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PubMed2001

Effect of nuclear matrix attachment regions on transgene expression in tobacco plants.

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants. The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer. This sMAR sequence was used to flank the beta-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121. Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens. Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs. This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene. However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used.

Agrobacterium tumefaciens↗

PubMed2001

[1.4 kb 5' flanking region of class I patatin directs tuber-specific gus expression in potato (Solanum tuberosum L.)].

Binary vectors pPATIs (with partial signal sequence) and pPATI (without signal sequence) were constructed by fusing 1.4 kb 5' flanking regions of Class I patatin gene with GUS. Transient GUS expression was observed in in vitro tuber slices bombarded with pPATI. These constructs were then introduced into potato (cv. Desiree) via Agrobacterium tumefaciens transformation. Transgenic potato plants were confirmed by X-Gluc staining and PCR. Using in vitro tuberization system, GUS activities were assayed by fluorescence. It was shown that, in plants transformed with PATI-GUS, GUS specific activities were about 10-20 fold higher in tubers than in stems. Increased sucrose concentration could not induce PATI-GUS expression, but light enhanced PATI-GUS expression in cultured shoots.

Carboxylic Ester Hydrolases↗

PubMed1999

[Deletion analysis and functional studies of the promoter from commelina yellow mottle virus].

Commelina Yellow Mottle Virus(CoYMV) is a double-stranded, circular DNA virus and its promoter could direct GUS gene specifically expressing in phloem tissue of transgenic tobacco plants. To determine the optimal promoter sequence for pholem-specific gene expression, CoYMV promoter was deleted from its 5 end to form promoter fragments with 5 different lengths. Chimeric GUS genes were constructed using the promoter deletion based on the binary vector pBI121. Transgenic tobacco plants evidenced by PCR analysis were obtained with each kind of chimeric GUS gene structure by Agrobacterium mediated transformation. The results of GUS activity assay and histo-chemical staining showed that most of the chimeric GUS genes were expressed in transgenic plants. The GUS activity with the promoter deleted to -870 bp was about 78% higher than that of the full length promoter(1040 bp) and was a little higher than that of the promoter deleted to -585 bp, but the difference is not significant. The GUS activity reduced significantly when the promoter was deleted to -447 bp or -232 bp, whereas the property of phloem-specific expression pattern was still retained. When the promoter was deleted to -44 bp, just upstream adjacent to the TATA box, its tissue-specificity was lost and the activity was reduced to undetectable level. These results suggest that the region between -870 bp-232 bp and downstream of -232 bp of CoYMV promoter could be responsible for promoter activity and tissue specific expression, respectively. A negative regulation sequence might exist upstream of -870 bp of the CoYMV promoter. Therefore, we recommend that the optional CoYMV promoter sequence for phloem specific expression could be downstream from -870 bp or -585 bp. In comparison with CaMV 35S promoter, the GUS activity when driven by -870 bp CoYMV promoter was about 70% of that when driven by the 35S promoter. Considering the fact that 35S promoter-GUS gene is constitutively expressed, while the CoYMV promoter-GUS gene is expressed only in phloem tissues, the activity of the latter in phloem may be the same with or even higher than that of the 35S promoter.

Badnavirus↗

PubMed2003

[A simple and highly efficient Agrobacterium-mediated rice transformation system].

A simple and highly efficient rice transformation system was established based on the studying of factors influencing the Agrobacterium-mediated rice transformation. Embryogenic calli derived from mature embryos were infected and cocultivated with A. tumefaciens EHA101 harboring binary vectors: pHQ9, pHQ10, pHQT3. The highest transformation frequency was about 100 hygromycin resistance calli per gram of calli explants, and above 85% of these calli could be regenerated into plants. The putative transgenic plants were confirmed by GUS assay and southern-blot analysis. Hygromycin resistance tests indicated that the segregation of transgene in T1 progeny corresponded to the Mendelian ratio, 3:1. This system will benefit the functional genomic study of rice by using T-DNA insertion mutagenesis and gene targeting.

Agrobacterium tumefaciens↗

PubMed2004

Agrobacterium-mediated transformation of Bangladeshi Indica rices.

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.

Agrobacterium tumefaciens↗

PubMed2003

Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens.

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.

Agrobacterium tumefaciens↗

PubMed2004

[Endosperm-specific expression of the ferritin gene in transgenic rice (Oryza sativa L.) results in increased iron content of milling rice].

