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1.
Analysis of Transgenic Tobacco Plants Carrying the Gene for the Surface Antigen of the Hepatitis B Virus 总被引:1,自引:0,他引:1
Rukavtsova E. B. Zolova O. E. Buryanova N. Ya. Borisova V. N. Bykov V. A. Buryanov Ya. I. 《Russian Journal of Genetics》2003,39(1):41-45
The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBs antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HBsAg gene under a single 35S promoter was 0.0001–0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002–0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants. 相似文献
2.
N. S. Zakharchenko S. V. Pigoleva A. A. Yukhmanova Ya. I. Buryanov 《Russian Journal of Genetics》2009,45(8):929-933
The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective
genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The
transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial
activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were
transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the
bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed. 相似文献
3.
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
4.
《Saudi Journal of Biological Sciences》2022,29(3):1559-1564
The Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans. HBV causes acute and chronic hepatitis. Chronic hepatitis leads to hepatocellular carcinoma, which is a significant cause of death. DNA-based immunization programs to control the spread of Hepatitis B in developing countries are costly and require special storage and transportation. The alternative way is to express Hepatitis B surface antigen (HBsAg) in plants to develop oral vaccines. In this study, HBsAg gene was isolated, cloned, and then transformed in tomato plants. The transgenic tomato plants were confirmed through RT-qPCR. HBsAg expression was analysed in mature green and red stages of tomato fruit through quantitative real-time PCR. It was observed that expression of HBsAg was high in matured red tomato as compared to mature green. The present study is the first step to developing Solanum lycopersicum as an edible vaccine production system in this world region. 相似文献
5.
E. B. Rukavtsova T. V. Abramikhina N. Ya. Shulga V. A. Bykov Ya. I. Bur’yanov 《Russian Journal of Plant Physiology》2007,54(6):770-775
The tobacco plants (Nicotiana tabacum L.) carrying the HBsAg gene controlled by (Aocs)3AmasPmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh1, maize alcohol dehydrogenase gene intron were obtained. The presence of the adh1 gene intron did not significantly change the level of expression of the HBsAg gene in plants. The analysis of expression of hepatitis B virus surface antigen (HBs-antigen) in transformed plants expressing the HBsAg under the control of different promoters was made. The level of HBs-antigen in plants carrying the HBsAg gene controlled by (Aocs)3AmasPmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to 0.01% of total amount of soluble protein. The level of HBs-antigen in plants carrying the HBsAg gene controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant and made up to 0.06% of the total amount of soluble protein. Hairy root and callus cultures of plants carrying the HBsAg gene and expressing the HBs-antigen were obtained. 相似文献
6.
7.
Olena S. Krasovska Oleh V. Stasyk Andriy A. Sibirny 《Applied microbiology and biotechnology》2013,97(23):9969-9979
Two methods of multicopy integrant selection in the methylotrophic yeast Hansenula polymorpha based on the use of heterologous yeast auxotrophic genes have been used to isolate effective overproducers of hepatitis B surface antigen (HBsAg). One selection marker was described earlier for this yeast, the Saccharomyces cerevisiae URA3 gene, whereas the second selection marker was developed by us, the Pichia pastoris ADE1 gene with shortened native promoter. Sequential use of both selection markers produced stable transformants containing up to 30 integration cassettes with HBsAg gene. Deletion of PEX3 gene coding for peroxine involved in the early step of peroxisome formation substantially increased the production of HBsAg in glucose medium as compared to the parental strain. Maximal production of HBsAg in Δpex3 strain was nearly 8–9 % of the total cell protein. 相似文献
8.
Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L.) 总被引:4,自引:0,他引:4
Vaccines produced by transgenic plants would have the potential to change the traditional means of production and inoculation of vaccines, and to reduce the cost of vaccine production. In the present study, an UreB antigen gene from a new Helicobacter pylori strain ZJC02 was cloned into the binary vector pBI121 which contains a CaMV35S promoter and a kanamycin resistance gene, and then transformed UreB into tobacco leaf-disc by Agrobacterium-mediated method. A total of 50 regenerated plants with kanamycin resistance were obtained in the selection media. The 35 putative transgenic individuals were tested and verified the presence and integration of the UreB into the nuclear genome of tobacco plants by PCR, PCR-southern, and Southern analyses. Expression of UreB gene in the tobacco plants was confirmed by RT-PCR and Western Blot analysis using polyclonal human antiserum. To our knowledge, this is the first report of the expression of Helicobacter pylori UreB antigen gene in a plant system, suggesting a major step in the production of plant-based vaccines for Helicobacter pylori. 相似文献
9.
