共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
Esmeralda Parra-Peralbo Manuel Pineda Miguel Aguilar 《Molecular biology reports》2009,36(8):2249-2258
A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net
charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent
AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity
to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light. 相似文献
3.
4.
Atsuko Uzura Fumiki Nomoto Akiko Sakoda Yukifumi Nishimoto Michihiko Kataoka Sakayu Shimizu 《Applied microbiology and biotechnology》2009,83(4):617-626
A novel nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, 3-quinuclidinone reductase, was isolated
from Rhodotorula rubra JCM3782. The enzyme catalyzes the asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol. The gene encoding the enzyme was also cloned and sequenced. A 819-bp nucleotide fragment was confirmed
to be the gene encoding the 3-quinuclidinone reductase by agreement of the internal amino acid sequences of the purified enzyme.
The gene encodes a total of 272 amino acid residues, and the deduced amino acid sequence shows similarity to those of several
short-chain dehydrogenase/reductase family proteins. An expression vector, pWKLQ, which contains the full length 3-quinuclidinone
reductase gene was constructed. Using Escherichia coli cells coexpressing the 3-quinuclidinone reductase and glucose dehydrogenase (cofactor regeneration enzyme) genes, 618 mM
3-quinuclidinone was almost stiochiometrically converted to (R)-3-quinuclidinol with an >99.9% enantiomeric excess within 21 h of reaction. 相似文献
5.
A gene encoding the xylanase from Bacillus subtilis strain R5 containing the native signal sequence was cloned and expressed in Escherichia coli. The heterologous expression of the gene resulted in the production of the recombinant protein in the cytoplasm as well as
its secretion into the culture medium. The xylanase activity in the culture medium increased with time after induction up
to 90% of the total activity in 14 h. Molecular mass and N-terminal amino acid sequence determinations of the purified recombinant
xylanase revealed that the native signal peptide was cleaved off by E. coli signal peptidases between Ala28 and Ala29. 相似文献
6.
The genome of Xenopus laevis codes for two genes of peroxiredoxin 6, i.e., xen1 (Acc. no. EMBL Data Bank-BCO54278) and xen2 (Acc. no. EMBL Data Bank-BCO54309). Both the genes were cloned and expressed in Escherichia coli. The amino acid sequences of Xen1 and Xen2 enzymes are identical by 95%, and they possess the same peroxidase activity as
well as similar optimums of temperature, pH, and thermostability. The genes of peroxiredoxin 6 of Xenopus laevis considerably differ in the period of expression during ontogenesis; i.e., xen2 is expressed during every stage of development, somewhat more intensively after stages 0–5; the expression of xen1 is initiated later, i.e., during the developmental stages of 47–48 h. Expression of xen2 increases after the incubation of embryos in a medium with hydrogen peroxide. Comparison of the amino acid sequences of
Xen1 and Xen2 proteins shows that only Xen2 can possess phospholipase activity because its amino acid sequence contain residues
of the phospholipase A2 active center: Ser31, His25, and Asp139. 相似文献
7.
8.
The purpose of this work was to characterize the cellular and proteomic responses of Escherichia coli JK-17 exposed to the rose flower extract (Rosa hybrida). The bacterial isolate was enriched and isolated from contaminated food. 16S rRNA sequence analyses revealed that the strain
was 99% similar to the E. coli species cluster; therefore, this strain was designated E. coli JK-17. The rose flower extract showed a dose-dependent antibacterial effect on E. coli JK-17. Treatment of E. coli JK-17 with 50 and 100 mg/mL of the rose flower extract completely inhibited growth within 12 and 6 h of incubation. The stress
shock proteins (SSPs) were induced with different concentrations of rose flower extract. The proteins were identified as 70-kDa
DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using anti-DnaK and anti-GroEL monoclonal antibodies. The levels of SSPs
induced by the rose flower extract increased when the exposure time to the rose flower extract was increased. SDS-PAGE with
silver staining revealed that the amount of lipopolysaccharide (LPS) in E. coli JK-17 increased or decreased with different concentrations and exposure times of the rose flower extract. To identify proteins
induced by the rose flower extract, 2-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of E. coli JK-17 cultures. In the pH range of 4 ∼ 7, more than 250 spots were detected on the silver stained gels. Notably, 15 protein
spots were increased or decreased after treatment with the rose flower extract. Twelve up-regulated proteins were identified
as chaperones (DnaK and GroEL) and porin proteins (PhoE, RfaI, RfaG, MdoH, and WzzE) by MALDITOF mass spectrometry, and three
down-regulated proteins were identified, including proteins involved in energy and DNA metabolism (SdhA and GyrB), and amino
acid biosynthesis (GltK). Using scanning electron microscopic analysis, some cells were shown to adopt irregular rod shapes
and wrinkled surfaces after treatment with the rose flower extract. These results provide clues for better understanding the
mechanism of rose flower extract-induced stress and cytotoxicity in E. coli JK-17. 相似文献
9.
The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of
PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed
a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned
into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type
PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100. 相似文献
10.
