水稻miR169o及其靶基因OsNF-YAs对缺水胁迫的早期表达模式

Micro RNAs(mi RNAs)是一类小的非编码RNA,在植物逆境胁迫应答中发挥重要的调控作用。mi R169可受干旱胁迫诱导表达,而过表达mi R169则可以增强植物对干旱的耐受性。然而,mi R169及其靶基因NF-YAs在水稻干旱胁迫条件下的表达动态至今尚不清楚。对水稻进行不同时间缺水处理,用q RT-PCR法定量测定了水稻根、茎、叶组织中mi R169o及其靶基因表达的动态变化。结果表明,随着缺水处理时间的增加,水稻不同组织中mi R169o表达量总体上有升高趋势;而靶基因(NF-YA1、NFYA2和NF-YA3)表达模式基本与mi R169o的表达模式相反,但并不全部对应。推测在水稻干旱胁迫早期反应中,mi R169o可能主要调控了部分特定靶基因的表达。此外,mi R169o在水稻根、茎、叶组织中的表达和丰度存在着明显的差异,具有组织特异性。     

麻竹miR172a靶基因DlAP2的克隆及其表达

为了解开花麻竹(Dendrocalamus latiflorus)的Dl AP2基因功能,采用RT-PCR和RACE技术克隆了mi R172a靶基因AP2同源序列c DNA全长,命名为Dl AP2。结果表明,Dl AP2基因c DNA全长为1729 bp,包含5′端非编码区81 bp、开放阅读框1464 bp、3′端非编码区160 bp和24个碱基的Poly A尾巴,在编码框靠近3′端130 bp处有1个高度匹配mi R172a的结合位点(CTGCAGCATCATCAGGATTCT)。Dl AP2编码487个氨基酸的蛋白,具有两个AP2结构域,属于AP2/ERF家族AP2亚家族的AP2组,与来自其它单子叶植物的AP2蛋白均有较高同源性。RLM-5′RACE分析表明,mi R172a主要在靶序列的第11~12个碱基之间剪切靶基因Dl AP2的mi RNA。q RT-PCR结果表明,麻竹花芽中Dl AP2基因的表达规律与mi R172a表达变化正好相反,证明mi R172a对Dl AP2基因的表达具有调控作用。     

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。     

SV40 PolyA序列及其AATAAA信号对上游GFP基因表达及转录终止的影响

SV40 PolyA(猴空泡病毒PolyA,简称PolyA)序列是有转录终止作用和使转录的mRNA添加PolyA尾的DNA序列(240 bp),含有AATAAA六核苷酸多腺苷化信号(Polyadenylation signal)。在pEGFP-C1质粒的GFP基因下游插入14个同向串联的Alu序列(Alu14),构建pAlu14质粒,瞬时转染HeLa细胞,用Northern blot检测和荧光显微镜观察GFP RNA和GFP蛋白表达,发现Alu串联序列强烈抑制GFP基因表达,该序列没有转录终止作用产生高分子量GFP融合RNA。又在pAlu14质粒GFP基因和Alu串联序列之间按正、反方向插入PolyA序列及去除AATAAA信号的PolyA序列,插入的这些PolyA序列均能部分解除Alu14对GFP基因的抑制作用;去除AATAAA信号的PolyA正、反序列仍然引起转录终止。将PolyA反序(PolyAas)分为4段每段60 bp,中间的2段分别称为2F2R和3F3R,将2F2R或3F3R插在pAlu14质粒的Alu串联序列的上游,随着插入2F2R片段拷贝数的增加转录的GFP融合RNA的分子量增加;2F2R的下游如果依然是2F2R那么2F2R可以支持转录延伸,如果2F2R下游是Alu串联序列则2F2R导致转录终止。无论插入一个3F3R或插入64个3F3R,均产生低分子量GFP RNA。     

黄瓜花叶病毒互补型载体可与CP转基因发生重组

为了探讨利用黄瓜花叶病毒(CMV)作为表达载体的可行性,克隆了山东株(SD)CMV RNA3的全长cDNA,并测定了全序列.采用定点突变的方法在衣壳蛋白(CP)基因起始密码子处改造出一个NsiⅠ位点.以绿色荧光蛋白(GFP)基因置换SD-CMV RNA3 cDNA的CP基因.将Fny株CMV RNA1,RNA2和嵌合SD-CMV RNA3的cDNA分别克隆在35 S启动子和终止子之间构建成表达载体.在烟草原生质体中验证了该表达载体可以表达GFP.然后将其接种到表达SD CP的转基因烟草上,试图构建成一个互补型载体.接种10d后,18棵植株中,有5棵的接种叶和其中1棵的系统叶上可检测到表达的GFP.然而1个月后,所有接种植株中都检测不到GFP.通过RT-PCR及序列分析,证实在互补系统中CMV载体与CP转基因发生了重组.上述结果对这种CMV互补型载体的可行性提出了质疑,同时也揭示了转CMV CP基因抗病毒植物的生物安全性中存在的新的问题.     

