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1.
The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants. 相似文献
2.
Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na+/H+ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na+ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected
by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants
when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single
transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na+, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
3.
A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization
of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed
for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression
of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results
indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the
problems related to the use of antibiotics diminished.
Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000 相似文献
4.
Vidadi M. Yusibov Pak Chun II Viacheslav M. Andrianov Eleonora S. Piruzian 《Plant molecular biology》1991,17(4):825-836
The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress. 相似文献
5.
An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration 总被引:9,自引:0,他引:9
L. Zhang J. J. Rybczynski W. G. Langenberg A. Mitra R. French 《Plant cell reports》2000,19(3):241-250
A method for producing large numbers of transgenic wheat plants has been developed. With this approach, an average of 9.7%
of immature embryo explants were transformed and generated multiple self-fertile, independently transformed plants. No untransformed
plants, or escapes, were regenerated. This transformation procedure uses morphogenic calli derived from scutellum tissue of
immature embryos of Triticum aestivum cv. Bobwhite co-bombarded with separate plasmids carrying a selectable marker gene (bar) and a gene of interest, respectively. Transformed wheat calli with a vigorous growth phenotype were obtained by extended
culture on media containing 5.0 mg/l bialaphos. These calli retained morphogenic potential and were competent for plant regeneration
for as long as 11 months. The bar gene and the gene of interest were co-expressed in T0 progeny plants. This wheat transformation protocol may facilitate quantitative
production of multiple transgenic plants and significantly reduce the cost and labor otherwise required for screening out
untransformed escapes.
Received: 15 June 1998 / Revision received: 6 April 1999 / Accepted: 26 April 1999 相似文献
6.
Overexpression of antifungal pathogenesis-related (PR) proteins in crop plants has the potential for enhancing resistance
against fungal pathogens. Thaumatin-like proteins (TLPs) are one group (PR-5, permatins) of antifungal PR-proteins isolated
from various plants. In the present study, a plasmid containing a cDNA of rice tlp (D34) under the control of the CaMV-35S promoter was introduced into tobacco plants through Agrobacterium-mediated transformation system. A considerable overproduction of TLP was observed in transformed tobacco plants by Western
blot analysis. There was a large accumulation of tlp mRNA in transgenic plants as revealed by Northern blot analysis. Southern blot analysis of the DNA from transgenic tobacco
plants confirmed the presence of the rice tlp gene in the genomic DNA of transgenic tobacco plants. Immunoblot analysis of intracellular and extracellular proteins of
transgenic tobacco leaves using a Pinto bean TLP antibody demonstrated that the 23-kDa TLP was secreted into the extracellular
matrix. T2 progeny of regenerated plants transformed with TLP gene were tested for their disease reaction to Alternaria alternata, the brown spot pathogen. Transgenic tobacco plants expressing TLP at high levels showed enhanced tolerance to necrotization
caused by the pathogen.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
7.
H. Fukuoka T. Ogawa I. Mitsuhara T. Iwai K. Isuzugawa Yoko Nishizawa Y. Gotoh Yaeko Nishizawa A. Tagiri M. Ugaki M. Ohshima H. Yano N. Murai Y. Niwa T. Hibi Y. Ohashi 《Plant cell reports》2000,19(8):815-820
The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin
phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of
interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the
control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests
that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene.
Received: 20 August 1999 / Revision received: 16 November 1999 / Accepted: 19 November 1999 相似文献
8.
X. Niu X. Li P. Veronese R. A. Bressan S. C. Weller P. M. Hasegawa 《Plant cell reports》2000,19(3):304-310
Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent
of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications
were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic
evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted
in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher
concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration
medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the
number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average
transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein,
osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is
a viable alternative to plant breeding and clonal selection for improvement of this crop.
Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999 相似文献
9.
为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。 相似文献
10.
