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1.
The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed
in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under
control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially
in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of
the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically
active lipase from a basidiomycete fungus. 相似文献
2.
Maksymilian Zienkiewicz Tomasz Krupnik Anna Drożak Wioleta Wasilewska Anna Golke Elżbieta Romanowska 《Plant molecular biology》2018,96(1-2):135-149
Key message
We have successfully produced single-cell colonies of C. merolae mutants, lacking the PsbQ’ subunit in its PSII complex by application of DTA-aided mutant selection. We have investigated the physiological changes in PSII function and structure and proposed a tentative explanation of the function of PsbQ’ subunit in the PSII complex.Abstract
We have improved the selectivity of the Cyanidioschyzon merolae nuclear transformation method by the introduction of diphtheria toxin genes into the transformation vector as an auxiliary selectable marker. The revised method allowed us to obtained single-cell colonies of C. merolae, lacking the gene of the PsbQ’ extrinsic protein. The efficiency of gene replacement was extraordinarily high, allowing for a complete deletion of the gene of interest, without undesirable illegitimate integration events. We have confirmed the absence of PsbQ’ protein at genetic and protein level. We have characterized the physiology of mutant cells and isolated PSII protein complex and concluded that PsbQ’ is involved in nuclear regulation of PSII activity, by influencing several parameters of PSII function. Among these: oxygen evolving activity, partial dissociation of PsbV, regulation of dimerization, downsizing of phycobilisomes rods and regulation of zeaxanthin abundance. The adaptation of cellular physiology appeared to favorite upregulation of PSII and concurrent downregulation of PSI, resulting in an imbalance of energy distribution, decrease of photosynthesis and inhibition of cell proliferation.3.
Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH4NO3 and containing 3.0 mg l−1 IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious
root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l−1 cefotaxime and 50 mg l−1 kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium
with 300 mg l−1 cefotaxime and 50 mg l−1 kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l−1) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production
of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of
adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can
be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng. 相似文献
4.
Dana Bernátová 《Biologia》2008,63(2):175-176
The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole
Western Carpathians till now. 相似文献
5.
Tamura M Togami J Ishiguro K Nakamura N Katsumoto Y Suzuki K Kusumi T Tanaka Y 《Plant cell reports》2003,21(5):459-466
Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants. 相似文献
6.
7.
Heike Helmholz Blair D. Johnston Christiane Ruhnau Andreas Prange 《Hydrobiologia》2010,645(1):223-234
Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact
many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1,
was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms
extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping
and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident
within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells
by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in
the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml−1 (corresponding to 0.2 μg 104 cells−1), and a total loss of activity was observed above 40.0 μg ml−1 (corresponding to 4.0 μg 104 cells−1). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 104 cells−1, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis
of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including
some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex
venoms and, in some cases, more potent venoms than smaller animals. 相似文献
8.
Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied
in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could
be an effective candidate in genetic engineering of plants for the control of leaf mold. 相似文献
9.
Matías Maggi Natalia Damiani Sergio Ruffinengo David De Jong Judith Principal Martín Eguaras 《Experimental & applied acarology》2010,50(3):269-279
We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell
width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of
worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading
female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells. 相似文献
10.
Labruna MB Pacheco RC Nava S Brandão PE Richtzenhain LJ Guglielmone AA 《Microbial ecology》2007,54(1):126-133
The tick species, Amblyomma neumanni (Acari: Ixodidae) is the most frequent tick parasitizing humans in northwestern Argentina. The present study evaluated the
rickettsial infection among 55 A. neumanni adult free-living ticks collected in Dean Funes, Córdoba Province. Ticks were individually processed by the hemolymph test
with Gimenez staining, isolation of rickettsia in Vero cell culture by the shell vial technique, and polymerase chain reaction
(PCR) targeting the citrate synthase rickettsial gene. Through the shell vial technique, rickettsiae were successfully isolated
and established in Vero cell culture from two ticks (ticks 4 and 13), which previously showed to contain Rickettsia-like organisms by the hemolymph test. These two Rickettsia isolates were designated as An4 and An13. Molecular characterization (partial DNA sequences of two to three rickettsial genes
were determined) of these two isolates and phylogenetic analyses identified them as Rickettsia bellii (isolate An4) and Candidatus “Rickettsia amblyommii” (isolate An13). After testing all A. neumanni ticks by PCR, the prevalence of Candidatus
R. amblyommii and R. bellii was 23.6% (13/55) and 3.6% (2/55), respectively. These two rickettsiae have been considered of unknown pathogenicity and
appropriate studies to test their pathogenicity to humans or animals need to be conducted. This is the first report of Rickettsia in ticks from Argentina, and also in the species A. neumanni. The results reinforce previous findings that R. bellii (and probably Candidatus
R. amblyommii) are widespread among some Neotropical Amblyomma species, suggesting that these ticks gained these bacterial agents from a common ancestor and/or by recent horizontal transmission
of rickettsiae between ticks. 相似文献
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12.