基于瞬时表达系统的水稻miRNA靶基因快速验证系统的建立

旨在建立一种准确且快速的基于瞬时表达系统的水稻mi RNA靶基因验证系统,为研究水稻miRNAs的生物学功能奠定基础。已有研究证实osa-miR169o剪切靶基因OsNF-YA4(LOC_Os03g48970.1)的mRNA,以此为参照,在烟草和水稻原生质体瞬时表达系统中将miR169o前体基因分别与LUC表达载体、LUC-48970和LUC-48970m3融合表达载体瞬时共表达,通过CCD活体成像和Luminometor分析了共表达后LUC活性的动态变化。利用茎环qRT-PCR对miR169o的表达水平进行分析,获得靶基因验证的适合体系。在水稻原生质体体系中,LUC活性和miR169o表达水平在原生质体转化后逐渐增高;24 h时LUC活性最高,相应miR169o表达水平上升10倍左右。综合分析,在原生质体转化后24 h-36 h为适宜检测时间段。在烟草瞬时表达体系中,农杆菌注射72 h后LUC活性最强,而miR169o的表达在48 h后即可上调20倍。因此,农杆菌注射后48 h-72 h为适宜检测时间段。本系统为水稻miRNA靶基因的验证提供了一种简便、快速,且更接近于体内真实情况的实验方法。     

应用于miRNA靶标检测的双荧光素酶报告载体的构建及鉴定北大核心CSCD

以牙鲆空通气孔同源框2基因(empty spiracles homeobox 2,emx2)为例,构建包含emx2 3'UTR区的野生型和突变型双荧光素酶重组报告表达载体,以期应用于mi RNA靶标的检测。利用Trizol法提取牙鲆成鱼精卵巢混合组织总RNA,参照已克隆出来的emx2基因c DNA序列,设计并合成emx2 3'UTR片段的引物并进行PCR扩增,将得到的基因片段和psi CHECK-2载体双酶切后,用T4 DNA Ligase酶进行连接反应,并转化入DH5α感受态细胞,筛选后得到野生型重组质粒;同时采用定点诱变法对emx2基因进行体外定点诱变并采用同样的方法形成突变型重组质粒。对野生型和突变型重组质粒进行双酶切、琼脂糖凝胶电泳鉴定及测序分析。成功克隆了emx2 3'UTR区,并将emx2 3'UTR区的mi RNA靶点序列GACTTGA突变为AGTCCAG,成功构建了野生型和突变型包含emx2 3'UTR区的mi RNA靶标检测载体。通过RT-PCR、基因重组及定点诱变技术成功构建了应用于mi RNA靶标验证的野生型和突变型双荧光素酶报告载体psi CHECK-emx2-3'UTR和psi CHECK-mutated-emx2-3'UTR。     

应用于miRNA靶标检测的双荧光素酶报告载体的构建及鉴定

以牙鲆空通气孔同源框2基因(empty spiracles homeobox 2,emx2)为例,构建包含emx2 3'UTR区的野生型和突变型双荧光素酶重组报告表达载体,以期应用于mi RNA靶标的检测。利用Trizol法提取牙鲆成鱼精卵巢混合组织总RNA,参照已克隆出来的emx2基因c DNA序列,设计并合成emx2 3'UTR片段的引物并进行PCR扩增,将得到的基因片段和psi CHECK-2载体双酶切后,用T4 DNA Ligase酶进行连接反应,并转化入DH5α感受态细胞,筛选后得到野生型重组质粒;同时采用定点诱变法对emx2基因进行体外定点诱变并采用同样的方法形成突变型重组质粒。对野生型和突变型重组质粒进行双酶切、琼脂糖凝胶电泳鉴定及测序分析。成功克隆了emx2 3'UTR区,并将emx2 3'UTR区的mi RNA靶点序列GACTTGA突变为AGTCCAG,成功构建了野生型和突变型包含emx2 3'UTR区的mi RNA靶标检测载体。通过RT-PCR、基因重组及定点诱变技术成功构建了应用于mi RNA靶标验证的野生型和突变型双荧光素酶报告载体psi CHECK-emx2-3'UTR和psi CHECK-mutated-emx2-3'UTR。     

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。     

氧化应激诱导内皮细胞凋亡microRNA表达改变的研究

目的:研究氧化应激诱导的内皮细胞micro RNA的表达变化。方法:ECM(Endothelial Cell Medium)培养人脐静脉内皮细胞,利用不同浓度双氧水(0μmol/L,200μmol/L,500μmol/L,800μmol/L)刺激24小时后应用流式细胞术检测其凋亡水平。提取细胞总RNA,利用实时定量PCR(Quantitive real-time PCR;q RT-PCR)检测micro RNA表达量变化,并利用生物信息学软件预测可能的靶基因。结果:加入不同浓度双氧水处理24 h后的内皮细胞总凋亡率均显著高于对照组,200μmol/L、500μmol/L和800μmol/L组的凋亡率分别为(13.31%vs 4.75%,35.9%vs 4.75%,89.75%vs 4.75%,P0.01)。200μmol/L的双氧水处理内皮细胞后,micro RNA的表达出现了明显的改变。其中mi R-92a、mi R-126的表达明显下调(P0.05),mi R-181a、mi R-217、mi R-34a和mi R-320的表达明显上调(P0.05)。靶基因预测显示mi R-320、mi R-92a可能调控多个和内皮细胞凋亡相关的基因表达。结论:在氧化应激诱导的内皮细胞凋亡中,mi RNA表达发生改变并可能参与调控内皮细胞功能。     

