共查询到20条相似文献,搜索用时 31 毫秒
1.
Kim TG Kim MY Kim BG Kang TJ Kim YS Jang YS Arntzen CJ Yang MS 《Protein expression and purification》2007,51(1):22-27
Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells (Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0-2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity. 相似文献
2.
Nguyen Hoang Loc Nguyen Van Song Nguyen Quang Duc Tien Tang Thuy Minh Phan Thi Quynh Nga Tae-Geum Kim Moon-Sik Yang 《Plant Cell, Tissue and Organ Culture》2011,105(1):39-45
A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum
retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB)
gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into
the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed
by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest
amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein.
GM1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized
LTB subunits formed biologically active pentamers. 相似文献
3.
Tae-Geum Kim Bang-Geul Kim Mi-Young Kim Jae-Kwon Choi Eun-Sun Jung Moon-Sik Yang 《Molecular biotechnology》2010,44(1):14-21
Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment.
Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The
synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated
transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis,
respectively. The binding activity of LTB protein expressed in transgenic rice callus to GM1-ganglioside, a receptor for biologically active LTB, was confirmed by GM1-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody
titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to GM1-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers.
Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response.
Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than
mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic
rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that
transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic
E. coli heat-labile toxin. 相似文献
4.
Oszvald M Kang TJ Tomoskozi S Jenes B Kim TG Cha YS Tamas L Yang MS 《Molecular biotechnology》2008,40(3):261-268
The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced
into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific
promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration
of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1%
of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced
only in the rice endosperm demonstrated strong affinity for GM1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in
transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine. 相似文献
5.
Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant
macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the
Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase.
Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase
accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds
of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing
a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication
problems. 相似文献
6.
Expression of the B Subunit of E. coli Heat-labile Enterotoxin in the Chloroplasts of Plants and its Characterization 总被引:6,自引:0,他引:6
Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals. 相似文献
7.
Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A 总被引:6,自引:0,他引:6
Ann Koning Aubrey Jones JoAnne J. Fillatti Luca Comai Michael W. Lassner 《Plant molecular biology》1992,18(2):247-258
Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated -glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting -glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile. 相似文献
8.
R. Nandakumar S. Babu K. Kalpana T. Raguchander P. Balasubramanian R. Samiyappan 《Biologia Plantarum》2007,51(1):142-148
Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed
varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to
5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and
in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic
plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to
rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was
correlated with sheath blight resistance. 相似文献
9.
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
10.
Field evaluation and risk assessment of transgenic indica basmati rice 总被引:11,自引:1,他引:10
Bashir Khurram Husnain Tayyab Fatima Tahira Latif Zakia Aks Mehdi Syed Riazuddin Sheikh 《Molecular breeding : new strategies in plant improvement》2004,13(4):301-312
We report the first field trial of different transgenic lines of Indica Basmati rice (B-370) expressing cry1Ac and cry2A genes. Different transgenic lines were grown under field conditions for two consecutive years, according to RCBD and Split Plot Design respectively. All the biosafety measures were taken into consideration. Sixty neonate larvae of yellow stem borer were artificially infested into each plant in three installments. Data was recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines exhibited inherent ability to protect rice plants from target insects (p<0.01). Natural infestations of rice skipper and rice leaf folder were also observed and transgenic plants were statistically superior to their untransformed counterparts. Green house whole plant bioassays were done by infesting two 2nd instar larvae of rice leaf folder per tiller. Transgenics were 96% more resistant than untransformed control plants. The presence of cry genes was observed with Dot blot, PCR and Southern blot analysis, while ELISA and Western blot analysis confirmed the expression of Cry proteins. All lines expressed higher level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits to kill the target insects. Morphological studies showed significant variation for days to maturity, plant height and panicle length. Cooking qualities of seeds harvested from these lines were compared with the untransformed control. The transgenic lines had no effect on non-target insects (insects belonging to orders other than diptera and lepidoptera) and germination of three local varieties of wheat. Chances of gene spread were calculated at a level of 0.18% cross pollination in experimental lines. 相似文献
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12.
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
13.
以基因枪介导获转ps1—barnase基因的工程雄性不育水稻植株 总被引:17,自引:0,他引:17
以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6%)、高不育(占15.6%)及半不育的个体(占43.7%)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。 相似文献
14.
A protocol has been developed to produce a cholera toxin B subunit (CTB) in tobacco tolerant to the herbicide phosphinothricin
(PPT) by means of in vitro selection. The synthetic CTB subunit gene was altered to modify the codon usage to that of tobacco
plant genes. The gene was then cloned into a plant expression vector and was under the control of the ubiquitin promoter and
transformed into tobacco plants by Agrobacterium-mediated transformation. Transgenic plantlets were selected in a medium supplemented with 5 mg/L PPT. Polymerase chain reaction
analysis confirmed stable integration of the synthetic CTB gene into a chromosomal DNA. A high level of CTB (1.8% of total
soluble protein) was expressed in transgenic plants, which was 18-fold higher than that under the control of the expressed
CaMV 35S promoter with native gene. The transgenic plants when transferred to a greenhouse proved to be resistant to 2% PPT. 相似文献
15.
编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7~14倍和6~16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。 相似文献
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17.
Summary Embryogenic soybean [Glycine max (L.) Merrill] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the
rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones
of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration inheritance, and expression of transgenes
were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were
inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that
the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes.
Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of
48kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants
expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode. 相似文献
18.
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
19.
20.
Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice 总被引:2,自引:0,他引:2
Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects.
Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe
grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of
cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on
plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the
results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection
of rice plants against the rice blast fungus M. grisea. 相似文献