Iron deficiency is the most widespread micronutrient deficiency world-wild, and is estimated to affect about 30% of the world population. To increase the iron content of rice in Chinese, the 764 bp cDNA of ferritin gene was cloned from soybean (Phaseolus limensis), and constructed between the 1 353 bp rice glutelin GluB-1 promoter and NOS terminator in a binary vector pCAMIBA1301. The constructed pYF1067 vector was introduced into Agrobacterium strain EHA105, and used for transformation of the primary callus derived from immature embryos of a high-yielding rice ( Oryza sativa L. ssp. japonica) variety Wuxiangjin 9. Under the selection on hygromycin-containing medium, seventeen independent transgenic rice lines, total more than 80 transgenic plants, were finally regenerated, and most of these transgenic rice plants grew normally. PCR and Southern blot analysis of total DNA from primary transformants confirmed that one to three copies of the transgenes integrated into the genome of most of the transgenic plants, and they could be stably transmitted into the progeny of the transgenic rice. Northern blot analysis showed that the ferritin gene could specifically express in the endosperm of transgenic rice with high level, while no or low expression in leaves. The expression level varied among different independent transgenic rice plants. There was a significant effect of the high-expression of ferritin on the increased iron content in transgenic rice, the iron content in the milling rice of transgenic rice was up to 64% higher than that of the untransformed wild-type plant, whereas no significant alteration of the zinc level occurred between these two type rice plants.

Ferritins↗

PubMed2004

[Transgenic soybean obtained with Agrobacterium tumefaciens-mediated transformation of embryonic tip of soybean mature seeds].

Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 63.3% (Table 1) and the transformation efficiency reached up to 6.4%-12.1% (Table 2). The effect of infection time on T-DNA delivery into soybean embryonic tips was determined (Table 1). We also discuss the effects of days of co-cultivation to transient expression and the effects of different AS concentrations to transient expression of gus gene (Figs. 1,2). These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.

Agrobacterium tumefaciens↗

PubMed2004

[Suppression of apple polyphenol oxidase by double-stranded RNA (RNAi)].

Antisense and sense gene fragments (710 base pairs) of apple polyphenol oxidase (APPO) gene were obtained by RT-PCR amplification, using the total RNAs isolated from ripen apple fruit as the template. These two fragments were ligated with a 1000 bp spacer, YYT (crtW+crtY fusion) gene, which is relative to carotenoid synthesization in subcocci. The full-length 2446 bp-target gene was then inserted into plant binary vector pYPX145 to generate the recombinant plasmid pYF7704, which carried the expression unit, of APPO dsRNA. pYF7704 was transformed to apple (Malus x domestica) var. Red Fuji via agrobacterium tumefaciens mediated leaf disc transformation. With the selection of Karamycin and GUS detection assays, transgenic shoots of A PPO dsRNA were obtained. The results of FQ-RT-PCR indicated that APPO mRNA level was suppressed to 91.69% in transgenic shoots compared to wide shoots. The data suggested that dsRNAi technology on apple polyphenol oxidase is feasible to be utilized in transgenic shoots.

Agrobacterium tumefaciens↗

PubMed2005

[Inducible expression of a promoter of the gene encoding barley beta-1, 3-glucanase isoenzyme GIII in transgenic rice].

A promoter of the gene encoding beta-1, 3-glucanase isoenzyme GIII was amplified from barley genomic DNA using PCR. The GIII gene promoter, designated P(GIII), was ligated upstream of the gus report gene and pGIII-gus fusion fragment was then cloned into a binary vector pCAMBIA1300 for Agrobacterium-mediated transformation of rice (Oryza sativa L. cv. Taipei 309). PCR analyses indicated that the fusion gene was present in all T0 transgenic plants. The integration of the gene into rice genomic DNA was further confirmed by Southern blot. Histochemical staining and spectrofluorophotometric analyses of transgenic rice leaves showed that the GUS activity was increased after treatment with SA and fungal elicitor. Northern blot analysis also showed expression of such induction. Histochemical staining of T(1) rice seeds displayed marked GUS activity after induction with SA and elicitor, while no GUS activity was detected in untreated T(1) rice seeds. The results presented here strongly suggested that P(GIII) could be pathogen-inducible promoter.

Blotting, Northern↗

PubMed2005

Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains.

An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which--although indispensable--was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta.

Aminobutyrates↗

PubMed2005

[Specific expression of the foreign gene regulated by the rice rbcS promoter in transgenic rice].

