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PubMed1995

A novel Ti-plasmid-convertible lambda phage vector system suitable for gene isolation by genetic complementation of Arabidopsis thaliana mutants.

A new lambda phage vector system, lambda TI, has been constructed to facilitate genetic complementation of higher plant mutations. The lambda TI vectors are stable, and by using the Cre-lox site-specific recombination, are automatically convertible into Ti-plasmid binary vectors which are capable of expressing genes in higher plants. Two lambda TI vectors were constructed: (i) lambda TI1, which can generate a Ti-plasmid that contains the cauliflower mosaic virus (CaMV) 35S promoter and is suitable for the expression of cDNA in transformed plants and (ii) lambda TI2, which can generate a Ti-plasmid with the multicloning site (MCS). cDNA and genomic libraries, which were constructed from the cruciferous plant Arabidopsis thaliana in these lambda TI vectors, can be probed by large DNA fragments of more than 100 kb, such as yeast artificial chromosomes (YACs), enabling the direct screening of the clones in the chromosome region containing a specified genetic locus. These libraries will certainly become powerful tools for the genetic complementation of Arabidopsis mutant phenotypes by quickly providing transformation-competent clones.

Agrobacterium tumefaciens↗
PubMed2006

Superexpression of tuberculosis antigens in plant leaves.

Recent developments in genetic engineering allow the employment of plants as factories for 1/foreign protein production. Thus, tuberculosis (TB) ESAT6 antigen was expressed in different plant systems, but the level of vaccine protein accumulation was extremely low. We describe the technology for superexpression of TB vaccine proteins (Ag85B, ESAT6, and ESAT6:Ag85B fusion) in plant leaves which involves: (i) construction of tobacco mosaic virus-based vectors with the coat protein genes substituted by those for TB antigens; (ii) Agrobacterium-mediated delivery to plant leaf tissues of binary vectors containing the cDNA copy of the vector virus genome; and (iii) replication of virus vectors in plant cells under conditions suppressing the virus-induced gene silencing. This technology enables efficient production of the TB vaccine proteins in plants; in particular, the level of Ag85B antigen accumulation was not less than 800 mg/kg of fresh leaves. Expression of TB antigens in plant cells as His(6)-tagged proteins promoted their isolation and purification by Ni-NTA affinity chromatography. Deletion of transmembrane domains from Ag85B caused a dramatic increase in its intracellular stability. We propose that the strategy of TB antigens superproduction in a plant might be used as a basis for the creation of prophylactic and therapeutic vaccine against TB.

Agrobacterium tumefaciens↗
PubMed2003

Construction of a genomic library of wild rice and Agrobacterium-mediated transformation of large insert DNA linked to BPH resistance locus.

Here we report the first genomic library of wild rice constructed on a plant-transformation-competent binary vector (BIBAC2) and transformation of the large insert DNA into rice via Agrobacterium. We selected Oryza officinalis for genomic library construction. The library consists of 55,296 clones and stored in one hundred forty-four 384-well plates. Random sampling of 140 clones indicated an average insert size of 71 Kb at a range of 15-235 Kb and 4.8% empty vectors. Four wheat chloroplast probes and four maize mitochondrial probes were hybridized separately to the library, showing that contamination with organellar DNAs is very low (0.61% and 0.04%, respectively). The binary bacterial artificial chromosome (BIBAC) library provides 5.3 haploid genome equivalents, implying a 99.5% probability of recovering any specific sequence of interest. A stability test indicated that the large DNA inserts were stable in this BIBAC vector both in host cells of Escherichia coli and Agrobacterium. Two restriction-fragment length polymorphism (RFLP) markers R288 and C820, which co-segregate with brown planthopper (BPH) resistance gene Qbp2, were used to screen the library, and identified seven and eight positive clones, respectively. The candidate clones of target gene isolated from the library are directly used to transform cultivated rice. After screening the Agrobacterium strains and helper plasmids, and using an improved procedure of transformation, a BIBAC clone with 120 Kb O. officinalis DNA insert was successfully transferred into the rice genome via Agrobacterium-mediated transformation. The system developed here should serve as source for gene discovery, gene cloning and genome-related research in wild rice.

