不同启动子调控外源基因在斑玉蕈中的表达分析

启动子是控制基因转录的重要顺式元件,也是遗传转化实验中驱动外源基因表达的重要工具.在同一真菌中,不同启动子驱动外源基因表达水平可能存在明显差异.因此,选择合适的启动子是提高外源基因表达水平的关键.本研究分别应用花椰菜病毒35S RNA (cauliflower mosaic virus 35S RNA,CaMV35S)...     

Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.     

Transgene Expression in the Vegetative Tissues of Apple Driven by the Vascular-specific rolC and CoYMV Promoters

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of -glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.     

大麦与油菜种皮特异启动子表达模式的比较

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件;转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高;油菜Bntt种皮特异启动子表达较弱;成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性;Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。     

绿色杜氏藻不同β-胡萝卜素羟化酶基因家族胁迫应答模式研究

β-胡萝卜素羟化酶(β-carotenoid hydroxylase, CHYB)是植物类胡萝卜素生物合成途径中的一个重要限速酶。本研究对绿色杜氏藻转录组测序数据进行分析,获得2条β-胡萝卜素羟化酶家族基因chyb1和chyb2。采用染色体步移法分别克隆并获得了绿色杜氏藻chyb1和chyb2基因的启动子序列,全长分别为1080 bp(GenBank登录号：KY012338)和1155 bp (GenBank登录号：KY012339)。利用Plantcare软件分析两个启动子的顺式作用元件,结果表明绿色杜氏藻chyb1基因启动子含有与甲基茉莉酸、花生四烯酸、水杨酸等非生物胁迫相关的顺式作用元件,而绿色杜氏藻chyb2基因启动子含有与光照相关的顺式作用元件。通过qRT-PCR分析了绿色杜氏藻CHYB基因家族在不同胁迫下的基因表达水平,结果表明该基因家族的基因表达水平与启动子调控相关,且不同的家族基因应答不同的胁迫。     

人工合成根特异启动子SRSP的功能分析

根负责吸收水分和养分,是重要的植物组织,但易受生物及非生物胁迫,影响作物的生长发育和产量。设计合成根特异启动子,可为与胁迫相关的抗性基因在作物根部的功能研究及高效表达提供候选启动子。文中将4拷贝的根特异性顺式作用元件(OSE1ROOTNODULE、OSE2ROOTNODULE、SP8BFIBSP8AIB和ROOTMOTIFAPOX1)以串联排列方式设计合成了一个根特异性模块(pro-SRS),并与来自CaMV35S启动子的最小启动子融合,合成一个人工合成启动子SRSP。通过替换CaMV35S启动子将SRSP启动子克隆到pCAMBIA2300.1中以驱动GUS表达。将携带SRSP启动子的构建体通过农杆菌介导的方法转化到烟草中。GUS组织化学染色分析和实时PCR (RT-PCR)分析显示SRSP启动子在转基因烟草中赋予根特异性表达。说明顺式作用元件重复排列可实现启动子预期功能,本研究为理性设计植物组织特异启动子奠定了理论基础。     

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.     

参与水稻光合作用的PsbR3基因启动子的功能分析

本实验旨在研究水稻光合作用蛋白中各基因的表达模式. 采用RT-PCR和定量real-time PCR数据分析水稻不同组织的mRNA表达水平.结果显示,PsaK和PsbR3基因仅在茎、叶等绿色组织表达,而胚、胚乳部分均不表达.通过其启动子克隆、植物表达载体构建,以及农杆菌介导转化后,GUS组织染化分析和GUS荧光定量分析表明,两启动子均为组织特异性优势表达,PsbR3启动报告酶GUS在叶片中的表达活性为Actin启动子的3.29倍,而PsaK启动报告酶GUS在叶片中的表达活性低于Actin启动子的.这些初步结果提示,PsbR3启动子决定水稻绿色组织茎叶的优势表达,PsbR3基因可能参与水稻光合作用.     

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.     

Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Constitutive promoters are the most common promoters used to drive the expression of various genes in monocots and dicots. Therefore, it is of intense interest to ascertain their expression patterns in various plant species, organs and during their ontogenic development. In this study, the activity of the CaMV 35S promoter in transgenic tobacco plants was assessed. In contrast to other studies, performed rather on the primary transformants (T₀ generation), here, individuals of T₁ and T₂ generations were used. The expression profiles of the CaMV 35S promoter were tracked within various plant organs and tissues using the GFP marker. Special attention was given to floral tissues for which the original data regarding the CaMV 35S expression were obtained. As expected, distinct developmental and organ/tissue specific expression patterns in a plant body were observed. CaMV 35S activity was detected in most of the plant tissues and during different developmental stages. The GFP signal was not visible in dry seeds only, but it became clearly apparent within 24–48 h after sowing onto the medium, what, among other things, enables the discrimination of transgenic and non-transgenic seeds/seedlings. Afterwards, the most pronounced GFP fluorescence intensity was usually visible in various vascular tissues of both, T₁ and T₂ plants, indicating the high promoter activity. A stable manifestation of the promoter was retained in the next T₂ generation without any evident changes or losses of activity, showing the expression stability of the CaMV 35S.     

