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1.
AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary
vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants
per 1×106 conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial
strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated
that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than
two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon. 相似文献
2.
Burns C Leach KM Elliott TJ Challen MP Foster GD Bailey A 《Molecular biotechnology》2006,32(2):129-138
There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to
establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article,
we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trip2 promoters. The Coprinus cinereus β-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective,
whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp 1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection
of both appropriate Agrobacterium strains, with ALG-1 yielding more than LBA1126 and by the choice of the binary vectors used
to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems. 相似文献
3.
A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5′ end of a reporter gene, β-glucuronidase
(GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the
promoter sequence revealed a myb response element. 相似文献
4.
Gene silencing using siRNA has been examined in the industrially-important fungus, Aspergillus niger. Protoplasts of an A. niger strain containing a single genomic copy of the Escherichia coli uidA gene, encoding β-glucuronidase (GUS), under control of the A. niger glaA promoter at the same genomic locus, were exposed to siRNA targeted against the uidA gene. Down-regulation of uidA mRNA
and GUS activity by siRNA was observed in mycelia that developed from the protoplasts. The down-regulation was transient and
was not carried over to conidiation. We concluded that gene silencing by siRNA provides a relatively quick method for analysis
of gene function in A. niger. 相似文献
5.
Terentiev Y Pico AH Böer E Wartmann T Klabunde J Breuer U Babel W Suckow M Gellissen G Kunze G 《Journal of industrial microbiology & biotechnology》2004,31(5):223-228
An Arxula adeninivorans
integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris. The vector harbours a conserved A. adeninivorans-derived 25S rDNA sequence for targeting, the A. adeninivorans-derived TEF1
promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants. Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene. The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level. 相似文献
6.
黑曲霉(Aspergillus niger)是一种重要的工业生产菌株,被广泛地应用于生产酶制剂和有机酸,但仍需要进行基因组改造提高它的应用潜力。CRISPR/Cas9技术是一种被广泛采用的黑曲霉基因组编辑技术,但由于需要在基因组中整合选择标记或基因编辑效率还有待提高,影响了其在工业菌株改造中的应用。本研究建立了一种基于CRISPR/Cas9技术的高效无选择标记的基因编辑方法。首先,利用5S rRNA启动子启动sgRNA的表达,构建了一个含有AMA1(autonomously maintained in Aspergillus)复制起始片段的sgRNA和Cas9共表达质粒;同时通过敲除kusA基因构建非同源末端连接(non-homologous end joining pathway,NHEJ)修复缺陷的高效同源重组菌株;最后利用含有AMA1片段质粒的不稳定性,通过无抗平板传代丢失含有sgRNA和Cas9共表达质粒。利用该方法,在采用同源臂长度仅为20bp的无选择标记供体DNA进行基因编辑时,基因编辑效率可达到100%。该方法为黑曲霉基因功能的研究和细胞工厂的构建奠定了基础。 相似文献
7.
Xin Lu Jibin Sun Manfred Nimtz Josef Wissing An-Ping Zeng Ursula Rinas 《Microbial cell factories》2010,9(1):23
Background
The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS. 相似文献8.
Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different
criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that
the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly
classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues.
The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence
of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92%
An erratum to this article is available at . 相似文献
9.
10.
Marta Rodríguez-Sáiz Ramiro P. Godio Vanessa Álvarez Juan Luis de la Fuente Juan F. Martín José Luis Barredo 《Molecular biotechnology》2009,41(2):165-172
The gdhA gene encoding the NADP-dependent glutamate dehydrogenase (GDH) activity from Xanthophyllomyces dendrorhous has been cloned and characterized, and its promoter used for controlled gene expression in this red-pigmented heterobasidiomycetous
yeast. We determined the nucleotide sequence of a 4701 bp DNA genomic fragment, showing an open reading frame of 1871 bp interrupted
by five introns with fungal consensus splice-site junctions. The predicted protein (455 amino acids; 49 kDa) revealed high
identity to GDHs, especially to those from the fungi Cryptococcus neoformans (70%), Sclerotinia sclerotiorum (66%), and several species of Aspergillus (66–67%). Gene phylogenies support the grouping of X. dendrorhous GDH close to those from the majority of the filamentous fungi. The promoter region of the gdhA gene (PgdhA) contains a TATA-like box and two large pyrimidine stretches. The use of PgdhA for gene expression was validated by electrotransformation of X. dendrorhous using an in-frame fusion with the hygromycin resistance gene (hyg
R) as a reporter. X. dendrorhous transformants were able to grow in YEME complex medium and in Czapek minimal medium supplemented with 50 μg/ml hygromycin,
but gene expression in Czapek medium was repressed when using ammonium acetate as a nitrogen source. PgdhA is a valuable tool for controlled gene expression in Basidiomycetes. 相似文献
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14.
