The growth promoting properties (nitrogen fixation, ammonia excretion, phosphate solubilization and IAA production) of the parent soil isolate and the gfp marked strain were found to be almost the same. All the quantitative wheat plant traits were significantly influenced by inoculation of A. chroococcum ABR 4G strain in sterilized and unsterilized soil. Inoculated bacterial counts increased gradually in wheat rhizosphere, reached maximum on 60 th d and declined on 80 th d. Fertility levels also affected survival of marked strain and the survival was comparable in sterilized and unsterilized soil. The growth promoting properties were also determined from the marked strain reisolated from wheat rhizosphere in both types of soil. Fig 1, Tab 2, Ref

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

采用GFP标记筛选抑制多血清型口蹄疫病毒3C基因表达的siRNA

为筛选出可抑制多种血清型口蹄疫病毒(FMDV)的小干扰RNA(siRNA),本试验通过多血清型口蹄疫病毒序列比对与siRNA设计分析,筛选出潜在抗多血清型siRNA位点;通过体外合成A型、O型和Asia I型病毒部分基因序列,制备绿色荧光蛋白(GPF)基因融合表达载体;将化学合成的siRNA与融合表达载体共转染,通过GFP观察、Western blot和实时荧光定量反转录PCR(qRT-PCR)方法检验目标siRNA对融合基因的抑制效率。生物信息分析发现,多血清型口蹄疫病毒3C基因高度保守并存在潜在siRNA作用靶位,GFP-3C融合表达载体成功构建,与化学合成的siRNA1-3C共转染293T细胞,GFP观察发现,siRNA1-3C可有效抑制来源于A型、O型和Asia I型3C基因的GFP-3C荧光信号且持续时间达72 h,Western blot检测证实此结果。qRT-PCR检测发现,siRNA1-3C对3个GFP-3C融合基因转录水平抑制效率超85%,且作用可维持72 h。本试验利用GFP作为筛选标记成功筛选出1个作用于FMDV 3C基因的siRNA,为开发多血清型口蹄疫抑制剂提供参考依据。

Soluble Expression and Rapid Quantification of GFP-hepA Fusion Protein in Recombinant Escherichia coli

To establish a rapid quantification method for heparinase I during its production in recombinant Es-cherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C ter-minus of a green fluorescent protein mutant (GFPmut1). As a result, not only was the functional recombinant ex-pression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluores-cence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.

Using of green fluorescent reporter gene (GFP) to monitor the fate of Fusarium moniliforme mycoparasitized by Trichoderma viride

Fusarium moniliforme Sheld.is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used biological control agent of plant disease. By geneticaly labelling F. moniliforme with the GFP reporter gene, we have studied the antagonistic action of Trichoderma viride against this pathogenic fungus. The binary GFP reporter vector pCHF3-35S∷GFP was constructed, which carries the gfp gene driven by the CaMv35S promoter. The vector was transformed into F. moniliforme via Agrobacterium.The mycoparasitism of T.viride against F.moniliforme was tested by dual culture and examined with fluorescence microscope. The result of the dual culture showed that the T.viride maintained a strong competitive ability against F. moniliforme , by growing on the top of the pathogen colony. Fluorescence microscope observation indicated that attacked hyphae of F. moniliform were distorted, swollen or broken. This indicate an enzymatic by T.viride to degrade the host cell walls and used the cell contents as a source of nutrients (Fig 1) .

基于mt-roGFP1探针研究除草化合物对拟南芥根尖细胞氧化还原电位的影响

【目的】研究几种代表性商品化除草剂以及植物源除草化合物小檗碱及其类似物对拟南芥根尖细胞氧化还原电位的影响。【方法】以靶向线粒体的氧化还原敏感绿色荧光蛋白(Mitochondria targeted redox-sensitive green fluorescent protein,mt-roGFP1)标记的拟南芥转基因植株为材料,采用不同质量浓度的化合物处理不同时间后,测定拟南芥根冠、分生区、过渡区和伸长区的细胞氧化还原电位的变化。【结果】经几种商品化除草化合物处理后,拟南芥根部分生区的细胞氧化还原电位最小。从分生区到伸长区氧化还原电位逐渐增大,呈现逐渐被氧化的趋势。其中,光系统II抑制剂(莠去津和环嗪酮)的氧化还原电位变化规律最为明显,说明mt-roGFP1荧光探针能较好地响应光系统II抑制剂。氨基酸生物合成抑制剂草甘膦对拟南芥根尖细胞氧化还原的影响具有明显的剂量−效应关系,随着草甘膦质量浓度增加,氧化还原电位变化量也逐渐增大,呈正相关关系(R^(2)=0.9956)。小檗碱及其类似物处理后,大多数处理组的拟南芥根尖细胞的氧化还原电位在分生区达到最大还原值,并从分生区开始逐渐被氧化。【结论】研究结果可以为应用roGFP荧光探针技术研究除草化合物对根系细胞线粒体的作用机制提供基础。

Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells

The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

把 ZJI 紧张基于纽卡斯尔疾病病毒(NDV ) 的完全的染色体顺序，七份教材被设计为构造 plasmid pNDV/ZJI 放大 cDNA 碎片，它包含了 NDV ZJI 紧张的全身的 cDNA。与三助手 plasmids， pCIneoNP， pCIneoP 和 pCIneoL， pNDV/ZJI 当时是进表示 T7 RNA 聚合酶的 BSR-T7/5 房间的 cotransfected。在进受胎的鸡肉的 transfected 房间文化上层清液的接种以后，从 specific-pathogen-free (SPF ) 的鸡蛋结队，传染 NDV ZJI 紧张成功地被救。格林荧光灯蛋白质(GFP ) 基因被放大并且插入了到 NDV 全身的 cDNA 产生标注 GFP 的 recombinant plasmid pNDV/ZJIGFP。在进 BSR-T7/5 房间的结果的 plasmid 和三支持 plasmids 的 cotransfection 以后， recombinant NDV， NDV/ZJIGFP，被救。特定的绿荧光在 BSR-T7/5 和鸡胚胎成纤维细胞(CEF ) 房间 48h 被观察感染以后，显示 GFP 基因被表示在一相对高级。NDV/ZJIGFP 被 oculonasal 线路接种进 10-day-old SPF 鸡。四天感染以后的、强壮的绿荧光能在肾和 tracheae 被检测，显示标注 GFP 的 NDV 能是的 recombinant 为 NDV 传播和致病的分析的一个很有用的工具。关键词纽卡斯尔疾病病毒(NDV )- 格林荧光灯蛋白质(GFP )- 营救 - 表示 CLC 数字 S831.7 基础条款：给中国(No.30630048 )

Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density Pichia pastoris bioreactor cultivations

The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.

GFP标记的球孢白僵菌在玉米中的定殖

为明确球孢白僵菌在玉米中的定殖分布规律以及不同施用方式和处理浓度对玉米生长发育的影响,本文利用绿色荧光蛋白(GFP)标记的球孢白僵菌,在激光扫描共聚焦显微镜下观测了球孢白僵菌在玉米根部、茎部和叶部的定殖情况,同时检测了不同接种方式在球孢白僵菌不同处理浓度下对玉米生长指标的影响。结果表明,球孢白僵菌在玉米不同组织部位上的定殖有着明显的差异,各球孢白僵菌接种处理中,均以叶部定殖的白僵菌孢子数最多,而茎部均未观测到孢子定殖;玉米根部仅在浸种和灌根处理中可见少量孢子。球孢白僵菌不同接种方式对其在玉米中定殖率的影响差异较大,灌根处理定殖率最高,为76.7%;其次为浸种处理,为73.3%;茎部注射处理及叶面喷施处理定殖率较低,分别为43.3%和36.7%。研究表明,不同施用方式及接种浓度球孢白僵菌孢悬液对玉米生长指标有一定的影响,球孢白僵菌可通过不同接种方式在玉米植株中定殖并扩散,对玉米的生长发育有一定的促进作用,其中以1×106～1×107孢子/mL球孢白僵菌孢悬液浸种或灌根,对玉米苗的促进生长作用最明显。

根癌农杆菌介导苎麻转绿色荧光蛋白(GFP)基因植株再生

以苎麻优良品系"5041-3"子叶作为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因的遗传转化研究,农杆菌菌株为EHA105,重组质粒为pBINm-GFP5-ER。经农杆菌浸染后的子叶外植体经过共培养、选择培养、伸长培养和生根培养后再生出抗性植株,对抗性植株的再生过程进行了GFP荧光检测,结果表明,GFP基因能在苎麻中强烈表达,证明GFP基因能够在苎麻遗传转化中得到应用。对抗性植株的Southern杂交检测证实外源基因已整合到苎麻基因组中。

用共聚焦激光扫描显微镜观测GFP标记的内生固氮菌Klebsiella oxytoca SA2侵染水稻根

用高效绿色荧光蛋白 (greenfluorescentprotein ,GFP)突变体EGFPmut2的基因标记内生固氮菌———产酸克雷伯氏菌 (Klebsiellaoxytoca (Fl櫣ｇｇｅ)Lautrop)SA2 ,用标记菌接种限菌培养条件下生长的水稻 (OryzasativaL .)幼苗 ,在接种后 1、2、4、8、12、16和 2 1d ,用共聚焦激光扫描显微镜对水稻鲜根进行光学切片 ,显示了标记菌从水稻根成熟区表面向根内入侵的过程。定殖在根表面的标记菌主要从侧根伸出的位置侵入侧根皮层 ,从邻近发生侧根的位置进入内皮层和维管柱。SA2还能从初生根成熟区无侧根伸出的位置侵入皮层向维管柱迁移。SA2入侵水稻根引发了水稻根局部的过敏反应以阻滞细菌进一步入侵 ,表现为侵入根内的细菌周围的根细胞细胞壁变厚 ,在蓝光下发出很强的黄绿荧光。

