共查询到20条相似文献,搜索用时 15 毫秒
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Li Sun Huaqing Cai Weihong Xu Yuanlei Hu Yin Gao Zhongping Lin 《Plant Molecular Biology Reporter》2001,19(4):383-384
Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient
procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established
forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom. 相似文献
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A genomic DNA sequence and cDNA encoding a putative manganese peroxidase were isolated from the white-rot basidiomycete Lentinula edodes. The gene, called lemnp1, consists of a 1985-bp open reading frame interrupted by 16 introns and was flanked by an upstream region having putative
CAAT, TATA, and heat shock elements and by a downstream region having polyadenylation signals. The lemnp1 gene encodes a protein of 364 amino acids that shows high sequence homology to manganese peroxidases of other basidiomycetes.
The deduced N-terminal amino acid sequence is different from the L. edodes manganese peroxidase reported previously. 相似文献
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Characterization, Molecular Cloning, and Differential Expression Analysis of Laccase Genes from the Edible Mushroom Lentinula edodes 总被引:3,自引:0,他引:3
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The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus. 相似文献
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《Mycoscience》2019,60(4):246-249
Lentinula edodes secretes laccase (Lcc: EC 1.10.3.2), an industrially useful enzyme. In this study, we introduced and expressed the L. edodes Lcc gene, lcc1, driven by L. edodes glyceraldehyde-3-phosphate dehydrogenase gene promoter into L. edodes. The resulting transformants showed 2-fold Lcc activity than that of the host strain, and expression of the recombinant lcc1 was confirmed by RT-PCR. 相似文献
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Yoshihiro Ozeki Yoshio Ito Nobuhiro Sasaki Mikiko Oyanagi Hirofumi Akimoto Yukie Chikagawa Junko Takeda 《Journal of plant research》2000,113(3):319-326
D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the
carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role
in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure
and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic
differentiation is discussed.
Received 10 June 2000/ Accepted in revised form 1 July 2000 相似文献
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The recombinant soluble human tumor necrosis factor-alpha (hTNF-α) was expressed in a yeastSaccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-α was placed under the control of two different promoters: a glyceraldehyde-3-phosphate
dehydrogenase (GPD) promoter and a yeast hybridADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-α existed among transformants,
the higher expression was obtained with theGPD promoter. Expressed hTNF-α protein (rhTNF-α) was successfully secreted into the culture medium, producing 2.5 mg per liter
of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast
cell line, with serial dilution of rhTNF-α, indicated that the secreted rhTNF-α was bioactive and its doseresponse was improved
eight to ten times over that of theE. coli-derived rhTNF-α. 相似文献
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Interband DNA of Drosophila melanogaster polytene chromosomes was studied using a novel approach based on the electron microscopic (EM) analysis of chromosome regions carrying DNA fragements of known molecular genetic composition, inserted by P element-mediated transformation. Insertion of such fragments predominantly into interbands makes it possible to clone interband DNA by constructing genomic libraries from transformed strains and probing them with the insert DNA. The transformed strain P[H-sp70:Adh](61C) has insertion in the 61 C7-8 interband on the left arm of chromosome 3. This DNA consists of part of the hsp70 gene promoter fused to the coding region of the Adh gene, and is flanked on either side by P element sequences. We constructed a genomic library from DNA of this strain and isolated a clone containing the insert and the interband DNA. Subsequently the genomic library of wild-type strain was probed with a subclone composed of interband DNA only. We have thus isolated a clone containing the entire native interband. 1289 by of interband DNA was sequenced and found to be AT-rich (53.4%) with numerous regions of overlapping direct and inverted repeats, regulatory sites, and two overlapping open reading frames (ORFs). 相似文献
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M. D. Kane †‡ J. A. Sringer N. V. Iannotti E. Gough † S. M. Johns § S. D. Schlueter M. S. Sepúlveda § 《Journal of fish biology》2008,72(9):2341-2353
The fathead minnow Pimephales promelas serves as a model organism for assessing the effects of environmental contaminants on early life stage growth and development. Yet, the utilization of genomic tools has been hindered by the lack of genome sequence and genomic information known from this model species. Utilizing published cDNA library sequences, the authors used sequence similarity to compare 4105 cDNAs isolated from fathead minnow fry (<14 days old) with over 250 000 adult cDNA sequences derived from whole body and various tissue types. The objectives of the computational subtraction were to (1) assess the extent of sequence similarity between developing and adult cDNA libraries and (2) predict which cDNA clones are expressed only in developing organisms. The results of the computational predictions were assessed through the construction of a development‐specific DNA microarray targeting all 4105 sequences in the fry cDNA library as well as 56 known mRNAs in P. promelas. Gene expression was determined by comparing total RNA isolated from fry with total RNA isolated from adult samples (whole animal, kidney, liver, brain, ovary and testes). The results showed that 1381 of the targeted fry cDNA sequences (34%) displayed expression across all sample comparisons, and of these, only 166 genes were found to harbour fry‐specific expression (i.e. no expression in adult samples). Of note, 69% of the genes computationally predicted to be fry specific were found across all experimental results; yet, only 27% of the computationally predicted fry‐specific sequences were experimentally confirmed to be fry specific. An important result was the identification of many novel mRNA sequences specific to the developing minnow, which lack homology with any other known sequence. In addition, the study results included tissue‐specific expression in adult samples. These results demonstrate the capabilities and limitations of inter‐library sequence comparisons as a predictor of gene activity in non‐sequenced organisms and tissues, as well as DNA microarray gene expression studies in non‐sequenced organisms. 相似文献
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The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T-cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Sp1 binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non-coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin-related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes. 相似文献
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Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger
The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form. 相似文献
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Toshitsugu Sato Toshikazu Irie Fumihiko Yoshino 《Bioscience, biotechnology, and biochemistry》2017,81(8):1553-1556
Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain. 相似文献
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