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1.
The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent 总被引:10,自引:0,他引:10
The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection. 相似文献
2.
Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry 总被引:20,自引:0,他引:20
A new method for the selection of transgenic plants has been developed. It is based upon selection of transgenic plant cells
expressing the xylA gene from Streptomyces rubiginosus, which encodes xylose isomerase, on medium containing xylose. The xylose isomerase selection system was tested in potato
and the transformation frequency was found to be approximately ten fold higher than with kanamycin selection. The level of
enzyme activity in the transgenic plants selected on xylose was 5- to 25-fold higher than the enzyme activity in control plants.
Potato transformants were stable over two generations in Southern blotting analysis. This novel selection system is more efficient
than the traditionally used kanamycin-based selection systems. In addition, the xylose isomerase system is independent of
antibiotic or herbicide resistance genes, but depends on an enzyme that is generally recognized as safe for use in the starch
industry and which is already being widely utilized in specific food processes.
Received: 13 August 1997 / Revision received: 26 November 1997 / Accepted: 15 December 1997 相似文献
3.
Kunze Irene Ebneth Marcus Heim Ute Geiger Michael Sonnewald Uwe Herbers Karin 《Molecular breeding : new strategies in plant improvement》2001,7(3):221-227
A novel selection marker for plant transformation alternative to antibiotic and herbicide resistance is described. The selective agent applied is 2-deoxyglucose (2-DOG) which in the cytosol of plant cells is phosphorylated by hexokinase yielding 2-DOG-6-phosphate (2-DOG-6-P). 2-DOG-6-P exerts toxic effects on overall cellular metabolism leading to cell death. We observed that constitutive expression of the yeast DOG
R1 gene encoding a 2-DOG-6-P phosphatase resulted in resistance towards 2-DOG in transgenic tobacco plants. This finding was exploited to develop a selection system during transformation of tobacco and potato plants. The lowest concentration of 2-DOG leading to nearly complete inhibition of regeneration of wild-type explants was found to range between 400 and 600 mg/l 2-DOG for tobacco, potato and tomato plants. After Agrobacterium tumefaciens-mediated transformation cells expressing the DOG
R1 gene were selected by resistance to 2-DOG. More than 50% of tobacco explants formed shoots and on average 50% of these shoots harboured the DOG
R1 gene. Similar results were obtained for potato cv. Solara. The acceptability of the resistance gene derived from baker's yeast, the unobjectionable toxicological data of 2-DOG as well as the normal phenotype of DOG
R1-expressing plants support the use of this selection system in crop plant transformation. 相似文献
4.
The selectable marker gene phospho-mannose isomerase (pmi), which encodes the enzyme phospho-mannose isomerase (PMI) to enable selection of transformed cell lines on media containing mannose (Man), was evaluated for genetic transformation of papaya (Carica papaya L.). We found that papaya embryogenic calli have little or no PMI activity and cannot utilize Man as a carbon source; however, when calli were transformed with a pmi gene, the PMI activity was greatly increased and they could utilize Man as efficiently as sucrose. Plants regenerated from selected callus lines also exhibited PMI activity but at a lower specific activity level. Our transformation efficiency with Man selection was higher than that reported using antibiotic selection or with a visual marker. For papaya, the PMI/Man selection system for producing transgenic plants is a highly efficient addition to previously published methods for selection and may facilitate the stacking of multiple transgenes of interest. Additionally, since the PMI/Man selection system does not involve antibiotic or herbicide resistance genes, its use might reduce environmental concerns about the potential flow of those genes into related plant populations. 相似文献
5.
Effective selectable markers are needed for basic research and commercial applications that do not involve antibiotic or herbicide
resistance. A novel selection system based on a feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2) as
selectable marker using either 4-methylindole (4MI) or 7-methyl-DL-tryptophan (7MT) as the selection agent was developed. We found that these two components were able to discriminate better
between ASA2 expressing and untransformed lines than the most commonly used analog 5-methyltryptopan (5MT) in the seedling
growth inhibition test. We successfully integrated an expression cassette containing an ASA2 cDNA driven by a cauliflower
mosaic virus 35S promoter into tobacco leaf discs by A. tumefaciens and selected transgenic plants on medium supplemented with 300 μM of 7MT or 4MI. Due to the expression of the feedback-insensitive
ASA2, the transgenic lines produced showed higher free tryptophan (Trp) concentrations than the untransformed WT control.
These results demonstrate the feasibility of the selection system with the ASA2 gene in combination with the use of Trp or
indole analogs as selective agent. 相似文献
6.