Alejandro López-Cortés Oliverio Rodríguez-Fernández Hever Latisnere-Barragán Humberto C. Mejía-Ruíz Getzabeth González-Gutiérrez Carlos Lomelí-Ortega 《World journal of microbiology & biotechnology》2010,26(1):109-118
Polyhydroxyalakanote (PHA) was produced by the marine bacteria Paracoccus seriniphilus Strain E71. Three methods were used for screening PHA in this strain: (1) microscopic analysis, (2) specifically designed
primers for amplify fragments of phaC gene from Gram negative bacteria, and (3) measurements using spectroscopy, calorimetry, thermogravimetry, and rheology. The
polyhydroxyalkanoic acid synthase gene (phaC) sequence had 77% identity with the phaC gene of P. denitrificans PD1222 strain. Additionally, the translated sequence showed an 86% similarity with the amino acid sequence of the phaC gene N-terminal portion of the P. denitrificans PD1222 strain. Our phaC sequence was closely related to two phaC sequences that correspond to P. denitrificans; therefore, this is the first report of a sequence of phaC that codifies a poly-(3-hydroxyalkanoate) synthase class I, specifically a poly-beta-hydroxybutyrate polymerase from the
marine bacteria Paracoccus seriniphilus. The polymer PHA of E71 melts at 167.86°C (T
m), which corresponded to the fusion of the crystalline polymer and thermally degrades at 296.52°C, indicating that the biopolymer
has good thermal stability. Rheology showed that this polymer behaves as a nonNewtonian fluid. All these characteristics suggest
that the E71 strain produces a PHA that corresponds to the crystalline thermoplastic polymer PHB type. 相似文献
11.
Hayakawa T Howlader MT Yamagiwa M Sakai H 《Applied microbiology and biotechnology》2008,80(6):1033-1037
Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes.
However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective
production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches. 相似文献
12.
13.
Ching-Nan Lin Wan-Jr Syu Wei-Sheng W Sun Jenn-Wei Chen Tai-Hung Chen Ming-Jaw Don Shao-Hung Wang 《Journal of biomedical science》2010,17(1):84
Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin
treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying
plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide
stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance
when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for
the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished
apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture
supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation. 相似文献
14.
Yu P 《Applied microbiology and biotechnology》2007,76(4):867-871
The gene for the Cu,Zn superoxide dismutase (Cu,ZnSOD) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli LMG194. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the E. coli periplasmic expression vector pBAD/gIIIA, yielding pBAD-1. E. coli was transformed using the constructed plasmid pBAD-1 and induced by adding 0.02% l-arabinose to express Cu,ZnSOD protein. The results indicated that Cu,ZnSOD enzyme activity in the periplasmic space was about
fivefold to sixfold higher in the recombinant E. coli strains bearing the sod gene than in the control strains. The yields of Cu,ZnSOD were about threefold higher at 48 h than at 24 h in the recombinant
E. coli cells. Significantly higher survival of strains was obtained in cells bearing the sod gene than in the control cells when the cells were treated by heat shock and superoxide-generating agents, such as paraquat
and menadione. 相似文献
15.
O. V. Sirotkina A. M. Shaydina T. V. Vavilova E. I. Schwartz 《Russian Journal of Genetics》2005,41(6):683-687
Glycoprotein IIb/IIIa complex, a platelet surface fibrinogen receptor, plays a key role in producing primary hemostasis. At present, only a single mutation in the GPIIIa gene, Leu33Pro, and a single mutation in the GPIIb gene, Ile843Ser, has been described. The mutations are known to enhance signaling functions of the receptor and are associated with the development of arterial thromboses. In the present study, we describe a novel GPIIIa mutation, which is T to G nucleotide substitution in position 1585, resulting in the replacement of Leu for Arg in position 40 of the amino acid sequence of the protein.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 838–843.Original Russian Text Copyright © 2005 by Sirotkina, Shaydina, Vavilova, Schwartz. 相似文献
16.
The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced
with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular
weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the
APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector
pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew. 相似文献
17.
Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted
plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration
of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration
system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine
(BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis
with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box
genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed
in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two
MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation. 相似文献
18.
DUF538 domain-containing protein family consists of several plant proteins of unknown functions. This protein family has already
been discovered by genome annotation tools and cloned as an inducible gene product under various environmental stress conditions.
For the first time, we presented a full length DUF538 cDNA (encoding 170 amino acid residues) clone, which was randomly isolated from Celosia cristata leaf cDNA library constructed under normal growth conditions and consistently amplified from leaf cDNA populations prepared
from nonstressed and drought-stressed leaves. We predicted that a DUF538 gene product can be a putative candidate for common stress-related protein (regulatory factor) in the plant system. The nucleotide
and deduced amino acid sequences of the isolated clone have been submitted to EMBL data bases under accession no. AJ535713. 相似文献
19.
A rod-shaped, gram-negative bacterium Stenotrophomonas sp. SMSP-1 was isolated from the sludge of a wastewater treating system of a pesticide manufacturer. Strain SMSP-1 could
hydrolyze methyl parathion to p-nitrophenol (PNP) and dimethyl phosphorothioate but could not degrade PNP further. Strain SMSP-1 was able to hydrolyze other
organophosphate pesticides, including fenitrothion, ethyl parathion, fenthion, and phoxim, but not chlorpyrifos. A 4395-bp
DNA fragment, including an organophosphorus hydrolase encoding gene ophc2, was cloned from the chromosome of strain SMSP-1 using the shotgun technique. Its sequence analysis showed that ophc2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase), and orf1 (encoding a CDF family heavy metal/H+ antiporter). The ophc2 gene was effectively expressed in E. coli. This is the second report of cloning the ophc2 gene and the first report of this gene from the genus of Stenotrophomonas. 相似文献
20.