Animals↗
PubMed2001

[Construction of plant expression vectors harboring a peptide antibiotic-apidaecin gene and resistance analysis of the transgenic tobacco].

Two plant expression vectors(pBinPRHbI and pBinPRSIHbI) were constructed: Firstly, apidaecin gene were fused to the signal peptide coding sequencing of a PR-protein, and cloned into a binary vector pBin438 to form pBinPRHbI. Then, the cassette consisting of 35S promoter, PR signal peptide coding sequencing and apidaecin gene was cut off from pBinPRHbI and inserted into another plant expression vector pBinPRSI to produce a bivalent plant expression vector pBinPRSIHbI. pBinPRSI was constructed previously in our lab and contained PR signal peptide and Shiva-I fusion gene under control of 35S promoter. The three plant expression vectors were introduced into tobacco by Agrobacterium-mediated transformation. The positive rate of PCR was 95% in all putative transgenic plants. Results from Southern blot indicated that foreign genes were integrated into tobacco genome and RT-PCR analysis proved that the foreign gene was transcribed in transgenic tobacco. The transgenic tobacco showed higher resistance to P. syringae pv tabaci, the causal agent of tobacco wild fire disease, than their original cultivars. From the disease index, the transgenic plants carrying apidaecin and Shiva-I genes had highest resistance among three kinds of transgenic plants, and the plants carrying Shiva-I gene alone had lowest resistance.

Anti-Infective Agents↗
PubMed2006

Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization.

Complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library, constructed with BamHI in the pECBAC1 vector, contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries combined contain 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3,000 Mb/1C), and provide a greater than 99% probability of obtaining a clone of interest. The frequencies of BAC and BIBAC clones carrying chloroplast or mitochondrial DNA sequences were estimated to be 2.35 and 0.04%, respectively, and insert-empty clones were less than 0.5%. To facilitate chromosome engineering and anchor the sunflower genetic map to its chromosomes, one to three single- or low-copy RFLP markers from each linkage group of sunflower were used to design pairs of overlapping oligonucleotides (overgos). Thirty-six overgos were designed and pooled as probes to screen a subset (5.1x) of the BAC and BIBAC libraries. Of the 36 overgos, 33 (92%) gave at least one positive clone and 3 (8%) failed to hit any clone. As a result, 195 BAC and BIBAC clones representing 19 linkage groups were identified, including 76 BAC clones and 119 BIBAC clones, further verifying the genome coverage and utility of the libraries. These BAC and BIBAC libraries and linkage group-specific clones provide resources essential for comprehensive research of the sunflower genome.

Chromosome Mapping↗
PubMed1994

Study on replicase (subunit) gene of papaya ringspot virus cloning, sequencing and construction of higher plant expression vector.

The ds-cDNA was synthesized using genomic RNA of papaya ringspot virus (PRSV) as template. The blunt-ended cDNA was cloned into the EcoRV site of vector pBluescript SK. From the recombinants, the NIb gene of PRSV was obtained, and its complete sequence was also determined. After the NIb gene was modified through PCR, a binary vector of PRSV-NIb gene was constructed for its expression in higher plants.

Amino Acid Sequence↗
PubMed2003

[Construction of expression vector with BG2 gene and its transformation in wheat].

beta-1,3-glucanase(BG2) is one of the pathogensis-related-proteins(PR). Study of these proteins and their related genes is one of the hot points in plant genetic engineering of disease resistance for a long time. In this research, specific primers were designed with the enzyme cleavage site of Spe I in its forward one and Not I site in the backward according to the BG2 gene sequence. Using this pair of primers, BG2 gene, which was contained in the plasmid of pRTL2, was amplified and confirmed by sequencing the amplified fragment inserted into T-easy vector. The positive clone containing BG2 gene was digested with the enzymes of Spe I/Not I and then BG2 gene was inserted into the Xba I/Not I sites of super expression binary vector pATC940. The reconstructed expression vector named as pATCBG2 was introduced into the wheat of Longfumai10 and Longfumai3 (Triticum aestivum L. em. Thell) through the particle gun transformation method. The Kanamysin resistant (Km') transformants were obtained. PCR, Dot-blotting and PCR-Southern hybridization analysis showed that the BG2 gene was integrated into the genome of wheat. Result of pathogen inoculation assay on the transgenic plants showed that the transgenic plants had a higher resistant disease score of 1-2 grade than the control.