Jingzhi Li Ruiqi Feng Zhihui Wen Aili Zhang 《Biotechnology and Bioprocess Engineering》2017,22(4):382-389
To investigate effects of different pyruvate decarboxylases on isobutanol titers in Saccharomyces cerevisiae, single-gene deletion of the three PDCs genes encoding pyruvate decarboxylases were constructed in this study. In addition, we over-expressed Ilv2, which catalyzed the first step in the valine synthetic pathway, and Bat2, which was the cytoplasmic branched-chain amino-acid aminotransferase that catalyzed L-valine to 2-ketoisovalerate, to increase isobutanol production in the genetically modified strains. Our results showed that knockout of PDC5 were one of the main factors among the three PDC genes for improving isobutanol titers in S. cerevisiae. Additionally, we found that deletion of PDC5 in strain carrying overexpressed ILV2 and ARO10 resulted in 8-fold higher isobutanol productivity as compared to the control strain in micro-aerobic fermentations. Our results also suggested that engineered strain pdc5ΔpILV2 pARO10 generated lower ethanol titers and higher acetate acid titers than the control strain, while the growth rate and glucose consumption rate of engineered strain pdc5ΔpILV2 pARO10 were slightly lower than that of the control strain. Meanwhile, the biomass concentration of pdc5ΔpILV2 pARO10 decreased dramatically than that of the control strain. 相似文献
13.
Konstantin V. Kiselev Anna V. Turlenko Yuri N. Zhuravlev 《Plant Growth Regulation》2009,59(3):237-243
14.
Nathaniel Liddy Peter E. Molloy Alan D. Bennett Jonathan M. Boulter Bent K. Jakobsen Yi Li 《Molecular biotechnology》2010,45(2):140-149
Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs).
The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative
would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm
of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and
DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that
the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen
binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid
and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries. 相似文献
15.
Jill A. Hollenbach Martha B. Ladner Koy Saeteurn Kent D. Taylor Ling Mei Talin Haritunians Dermot P. B. McGovern Henry A. Erlich Jerome I. Rotter Elizabeth A. Trachtenberg 《Immunogenetics》2009,61(10):663-671
In the present study, we investigated the relationship between the KIR loci and the genes encoding their HLA ligands and genetic
susceptibility to Crohn’s disease (CD). Analyses of the interactions between KIR3DL1, KIR2DL1, KIR2DL2, and KIR2DL3 with their
respective HLA ligands indicate that there is a protective effect for KIR2DL2 in the absence of its HLA ligand C1. Given that
KIR2DL2 and KIR2DL3 segregate as alleles, we compared their genotypic distributions to expectations under Hardy–Weinberg Equilibrium
(HWE) with regard to the HLA ligand C1 status. While all the genotypic distributions conform to expectations under HWE in
controls, in C2 ligand homozygous cases there is significant deviation from HWE, with a reduction of KIR2DL2, KIR2DL3 heterozygotes.
KIR2DL2, KIR2DL3 heterozygosity is the only genotypic combination that confers protection from CD. In addition to the protective
effect (OR = 0.44, CI = 0.22–0.87; p = 0.018) observed in C2 ligand homozygotes, the KIR2DL2, KIR2DL3 genotype is predisposing (OR = 1.34, CI = 1.03–4.53; p = 0.031) in the presence of C1 ligand. A test for trend of HLA class I C ligand group genotypes with KIR2DL2, KIR2DL3 heterozygosity
in cases and controls indicates that C1, C2 ligand group heterozygotes have an intermediate effect on predisposition. These
results show for the first time that disease susceptibility may be related to heterozygosity at a specific KIR locus, and
that HLA ligand genotype influences the relative effect of the KIR genotype. 相似文献
16.
17.
Strigolactones (SLs) are a recently discovered type of plant hormone that controls various developmental processes. The DWARF53 (D53) protein in rice and the SMAX1-LIKE (SMXL) family in Arabidopsis repress SL signaling. In this study, bioinformatics analyses were performed, and 236 SMXL proteins were identified in 28 sequenced plants. A phylogenetic analysis indicated that all potential SMXL proteins could be divided into three groups and that the SMXL proteins may have originated in Bryophytes. An analysis of the SMXL chromosomal locations suggested that gene duplication events at different times led to expansion of the SMXL family members in Angiospermae. Subsequently, the gene structure and protein modeling of MdSMXLs showed that they are highly conserved. The expression patterns of MdSMXLs indicated that they were expressed in different organs of apple (stems, roots, leaves, flowers, and fruits) at varying levels and that MdSMXLs may participate in the SL signaling pathway and the response to abiotic stress. This study provides a valuable foundation for additional investigations into the function of the SMXL gene family in plants. 相似文献
18.
Razi H Howell EC Newbury HJ Kearsey MJ 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2008,116(2):179-192
Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping.
They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed
among Brassica
FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic
regions of BoFLC paralogues with Arabidopsis
FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known
to be essential in regulating FLC expression. Four B. oleracea
FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population.
All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms
were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence
against any of the 4 BoFLC being FT-QTL candidates in this population. 相似文献
19.
Abdul Ghaffar Sher Afzal Khan Zahid Mukhtar Muhammad Ibrahim Rajoka Farooq Latif 《Molecular biology reports》2011,38(5):3227-3233
We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml−1 while that of recombinant without intron (xyn669) was 1.26 U ml−1 after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant
xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite
lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis
than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was
quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications. 相似文献