New allele of the <Emphasis Type=Italic>COCHLEATA</Emphasis> gene in pea <Emphasis Type=Italic>Pisum sativum</Emphasis> L.

Analysis with the polymerase chain reaction showed that the Khlorofill-4 pea Pisum sativum chlorophyll-deficient mutant with reduced stipules has an altered structure of the COCHLEATA (COCH) gene, carrying a new mutant COCH allele. The phenotype of the mutant was described in comparison with another form having reduced stipules (stipules reduced) and the control. Leaves of the coch mutant are smaller and have other proportions than in the control; stipules are absent from leaves of the first nodes and are narrow, bandlike, or spoonlike at later ontogenetic stages. It was concluded that the cell number in the stipule epidermis is reduced in the st and coch mutants compared to the wild type.     

Isolation of mixis-related genes from the rotifer <Emphasis Type=Italic>Brachionus plicatilis</Emphasis> using subtractive hybridization

The monogonont rotifer Brachionus plicatilis produces resting eggs through sexual reproduction (mixis), which is affected by external and internal factors. We collected resting eggs from rotifers cultured at 15 and 25°C and hatched them with and without 14-day dormancy in the dark. Stem females hatched from both conditions were further cultured at 15, 20 and 25°C. We obtained two phenotypes, one with high mixis (more than 50%), which was hatched from resting eggs formed at 15°C without dormancy, and another in which sexual reproduction occurred at 25°C and resting eggs were formed at 15°C with a 14-day dormancy. In the latter phenotype, mictic females appeared at 15°C, but not at 25°C. Using subtractive hybridization, we isolated one gene from the latter phenotype of females that had no significant similarity to known genes in BLAST searches. We propose that this gene is unique to rotifer mictic reproduction. Ongoing characterization of this gene attempts understand its role in mixis.     

Molecular cloning,expression in <Emphasis Type=Italic>Escherichia</Emphasis><Emphasis Type=Italic>coli</Emphasis> of Attacin A gene from <Emphasis Type=Italic>Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Cloning of a novel lipase gene, <Emphasis Type=Italic>lipJ08,</Emphasis> from <Emphasis Type=Italic>Candida rugosa</Emphasis> and expression in <Emphasis Type=Italic>Pichia pastoris</Emphasis> by codon optimization

A novel lipase gene, lipJ08, was cloned from Candida rugosa ATCC14830, along with the already reported five lipase genes (lip1–lip5). Nucleotide sequencing indicated that the lipJ08 gene contains a 1650 bp open reading frame (ORF) without introns. The deduced amino acid sequence corresponds to 534 amino acid residues, including a putative signal sequence of 15 amino acid residues. Seventeen of the non-universal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by PCR-based mutagenesis. The native and codon-optimized lipJ08 genes were expressed in Pichia pastoris. The hydrolytic activity of the recombinant LIPJ08 was 4.7 U/ml, whereas the activity of the recombinant wild-type lipase could not be detected.     

Enhanced proline accumulation and salt stress tolerance of transgenic <Emphasis Type=Italic>indica</Emphasis> rice by over-expressing <Emphasis Type=Italic>P5CSF129A</Emphasis> gene

Δ¹-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T₁) under 150 mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.     

High-level expression of <Emphasis Type=Italic>Camelid</Emphasis> nanobodies in <Emphasis Type=Italic>Nicotiana benthamiana</Emphasis>

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative ‘universal’ nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.     

Photosynthetic and transpiration responses of <Emphasis Type=Italic>in vitro</Emphasis>-regenerated <Emphasis Type=Italic>Solanum nigrum</Emphasis> L. plants to <Emphasis Type=Italic>ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

Identification of nitrogen-fixing <Emphasis Type=Italic>Paenibacillus</Emphasis> from different plant rhizospheres and a novel <Emphasis Type=Italic>nifH</Emphasis> gene detected in the <Emphasis Type=Italic>P. stellifer</Emphasis>

A total of 534 isolates were selectively obtained from different plant rhizospheres based on their growth on nitrogen-free medium and their resistance to 80°C for 15 min. Of the 534 isolates, 23 isolates had nifH gene and exhibited nitrogenase activities. Based on 16S rDNA sequence, G + C content assay and DNA-DNA hybridization, the 23 isolates which divided into four monophyletic clusters were all belonged to the Paenibacillus genus. nifH gene deduced amino acid alignment aLnalysis revealed that cluster I, including 15 isolates, showed the highest NifH identity with Paenibacillus genus; while cluster II identified as P. stellifer by DNA-DNA hybridization was consistent with four uncultured bacterial clones. This study suggested that the nitrogen-fixing Paenibacillus were distributed in various ecosystems and prevalent in different plant rhizospheres. It was the first demonstration that nitrogen fixation existed in P. jamilae and P. stellifer. In eight isolates identified as P. stellifer species, a novel nifH gene was detected in Paenibacillus.     

Analysis of a <Emphasis Type=Italic>c</Emphasis><Subscript>0</Subscript><Emphasis Type=Italic>t-1</Emphasis> library enables the targeted identification of minisatellite and satellite families in <Emphasis Type=Italic>Beta vulgaris</Emphasis>

Background  

Repetitive DNA is a major fraction of eukaryotic genomes and occurs particularly often in plants. Currently, the sequencing of the sugar beet (Beta vulgaris) genome is under way and knowledge of repetitive DNA sequences is critical for the genome annotation. We generated a c ₀ t-1 library, representing highly to moderately repetitive sequences, for the characterization of the major B. vulgaris repeat families. While highly abundant satellites are well-described, minisatellites are only poorly investigated in plants. Therefore, we focused on the identification and characterization of these tandemly repeated sequences.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.