Ascomycota↗
PubMed2006

Removal of the selectable marker gene from transgenic tobacco plants by expression of Cre recombinase from a tobacco mosaic virus vector through agroinfection.

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus-host plant systems.

Base Sequence↗
PubMed1992

Genomic amplification and expression of delta-endotoxin fragment of Bacillus thuringiensis.

delta-Endotoxin gene of Bacillus thuringiensis HD-1 var kurstaki codes for the insecticidal crystal protein (ICP) specific for lepidopteran insects. Since the N-terminal half of the toxin is sufficient both for insect specificity and toxicity, the coding sequence of this part of the gene CryIA(b) was amplified by PCR and cloned in pUC19. As there was no expression of immunologically detectable delta-endotoxin in this clone in E. coli, the amplified ICP gene was transferred to an expression vector pGEx2T. Restriction mapping and immunoblotting confirmed the presence and expression of the CryIA(b) gene. This insert should be suitable for expression in plant system if it is mobilized into a plant binary vector.

Bacillus thuringiensis↗
PubMed2006

[Generation of selectable marker-free and vector backbone sequence-free Xa21 transgenic rice].

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.

DNA, Bacterial↗
PubMed1994

Versatile transformation vectors to assay the promoter activity of DNA elements in plants.

A convenient vector system was developed to evaluate transcriptional promoter activities in plants. Two primary vectors, optionally containing the cauliflower mosaic virus (CaMV) 35S -47 or -90 minimal promoters, offer multiple sites for cloning the sequence of interest upstream from the beta-glucuronidase gene (gusA). The promoter-gusA cassette can be transferred to a binary vector containing the selectable neomycin phosphotransferase II-encoding gene (nptII) next to the left border. In addition, the transferred DNA (T-DNA) contains the chloramphenicol acetyltransferase gene (cat) driven by the CaMV 35S promoter. Activity of cat can serve as a reference for gusA expression to correct for effect of chromosomal position or T-DNA copy number.

Base Sequence↗
PubMed1999

In situ localization of cacao swollen shoot virus in agroinfected Theobroma cacao.

Cacao swollen shoot virus (CSSV) is a small non-enveloped bacilliform virus with a double-stranded DNA genome. A very restricted host range and difficulties in transmitting the virus, either mechanically or via its natural vector, have hindered the study of cacao swollen shoot disease. As an alternative to the particle-bombardment method previously reported, we investigated another approach to infect Theobroma cacao. A greater-than-unit length copy (1.2) of the CSSV DNA genome was cloned into the Agrobacterium binary vector pBin 19 and was transferred into young plants via Agrobacterium tumefaciens. Typical leaf symptoms and stem swelling were observed seven and eleven weeks post inoculation, respectively. Viral DNA, CSSV coat protein and virions were detected in leaves with symptoms. Agroinfected plants were used to study the in situ localization of CSSV and its histopathologic effects in planta. In both leaves and petioles, virions were only seen in the cytoplasm of phloem companion cells and of a few xylem parenchyma cells. Light microscopy showed that stem swelling results from a proliferation of the xylem, phloem and cortex cells.

Badnavirus↗
PubMed2004

[Construction and expression of vector with aroA-in gene and its transformation in tobacco].