慢病毒介导的GFP转染对小鼠骨髓间充质干细胞表型、增殖和分化能力的影响

运用慢病毒(Lentivirus)载体构建绿色荧光蛋白(GFP)在小鼠骨髓间充质干细胞(MSCs)中的转染体系,检测转染后MSCs的表型和增殖能力,并诱导其向成肌细胞分化,检测此转染体系对MSCs细胞表型、增殖和分化能力的影响.骨髓细胞悬液破红后贴壁法培养MSCs,采用流式细胞术鉴定细胞表面标记,鉴定MSCs纯度;Lentivirus-GFP以1、10、50和100的感染复数(MOI)转染MSCs,作用48h后流式细胞术及免疫荧光法检测转染效率和表达的荧光强度;MTT法检测转染后MSCs的细胞活力.诱导MSCs-GFP向成肌细胞分化,蛋白印记法(WB)检测成肌细胞特异性蛋白desmin和α-SMA的表达.流式细胞术检测结果显示,培养的MSCs表达CD44、CD90和CD105,不表达CD34、CD45和CD188,符合干细胞特性.MOI为1、10、50和100的转染效率分别为23.45%、93.51%、95.44%和95.55%,MOI为10时,转染效率较高,且对MSCs活力无明显影响.MSCs-GFP体外经成肌细胞诱导分化后,表达特异性抗原desmin和α-SMA.表明本研究成功构建了小鼠骨髓来源MSCs的Lentivirus-GFP转染体系,有效标记了MSCs细胞,且此标记对MSCs的表型、增殖和分化能力等细胞生物学特性无明显影响.

农杆菌介导稻瘟病菌绿色荧光蛋白(GFP)遗传转化研究

稻瘟病是水稻生产中的主要病害,抗病品种选育和利用是控制稻瘟病最有效措施。研究稻瘟病菌与水稻品种互作机制是培育抗病品种的基础。GFP(Green fluorescent protein)基因因其具有片段小、易与多种不同蛋白质N端或C端融合等特点,广泛应用于病菌与寄主植物互作研究中。研究以p BGg Hg作为转化载体,根癌农杆菌C58C1作为转化介体,转化稻瘟病菌的强致病菌株py1022。研究发现,250μg·m L-1潮霉素B能完全抑制稻瘟病菌生长,利用根癌农杆菌介导稻瘟病菌遗传转化的最佳条件为:稻瘟病菌分生孢子浓度为1×105个·m L-1,共培养时间为4 d,共培养AS浓度为200μmol·m L-1,共培养温度为28℃。随机挑取8个转化子分别进行PCR扩增后测序、荧光显微镜下观察、致病力及稳定性测定,结果表明GFP成功转化到稻瘟病菌中,可为稻瘟病菌与水稻品种的互作机制研究提供技术支撑。

花粉介导法将TaPHR1∷GFP融合蛋白基因导入玉米

利用超声波辅助花粉介导基因转化法,将小麦耐低磷调控基因TaPHR1和GFP构建成的融合蛋白基因(TaPHR1∷GFP)导入3个玉米自交系郑58、昌7-2和PH6WC中.结果表明:3个自交系的转化效果存在基因型差异,郑58是很好的转化受体材料,平均结籽穗率、每穗平均结籽数、BASTA除草剂抗性和转基因植株PCR阳性率均优于昌7-2和PH6W;对T1代植株进行Southern杂交表明,TaPHR1∷GFP融合蛋白基因已整合到玉米基因组中;RT-PCR结果显示,TaPHR1∷GFP融合蛋白基因得到有效转录;通过对发芽籽粒进行GFP荧光观察表明,TaPHR1∷GFP融合蛋白基因得到了表达.

GFP基因在棉花转化中的应用

以绿色荧光蛋白GFP基因为报道基因，用花粉管通道和农杆菌介导的转化方法将外源基因导入棉花(Gossypium hirsutum L.)，分别获得转化幼胚、幼苗和转化愈伤组织。用手持紫外灯结合显微镜检术能够快速地对转化子进行活体筛选鉴定，比用GUS检测方法有明显的优越性。本研究不但为花粉管通道转化法的可行性提供了新的证据，同时也建立了GFP用于棉花基因工程研究的检测技术体系。

生防枯草芽孢杆菌Kct99的GFP标记及其在甘蓝根部的定殖示踪

将含有绿色荧光蛋白基因的质粒pGFP4412,电击转入生防菌株枯草芽孢杆菌Kct99,获得具有良好发光表型的荧光标记菌株gfp-Kct99。经稳定性测定表明,质粒pGFP4412在gfp-Kct99中的遗传稳定性为89%。平板对峙培养和温室盆栽接种试验显示,标记菌株gfp-Kct99对甘蓝枯萎病菌保持了原有的拮抗活性;以标记菌株和野生型菌株培养液对甘蓝盆栽苗灌根处理5 d后再接种病原菌,对甘蓝枯萎病的防治效果分别为87.7%,90.2%,二者无显著性差异,但均显著高于同时接种生防菌和病原菌、接种病原菌5 d后再接种生防菌2种处理,说明,绿色荧光标记菌株gfp-Kct99抗甘蓝枯萎病的活性未受标记基因的影响。借助荧光显微镜观察表明,标记菌株可以在甘蓝根部表皮内大量定殖,从而阻止病原菌的侵入。