Kidney-Specific Activity of the Bovine Uromodulin Promoter

A 10-kilobase (kb) bacteriophage bovine genomic clone containing 5.4 kb of the 5-flanking region, exons, and introns of bovine uromodulin gene was isolated. Transgenic mice containing 3.9 kb of the bovine uromodulin promoter and a lacZ reporter gene were generated by pronuclear microinjection. RT-PCR and northern blot analyses of transgene expression in various tissues of founder and F1 mice showed that the transgene was expressed exclusively in the kidney. In situ hybridization and histochemistry for lacZ demonstrated that transgene expression was restricted to tubule epithelial cells of the loop of Henle in the kidney. Stepwise 5 deletion analysis revealed that transfection of luciferase reporter constructs fused to various proximal 5-flanking regions of the bovine uromodulin gene markedly increased luciferase activity in mouse renal epithelial cells but not in mesenchymal cells and that the most critical cis elements of the uromodulin gene are located within the 600 bp upstream region.     

The Expression of a Cytosolic Cyclophilin Promoter from Periwinkle in Transgenic Tobacco Plants

The cloning of a 465 bp fragment from the 5-flanking region of the gene encoding a cytosolic cyclophilin from periwinkle was achieved through inverse polymerase chain reaction. The DNA fragment was fused to a gusA-intron marker then introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Histochemical analysis of the transgenic shoot cultures demonstrated that the construct was able to drive GUS expression in stomata guard cells, but not in mesophyll cells when shoots were still attached to the callus from which they were initiated. In separated transgenic shoots and in seedlings, GUS was expressed in external and internal phloem and root hairs, respectively. GUS activity in transgenic tobacco seedlings was also investigated by fluorimetric assays. Treatments with NaCl or ABA decreased promoter activity whereas treatment with yeast extracts increased it.     

番茄rbcS3A启动子控制的GUS融合基因在转基因水稻中的表达

为研究不同启动子用于转基因水稻,克隆了番茄Rubisco小亚基rbcS3A基因的5′上游调控区,构建了由rbcS3A启动子引导的GUS嵌合基因,并经农杆菌介导导入到水稻中。对转基因水稻植株中GUS活性的定性与定量测定结果表明,rbcS3A启动子可驱动GUS报告基因在转基因水稻植株茎和叶组织中高效表达,而在根和种子等器官中不表达或表达活性极弱,表现出一定的组织特异性。在转基因水稻中,番茄rbcS3A启动子驱动外源基因的表达不受光诱导。     

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

Summary A new procedure is described to recombine plasmid-bornelacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flankinglacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncatedlacZ andaadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carryinglacZ andaadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy inPseudomonas putida the regulatory elements controlling expression of the Xy1S-activatedPm promoter of the TOL catabolic plasmid pWWO. We show here that thePm promoter undergoes a Xy1S-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.     

转基因动物乳腺生物反应器的发展概况及人工染色体在其中的应用

转基因动物乳腺生物反应器位点效应的影响是制备转基因动物乳腺生物反应器过程中的主要问题。酵母人工染色体（YAC）和细菌人工染色体（BAC）具有容量大的特性,可以将乳蛋白的整个基因座包括所有调控序列全部装载进去,有可能克服位点效应的影响,是一种理想的载体。YAC和BAC载体转基因技术可能成为避开基因打靶获得高效表达的转基因动物乳腺生物反应器的另一途径.     

基于启动子捕获系统的水稻基因的分离

为了分离水稻的基因及其启动子,该实验室构建了T-DNA(GUS)结构的水稻启动子捕获系统,对其中编号为113#、T-DNA单拷贝插入、GUS报告基因为组成型表达的阳性捕获系进行了进一步分析。潮霉素筛选结合GUS组织化学染色获得了T-DNA插入的纯合株(113#-22和113#-26);Inverse法分离得到T-DNA插入位点水稻基因组DNA旁邻序列,测序和BLAST结果表明,T-DNA反方向插在水稻基因组4号染色体预测基因的内含子中;扩增T-DNA插入位点上游2kb左右DNA片段,构建启动子分析质粒转化水稻‘中花11’胚性愈伤组织,获得转基因植株,GUS组织化学染色模式与113#阳性株系一致。结果表明,该预测基因及其启动子是利用启动子捕获系统所捕获到的候选基因。     

Production of Marker-free Transgenic Tobacco Plants by FLP/frt Recombination System

Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/frt site-specific system of 2-μm plasmid from Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target (frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/frt site-specific recombination system was constructed, which included the frt-containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt. The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat-shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41 % re-transgenic tobacco plants, which indicated that this system could make a great contribution obtaining the marker-free transgenic plants.     

拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

cry3A和vhb基因在转基因马铃薯中的表达

分别构建了含cry3A和cry3A+vhb基因的植物表达载体pBCry3A和pBC₃Vhb,并通过根癌农杆菌介导转化了马铃薯. 对转化再生植株进行PCR和DNA印迹分析表明,外源基因已整合到马铃薯基因组中, 且连续三代无性繁殖后转基因仍存在. ELISA分析表明cry3A基因在转基因植株中得到了高效表达, 在单转cry3A植株中最高表达量达0.1%, 转cry3A与vhb双基因株系中为0.065%. 水涝试验显示,转双基因且vhb mRNA的RT-PCR呈阳性的马铃薯植株,对低氧胁迫有较好的耐受性, 表明获得的上述转双基因马铃薯株系可能会具有很好的抗虫和耐涝性能.     