芜菁原生质体的分离及绿色荧光蛋白的瞬时表达

目的：分离芜菁叶片原生质体,建立蛋白质在芜菁原生质体的瞬时表达系统。方法：以津田芜菁成叶为试材,酶解分离原生质体;通过PEG介导的转化,将编码绿色荧光蛋白（GFP）的瞬时表达载体转入原生质体中,用激光扫描共聚焦显微镜检测原生质体中GFP的表达情况。结果：分离出大量的津田芜菁原生质体,并获得了较高的转化效率,GFP在整个原生质体中都有表达。结论：建立了津田芜菁原生质体瞬时表达系统。     

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.     

小麦原生质体高效转化体系的建立

植物原生质体广泛应用于植物基因功能研究中,包括瞬时基因表达、亚细胞定位、蛋白互作和蛋白活性分析等。当前,小麦基因的亚细胞定位和功能分析,大多利用模式植物拟南芥等异源的原生质体,易于造成研究结果的不准确。为避免这种情况,小麦原生质体制备及高效转化体系的建立与应用是必需的。在PEG介导的小麦原生质体转化过程中,原生质体分泌的核酸酶大量降解质粒DNA,转化效率的提高因此受到阻碍。为了建立小麦原生质体的高效转化体系,本文测试了抑制胞外核酸酶活性的因素和提高质粒DNA浓度等多个条件对转化效率的影响。结果表明,转化过程中加入双倍用量的质粒DNA进行转化,且始终保持低温环境(1℃)用以抑制核酸酶酶活性,可以使小麦原生质体的转化效率提高至85%。本文还将该系统成功地应用于2个小麦抗病相关蛋白的亚细胞定位研究,证明了该系统的高效性和实用性。该研究对未来相关研究有一定参考价值。     

雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

为探索药用植物雷公藤(Tripterygium wilfordii)悬浮细胞原生质体提取的最优条件,并建立雷公藤原生质体瞬时转化体系,以雷公藤悬浮细胞为材料,对酶解液配比、酶解时间、甘露醇浓度及处理转速进行考察。用PEG介导的瞬时转化法将外源基因转化到雷公藤原生质体中。结果表明,以雷公藤悬浮细胞为材料提取原生质体的最佳条件是酶液配比为2.0%纤维素酶+0.5%果胶酶+0.5%离析酶,甘露醇浓度为0.6 mol·L–1,酶解10小时,处理转速为67×g;用PEG介导法将含有编码GFP的植物表达载体转化雷公藤悬浮细胞原生质体,激光共聚焦扫描显微镜下细胞显示绿色荧光。通过实验筛选得到雷公藤悬浮细胞原生质体的最佳提取条件,建立了雷公藤悬浮细胞原生质体的瞬时转化体系,为进一步开展雷公藤功能基因及合成生物学研究奠定了基础。     

Electroporation of rapeseed protoplasts – transient and stable transformation

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10⁻⁵ and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.     

Factors influencing transient gene expression in electroporated tall fescue protoplasts

For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 × 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3- and 5-day-old suspensions. These results show that monocot and dicot protoplasts respond similarly in electroporation.     

Transient expression for functional gene analysis using Populus protoplasts

Despite the availability of the Populus genome sequence and the development of genetic, genomic, and transgenic approaches for its improvement, the lengthy life span of Populus and the cumbersome process required for its transformation have impeded rapid characterization of gene functions in Populus. Protoplasts provide a versatile and physiologically relevant cell system for high-throughput analysis and functional characterization of plant genes. Here, a highly efficient transient expression system using Populus mesophyll protoplasts was developed based on the following three steps. The first step involved formulating a new enzyme cocktail containing 2 % Cellulase C2605 and 0.5 % Pectinase P2611, which was shown to enable efficient large-scale isolation of homogenous Populus mesophyll protoplasts. The second step involved optimization of transfection conditions, such as the polyethylene glycol concentration and amount of plasmid DNA to ensure a >80 % transfection efficiency for Populus protoplasts. The third step involved using the Populus protoplast transient expression system to successfully determine the subcellular localizations of proteins, emulate signaling events during pathogen infection, and prepare protein extracts for Western blotting and protein–protein interaction assays. This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways in Populus.     

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.     

Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

Background

Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.

Methodology/Principal Findings

We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.

Conclusions/Significance

This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.