转GFP::cDNA基因拟南芥筛选及相关基因的功能分析

本研究以随机GFP::cDNA融合基因转基因拟南芥为材料,筛选到在细胞核或在细胞核和细胞质中均表达GFP信号的转基因株系58个。对这些转基因株系中的cDNA插入片段进行克隆,获得4株插入片段能按原初编码框进行编码的转基因株系。对插入片段为编码富含甘氨酸蛋白AtGRP8 C-末端(富含甘氨酸的结构域)的转基因株系R2的表型分析发现,连续白光、红光或蓝光下其幼苗的下胚轴比野生型的要短,且较低光照强度白光(低于100μmol m-2s-1)、蓝光(低于75μmol m-2s-1)下的差异更加明显,但是黑暗中其幼苗的下胚轴与野生型相比无明显差异,表明AtGRP8蛋白可能通过其C-末端功能域参与调控拟南芥的光形态建成反应。     

拟南芥3个蛋白磷酸酶2C基因编码蛋白的亚细胞定位及组织表达分析

利用gateway技术从拟南芥中克隆了3个蛋白磷酸酶2C基因At5G66080、At1G68410和At5G06750,3个基因的ORF全长分别为1 158 bp、1 311 bp和1 182 bp,分别编码一条385、376和393个氨基酸残基的多肽.构建了3个基因的植物表达载体35S:GFP:At5G66080、35S:GFP:At1G68410和35S:GFP:At5G06750,采用基因枪法进行的洋葱表皮细胞GFP瞬时表达实验表明,荧光信号主要分布在细胞核上,显示这3个基因的产物可能在细胞核上发挥作用.利用实时荧光定量PCR研究At5G66080、At1G68410和At5G06750基因在不同组织中的表达特性,结果表明:3个基因在各个器官均有表达,但表达量不同;At5G66080、At1G68410和At5G06750基因在花中表达量最大;At5G66080和At5G06750基因在根、叶和叶柄中的表达量次之,在茎中的表达量最低;At1G68410基因在根中的表达量次之,在茎、叶和叶柄中的表达量较低.     

两种瞬时表达体系研究拟南芥Profilin-1的亚细胞定位

使用两种瞬时表达方法研究Profilin-1(PRF1)的亚细胞定位,并比较了2种瞬时表达体系在亚细胞定位研究中的优缺点。利用拟南芥幼叶作为材料,提取叶片的RNA,采用特异性引物RT-PCR的方法克隆PRF1基因,连接到p CAMBIA1300-GFP的改造载体上,成功的构建p CAMBIA1300-GFP-PRF1的表达载体。然后分别利用PEG转化拟南芥原生质体、农杆菌浸染烟草叶片两种技术进行了瞬时表达,并在激光共聚焦显微镜下观察绿色荧光蛋白(GFP)融合蛋白的表达。研究结果表明,将PRF1基因导入拟南芥的原生质体和烟草表皮细胞后,融合蛋白绿色荧光均能被观察到,PRF1基因与GFP融合蛋白的产物在烟草表皮细胞中主要定位在细胞质和外周细胞器中,在拟南芥的原生质体中的细胞核和细胞质中都有定位。两种不同的瞬时表达体系中PRF1蛋白的定位出现了不同,这可能与同源或异源表达的植物的特性相关。     

拟南芥酪蛋白激酶1A的原核表达及纯化

拟南芥酪蛋白激酶1A(Arabidopsis casein kinase 1A,At CK1A)属于丝氨酸/苏氨酸蛋白激酶家族,其可能参与了蓝光信号转导并调控植物开花时间。通过对At CK1A的全长序列进行生物信息学分析得到了其激酶区;采用RT-PCR的方法从拟南芥c DNA中扩增At CK1A激酶区,并构建至p ET28a原核表达载体;将表达载体导入BL21(DE3)菌株后,经IPTG诱导表达和Ni亲和层析、阳离子交换层析以及凝胶过滤层析纯化,得到高纯度的重组蛋白;最后利用His标签抗体对重组蛋白进行了验证。     

发育时期和光诱导对拟南芥AtSUC2基因表达的影响

蔗糖是高等植物中碳水化合物最主要的转运形式,对于植物的生长发育至关重要.植物体内蔗糖的转运主要依赖蔗糖转运蛋白,因此对于蔗糖转运蛋白基因的研究具有重要意义.拟南芥蔗糖转运蛋白AtSUC2在蔗糖装载中起主要作用,通过半定量RT-PCR测定拟南芥叶片不同发育时期和不同光强下AtSUC2基因的表达量,研究拟南芥特定发育阶段和光诱导作用下AtSUC2基因表达的影响.结果表明,在野生型拟南芥叶片中,AtSUC2基因在16 d幼叶、30 d营养期叶片、生殖期叶片中均表达,在16 d幼叶和生殖期叶片中表达强度较弱,在营养生长旺盛时(30 d叶龄)表达较高.同时,植株在暗处理12 h时,AtSUC2基因表达量降低,在强光处理12 h时,AtSUC2基因表达量与对照差异不显著,可能AtSUC2基因的表达受光诱导但与光强无关.     

拟南芥DTX31基因表达研究

以模式植物拟南芥为材料,通过PCR和RT-PCR在DNA和RNA水平上筛选鉴定了DTX31基因对应的T-DNA插入突变体,对其表型变化进行了观察.通过半定量RT-PCR分析检测了DTX31基因在拟南芥不同器官及环境胁迫响应中的表达情况,结果发现:DTX31基因在根中表达最高,而在茎、叶、叶柄、花中的表达则较弱;盐和赤霉素使DTX31基因的表达迅速升高,盐胁迫2 h后的表达量达到最高峰,GA处理1 h时就达到最高峰,热激使DTX31基因的表达变化不明显.因此,推测该基因可能是盐和GA信号传导通路中的一个重要调控因子.     

