cDNA文库构建方法的进展

cDNA文库构建是基因克隆的重要方法之一。从cDNA文库中能够筛选到所需的目的基因,并直接用于该目的基因的表达,它是发现新基因和研究基因功能的基础工具,本文介绍几种cDNA文库构建中的改进技术,并着重阐述其原理和特点。     

cDNA文库的构建策略及其应用

cDNA文库在基因分离和克隆中具有重要的作用。从cDNA文库中能筛选出所需要的目的基因,并直接用于该目的基因的表达。cDNA文库是发现新基因和研究基因功能的基础工具。随着分子生物学技术的发展。cDNA文库构建方法有了很大改进和提高,就cDNA文库的构建方法及其应用进行综述。     

cDNA文库及其在原虫研究中的应用

cDNA文库构建和筛选是基因克隆的重要方法之一,它是目前发现新基因和研究基因功能的基本工具.从cDNA文库中可以筛选到目的基因,并直接用于该基因的表达.由于cDNA文库在基因分离和克隆中具有重要作用,因此其应用也日益广泛.简要介绍自cDNA文库创建以来,发展起来的各类文库及其构建cDNA文库的方法.作者重点阐述了弓形虫、利什曼原虫、阴道毛滴虫、疟原虫等原虫cDNA文库的构建及其应用.     

干旱胁迫下刚毛柽柳消减文库的构建及分析

以干旱胁迫下的刚毛柽柳根部组织cDNA为 tester,正常生长的刚毛柽柳根部组织cDNA为driver,利用抑制性消减杂交技术(SSH)构建了干旱胁迫下刚毛柽柳根部组织的消减文库.文库克隆的重组率为 95%,插入片段大部分集中在250～ 600 bp之间.通过对文库阳性克隆的随机测序,获得了如Mn-SOD、myb相关蛋白、锌指蛋白等17种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面.所得EST序列已被GenBank收录.实验为抗逆基因克隆和系统研究干旱胁迫下柽柳根部基因的表达奠定了基础.     

绒毛白蜡FvNCED3基因植物表达载体构建及功能分析

[目的]构建绒毛白蜡FvNCED3基因的植物过表达载体并对其基因功能进行分析。[方法]采用双酶切法将绒毛白蜡FvNCED3基因插入pROKⅡ载体中,构建pROKⅡ-FvNCED3植物表达载体;浸花法转化拟南芥,抗性种子经Kan筛选后进行PCR检测;测定不同盐浓度培养基中转基因幼苗和对照植株的鲜重与根长,鉴定其基因功能。[结果]双酶切重组质粒得到1 823 bp的目的片段,表明成功构建pROKⅡ-FvNCED3植物表达载体;转基因种子经50mg/L Kan筛选培养6 d时效果最好,转化率达4.97‰。耐盐性分析表明,盐胁迫下,转基因株系根长显著长于对照,且盐浓度为100 mmol/L、150 mmol/L时,其鲜重分别为对照的1.43倍、2.06倍。[结论]成功构建pROKⅡ-FvNCED3植物表达载体,经PCR证明FvNCED3基因整合到植物基因组中。转基因后代的耐盐性分析初步表明FvNCED3基因具有增强植物耐盐性的功能。     

干旱胁迫下黄檗幼苗cDNA消减文库的构建和分析

以干旱胁迫下的黄檗幼苗cDNA 为tester, 正常生长的黄檗幼苗cDNA为driver, 利用抑制性消减杂交技术(suppression subtractive hybridization, SSH)构建了干旱胁迫下黄檗幼苗的消减文库并对其进行了EST序列分析。从消减文库中随机挑取20个阳性克隆, 提取质粒进行酶切和PCR鉴定, 显示文库克隆的重组率大于95%, 插入片段大小大部分集中在300~800bp之间。随机挑取816个克隆进行测序, 得到265个基因。将其进行同源性分析, 划分为16类。获得了热激蛋白70、脱水响应蛋白(RD22)、通用胁迫蛋白、金属硫蛋白(MTII), 晚期胚胎丰富蛋白(LEA14)等44种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。本研究为抗逆基因克隆和系统研究干旱胁迫下黄檗基因的表达奠定了重要的理论基础。     