To use different types of promoters in transgenic rice research, the 5'-upstream regulation region of rice Rubisco small subunit gene (rbcS) was cloned from a Chinese cultivar Wuyunjing 8, and its sequences were confirmed by comparison with the known genome sequences of both japonica and indica rice. The cloned rbcS promoter was fused to the 5'-upstream of GUS (beta-glucuronidase) coding region in a binary vector, and introduced into rice by Agrogacterium-mediated transformation. The integration of the rbcS-GUS fusion gene in transgenic rice was confirmed by PCR analysis. The results of both histochemical staining and quantitative analysis of GUS activity showed that the expression level of GUS fusion gene was significantly stronger in leaf blade and sheath than in other organs of transgenic rice plants, and the GUS activity was restricted to the mesophyll cells of leaf tissue, which showed that the rice rbcS promoter could control not only the tissue- but also the cell-specific expression of foreign genes in transgenic rice. The present results also demonstrated that light induction had a significant effect on the enhancement of transgene's expression when regulated by the rice rbcS promoter in transgenic rice. Our results showed that the rice rbcS promoter might be very useful for the expression of target genes in transgenic rice, with particularly high efficiency in leaf tissues.

Gene Expression Regulation, Plant↗

PubMed2003

[Optimization of T-dNA insertional mutagenesis and analysis of mutants of Magnaporthe grisea].

The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M. grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHtl constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M. grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A. tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600microg/mL among different M. grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1 x 10(6) spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.

Agrobacterium tumefaciens↗

PubMed2004

[Using green fluorescent protein as a reporter to monitor elimination of selectable marker genes from transgenic plants].

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.

Genetic Markers↗

PubMed2003

[Relativity betweeen expression level of potato virus X coat protein and the codon usage of wobble codon].

The relationship between the quantities of rare codons and the expression levels of foreign genes is very important in gene engineering. But up to now, there is few experiment on it in multi-cellular eukaryotes, especially in plants. To study the effect of rare codons on the expression level of potato virus X (PVX) coat protein (CP) in transgenic tobacco, some preferred codons in PVX coat protein gene were substituted with synonymous rare codons without changing the encoded amino acid sequence using site-specific mutation. The modified coat protein gene (cpm) and non-modified coat protein gene (cpw) were inserted into binary vector under the control of CaMV35S promoter, and these two plant expression constructs were transferred into tobacco (Nicotiana tabacum cv. Xanthi) genomes via Agrobacterium mediated method and transgenic plants were generated. Western blot and ELISA of these transgenic tobaccos showed that the expression level of modified CP is about one third to one sixth of that of non-modified CP. Northern blot analysis of RNAs from some transformants indicated that the transcriptional levels of cpm are the same as that of cpw, which implicated that the quantity of rare codons in foreign gene was a possible limited factor in foreign gene expression process. Changing the quantity of rare codons maybe an effective method for organism to control gene expression.

Amino Acid Sequence↗

PubMed2005

[Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1].

Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F1 and F2 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.

Animals↗

PubMedDate not supplied

Transgenic plants regenerated from hairy roots of Nicotiana benthamiana: a promising host for transient expression of foreign proteins.

Hairy root cultures of Nicotiana benthamiana have been obtained by co-cultivation of leaf explants with Agrobacterium rhizogenes strain A4 harboring a binary vector plasmid, and transgenic nature of the obtained cultures was confirmed by PCR analysis. Transgenic plants were regenerated from hairy roots. The biomass yield of transgenic plants grown in vitro was almost two-fold higher than those of wild-type N. benthamiana plants. They differed from untransformed plants by short internodes, reinforced stem, thick and wrinkled leaves and more developed root system. The level of Agrobacterium-mediated transient expression of green fluorescent protein (GFP) in the regenerated plants was similar to that of untransformed plants.

Agrobacterium tumefaciens↗

PubMed2005

[Expression and immunization testing of fusion protein of Newcastle disease virus in leaf tissue of transgenic rice].

Transgenic plant is an attractive bioreactor to produce recombinant protein, such as safe and economic vaccine. In present study, a plant expression binary vector pUNDV, containing the 1.7 kb fusion protein gene of Newcastle Disease Virus (NDV F) under the control of maize ubiquitin (Ubi) promoter and nos terminator was constructed, in which, the Hygromycin phosphotransferase (HPT) gene was used as the selectable marker. The expression cassette was introduced into a japonica rice variety by Agrobacterium-mediated transformation, and 6 independent transgenic rice lines were regenerated and verified by hygromycin resistance selection. The integration of target gene in transgenic plants was confirmed by PCR analysis. The results from ELISA and Western blot analyses revealed that NDV F could be expressed in the leaf of several transgenic plants. The total salt soluble proteins were extracted from leaf of transgenic plant F5, which contained the highest expressing level of NDV F protein, and used in immunization of BALB/c mice. The specific antibodies could be elicited in mice immunized with NDV F protein expressed in transgenic rice.

Animals↗