Ascorbate peroxidase plays a key role in scavenging reactive oxygen species under environmental stresses and in protecting
plant cells against toxic effects. The Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected
for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that StAPX was transferred into the tobacco genome and StAPX was induced by salt and osmotic stresses in tomato leaves. Over-expression of StAPX in tobacco improved seed germination rate and elevated stress tolerance during post-germination development. Two transgenic
lines showed higher APX activity and accumulated less hydrogen peroxide than wild-type plants after stress treatments. The
photosynthetic rates, the root lengths, the fresh and dry weights of the transgenic lines were distinctly higher than those
of wild-type plants under stress conditions. Results indicated that the over-expression of StAPX had enhanced tolerance to salt stress and osmotic stress in transgenic tobacco plants. 相似文献
7.
Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner 总被引:1,自引:0,他引:1
Xiaohui Zhang Hanxia Li Junhong Zhang Chanjuan Zhang Pengjuan Gong Khurram Ziaf Fangming Xiao Zhibiao Ye 《Transgenic research》2011,20(3):569-581
Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring
virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3′ untranslated
region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs
displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV
infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated
resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular
system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective
to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3′ UTR of viral genome did. Our work
provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly
in other crops. 相似文献
8.
Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/frt site-specific system of 2-μm plasmid from Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target (frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/frt site-specific recombination system was constructed, which included the frt-containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt. The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat-shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41 % re-transgenic tobacco plants, which indicated that this system could make a great contribution obtaining the marker-free transgenic plants. 相似文献
9.
Summary A novel principle for selection of transgenic plant cells is presented. In contrast to traditional selection where the transgenic cells acquire the ability to survive on selective media while the non-transgenic cells are killed (negative selection), this selection method actively favours regeneration and growth of the transgenic cells while the non-transgenic cells are starved but not killed. Therefore, this selection strategy is termed positive selection. TheE. coli -glucuronidase gene was used as selectable (as well as screenable) gene and a glucuronide derivative of the cytokinin benzyladenine as selective agent which is inactive as cytokinin but, upon hydrolysis by GUS, active cytokinin is released stimulating the transformed cells to regenerate. Selection ofAgrobacterium tumefaciens inoculated of tobacco leaf discs on benzyladenine N-3-glucuronide (7.5–15 mg/l) resulted in 1.7–2.9 fold higher transformation frequencies compared to kanamycin selection. A significant advantage of this selection procedure is the elimination of the need for herbicide and antibiotic resistance genes. 相似文献
10.
植物转化中的安全标记基因 总被引:8,自引:0,他引:8
赵艳 《生物化学与生物物理进展》2002,29(3):352-354
转基因植物中的除草剂或抗生素抗性标记基因的生态环境和食用安全性一直颇有争议.糖类分解代谢酶基因作为安全标记基因,近年来在植物转化中显示了巨大应用潜力.这类标记基因编码产物是筛选剂糖类的分解代谢酶,使转化细胞能利用筛选剂糖类作为主要碳源从而获得优势生长,非转化细胞则因饥饿生长被抑制但不被杀死,故称为正筛选系统(positive selection system).目前木糖异构酶基因(xylose isomerase,xylA)和磷酸甘露糖异构酶基因(phosphomannose isomerase,pmi)等安全标记基因已成功应用于植物转化. 相似文献
11.
In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate
peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel
blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase
activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical
efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and
heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants. 相似文献
12.
利用FLP/frt重组系统产生无选择标记的转基因烟草植株 总被引:3,自引:0,他引:3
在植物转基因植株产生过程中,对转化细胞进行抗性筛选是通用程序,转化细胞的抗性一般是抗生素抗性或除草剂抗性,将赋予转化细胞抗性的选择标记基因删除是提高转基因植物生物安全性的重要措施。来自于啤酒酵母的FLP/frt位点特异性重组系统可有效删除同向定点重组位点frt之间的基因。通过多步骤重组,建立了可在植物中广泛应用的FLP/frt位点特异性重组系统。该系统包括含有frt位点的植物表达载体pCAMBIA1300-betA-frt-als-frt和含有由热诱导启动子hsp启动的FLP重组酶基因的植物表达载体pCAMBIA1300-hsp-FLP-hpt。利用二次转化的方式将二者先后转入烟草植株,热激处理后,热诱导型启动子hsp调控的重组酶FLP基因的表达催化位于选择标记基因als两侧同向frt位点间的重组反应,有效地删除了选择标记基因als。41%的经热激处理的二次转化植株发生了选择标记基因的删除,表明该系统在获得无选择标记基因的转基因植株中有很好的应用价值。 相似文献
13.
Gunvant Patil Amit Deokar P. K. Jain R. J. Thengane R. Srinivasan 《Plant cell reports》2009,28(11):1669-1676
To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose
isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented
medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed
using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of
20 g l−1 mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection
pressure of 15 g l−1 mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been
transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into
the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker
genes. 相似文献
14.