Changes in the promoter of a defender against apoptotic cell death gene affect its expression in upland cotton

The DAD1 (defender against apoptotic cell death) gene is a negative regulator of programmed cell death. It plays important roles in plant growth, development, environmental adaptation, and aging. We examined whether and how the expression of DAD1 gene is affected by the promoter changes in the allotetraploid upland cotton (Gossypium hirsutum L.). We compared the expression status of the genes in the leaves of an elite cultivar and a wild variety of G. hirsutum, and cloned and analyzed a 1.3-kb promoter region of the genes in two cotton types. Results revealed that the expression of a subgenomic homoeolog of DAD1 (DAD1 _At ) was significantly upregulated in leaves of the cultivated cotton when compared with that in a wild cotton. In addition, we identified 18 variations, including 14 single nucleotide polymorphisms and four indels between the promoters of the two cotton types. We detected at least 10 of 18 variations in the promoters related to possible cis-regulatory elements. Further transient tobacco assays using heterogeneous DAD1 _At promoter::GUS fusion showed that the activity of the DAD1 _At promoter in the cultivated cotton was stronger than that in the wild cotton, which suggested a dynamic change in the DAD1 _At promoter during genetic divergence of the two cottons. This study provides an example of how gene expression could be affected by specific changes in the gene promoter.     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.     

Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17.5C控制Cre定位重组酶介导的DNA删除系统.在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S-GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除.通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率.结果显示在两个小时热激处理后转基因烟草中有41%的CaMV35S-GUS片段被删除.由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作.     

马铃薯StCRKs基因家族的鉴定分析及响应逆境信号的表达

为鉴定马铃薯（Solanum tuberosum）中富含半胱氨酸的类受体激酶（cysteine-rich receptor-like kinase,CRKs）,利用Pfam等工具对马铃薯蛋白质组和基因组序列进行分析,共鉴定到18个新的马铃薯StCRKs家族基因,这些基因分布在1、2、11和12号染色体上,均具有典型保守的CRK结构域。StCRKs基因的启动子区具有响应5种植物激素、昼夜节律、生物胁迫、非生物胁迫及种子特异性的响应元件。利用qRT-PCR方法对马铃薯盆栽苗开花期的根、茎、叶和花中的18个StCRKs基因进行组织特异性表达分析,结果显示不同基因的表达部位不同。分别用水杨酸类似物BTH和4 ℃低温处理马铃薯,有13个StCRKs基因能够响应低温信号,10个StCRKs基因能够被水杨酸类似物BTH诱导。为进一步深入研究StCRKs在生物胁迫及非生物胁迫中的功能提供了线索。     

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.     

Isolation and Characterization of Three New Promoters from Gossypium hirsutum that Show High Activity in Reproductive Tissues

Engineering of plant protection requires well-characterized tissue-specific promoters for the targeted expression of insecticidal resistance genes. Herein, we describe the isolation of five different fragments of promoters of three distinct flower-specific cotton (Gossypium hirsutum) genes. Expression analyses of the three genes GhPME-like1, GhβGal-like1 and GhPL-like1 revealed that they are expressed highly in flowers buds ranging from 4 to 12 mm in size. Several putative regulatory cis-elements were identified in the promoter regions, including elements involved in the control of tissue-specific gene expression in pollen grains and fruits. In vivo analyses of these promoters were performed using the heterologous plant system Arabidopsis thaliana by fusing them with the gene uidA (GUS). GUS staining in Arabidopsis tissues revealed that their expression was restricted to anthers, with the majority of expression in pollen grains and in the upper portion of the carpels and siliques. A comparison between a CaMV35S::GUS constitutive promoter and the promoters isolated in this study revealed that the cotton promoters were more active and were specific to flowers and fruits, which are organs that are preferentially attacked by important pest insects such as the boll weevil (Anthonomus grandis). The activity of the promoters was also confirmed using transient expression assays in flower buds of G. hirsutum. The promoters of GhPME-like1, GhβGal-like1 and GhPL-like1 are specific to reproductive tissues and could represent important biotechnological tools for controlling insect pests, in particular the cotton boll weevil, which attacks floral and fruit tissues.     

Transgene expression driven by heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase small-subunit gene promoters in the vegetative tissues of apple (Malus pumila Mill.)

It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max [h.] Mers.), to drive expression of the β-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approximately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple. Received: 15 June 1999 / Accepted: 12 August 1999     

利用GFP/RFP双荧光指示载体鉴定特异性启动子功能

在基因表达定位或启动子调控模式的研究中, 多以gusA作为报告基因。但由于部分组织中高内源GUS背景活性或转化手段的限制, 使判断基因表达定位或调控时存在很大误差。为了解决上述问题, 本实验将报道基因绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)融合构建双荧光标记瞬时表达载体pBI221-RFP/GFP。该载体以CaMV35S启动子驱动GFP确定转化效率, 通过鉴定阳性个体的红色荧光活性分析目的基因或启动子的表达模式。并通过番茄E8和西瓜AGPL1果实特异启动子验证了该载体在启动子调控模式研究中的应用可行性。结果表明pBI221-RFP/GFP是一个可以在基因和启动子功能验证中应用的高效瞬时表达载体。