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Lohar DP Schuller K Buzas DM Gresshoff PM Stiller J 《Journal of experimental botany》2001,52(361):1697-1702
Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar encodes for Phosphinothricin Acetyl Transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenic L. japonicus plants resistant to PPT were positive upon PCR by bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA insertion into the plant genome. All regenerated plants were fertile and void of visible somaclonal abnormalities contrary to 14% infertility when antibiotic selectable markers were used. The lack of somaclonal variation, ease of PPT application and low cost of PPT makes this protocol an attractive alternative for the regeneration of transgenic L. japonicus. The production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production. 相似文献
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Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce. 相似文献
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Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules. 相似文献
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A study of bacterial surface oligosaccharides were investigated among
different strains of Neisseria gonorrhoeae to correlate structural features
essential for binding to the MAb 2C7. This epitope is widely expressed and
conserved in gonococcal isolates, characteristics essential to an effective
candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared
by a modification of the hot phenol-water method from which de-O-acetylated
LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and
ES-MSnin a triple quadrupole and an ion trap mass spectrometer,
respectively. Previously documented natural heterogeneity was apparent from
both LOS and OS preparations which was admixed with fragments induced by
hydrazine and mild acid treatment. Natural heterogeneity was limited to
phosphorylation and antenni extensions to the alpha-chain. Mild acid
hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic
linkage of lipid A. OS structures were determined by collisional and
resonance excitation combined with MS and multistep MSn which provided
sequence information from both neutral loss, and nonreducing terminal
fragments. A comparison of OS structures, with earlier knowledge of MAb
binding, enzyme treatment, and partial acid hydrolysis indicates a generic
overlapping domain for 2C7 binding. Reoccurring structural features include
a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the
nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc
(gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the
central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain),
moiety is required although extensions to this residue appear unnecessary.
相似文献
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Thomas GH; Newbern EC; Korte CC; Bales MA; Muse SV; Clark AG; Kiehart DP 《Molecular biology and evolution》1997,14(12):1285-1295
Many structural, signaling, and adhesion molecules contain tandemly
repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin
superfamily of F-actin-crosslinking proteins contains an array of triple
alpha-helical motifs (spectrin repeats). We present here the complete
sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H).
The sequence of beta H supports the origin of alpha- and beta-spectrins
from a common ancestor, and we present a novel model for the origin of the
spectrins from a homodimeric actin-crosslinking precursor. The pattern of
similarity between the spectrin repeat units indicates that they have
evolved by a series of nested, nonuniform duplications. Furthermore, the
spectrins and dystrophins clearly have common ancestry, yet the repeat unit
is of a different length in each family. Together, these observations
suggest a dynamic period of increase in repeat number accompanied by
homogenization within each array by concerted evolution. However, today,
there is greater similarity of homologous repeats between species than
there is across repeats within species, suggesting that concerted evolution
ceased some time before the arthropod/vertebrate split. We propose a
two-phase model for the evolution of the spectrin repeat arrays in which an
initial phase of concerted evolution is subsequently retarded as each new
protein becomes constrained to a specific length and the repeats diverge at
the DNA level. This evolutionary model has general applicability to the
origins of the many other proteins that have tandemly repeated motifs.
相似文献
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Dasharath Lohar Jiri Stiller Jason Kam Gary Stacey Peter M. Gresshoff 《Annals of botany》2009,104(2):277-285
Background and Aims
Transgenics are used to demonstrate a causal relationship between ethylene insensitivity of a seedling legume plant, the level of ethylene receptor gene expression, lateral root growth and Mesorhizobium loti-induced nodule initiation.Methods
Lotus japonicus plants expressing the dominant etr1-1 allele of the Arabidopsis thaliana gene encoding a well-characterized mutated ethylene receptor were created by stable Agrobacterium tumefaciens transformation. Single insertion, homozygous lines were characterized for symbiotic properties.Key Results
Transgenic plants were ethylene insensitive as judged by the lack of the ‘Triple Response’, and their continued ability to grow and nodulate in the presence of inhibitory concentrations of ACC (1-aminocyclopropane-1-carboxylic acid; an ethylene precursor). Transgenic plants with high insensitivity to ACC had significantly fewer lateral roots and exhibited increased nodulation while showing no altered nitrate sensitivity or lack of systemic autoregulation. Whereas ACC-insensitive shoot growth and nodulation were observed in transformants, root growth was inhibited similarly to the wild type. Increased nodulation was caused by increased infection and a seven-fold increase in nodules developing between xylem poles. Bacteroid numbers per symbiosome increased about 1·7-fold in ethylene-insensitive plants.Conclusions
The study further demonstrates multiple roles for ethylene in nodule initiation by influencing root cell infections and radial positioning, independent of autoregulation and nitrate inhibition of nodulation.Key words: Ethylene insensitivity, Lotus japonicus, symbiosis, phytohormone, nodulation, signal transduction 相似文献9.
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Sedentary plant-parasitic nematodes engage in complex interactions, and induce specialized feeding structures by redirecting plant developmental pathways, and parallels have been observed with rhizobial nodule development on legumes. A model legume would greatly facilitate a better understanding of the differences between parasitic (nematode) and mutualistic (rhizobia and mycorrhizae) symbioses, and we have developed Lotus japonicus as such a model. Conditions for efficient parasitism by root-knot nematode (Meloidogyne spp.) of the widely used Lotus "Gifu" ecotype were established. Features of Lotus biology, such as thin and translucent roots, proved ideal for monitoring the progress of nematode infection both on live specimens and post-staining. We examined L. japonicus mutants with nodulation phenotypes. One, har1, which is a hypernodulated mutant defective in a CLAVATA1-like receptor kinase gene, was found to be hyperinfected by M. incognita. However, another hypernodulated Lotus mutant exhibited the same level of M. incognita infection as wild-type plants. We also established conditions for infection of Lotus by soybean cyst nematode (Heterodera glycines). In contrast to the response to root-knot nematode, the Gifu ecotype is resistant to H. glycines, and elicits a hypersensitive response. This pattern of resistance recapitulates that seen on nematode-resistant soybean plants. We conclude that L. japonicus is a powerful model legume for studying compatible and incompatible plant-nematode interactions. 相似文献