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.     

沙柳SpsLAS基因超表达对拟南芥营养生长的影响

用沙柳SpsLAS基因构建35S∷SpsLAS超表达载体并转化野生型拟南芥,对转基因拟南芥进行表型观察,利用荧光定量PCR,对分枝、生长素及细胞分裂素相关基因进行表达分析。结果显示:(1)成功构建35S∷SpsLAS超表达载体,并获得9株纯合转基因株系,且转基因株系的萌芽速率快于野生型(对照),生活周期也较长;其中7个株系表现为生长迅速、株高增加、莲座叶叶片增大、分枝增加,2个株系表现为矮化、分枝增加、育性降低等一系列变化。(2)荧光定量PCR显示,与对照相比24h时转基因株系幼苗生长素及细胞分裂素途径关键基因无明显变化,4d时各基因在各转基因株系呈上调趋势;30d时分枝相关基因RAX1、RAX3表达量均上调,而MAX1、MAX3、REV、AXR1无明显变化。研究表明,SpsLAS基因过表达对拟南芥株型、莲座叶有明显影响,该研究结果为进一步研究该基因对分枝调控机制奠定了基础。     

基于农杆菌介导瞬时转化法的AtGLR1.4亚细胞定位分析

将拟南芥基因AtGLR1.4启动子驱动的AtGLR1.4基因与绿色荧光蛋白(GFP)基因融合后,利用根瘤农杆菌介导瞬时转化法(Fast Agro-mediated Seedling Transfomation,FAST)浸染拟南芥幼苗,对其进行亚细胞定位的研究。转基因植株通过激光共聚焦扫描显微镜的观察,发现GFP绿色荧光在叶片表皮细胞的细胞膜上特异表达,表明At-GLR 1.4蛋白定位于细胞质膜上,为其后续的功能研究提供了线索。     

头孢霉素对拟南芥主根生长发育的影响

以拟南芥野生型和相关转基因株系为材料,设置0、50、100、200和400μg/mL头孢霉素处理,考察头孢霉素对主根伸长生长、根尖分生组织活性、生长素分布运输以及干细胞活性的影响,探究头孢霉素对拟南芥主根生长发育的毒性作用机制。结果显示:(1)头孢霉素能以浓度依赖的方式抑制拟南芥主根的生长,并抑制分生组织长度和CYCB1;1基因的表达,说明它能抑制根尖分生组织活性。(2)头孢霉素能降低根尖生长素报告基因DR5∷GUS、DR5∷GFP和生长素极性运输蛋白PIN1、PIN2、PIN3、PIN7和AUX1的表达,说明它能抑制根尖生长素的分布和极性运输。(3)头孢霉素能下调根尖静止中心标记系WOX5∷GFP、QC25和QC46的表达,以及SHR和SCR蛋白的表达,说明它能抑制根尖干细胞活性。研究表明,头孢霉素能通过抑制根尖分生组织活性、生长素的分布和极性运输以及干细胞活性,从而调节拟南芥主根的生长发育。     

拟南芥未知功能基因At3g61870编码蛋白的叶绿体定位研究

利用反向遗传学研究方法对1个预测的拟南芥叶绿体未知功能基因At3g61870编码蛋白进行了亚细胞定位研究.通过克隆At3g61870基因5′端长229 bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-CP-TP-GFP,经农杆菌介导转化拟南芥.转基因植株的叶肉细胞经激光共聚焦显微镜观察,叶绿素自发荧光与GFP荧光共定位于叶绿体中.结果表明,未知功能基因At3g61870编码的蛋白质为叶绿体蛋白质.     

地上部特异性启动子驱动番茄原系统素表达的载体构建及转基因植株的获得

克隆地上部特异表达的启动子——cab2(chlorophyll a/b binding protein 2,cab2)基因的启动子,构建该启动子驱动下的番茄原系统素(Prosystemin;PS)与GFP融合的植物表达载体并获得转基因植株。利用农杆菌介导法转化拟南芥,通过RT-PCR的方法及激光共聚焦显微镜观察启动子驱动PS-GFP的表达及其亚细胞定位。以拟南芥基因组为模板,利用高保真聚合酶获得了cab2启动子的目的片段,并将其与接GFP的番茄原系统素载体(SlPS)融合,激光共聚焦显微镜观察表明,该启动子驱动的基因正常表达和并定位于细胞质中。克隆获得到了cab2基因的启动子,该启动子能够驱动番茄原系统素和GFP的融合蛋白正常表达和定位。     

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.     