Inteins are intervening protein sequences that undergo self-excision from precursor protein with concomitant joining of flanking sequences. Here, we demonstrated intein cis-splicing in Nicotiana tabacum nuclear genomes by using artificial cis Ssp DnaB and Rma DnaB intein. We want to test whether protein splicing can occur in higher eucaryotic cell,which would play an important role in transgene containment in transgenic plants. Glyphosate-resistant Salmonella typhimurium aroA gene was divided at position 235/236 aa within EPSPS by inverse PCR from pLEPSPS. Amplified gene products with artificial cis-Ssp DnaB/Rma DnaB intein and split-Ssp DnaB/Rma DnaB intein were inserted at position 235 of EPSPS respectively to construct plasmid pLEBC, pLERC, pLEBT and pLERT. Above four aroA-In gene fusions were ligated into pET-32 to obtain E. coli expression vectors termed pETLEBC, pETLEBT, pETLERC and pETLERT. E. coli DE3 cells containing individual recombinant plasmids described above were induced by IPTG to produce corresponding protein products. Detectable spliced EPSPS and unspliced precursor demonstrated that splicing occurred in bacteria. aroA-cis SSp DnaB and aroA-cis Rma DnaB were ligated into Agrobacterium tumefaciens binary vector pLYM. Then A. tumefaciens containing EPSPS-(cis) intein cassettes were used for leaf disk transformation in N. tabacum. Integration of aroA-In gene into plant genome was confirmed by genomic PCR analyses. To verify the expression of fusion genes at transcriptional level, RT-PCR analyses were performed and the expected products were identified. These results suggested that plant cells support expression of S. typhimurium aroA-In fusion gene in nulear genomes. Thus,we speculated the existence of protein-splicing activity in plant cells. This opens the possibility of applying intein trans-splicing technique to reduce/prevent gene transfer by way of pollen in transgenic plants.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2004

Transactivator and structurally optimized inducible lentiviral vectors.

Lentiviral vectors offer well-recognized advantages as a gene delivery system both for the analysis of gene function and as a vehicle for gene therapy. In the present study optimized HIV-1-based vector systems that display efficient doxycycline (Dox)-dependent transgene expression in vitro and in vivo have been developed through the modification of factors that contribute to basal activity levels. Dissection of HIV-1 vectors harboring a tTA-dependent transgene expression cassette revealed several mechanisms that account for Dox-independent transgene expression, including those mediated by an internal CMV promoter, as well as a potential contribution from fusion proteins generated by translational readthrough. A precipitous reduction in basal activity levels was accomplished by separating the transactivator and the transgene cassettes into a binary vector system and by relocating the inducible promoter to the U3 region of the LTR. In addition, substituting the VP16 portion of tTA with the human p65 transactivating domain improved Dox-dependent transgene expression in a number of cell types. Optimizing HIV-1-based vectors culminated in a "toolbox" of vectors suitable for transgene delivery in vitro and in vivo, as conveyed by our ability to control the Dox-dependent differentiation of embryonic fibroblasts into muscle cells in vitro and transgene expression in rat brains.

Animals↗
PubMedDate not supplied

Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture.

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefaciens was achieved.

Agrobacterium tumefaciens↗
PubMed2006

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector.

We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

Arachis↗
PubMed1992

Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants.

Progress in plant molecular biology has depended heavily on the availability of effective vectors for plant cell transformation and heterologous expression. In this paper we describe the structures of a wide array of plasmids which have proved extremely effective in (a) plant transformation, (b) expression of heterologous genes and (c) assaying the activity of transposons in transgenic plants. Constructs that confer resistance to kanamycin, hygromycin, streptomycin, spectinomycin and phosphinotricin, or that confer beta-glucuronidase (GUS) gene expression are presented. Binary vector constructs that carry polylinkers of the pUC and Bluescript types are also described. Plasmids that permit the expression of any heterologous reading frame from either nopaline synthase (nos) or octopine synthase (ocs) promoters, as well as the cauliflower mosaic virus 35S promoter, using either the nopaline synthase or octopine synthase 3' polyadenylation sequences, are presented. These constructs permit a choice of orientation of the resulting transgene of interest, relative to the orientation of the selection marker gene. Most of the plasmids described here are publicly available.

Amino Acid Oxidoreductases↗