Expression of the Arabidopsis thaliana Histone Gene AtHTA1 Enhances Rice Transformation Efficiency

We expressed the Arabidopsis thaliana histone AtHTA1 in rice under the control of the maize ubiquitin promoter. Transformation efficiencies of rice plants that constitutively expressed AtHTA 1 were 28-44% higher than calli containing an empty vector control. Furthermore, co-infection of rice calli with a vector containing AtHTA 1 and another vector with the target gene increased transformation by 27-50%. Thus, expression of AtHTA 1 either transiently or in stably transformed cells improved rice transformation efficiency.     

High-throughput Binary Vectors for Plant Gene Function Analysis

A series of high-throughput binary cloning vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing, and green fluorescent protein fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for λ phage integrase/excisionase. In addition, unique restriction sites are incorporated in a multiple cloning site and enable promoter replacement. The entire vector series are available with complete sequence information and detailed annotations and are freely distributed to the scientific community for non-commercial uses.     

New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.     

NPR1-dependent salicylic acid signaling inhibits Agrobacteriummediated transient expression in Arabidopsis

正Dear Editor,Arabidopsis thaliana,as a model plant,is the most well-studied plant species.One of the advantages of using Arabidopsis is that obtaining stably transformed plants using flower-dipping method is easy.However,generating transgenic plants is still time-consuming.Therefore,transient expression is frequently used to characterize protein functions.Several transient expression assays have been developed,including protoplast transfection,biolistic bombardment,and Agrobacterium-mediated transient expression.Among these assays,the Agrobacterium-mediated     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.     

Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress （Thellungiella halophila）, a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress(Thellungiella halophila),a close relative of the model plant Arabidopsis thaliana L.,is an extremophile that isadapted to harsh saline environments.To mine salt-tolerance genes from this species,we constructed an entry cDNA libraryfrom the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombination-assisted cDNA library construction method that is completely free of manipulations involving restriction enzymes andDNA ligase.This cDNA library construction procedure is significantly simplified and the quality of the cDNA library isimproved.This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for planttransformation and expression via recombination-assisted cloning.The library is plant transformation ready and is used totransform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS—1 proteins

Human gastric cancer MKN-45 cells were transfected with pULB 3238,a plasmid carrying MVMp MS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR.After the integration and expression of NS-1 gene,some of the transfectants died,while others remained alive,but the growth features of survived cells were changed.For further study on the antineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established by conventional method.Among 4 founders,one of them was found to be able to transmit the transgene to around 50% of their offsprings.RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues.The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.     

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic     

Construction and Validation of a Dual-Transgene Vector System for Stable Transformation in Plants

In this study,we constructed dual-transgene vectors(pDT1,pDT7,and pDT7G) that simultaneously co-expressed two genes in plants.ACTIN2 and UBQ10 promoters were used to control the expression of these two genes.The 4×Myc.3×HA,and 3×Flag reporter genes allowed for the convenient identification of a tunable co-expression system in plants,whereas the dexamethasone(Dex) inducible reporter gene C-terminus of the glucocorticoid receptor(cGR) provided Dex-dependent translocation of the fusion gene between the nucleus and cytoplasm.The function of pDT vectors was validated using four pairwise genes in Nicotiana benthamiana or Anihidopsis thaliana.The co-expression efficiency of two genes from the pDT1 and pDT7 G vectors was 35%and 42%,respectively,which ensured the generation of sufficient transgenic materials.These pDT vectors are simple,reliable,efficient,and time-saving tools for the co-expression of two genes through a single transformation event and can be used in the study of protein-protein interactions or multi-component complexes.     

An Integrative Analysis of the Effects of Auxin on Jasmonic Acid Biosynthesis in Arabidopsis thaliana

Auxin and jasmonic acid （JA） are two plant phytohormones that both participate in the regulation of many developmental processes. Jasmonic acid also plays important roles in plant stress response reactions. Although extensive investigations have been undertaken to study the biological functions of auxin and JA, little attention has been paid to the cross-talk between their regulated pathways. In the few available reports examining the effects of auxin on the expression of JA or JA-responsive genes, both synergetic and antagonistic results have been found. To further investigate the relationship between auxin and JA, we adopted an integrative method that combines microarray expression data with pathway information to study the behavior of the JA biosynthesis pathway under auxin treatment. Our results showed an overall downregulation of genes involved in JA biosynthesis, providing the first report of a relationship between auxin and the JA synthesis pathway in Arabidopsis seedlings.     

Dexamethasone-inducible green fluorescent protein gene expression in transgenic plant cells

Genomic research has made a large number of sequences of novel genes or expressed sequence tags available. To investigate functions of these genes, a system for conditional control of gene expression would be a useful tool. Inducible transgene expression that uses green fluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines of cotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir (NOR; Abies nordmanniana Lk.), and rice (RIC; Oryza sativa L. cv. Radon). Transgenic cell lines were used to test the function of the chemical inducer dexamethasone. Inducible transgene expression was observed with fluorescence and confocal microscopy, and was confirmed by northern blot analyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h after treatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgenic cells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducer for optimum inducible gene expression system varied among transgenic cell lines. The inducible gene expression system described here was very effective and could be valuable in evaluating the function of novel gene.     

表达尾穗苋凝集素基因的转基因烟草对桃蚜(Myzus persicae)繁殖的影响(英文)

To investigate the possible function of the agglutinin from Amaranthus caudatus L. (ACA) in plant defending against insect pests, ACA cDNA was cloned by RT-PCR and the 5‘ and 3‘ sequences were confirmed by rapid amplification of cDNA ends (RACE). The phloem-specific expression vector of ACA gene, pBCACAc, was constructed based on the plant binary vector pBC438 and transfered into tobacco plants via Agrobacterium-mediated transformation method. Results from PCR and Southern blotting analysis showed that AOA gene was integrated into the genomes of transformed plants and the transgene integration varied from one to four estimated copies per genome. Western blotting analysis indicated that ACA gene was transcribed and translated in the transgenic plants. The bioassay of Myzus persicae Sulzer on detached leaves demonstrated that the 78% transgenic tobacco plants displayed an average aphid-resistant rate of more than 75%. Some apterous progeny of M. persicae were found dead on the resistant plants. These results indicate that ACA gene should be an effective aphid-resistant gene and could be valuable for application in crop breeding for aphid resistance.     

Defining the Functional Network of Epigenetic Regulators in Arabidopsis thaliana

Development of ChiP-chip and ChlP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting （CC） Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator＇s expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.     

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.