互花米草NHX2基因的克隆与功能鉴定

NHX2属于CPA1基因家族,编码Na~+/H~+逆向转运蛋白,控制液泡膜中活性K~+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。     

水稻C2H2型锌指蛋白基因RZF71的克隆与表达分析

利用生物信息学和RT-PCR方法从水稻幼苗组织中分离了1个新的C2H2型锌指蛋白基因RZF71, 该基因编码一条250个氨基酸残基的多肽, 含有两个典型的C2H2型锌指结构。半定量RT-PCR分析表明: RZF71在根、茎、叶和幼穗中呈组成性表达, 在根中的表达丰度略高; 在高盐和PEG6000胁迫的水稻幼苗组织中, RZF71的表达显著增强, 但低温和ABA处理对该基因的表达量影响不大。农杆菌介导的洋葱表皮细胞GFP瞬时表达实验表明: RZF71定位于细胞核内。讨论了RZF71可能作为一个转录调控因子在水稻耐高盐和渗透胁迫中的作用。     

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.     

Analysis of the function of the photoreceptors phytochrome B and phytochrome D in Nicotiana plumbaginifolia and Arabidopsis thaliana

To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.     

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.     

Nuclear localization of the Arabidopsis blue light receptor cryptochrome 2

The cryptochrome blue light photoreceptor family of Arabidopsis thaliana consists of two members, CRY1 and CRY2 (PHH1). CRY2 contains a putative nuclear localization signal (NLS) within its C-terminal region. We examined whether CRY2 is localized in the nucleus and whether the C-terminal region of CRY2 is involved in nuclear targeting. Total cellular and nuclear protein extracts from Arabidopsis were subjected to immunoblot analysis with CRY2-specific antibodies. Strong CRY2 signals were obtained in the nuclear fraction. Fusion proteins consisting of the green fluorescent protein (GFP) and different fragments of CRY2 were expressed in parsley protoplasts and the localization of the fusion proteins was determined by fluorescence and confocal laser scanning microscopy. GFP-fusions containing the entire CRY2 protein or its C-terminal region were found exclusively in the nucleus. We conclude from these results that CRY2 is localized in the nucleus and that nuclear localization is mediated by the C-terminal region of CRY2.     

Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.     

Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants

Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.     

A maltose transporter from apple is expressed in source and sink tissues and complements the Arabidopsis maltose export-defective mutant

Prior to the cytosolic synthesis of transport sugars during transitory starch utilization, intermediate products of starch breakdown, such as maltose, must be exported from chloroplasts. Recent work in Arabidopsis indicates that a novel transporter mediates maltose transfer across the chloroplast inner envelope membrane. We cloned a gene from an apple cDNA library that is highly homologous with the Arabidopsis maltose transporter, MEX1. Expression levels of MdMEX determined by real-time PCR were low in the tips of growing shoots, higher in expanding leaves and maximal in mature leaves. Expression was also detected in fruits and roots, indicating a role for MdMEX in starch mobilization in sink tissues. The cDNA from apple was subcloned into an expression cassette between the cauliflower mosaic virus 35S promoter and the sGFP (green fluorescent protein) coding sequence. Plants of the Arabidopsis maltose excess1-1 mutant, which is homozygous for a defective MEX1 allele, were transformed with the 35S:MdMEX:GFP construct. Fluorescence of GFP was localized to chloroplasts, indicating that Arabidopsis recognized the predicted 55 amino acid chloroplast transit peptide in the apple protein. The phenotypes of several independently transformed lines were analyzed. The complemented plants were relieved of the severe stunting and chlorosis characteristic of mex1-1 plants. Furthermore, starch levels and concentrations of soluble sugars, leaf chlorophyll content and maximum quantum efficiency of PSII were restored to wild-type levels. MdMEX (Malus domestica maltose transporter) is the second member of the unique maltose transporter gene family.     

Differential tissue-specific response to sulfate and methionine of a soybean seed storage protein promoter region in transgenic Arabidopsis

Expression of the gene encoding the beta subunit of beta-conglycinin, a major soybean seed storage protein, is upregulated by sulfur deficiency and downregulated by methionine (Met). The tissue-specificity of these regulatory mechanisms was studied using a sulfate-responsive region (beta(SR)) from the beta subunit gene promoter. Transgenic Arabidopsis thaliana lines were generated carrying a green fluorescent protein (GFP) reporter gene under control of the cauliflower mosaic virus 35S RNA promoter with a tandem repeat of the beta(SR) element, referred to as the P35S::beta(SR)x3: GFP transgene. Upregulation of P35S::beta(SR)x3:GFP by sulfur deficiency was strongest in leaf margins, where symptoms of sulfur deficiency first appear. P35S::beta(SR)x3:GFP was also upregulated at 2 d after a medium shift from sulfur-sufficient to sulfur-deficient conditions, suggesting that the chimeric promoter is an efficient indicator of sulfur nutritional status. Analysis of transgene expression in a Met-overaccumulating mto1-1 mutation background revealed that the beta(SR) region carries sufficient information for downregulation of promoter activity by Met in developing seeds, but not in young rosettes. Comparisons with another transgenic line, in which the full-length beta promoter is active in non-seed tissues, also suggested that at least two separate tissue-specific mechanisms exist for the downregulation of the beta promoter by Met.