Chandrasekhar Gurramkonda Ahmad Adnan Thomas Gäbel Heinrich Lünsdorf Anton Ross Satish Kumar Nemani Sathyamangalam Swaminathan Navin Khanna Ursula Rinas 《Microbial cell factories》2009,8(1):13-8
Background
Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. 相似文献10.
乙肝病毒表面抗原基因在胡萝卜中的克隆及表达 总被引:4,自引:0,他引:4
构建HBV表达抗原(HBsAg)植物表达载体并在胡萝卜植株中表达。采用自行构建adr亚型HBsAg基因克隆T-adr,再次酶切获得860bp含PreS2的HBsAg基因片段,将其插入到植物表达载体pBPC55,新质粒命名为pBPC91adr。将其与含除草剂抗性基因及GUS蛋白基因的筛选质粒pBPC93共同经基因枪(PDS-1000/He)转化胡萝卜悬浮细胞,经含除草剂(Biolaphos)的培养基筛选及植物激素诱导分化,获得除草剂抗性胡萝卜幼苗,结果为转化后8周,自胡萝卜细胞中分化出除草剂抗性胡萝卜幼苗,提取新分化幼苗总DNA,特异性引物PCR扩增后可见860bp扩增带;Southern-Blot证明有HBsAg基因整合,胡萝卜蛋白萃取物的Western-Blot及ELISA检测证实有HBsAg蛋白表达。利用基因枪转化使质粒pBPC91adr中HBsAg基因在胡萝卜幼苗内整合并表达,提示以植物生产疫苗具有可行性。 相似文献
11.
Expression of hepatitis B surface antigen in transgenic banana plants 总被引:16,自引:0,他引:16
12.
M.-L. Wang G. Uruu L. Xiong X. He C. Nagai K. T. Cheah J. S. Hu G.-L. Nan B. S. Sipes H. J. Atkinson P. H. Moore K. G. Rohrbach R. E. Paull 《In vitro cellular & developmental biology. Plant》2009,45(2):112-121
A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from
Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing
of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from
two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided
the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated
by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated
from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent
lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic
evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement. 相似文献
13.
Madhurima Bhatnagar Kalyani Prasad Pooja Bhatnagar-Mathur M. Lakshmi Narasu Farid Waliyar Kiran K. Sharma 《Plant cell reports》2010,29(5):495-502
Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation for selecting the primary transgenic events. However, these become redundant once the transgenic plants have been developed and identified. Although, there is no evidence that the selectable marker genes are unsafe for consumers and the environment, it would be desirable if the marker genes can be eliminated from the final transgenic events. The availability of efficient transformation methods can enable the possibility of developing transgenic events that are devoid of the marker gene/s upfront. Taking advantage of the high and consistent transformation potential of peanut, we report a technique for developing its transgenics without the use of any selectable marker gene. Marker-free binary vectors harboring either the phytoene synthase gene from maize (Zmpsy1) or the chitinase gene from rice (Rchit) were constructed and used for Agrobacterium tumefaciens-mediated transformation of peanut. The putative transgenic events growing in vitro were initially identified by PCR and further confirmed for gene integration and expression by dot blots assays, Southern blots, and RT-PCR where they showed a transformation frequency of over 75%. This system is simple, efficient, rapid, and does not require the complex segregation steps and analysis for selection of the transgenic events. This approach for generation of marker-free transgenic plants minimizes the risk of introducing unwanted genetic changes, allows stacking of multiple genes and can be applicable to other plant species that have high shoot regeneration efficiencies. 相似文献
14.
Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation 总被引:2,自引:0,他引:2
Heidi F. Kaeppler A. R. Carlson G. K. Menon 《In vitro cellular & developmental biology. Plant》2001,37(2):120-126
Summary Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to
overcome drawbacks sometimes associated with use of existing markers. A useful alternative to chemical-based selection systems
would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the green
fluorescent protein (gfp) gene could be utilized alone as a visual screenable marker to produce stably transformed, fertile oat plants. Twelve experiments
were conducted in which gfp-based selection was utilized to obtain routinely stable transgenic lines in oat. A synthetic gfp gene under the control of the maize ubiquitin promoter was delivered into embryogenic oat callus via microprojectile bombardment. Cell clusters (1–3 mm) expressing gfp were visually identified using epifluorescence microscopy and physically isolated approximately 3 wk post-bombardment. Fertile,
gfp-expressing T0 plants were regenerated from 78% of the glowing cell sectors placed on regeneration medium. T0 plants from
55% of the events produced gfp-expressing progeny in a 3∶1 Mendelian ratio. Southern blot and PCR analysis confirmed transgene integration and transmission
to progeny. Expression of gfp did not reduce plant growth or fertility. Transgene copy number and integration patterns were similar to those in transgenic
plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile events obtained
per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening
for expression of gfp, demonstrating efficiency and repeatability of the selection system.
Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the University of Wisconsin or
the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. 相似文献
15.
The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous
presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation)
vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic
plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium
tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35
explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest
and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in
vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free
MS medium. 相似文献
16.
We report an efficient whole plant transformation system for Hyoscyamus muticus, an important medicinal plant of the Solanaceous family. We developed a system using a plasmid carrying the nptII and gusA genes, which was delivered into leaf explants by particle bombardment. Ten percent of bombarded leaf explants formed kanamycin-resistant callus, from which putative transgenic plants were recovered. The nptII gene conferring kanamycin resistance was found to be incorporated into the genome of all transgenic plants screened. Over 50% of the kanamycin resistant plants showed strong expression of the non-selected gusA gene. The majority of transgenic plants reached maturity, could be self pollinated, and produced fertile seed. A simple and efficient whole plant transformation system for this medicinal plant is an important step in furthering our understanding of tropane alkaloid production in plants. 相似文献
17.
Young Im Choi Eun Woon Noh Hyo Shin Lee Mu Seok Han Jae Soon Lee Kwan Sam Choi 《Journal of Plant Biology》2005,48(4):351-355
A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments.
So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance.
However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic
plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation.
The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium
containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance
to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve
as an excellent selectable marker for plant transformation. 相似文献
18.
Analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis B virus 总被引:1,自引:0,他引:1
Rukavtsova EB Zolova OE Bur'ianova NIa Borisova VN Bykov VA Bur'ianov IaI 《Genetika》2003,39(1):51-56
The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBS antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HbsAg gene under a single 35 S promoter was 0.0001-0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002-0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants. 相似文献
19.
利用Ac/Ds转座子系统在水稻中获得无选择标记转基因植株的方法 总被引:7,自引:0,他引:7
获得无选择标记转基因植株是进行重复转基因及消除转基因植株中标记基因潜在危害性的关键。实验采用了Ac/Ds转座子系统在水稻(Oryza sativa,L.)中进行无hpt选择标记的转基因。将含有目的基因bar的Ds元件和hpt标记基因置于同一个T-DNA中,通过农杆菌(Agrobacterium tumefaciens)EHAl05介导将Ac-T-DNA及Ds-T-DNA分别转入到不同的水稻植株,再将单拷贝的Ac-T-DNA植株与单拷贝的Ds-T-DNA植株杂交得到同时含有Ac和Ds元件的F1植株,Fl自交产生F2后代,F2植株中转座后的Ds元件与T-DNA独立分离,在总共100株F2水稻植株中筛选得到2株只含有Ds元件插入而无hpt标记基因的转基因水稻植株。结果表明,利用Ac/Ds转座子系统在水稻中获得无选择标记的转基因植株是可行的。 相似文献
20.
High molecular weight (HMW) glutenin polypeptides are critical contributors to the visco/elastic properties responsible for
the processing characteristics and utilizations of wheat flour. In order to improve bread making quality of flour and produce
transgenic plants free of selectable markers, a linear DNA construct consisting of a minimal expression cassette with the
HMW-GS 1Bx14 gene was transformed into wheat cultivar Mianyang19 by microprojectile bombardment. The transform ants were selected by PCR
instead of herbicidal markers. Seven transgenic plants were identified from a total of 1219 transformants, yielding a transformation
frequency of 0.28%. An SDS-PAGE analysis confirmed that the 1Bx14 gene was expressed in three T1 seeds of the transgenic plants. Our results demonstrated that it is feasible to obtain marker-free
trans-formants using the linear-expression-cassette-transformation approach coupled with PCR selection. 相似文献