The <Emphasis Type="Italic">rolB</Emphasis> gene promotes rooting <Emphasis Type="Italic">in vitro </Emphasis>and increases fresh root weight <Emphasis Type="Italic">in vivo</Emphasis> of transformed apple scion cultivar ‘Florina’

Transgenic apple (Malus × domestica Borkh.) Florina plants were obtained by Agrobacterium-mediated transformation. The efficiency of gene transfer was 7.9%, calculated as a number of explants producing at least one transgenic shoot, after co-cultivation of leaf explants from in vitro-grown shoots in a thin layer of the A. tumefaciens C58C1 strain with the binary vector pCMB-B:GUS. Polymerase chain reaction revealed that all the clones contained the nptII and rolB genes, while four of them did not contain the gus gene. Southern blot analysis confirmed the integration of the nptII and rolB genes, with one to three copies per genome being present. All independent rolB-transgenic lines were able to produce roots in vitro on the hormone free medium, while the plants, transformed with the vector pIB16.1, or untransformed control plants did not root, and only half of shoots of MM106 rootstock rooted on this medium. The average root number in the rolB-transgenic clones ranged from 4 to 7.7. Pretreatment with indole-3-butyric acid caused root formation in all transgenic and control plants and significantly increased root number in the rolB-transgenic lines, compared to untransformed plants. RolB-transgenic plants, grown in vivo in greenhouse for 2 years, did not differ phenotypically from the wild type line with the exception of root parts. All rolB-transformed plants produced altered root systems containing more fine roots leading to significantly increased fresh root weight in five plant lines.     

Mating system and seed variation of <Emphasis Type="Italic">Acacia</Emphasis> hybrid (<Emphasis Type="Italic">A. mangium</Emphasis> × <Emphasis Type="Italic">A. auriculiformis</Emphasis>)

The mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean±s.e. t _m = 0.86±0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%–70%), followed by among pods within inflorescence (29%–37%), and the least variation among inflorescences within tree (<1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (<Emphasis Type="Italic">Smhppd</Emphasis>) from <Emphasis Type="Italic">Salvia</Emphasis> <Emphasis Type="Italic">miltiorrhiza</Emphasis> hairy root cultures

A novel 4-hydroxyphenylpyruvate dioxygenase gene (designated as Smhppd) was cloned from hairy roots of Salvia miltiorrhiza Bung. The full-length cDNA of Smhppd was 1,736 bp long with an ORF (open reading frame) that putatively encoded a polypeptide of 481 amino acids, with a predicted molecular mass of 52.54 kDa. The deduced amino acid sequence of the Smhppd gene shared high homology with other known HPPDs. Analysis of Smhppd genomic DNA revealed that it contained two exons and one intron. The analysis of Smhppd promoter region was also presented. Southern-blot analysis revealed that the Smhppd was a low-copy gene in S. miltiorrhiza. Real-time quantitative PCR analysis indicated that Smhppd was constitutively expressed in roots, stems and leaves of S. miltiorrhiza, with the high expression in roots. In addition, Smhppd expreession level under different stress condition was also analyzed during the hairy root culture period, including signaling components for plant defence responses, such as methyl jasmonate and salicylic acid, as well as an abiotic elicitor, Ag⁺ and a biotic elicitor, yeast extract. This study will enable us to further understand the role Smhppd plays in the synthesis of active pharmaceutical compounds in S. miltiorrhiza at molecular level.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Deletion of <Emphasis Type="Italic">psbQ</Emphasis>’ gene in <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis> reveals the function of extrinsic <Emphasis Type="Italic">PsbQ</Emphasis>’ in PSII

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.

     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

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.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

In-fusion expression and characterization of <Emphasis Type="Italic">β</Emphasis>-xylanase and <Emphasis Type="Italic">β</Emphasis>-1,3-1,4-glucanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower K_n higher k_cat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

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.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

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.     

Cloning and expression of <Emphasis Type="Italic">β</Emphasis>-glucosidase gene from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> into <Emphasis Type="Italic">E. coli</Emphasis> BL 21 (DE3)

A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50°C and pH 6. K _m and V _max were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy E_a, enthalpy of activation ?H and entropy of activation ΔS were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pK_a1 and pK_a2 of the ionizable groups of active site residues involved in V_max were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Influence of <Emphasis Type="Italic">RAR</Emphasis> α gene on <Emphasis Type="Italic">MDR1</Emphasis> expression and P-glycoprotein function in human leukemic cells

Background  

Multidrug resistance (MDR) phenotype of malignant cells is the major problem in the chemotherapy of neoplasia. The treatment of leukemia with retinoids is aimed on the induction of leukemic cells differentiation. However the interconnections between retinoid regulated differentiation of leukemic cells and regulation of MDR remains unclear.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

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.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.     

Applications of PCR-RFLPs for differentiating two freshwater sponges: <Emphasis Type="Italic">Ephydatia </Emphasis><Emphasis Type="Italic">fluviatilis</Emphasis> and <Emphasis Type="Italic">Ephydatia mülleri</Emphasis>

The two freshwater sponges Ephydatia fluviatilis and Ephydatia mülleri belong to the widespread Spongillidae family. Their morphological tracts are very similar and can be distinguished mainly on the basis of their gemmuloscleres. However, when gemmules are absent it is essential to have an unambiguous species attribution for a population genetic study based on fresh tissues and historical collections. This article reports a simple Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, applied to DNA extracted from both gemmules and fresh tissues in order to discriminate between the two congeneric E. fluviatilis and E. mülleri. Such a biomolecular method is based on the discriminative enzymes’ digestion of each of the three amplified fragments 5.8S-ITS2-28S, D3 domain of the 28S subunit and COI. Two restriction enzymes were tested for a 620–642 bp fragment of 5.8S-ITS2-28S and for a 342 bp fragment of the D3 domain of the 28S, one restriction enzyme was tested for a 681 bp fragment codifying for COI. Obtained digestion patterns were diagnostic for each of the two species, providing a relatively simple, fast and cheap method for species attribution compared to sequencing. Handling editor: C. Sturmbauer