干旱胁迫下黄檗幼苗cDNA消减文库的构建和分析

以干旱胁迫下的黄檗幼苗cDNA 为tester, 正常生长的黄檗幼苗cDNA为driver, 利用抑制性消减杂交技术(suppression subtractive hybridization, SSH)构建了干旱胁迫下黄檗幼苗的消减文库并对其进行了EST序列分析。从消减文库中随机挑取20个阳性克隆, 提取质粒进行酶切和PCR鉴定, 显示文库克隆的重组率大于95%, 插入片段大小大部分集中在300~800bp之间。随机挑取816个克隆进行测序, 得到265个基因。将其进行同源性分析, 划分为16类。获得了热激蛋白70、脱水响应蛋白(RD22)、通用胁迫蛋白、金属硫蛋白(MTII), 晚期胚胎丰富蛋白(LEA14)等44种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。本研究为抗逆基因克隆和系统研究干旱胁迫下黄檗基因的表达奠定了重要的理论基础。     

渗透胁迫下白花柽柳cDNA文库的构建及EST分析

目的：寻找渗透胁迫相关基因EST片断,方法：构建白花柽柳cDNA文库,对文库阳性克隆随机测序,并进行网上比对分析。结果：该cDNA文库克隆的重组率大于95%,插入片段主要集中在250～1000bp之间,并获得了如脯氨酸丰富蛋白基因,钙依赖蛋白激酶基因,丝氨酸／苏氨酸激酶基因,过氧化氢酶基因,膜转运蛋白等基因。结论：渗透胁迫基因是多基因参与的系统调控过程,它们涉及了植物细胞壁合成、信号传递、基因调控、氧化物质清除、渗透调节物质的转运等方面。     

通过cDNA微阵列鉴定水稻(Oryza sativa L.) 盐胁迫应答基因

利用水稻耐盐品种兰胜构建了一个高质量的cDNA文库以鉴定水稻盐胁迫应答基因, 从cDNA文库中提取约15000个质粒, 并用Biomek 2000 高密度点阵系统或手工操作, 将这些质粒点于尼龙膜上. 通过这种方法鉴定了30个盐应答基因, 对其中的12个基因通过Northern杂交进行表达分析, 确证了cDNA微阵列的杂交结果. 30个基因中, 18个基因受盐诱导, 另外12个基因受盐抑制, 其中27个在GenBank数据库中有同源序列. 根据功能, 这些基因大致可以分为5类：光合作用相关基因、物质运输相关基因、代谢相关基因、耐逆相关基因以及其他未分类的基因. 研究结果表明, 盐胁迫影响了植物生长发育的多个方面, 其中有些基因可能在植物体的耐盐过程中具有重要作用.     

香蕉MuMADS1基因表达产物的亚细胞定位

MuMADS1是从香蕉果实cDNA文库中筛选分离到的一个MADS—box基因．通过生物信息学分析表明,该基因编码的蛋白可能作为转录因子定位于细胞核中,而且芯片分析表明：该基因在果实成熟早期表达上调．是乙烯的上游调控因子,可能与花的发育、果实发育及成熟相关．为进一步深入研究该基因功能。构建了以绿色荧光蛋白（Green fluorescent protein．GFP）为报告基因的融合植物表达载体pCAMBIA1304 MuMADS1．利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达．荧光显微镜检测结果表明。该基因表达产物定位于细胞核中．符合转录因子特性．     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