The DIANTHIN gene encoding a ribosome-inactivating protein (RIP) from Dianthus caryophyllus L. was tested for negative selection in tobacco and rice. Tobacco leaf discs and scutellum-derived callus of rice were transformed
with Agrobacterium tumefaciens strain LBA4404 (pSB1, pJAS1). pJAS1 harbors the DIANTHIN gene under the control of the CaMV 35S promoter. Tobacco transformation efficiency, in comparison to pCAMBIA1301, was reduced
by 87 % in pJAS1-transformed leaf discs. The DIANTHIN gene proved to be completely toxic to tobacco as all the recovered hygromycin-resistant transgenic plants harbored truncated
T-DNAs with deletions of the DIANTHIN gene. Transformation of the DIANTHIN gene under a Mungbean yellow mosaic virus (MYMV)-inducible promoter did not cause any toxicity in tobacco as reflected by the recovery of transgenic tobacco plants
with the complete DIANTHIN gene. Transformation efficiency of pJAS1 did not decline in rice. Interestingly, all transgenic rice plants harbored the
complete DIANTHIN gene and expressed the gene. The T1 transgenic lines showed reduction of sheath blight symptom in the range of 29 to 42 %. The difference in the sensitivity
to DIANTHIN between tobacco and rice provides a new direction to study the mechanisms underlying RIP toxicity in plants. 相似文献
11.
Marker genes are essential for the selection and identification of rarely occurring transformation events generated in biotechnology. This includes plastid transformation, which requires that multiple copies of the modified chloroplast genome be present to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here, we demonstrate the precise excision of attP‐ and attB‐flanked DNA from the plastid genome mediated by the large serine recombinase Bxb1. We transformed the tobacco plastid genome with the pTCH‐PB vector containing a stuffer fragment of DNA flanked by directly oriented nonhomologous attP and attB recombinase recognition sites. In the absence of the Bxb1 recombinase, the transformed plastid genomes were stable and heritable. Nuclear‐transformed transgenic tobacco plants expressing a plastid‐targeted Bxb1 recombinase were crossed with transplastomic pTCH‐PB plants, and the T1 hybrids exhibited efficient excision of the target sequence. The Bxb1–att system should prove to be a useful tool for site‐specifically manipulating the plastid genome and generating marker‐free transplastomic plants. 相似文献
12.
T. Leroy A. -M. Henry M. Royer I. Altosaar R. Frutos D. Duris R. Philippe 《Plant cell reports》2000,19(4):382-385
A synthetic version of the cry1Ac gene of Bacillus thuringiensis has been used for the transformation of coffee species (Coffea canephora and C. arabica) to confer resistance to an important pest, the coffee leaf miner (Perileucoptera coffeella and other Leucoptera spp). Somatic embryos were co-cultivated with the LBA4404 strain of Agrobacterium tumefaciens containing the cry1Ac gene. More than 100 transformed plants from independent transformation events were obtained for each coffee genotype.
The integration and expression of the cry1Ac gene was studied, and effective resistance of transgenic plants against leaf miner was verified in bioassays with the
insects. These plants could represent a good opportunity to analyse the impact of genetic engineering of perennial crops for
sustainable resistance to an obligate endocarpic pest using a B. thuringiensis insecticidal protein.
Received: 7April 1999 / Revision received: 20 July 1999 / Accepted: 22 July 1999 相似文献
13.
S. Krasnyanski R. A. May A. Loskutov T. M. Ball K. C. Sink 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(3-4):676-682
Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint
plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis
through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts,
they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced
with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both
transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types
of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations.
Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert
during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived
both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone,
low-menthol, high-menthofuran and –pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly,
around 1.2%, in transgenic plants produced by both methods.
Received: 22 November 1998 / Accepted: 4 Januar 1999 相似文献
14.
De Bolle MF Butaye KM Goderis IJ Wouters PF Jacobs A Delauré SL Depicker A Cammue BP 《Plant molecular biology》2007,63(4):533-543
Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression
of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene
expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why
chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs
on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs
do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results
show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have
an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene
expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression
in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.
Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work 相似文献
15.
High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures 总被引:6,自引:0,他引:6
K. Rajasekaran R. L. Hudspeth J. W. Cary D. M. Anderson T. E. Cleveland 《Plant cell reports》2000,19(6):539-545
Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and
stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment,
was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable
expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed
cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum
transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures
less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with
abnormal morphology and a high degree of sterility.
Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999 相似文献
16.
A new plant expression vector (pBSbtCry1Ac-GNA) containing two insect resistant genes, a synthetic chimeric gene SbtCry1Ac encoding the insecticidal protein CrylAc and a gene GNA encoding snowdrop lectin (Galanthus nivalis agglutinin) was constructed. Transgenic tobacco plants containing these two genes were obtained through Agrobacterium-mediated transformation of tobacco leaf discs. Results from PCR detection and genomic DNA Southern blot analysis indicated
that both SbtCrylAc gene and GNA gene were integrated into the genome of these plants. Results of Western blot analysis indicated that these two proteins
were expressed in the analyzed plants. Bioassays of Myzus persicae and Helicoverpa assulta on detached leaves of transformed tobacco plants were carried out. The average aphid inhibition rate of these plants tested
at 12 d post-infestation was 71.9 %. The average H. assulta mortality of these plants tested at 6 d post-infestation was up to 89.8 %. The kanamycin resistance of the T1 progeny of these transgenic plants was analyzed and a typical 3:1 segregation was observed. 相似文献
17.
Zheng Lu Jin Joon Ki Hong Dae-Jin Yun Sang Yeoi Lee Young Ju Choi Jeong Dong Bahk Roger N. Beachy Moo Je Cho Chae Oh Lim 《Journal of Plant Biology》2002,45(2):77-82
Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), usingAgrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV
MP when infected with chimeric TMVs containing defective MR These transgenic plants displayed stunting, pale-green leaves,
and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement. 相似文献
18.
Suk-Min Ko Byung-Ho Yoo Jong-Min Lim Kwang-Hoon Oh Jae-Il Liu Suk-Weon Kim Jang-Ryol Liu Kwan-Sam Choi Eui-Soo Yoon 《Plant Molecular Biology Reporter》2009,27(4):448-453
The earthworm fibrinolytic enzyme, which belongs to a group of serine proteases with strong fibrinolytic activity, has been
used as an oral drug for prevention and treatment of thrombosis in East Asia. Fibrizyme is a fibrinolytic enzyme isolated
from the earthworm Eisenia andrei. Here we report genetic engineering of tobacco plastids with stable integration of the fibrizyme gene into the tobacco chloroplast
genome. A plastid transformation vector was constructed by introducing various regulatory elements into fibrizyme cDNA. This
vector was delivered by particle bombardment into tobacco leaf explants and plastid-transformed plants were subsequently regenerated
into whole plants through several rounds of selection. We confirmed stable integration of the fibrizyme gene into the tobacco
plastid genome by PCR and Southern blot analyses. Northern and Western blot analyses revealed that mRNA and protein of recombinant
fibrizyme were highly expressed in transformed tobacco plants. 相似文献
19.
Generation of fertile transplastomic soybean 总被引:26,自引:0,他引:26
Dufourmantel N Pelissier B Garçon F Peltier G Ferullo JM Tissot G 《Plant molecular biology》2004,55(4):479-489
We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean. 相似文献
20.
N. Sentoku M. Taniguchi T. Sugiyama K. Ishimaru R. Ohsugi F. Takaiwa S. Toki 《Plant cell reports》2000,19(6):598-603
Expression of Panicum miliaceum L. (proso millet) mitochondrial and cytosolic aspartate aminotransferase (mAspAT and cAspAT, respectively) genes in transgenic
tobacco plants (Nicotiana tabacum) and their influences on protein synthesis were examined. The mAspAT- or cAspAT-transformed plants had about threefold or
3.5-fold higher AspAT activity in the leaf than non-transformed plants, respectively. Interestingly, the leaves of both transformed
plants had increased levels of phosphoenolpyruvate carboxylase (PEPC) and transformed plants with cAspAT also had increased levels of mAspAT in the leaf. These results
suggest that the increased expression of Panicum cAspAT in transgenic tobacco enhances the expression of its endogenous mAspAT and PEPC, and the increased expression of Panicum mAspAT enhances the expression of its endogenous PEPC.
Received: 6 February 1998 / Revision received: 6 August 1999 · Accepted: 6 September 1999 相似文献