拟南芥砷诱导基因At4g13180的表达模式研究

拟南芥(Arabidopsis thaliana)砷诱导基因At4g13180编码蛋白是短链脱氢酶(Short-Chain Dehydrogenase/Reductase Superfamily,SDR)家族的成员之一,其过表达可以增强植物对过氧化氢的耐受性。该实验通过半定量RT-PCR,构建ProAt4g13180:GUS、At4g13180-EGFP和At4g13180-OE表达载体,获得At4g13180基因过表达转基因株系,并研究了At4g13180基因的表达模式及其编码蛋白的亚细胞定位。结果显示,At4g13180基因在根尖、叶脉、萼片和花丝等组织都强烈表达,该基因编码蛋白主要定位于胞质和核中。该研究结果为深入探究拟南芥砷诱导基因At4g13180的功能奠定了一定的基础。     

拟南芥未知功能基因At4g22890 编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890 编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208 bp 的DNA 片段, 与绿色荧光蛋白(GFP) 基因构建重组表达载体pMON530-cTP-GFP, 经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察, GFP 荧光仅在叶绿体中观察到, 表明所克隆的DNA 序列编码的多肽能够将At4g22890 编码蛋白质引导进入叶绿体, 由此推测该蛋白质为叶绿体蛋白质。     

马铃薯Sgt1基因启动子的结构及功能分析

糖苷生物碱(steroidal glycoalkaloids,SGAs)是一类存在于茄科和某些百合科植物的重要次生代谢物,与植物的抗逆性和产品品质有密切关系．茄啶半乳糖基转移酶（solanidine galactosyltransferases,SGT1）是SGAs合成代谢途径的末端关键酶之一,研究其编码基因的启动子序列对于SGAs生物合成代谢调控有重要的作用和意义．研究采用染色体步移技术(Genome walking),首次克隆到马铃薯Sgt1基因起始密码子上游2 183 bp的启动子序列,已注册到GenBank（注册号：KC759163）．构建该启动子驱动融合报告基因gfp::gus的植物双元表达载体p1304Sgt1p,转化野生型烟草获得Sgt1p::gfp::gus转基因植株,通过GUS组织化学染色分析Sgt1p::gfp::gus转基因植株中Sgt1p启动子的活性．结果表明,gus基因在转基因烟草的根、茎和叶中均表达,在叶中Sgt1p启动子的活性低于CaMV35S启动子,而在根和茎中二者基本相同;光诱导结果显示,光照处理明显增强了Sgt1p::gfp::gus转基因烟草叶片中Sgt1p启动子的活性,表明庄薯3号马铃薯 Sgt1p启动子是一种光诱导型启动子．     

植物多基因干扰载体体系构建与效用分析

多数重要的功能基因属于多基因家族,这些家族成员间存在功能冗余,高效的多基因干扰体系对研究多基因家族成员的生物学功能及其分子调控机制具有重要意义。对pCAMBIA1301载体改造,构建了适用于植物的多基因干扰体系pCAMBIA1301m和pCAMBIA1301s。使用该多基因干扰体系构建了四基因的干扰载体pCAMBIA1301m：35S∷SlPP2C1-2-3-4,4个目标基因为来源于番茄PP2C家族A组的PP2C1、PP2C2、PP2C3和PP2C4,并通过遗传转化导入番茄,用GUS染色和PCR检测转基因阳性植株,再利用RT-qPCR技术检测T₁和T₂代转基因植株中目标基因的干扰效率,用T₂代种子分析转基因番茄对ABA敏感性。结果表明,应用该干扰体系成功获得了四基因干扰的转基因植株35S∷SlPP2C1-2-3-4。在转基因番茄中4个目标基因的表达量显著低于野生型,其干扰效率均高于70%,转基因番茄种子萌发具有强烈的ABA不敏感性。多基因干扰体系能高效地同时沉默多个目标基因。     

Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO(2) limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH(3), and CO(2) source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S::GLN2::green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2::GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration.     

Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination be     

The green fluorescent protein as a marker to visualize plant mitochondria in vivo

To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated. The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue. For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary. Confocal or two-photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP. In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm. It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts. In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co-localization with a mitochondrial-specific dye. This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development. Three-dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.     

用绿色荧光蛋白监测转基因植物中选择标记基因的消除

绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。     

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.Abbreviations ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - GFP Green fluorescent protein Communicated by M.C. Jordan