新型双基因表达盒真核载体的构建及功能验证

目的: 构建含双基因表达盒的新型基因疫苗真核表达载体pVAX2,并对其功能进行验证。方法: 设计一个包含人巨细胞病毒启动子(CMV promoter)、T7启动子、信号肽基因、血凝素A表位基因(HA)、多克隆位点区域(MCS)、c-myc抗原表位、血小板来源的生长因子受体跨膜区域(PDGFR TM)及牛生长激素多腺苷酸化信号(BGH polyA)等八种元素的表达盒Ⅱ(Expressing cassette Ⅱ),在其上下游添加Nru Ⅰ酶切位点后,进行人工化学合成,连接到克隆载体pGH中得pGH-Ⅱ;用Nru Ⅰ酶切pGH-Ⅱ,回收1 300bp左右的片段,去磷后插入到pVAX1的相应位点,Nru Ⅰ及Bgl Ⅱ/Pst Ⅰ酶切鉴定,获得新型基因疫苗真核表达载体pVAX2;然后以增强型绿色荧光蛋白(EGFP)为报告基因,将其分别构建至两个不同的表达盒内,脂质体转染BHK-21细胞,利用RT-PCR及荧光显微镜技术进行该载体的功能验证。结果: 两个表达盒内的EGFP基因在BHK-21细胞均能高效表达,相互间不受影响,且新构建的表达盒具有蛋白展示功能。结论: 成功构建含双基因表达盒的新型基因疫苗真核表达载体pVAX2,为多价DNA疫苗的研究奠定了坚实基础。     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.     

Development of a Gateway-compatible two-component expression vector system for plants

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator–reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

     

A novel approach for the construction of plant amiRNA expression vectors

Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60 bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60 bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.     

MicroRNA-based RNA Interference Vector for Gene Silencing in Plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes. In this study a microRNA 159a-based binary vector was constructed which can be used for hpRNA expression. Hairpin (hp) RNA expression cassettes carrying the gene sequences are typically constructed on binary plasmid and delivered into plant cells by Agrobacterium-mediated genetic transformation. This system allows simple insertion of 21- nt target gene sequences into microRNA backbone, to facilitate the processing of microRNA hpRNA by the endogenous machinery of host, thereby producing artificial microRNA carrying the sequence of target gene(s). The functionality of new vector system was tested by silencing viral gene in transgenic plants. Strong down regulation of viral gene was observed in virus infected tobacco plants transformed with pAmiR₁₅₉ vector. The processing of amIRNA leading to viral-specific sIRNA was confirmed by northern blotting. This vector system provides an important addition to the plant molecular biologists’ toolbox, which will significantly facilitate the use of RNAi technology for analyses of various gene functions in plant cells.     

三个方便实用的植物表达载体构建与验证

为满足植物功能基因组学研究及转基因安全性需要,本研究根据一些国内外引进或商业化的植物表达载体及其相关元件,构建了3个适合于植物,尤其是单子叶植物转化的表达载体,即p AH006、p WMB022和p WMB025。p AH006载体包含由玉米泛素ubi启动子调控的GUS基因和bar基因的完整T-DNA区域,此区段能够被酶切回收,可用于单子叶植物农杆菌介导转化效率评价及基因枪介导线状DNA转化效果研究;p WMB022载体携带由双35S启动子调控的玉米色素基因Lc和C1,可用作基因枪介导的共转化筛选标记,直观筛选含目标基因转基因材料;p WMB025载体携带由ubi启动子调控的、商业化转基因植物中广泛利用的EPSPS基因,可用于禾谷类作物农杆菌或基因枪介导的遗传转化,载体多克隆位点可通过酶切方式更换目标基因。酶切鉴定结合农杆菌或基因枪介导的小麦幼胚愈伤组织或叶片转化验证此3个载体表明,载体构建正确,其标记基因、可视化基因和报告基因均能正常表达。这3个载体的构建对于小麦等植物转化效率提升、安全型转基因作物获得和植物功能基因组学研究等具有重要意义。     

Graft‐accelerated virus‐induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS', where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR_1, RhA G, and RhNUDX_1 in rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.