A recombinase-mediated transcriptional induction system in transgenic plants

We constructed and tested a Cre-loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed. The system developed here uses a heat-shock-inducible Cre to excise a DNA fragment flanked by lox sites, thereby generating a constitutive GUS reporter gene under control of the CaMV 35S promoter. Heat-shock-mediated excision of several, independent lines resulted in varying degrees of recombination-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over-expression, or under-expression by antisense, would otherwise affect embryo, seed or seedling viability.     

Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F₁ mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F₁ mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F₁ fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F₁ mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.     

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.

     

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.     

分泌蛋白特异性基因陷阱的设计与验证

蛋白质分子向细胞外分泌的过程有赖于信号肽的介导.基因陷阱是功能性基因克隆的一种有效方法,经典的基因陷阱以geo作为报告基因,对所克隆的基因类型没有选择性.将绿脓杆菌外毒素、2A序列、IL-2受体穿膜区以及新霉素磷酸转移酶的基因依次融合构建新型报告基因——peo,该报告基因能够特异性地甄别具有信号肽编码序列的基因.为验证peo对信号肽编码序列的特异性甄别作用,以peo为报告基因,构建了3种质粒载体,分别模拟用基因陷阱载体进行筛选时可能出现的3种情况,通过转染HeLa细胞、G418筛选以及碱性磷酸酶检测,证明peo能够有效地区分信号肽和非信号肽编码序列,以peo为报告基因改造的基因陷阱载体可以用于分泌蛋白基因的筛选.     

New plant promoter and enhancer testing vectors

We describe here a set of binary vectors suitable forAgrobacterium-mediated gene transfer and specially designed for studying plant promoters. These vectors are based on the use of thegus reporter gene, contain multiple unique restriction sites upstream of thegus gene, and minimal promoters for testing the effect of enhancers or activator elements. In addition, an intron-containinggus (uidA) gene was introduced into one of these vectors in order to examine reporter gene activity in tissues whereAgrobacterium contamination may be a problem or in transient expression assays.     

Gene trapping of the Arabidopsis genome with a firefly luciferase reporter

Experiments with gene-trap vectors containing the firefly luciferase (LUC) reporter genes were carried out with the aim of analyzing functions of the Arabidopsis genome. Studies with protein fusion-type trap vectors as well as an internal ribosome entry site (IRES)-assisted non-fusion-type vector revealed that both types of vectors were suitable for gene trapping in Arabidopsis, although there were some differences in trapping efficiencies. The established trap lines were subjected to analyses for light responses, demonstrating the powerful and unique applications of a LUC-trapping system. A systematic survey of the insertion sites of the T-DNAs in LUC-expressing lines revealed 12-41% gene-trapping efficiencies depending on the vector. We demonstrate that the LUC-trapping system provides a unique system with which to monitor temporal expression of plant genes.     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation

The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

pGFPGUSPlus, a new binary vector for gene expression studies and optimising transformation systems in plants

A binary vector containing two reporter gene cassettes has been developed. This vector is ideal for optimising new plant transformation systems. Following optimisation, one of the reporter genes can be replaced with a gene of interest; the second can be used as a marker to confirm transgenic lines, and to estimate locus number and determine zygosity. This allows simple, efficient and economical screening for homozygous single-insert lines and azygous controls.     

Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum

We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using beta-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of beta-glucuronidase in both L. sakei and L. plantarum.     

Efficient transformation of<Emphasis Type="Italic"> Medicago truncatula</Emphasis> cv. Jemalong using the hypervirulent<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain AGL1

The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter-gus/gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4–5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T₁ descendents.Communicated by R.J. Rose