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Qiulei Lang ChunZhi Jin Leiyu Lai Junli Feng Shaoning Chen Jishuang Chen 《Molecular biology reports》2011,38(3):1523-1531
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N. Čeřovská H. Hoffmeisterová T. Moravec H. Plchová J. Folwarczna R. Hadámková 《Biologia Plantarum》2008,52(1):184-186
We describe the optimized storage conditions of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV-16). Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope
derived from E7 oncoprotein was fused to its C-termini. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. The effect of storage conditions on the serological activity of L2ACPE7 was studied by ELISA using
IgG anti PVX, PVA and L2. Purified L2ACPE7 stored freeze-dried (at −20 °C), frozen at various temperatures (−20 °C, −70 °C)
and at +4 °C were tested. Purified L2ACPE7 was most stable as lyophilized material stored at −20 °C. Our study demonstrates
suitable way for the storage of plant material containing foreign viral epitopes for the purposes of edible vaccination. 相似文献
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Liu K Kang BC Jiang H Moore SL Li H Watkins CB Setter TL Jahn MM 《Plant molecular biology》2005,58(4):447-464
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Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP)
gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the
activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of
enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results
indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes. 相似文献
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Cerovska N Hoffmeisterova H Moravec T Plchova H Folwarczna J Synkova H Ryslava H Ludvikova V Smahel M 《Journal of biosciences》2012,37(1):125-133
Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens
that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present
study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX
CP- L2108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue.
Immunogenicity of L2108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In
animal sera the antibodies against the PVX CP and the L2108-120 epitope were found after both methods of vaccine delivery. 相似文献
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Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l–1 thidiazuron, 0.5 mg l–1 -naphthaleneacetic, 10 mg l–1 kanamycin (Km), and 250 mg l–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m–2 s–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy. 相似文献
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Xiaoping Chen Zhangying Wang Jianhua Wang Maoyan Wang Li Zhao Guoying Wang 《Plant Cell, Tissue and Organ Culture》2007,88(1):11-20
ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region
preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied
via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb
fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking
fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous
system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression
in embryos, which is different from similar promoters tested in maize. 相似文献
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Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was
aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence
the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the
most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under
short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under
SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under
the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly
decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including
Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01%
Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation
of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs
at high relative humidity are necessary for maximal levels of expression. 相似文献
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Melhorn V Matsumi K Koiwai H Ikegami K Okamoto M Nambara E Bittner F Koshiba T 《Journal of plant research》2008,121(1):125-131
Abscisic acid (ABA) regulates stomatal closure in response to water loss. Here, we examined the competence of guard cells
to synthesize ABA, using two Arabidopsis ABA biosynthetic enzymes. 35S pro::AtNCED3-GFP and AAO3-GFP were introduced into guard cells of broad bean leaves. AtNCED3-GFP expression was detected at the chloroplasts, whereas green
fluorescent protein (GFP) and AAO3-GFP were in the cytosol. The stomatal aperture was decreased in AtNCED3-GFP- and AAO3-GFP-transformed guard cells. This indicated that ABA biosynthesis is stimulated by heterologous expression of AtNCED3 and Arabidopsis
aldehyde oxidase 3 (AAO3) proteins, which both seem to be regulatory enzymes for ABA biosynthesis in these cells. Furthermore,
stomatal closure by the expression of AtNCED3 and AAO3 suggested that the substrates of the enzymes are present and native
ABA-biosynthesis enzymes are active in guard cells.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
V. Melhorn and K. Matsumi contributed equally to this work. 相似文献
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Richard H. Baltz 《Journal of industrial microbiology & biotechnology》2010,37(8):759-772
Natural products discovery from actinomycetes has been on the decline in recent years, and has suffered from a lack of innovative
ways to discover new secondary metabolites within a background of the thousands of known compounds. Recent advances in whole
genome sequencing have revealed that actinomycetes with large genomes encode multiple secondary metabolite pathways, most
of which remain cryptic. One approach to address the expression of cryptic pathways is to first identify novel pathways by
bioinformatics, then clone and express them in well-characterized hosts with known secondary metabolomes. This process should
eliminate the tedious dereplication process that has hampered natural products discovery. Several laboratory and industrial
production strains have been used for heterologous production of secondary metabolite pathways. This review discusses the
results of these studies, and the pros and cons of using various Streptomyces and one Saccharopolyspora strain for heterologous expression. This information should provide an experimental basis to help researchers choose hosts
for current application and future development to express heterologous secondary metabolite pathways in yields sufficient
for rapid scale-up, biological testing, and commercial production. 相似文献
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Konstantin V. Kiselev Anna V. Turlenko Yuri N. Zhuravlev 《Plant Cell, Tissue and Organ Culture》2009,99(2):141-149
A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium
(MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained
by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture
in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in
darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation
and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic
acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed
a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development. 相似文献
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Using a direct amplification of genomic DNA from two Brassica rapa forms, we obtained two homologs of the CONSTANS gene, which controls the photoperiodic induction of flowering in Arabidopsis plants. The cloned fragments of B. rapa genome were identified as members of the CONSTANS-LIKE1 class. By aligning the nucleotide sequences of the CONSTANS gene and its homologs, three classes, CONSTANS, CONSTANS-LIKE1, and CONSTANS-LIKE2, were distinctly discerned by their primary structure. The pattern of restriction fragment length polymorphisms (RFLP) of the CONSTANS homologs in B. carinata, B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa were genome-specific; in addition, the CONSTANS homologs were classified by plant geographic origin, and we assume that such classification is related to plant photoperiodic response.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 274–281.Original Russian Text Copyright © 2005 by Martynov, Khavkin.This revised version was published online in April 2005 with a corrected cover date. 相似文献
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Hughes SL Hunter PJ Sharpe AG Kearsey MJ Lydiate DJ Walsh JA 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2003,107(7):1169-1173
A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC1 population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus. 相似文献
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Fernanda Laroza Paganelli Eliana Gertrudes de Macedo Lemos Lúcia Maria Carareto Alves 《World journal of microbiology & biotechnology》2011,27(4):773-778
Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These
polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These
polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have
thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of
growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different
growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect
the increase or decrease in PHB accumulation. 相似文献