The Production of Transgenic Scots Pine (Pinus Sylvestris L.) via the Application of Transformed Pollen in Controlled Crossings

The study demonstrates the production of a transgenic Scots pine (Pinus sylvestris L.) seedling through the application of transformed pollen in controlled crossings. The pollen lots were transformed by particle bombardment, resulting in transient transformation frequencies varying from 15 to 49% of the germinated pollen grains, and bombarded pollen was used to pollinate megasporangiate strobili. Progeny was screened by histochemical, GUS assays, and selected seedlings were further analysed by PCR. PCR amplification revealed the presence of both the nptII and gusA genes in one seedling (23/237). Results were confirmed by Southern blot analysis. The morphology and growth of this transgenic seedling was normal. Although the transformation frequency of recovered plants was very low (1/14999), the present protocol suggests that production of transgenic Scots pine is possible without the use of any tissue culture methods or the involvement of marker genes, for selection of transformants.     

The Promoter of a Metallothionein-Like Gene from the Tropical Tree Casuarina Glauca is Active in Both Annual Dicotyledonous and Monocotyledonous Plants

A chimeric gene consisting of the -glucuronidase (gusA) reporter gene under the control of the metallothionein-like promoter cgMT1 from the tropical tree Casuarina glauca was introduced into Nicotiana tabacum via Agrobacterium tumefaciens and into Oryza sativa by particle bombardment. The strongest histochemical staining for GUS activity was observed in the root system of the transgenic plants, and especially in lateral roots. In contrast, a relatively low level of reporter gene expression was seen in the aerial tissues and GUS staining was located mainly in the plant vascular system. The average ratio of GUS activity between root and leaf was found to be 13:1 in tobacco and 1.5:1 in rice. The pattern of cgMT1 promoter activity in floral organs was found to be different in tobacco and rice. High levels of gusA gene expression were detected in the ovules, pollen grains and tapetum, whereas in rice PcgMT1 directs expression to the vascular system of the floral organs. These results suggest that PcgMT1 is potentially useful in molecular breeding to express genes of interest whose products are preferentially needed in roots.     

龙眼DCL家族基因的启动子分析及时空表达

为了解龙眼DCL基因的功能,该试验对龙眼基因组数据提取的DlDCL1、DlDCL2、DlDCL3和DlDCL4基因序列进行启动子顺式作用元件及其受miRNA调控的分析;并以龙眼胚性愈伤组织为材料,研究了DlDCLs不同基因成员在非生物胁迫和外源激素处理下的表达情况。结果显示:(1)龙眼DCL基因启动子中除了TATA和CAAT外,还具有大量的光反应元件、激素应答元件、胁迫响应元件、组织特异性调控元件及植物生长发育相关的顺式调控元件,提示龙眼DCL基因启动子转录活性可能受到光、激素信号及逆境胁迫因素的诱导。(2)对调控龙眼DCL基因的miRNA进行筛选,结果显示DlDCL1受miR162和miR1024调控,DlDCL4受miR390和miR396调控。(3)实时荧光定量PCR显示,在一定浓度范围内,外源激素GA3、ABA和ETH均能下调DlDCLs基因的表达,而高浓度ETH处理则显著上调DlDCLs的表达。(4)高浓度蔗糖(6%)处理时DlDCL2、DlDCL3和DlDCL4显著上调表达,而低浓度(0.1%)处理时DlDCL1显著上调表达;不同温度处理下,DlDCL1在34℃时显著上升,DlDCL3随着温度的提高相对表达量逐渐减低;而DlDCL2和DlDCL4表达量差异不明显;NaCl胁迫处理下,DlDCLs在1h处理时表达量下调,但在其他不同时间点则上调表达。研究表明,龙眼DCL基因在外源激素及非生物胁迫处理下,并非是简单的一对一响应,而是存在较为复杂的响应机制。     

Controlled Expression of Recombinant Proteins in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using β-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 °C. In contrast, a short non-damaging heat-treatment at 38 °C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 °C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.     

A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta™Mouse)

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta™Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50–200 mg/kg) and EMS (100–400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0–4.6x10⁻⁶. MF in bone marrow was increased by ENU to 3.4x10⁻⁵ at 200 mg/kg and induced by EMS to 1.8x10⁻⁵ at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O⁶-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O⁶-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.     

拟南芥AtCIPK23基因对烟草抗旱能力的影响

为了探索拟南芥AtCIPK23基因对烟草耐旱能力的影响,对3个转AtCIPK23基因阳性纯合株系KA13、KA14和KA44与野生型烟草K326（对照）进行了自然干旱处理,测定离体叶片的失水速率、叶绿素含量、相对电导率、脯氨酸和可溶性糖含量,并分析了转基因及野生型材料对活性氧的清除能力,对活性氧清除基因NtSOD、NtCAT和NtAPX及干旱胁迫相关基因NtDREB、NtLEA5和NtCDPK2的表达量进行检测。结果表明：（1）转基因烟草离体叶片的失水速率明显低于K326;自然干旱7 d后,野生型K326出现了明显的干旱胁迫症状;干旱7 d进行复水后,转基因株系的复水存活率明显高于K326。（2）转基因株系中的叶绿素、脯氨酸及可溶性糖含量比K326显著提高,电导率则明显降低。（3）野生型烟草K326中H₂O₂的积累量明显高于3个转基因株系,转基因株系中ROS清除机制的3个关键基因NtSOD、NtCAT和NtAPX被诱导上调表达。（4）抗旱相关基因NtDREB、NtLEA5和NtCDPK2仅在转基因烟草中受干旱诱导。研究认为,AtCIPK23基因可能具有提高植物抗旱能力的功能。