M. K. Saroha P. Sridhar V. S. Malik 《Journal of plant biochemistry and biotechnology.》1998,7(2):65-72
This review focuses on the genes for the enzymes 5-enolpyruvyl-3-phosphoshikimlc acid synthase (EPSPS) and the glyphosate oxidoreductase (GOX). These genes have been used to genetically engineer plants that are resistant to the herbicide glyphosate. Overproduction of glyphosate-insensitive.EPSPS in transgenic crops has been used to overcome the deleterious effuts of this herbicide. The introduction into plants of GOX also confers glyphosate tolerance to plants and augments the tolerance of transgenic plants already expressing a glyphosate tolerant EPSPS. These genes also provide a method for selecting transformed plant tissue using the glyphosate tolerance as the selectable marker in the presence of inhibitory concentrations of glypllosate. Glyphosate tolerant transgenic plants of beet, corn, cotton, lettuce, poplar, potato, rapeseed. soybean, tobacco, tomato, and wheat have already been field tested and are entering agriculture. 相似文献
15.
16.
Plant transformation is an important tool for basic research and agricultural biotechnology. In most cases, selection of putative transformants is based on antibiotic or herbicide resistance. Overexpression of plant genes that provide protection from abiotic or biotic stresses can result in a conferred phenotype that can be used as a means for selection. We have demonstrated herein that specific methionine sulfoxide reductase B (MsrB) genes that are overexpressed in transgenic plants may constitute a new selectable marker with concomitantly increased tolerance to methyl viologen (MV) treatment. Arabidopsis transformants overexpressing cytosolic MsrB7, MsrB8 or MsrB9 are viable and survive after MV selection. To establish whether these native plant origin genes serve as new non-antibiotic markers that can be applied to crop transformation, tomato cotyledons were used as transformation materials. MsrB7 transgenic tomato plants were successfully obtained by Agrobacterium-mediated transformation and selection on medium supplemented with MV. We suggest that specific MsrB genes that are overexpressed in transgenic plants may constitute a new selectable marker with increased tolerance to oxidative stress concomitant with MV treatment. 相似文献
17.
Hye Jin Choi Thummala Chandrasekhar Hyo-Yeon Lee Kyung-Moon Kim 《Plant Cell, Tissue and Organ Culture》2007,91(3):235-242
Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l−1 PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR,
and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each
transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing
900 mg l−1 of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application
which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene
were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop
new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide
Basta. 相似文献
18.
E. B. Rukavtsova A. R. Gayazova E. N. Chebotareva Ya. I. Buryanov 《Russian Journal of Genetics》2009,45(8):924-928
The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants.
A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting
transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco
and tomato transgenic lines synthesizing this antigen at a level of 0.01–0.05% of the total soluble protein were obtained.
The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe
edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers. 相似文献
19.
Yamada T Ohashi Y Ohshima M Inui H Shiota N Ohkawa H Ohkawa Y 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(2-3):308-314
A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide
tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order
to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were
obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA
corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 μmol/pot), whereas no accumulation of the corresponding
mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves
by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron
and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides
to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1
cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed
a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides.
These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study
demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred
chemical-inducible herbicide tolerance on potato.
Received: 15 March 2001 / Accepted: 14 June 2001 相似文献
20.
Eliseu S. Figueira Filho Lúcio F. A. Figueiredo Damares C. Monte-Neshich 《Plant cell reports》1994,13(12):666-670
Summary In search of establishing a system for genetic transformation of Brazilian potato cultivars, Agrobacterium tumefaciens carrying the plasmid pGV1040, was used to transform leaf discs of three cultivars of local importance, i.e., Aracy, Baronesa and Mantiqueira. This plasmid contains marker genes for resistance to kanamycin and phosphinothricin plus the gene for the enzyme -glucuronidase. A two step regeneration/selection procedure produced shoots of potato cultivar Mantiqueira with in vitro resistance to kanamycin and to phosphinothricin. After transfer to the greenhouse, the potentially transgenic plants, sprayed with the herbicide Finale® (20% a.i.; Hoechst®) remained green as compared to control clones that died immediately afterwards. Southern blot analysis and histochemical and fluorimetric assay for -glucuronidase indicated that the gene coding for the enzyme was integrated in the potato genome and could be expressed in potato tissues. No success was obtained for transformation of cultivars Aracy and Baronesa using this procedure.Abbreviations NAA
Naphthalene Acetic Acid
- BAP
Benzyl-aminopurine
- GA3
Gibberellic Acid
- PPT
Phosphinothricin
- PAT
Phosphinothricin Acetyl Transferase 相似文献