Tadanori Aimi Hiroyuki Taguchi Yoshikazu Tanaka Yutaka Kitamoto Tsutomu Morinaga 《Mycoscience》2005,46(1):27-31
Hygromycin B resistance was conferred to the mycelium of the white root rot fungus Rosellinia necatrix by transformation with the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. In all three transformants, the presence of hph and single-copy integrations of the marker gene were demonstrated by Southern analysis. This is the first report describing A. tumefaciens-mediated transformation of R. necatrix 相似文献
15.
A. M. Rozhkova A. S. Sereda N. V. Tsurikova A. K. Nurtaeva M. V. Semenova L. V. Rimareva E. A. Rubtsova I. N. Zorov O. A. Sinitsyna A. P. Sinitsyn 《Applied Biochemistry and Microbiology》2011,47(3):279-287
A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of glucoamylase. Vector pGa was prepared using promoter and terminator areas of the glucoamylase
gene, and Aspergillus niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant
proteins to be within the 0.6–14% range of total protein. 相似文献
16.
Four male cone-specific promoters were isolated from the genome of Pinus radiata D. Don, fused to the -glucuronidase (GUS) reporter gene and analysed in the heterologous host Arabidopsis thaliana (L.) Heynh. The temporal and spatial activities of the promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1 during seven anther developmental stages are described in detail. The two promoters PrMC2 and PrMALE1 confer an identical GUS expression pattern on Arabidopsis anthers. DNA sequence analysis of the PrMC2 and PrMALE1 promoters revealed an 88% sequence identity over 276 bp and divergence further upstream (<40% sequence identity). GUS expression driven by a 276-bp PrMALE1 promoter fragment showed the same pattern in Arabidopsis anthers as observed for the full-length PrMALE1 promoter. Within the 276-bp promoter fragment a region of high homology to a previously described 16-bp anther-box was identified. In gain-of-function experiments the putative PrMALE1 anther-box was fused upstream of a 90-bp CaMV 35S minimal promoter, as a single copy in the sense direction and as an inverted repeat. No GUS expression was conferred to Arabidopsis anthers by either of these two constructs. In a loss-of-function experiment a 226-bp PrMALE1 deletion construct, which did not contain the putative PrMALE1 anther-box, still maintained the originally observed PrMALE1 GUS expression pattern. Hence, gain-of-function as well as loss-of-function experiments consistently showed that the putative anther-box of the PrMALE1 promoter is non-functional in the Arabidopsis genetic background. For the analysis of the four full-length pine promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1, transformation vectors based on pCAMBIA2200 and pCAMBIA1302 were used. It will also be demonstrated in this article that sequences within the T-DNA borders of these vectors caused a characteristic histological background expression in Arabidopsis, with staining observed in vascular tissue of leaves, sepals, roots, filaments of stamens and in stems and pistils.Abbreviation GUS -glucuronidaseGenBank accession numbers for the analysed promoters: AF 337656 (PrCHS1), AF 337655 (PrLTP2), AF 337657 (PrMC2) and AF 337658 (PrMALE1). 相似文献
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18.
The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for
the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity
assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes
AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle. 相似文献
19.
The filamentous fungus Penicillium paxilli contains two distinct geranylgeranyl diphosphate (GGPP) synthases, GgsA and GgsB (PaxG). PaxG and its homologues in Neotyphodium lolii and Fusarium fujikuroi are associated with diterpene secondary metabolite gene clusters. The genomes of other filamentous fungi including Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Fusarium graminearum also contain two or more copies of GGPP synthase genes, although the diterpene metabolite capability of these fungi is not
known. The objective of this study was to understand the biological significance of the presence of two copies of GGPP synthases
in P. paxilli by investigating their subcellular localization. Using a carotenoid complementation assay and gene deletion analysis, we
show that P. paxilli GgsA and PaxG have GGPP synthase activities and that paxG is required for paxilline biosynthesis, respectively. In the ΔpaxG mutant background ggsA was unable to complement paxilline biosynthesis. A GgsA-EGFP fusion protein was localized to punctuate organelles and the
EGFP-GRV fusion protein, containing the C-terminus tripeptide GRV of PaxG, was localized to peroxisomes. A truncated PaxG
mutant lacking the C-terminus tripeptide GRV was unable to complement a ΔpaxG mutant demonstrating that the tripeptide is functionally important for paxilline biosynthesis.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
20.
Martin Schindler Robert L. Mach Sabine K. Vollenhofer Regina Hodits Franz Gruber Jaap Visser Leo De Graaff Christian P. Kubicek 《Gene》1993,130(2)
The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi. 相似文献