PCR Southern Blot Analysis Research Articles

A practical random mutagenesis system for Ralstonia solanacearum strains causing bacterial wilt of Pogostemon cablin using Tn5 transposon.

A practical random mutagenesis system of Ralstonia solanacearum by electroporation with Tn5 transposon was established, which may be utilized to provide genetic approach to study virulence genes of R. solanacearum strains and create nonpathogenic mutants for biological control of bacterial wilt in Pogostemon cablin. R. solanacearum strain PRS-84 used in this study was isolated from P. cablin plants infected with bacterial wilt. The bacterial suspension of R. solanacearum strain PRS-84 was mixed with Tn5 transposome complex and the mixture was transformed by electroporation. The electroporated cells were then spread on the 2, 3, 5-triphenyltetrazolium chloride agar plates containing kanamycin to select the kanamycin-resistant colonies. Several factors which determined the bacterial transformation efficiency were optimized. The transformation process was shown to be optimal at the electric field strength of 12.5kVcm-1. Bacterial cells harvested at mid-exponential phase gave the highest transformation efficiency. 10µgmL-1 kanamycin was found to be the optimal concentration for transformant selection. Tn5 insertion mutants of R. solanacearum strain PRS-84 were identified by PCR amplification and Southern blot analysis. Mutants subcultured for 100 passages were also detected by PCR amplification and Southern blot analysis. Furthermore, pathogenicity screening test of mutants was performed by inoculating in vitro regenerated patchouli plants. Results revealed that mutants with a single Tn5 insertion in their genomes were obtained from R. solanacearum strain PRS-84, and the Tn5 insertion could be stably inherited in the mutants. Then, mutants with reduced pathogenicity were selected.

Unraveling unusual X-chromosome patterns during fragile-X syndrome genetic testing

BackgroundFragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID). Together with fragile X-associated tremor and ataxia (FXTAS) and fragile X-associated premature ovarian failure (POF)/primary ovarian insufficiency (POI), FXS depends on dysfunctional expression of the FMR1 gene on Xq27.3. In most cases, FXS is caused by a >200 CGG repeats in FMR1 5′-untranslated region (UTR) and by promoter hypermethylation that results in gene silencing. Males and females with unmethylated premutated alleles (repeats between 55 and 200) are at risk for FXTAS and POF/POI. MethodsFXS molecular testing relied on PCR and methylation-specific Southern blot analysis of the FMR1 5′UTR. Atypical Southern blot patterns were studied by X-chromosome microsatellite analysis, copy number dosage at DMD locus, amelogenin gender-marker analysis and array-comparative genomic hybridization (array-CGH). ResultsSix men affected by ID and three women affected by ID and POF/POI underwent FXS molecular testing. They had normal FMR1 CGG repeats, but atypical X chromosome patterns. Further investigations revealed that the six males had Klinefelter syndrome (XXY), one female was a Turner mosaic (X0/XX) and two women had novel rearrangements involving X chromosome. ConclusionsDiagnostic investigation of atypical patterns at FMR1 locus can address patients and/or their relatives to further verify the condition by performing karyotyping and/or array-CGH.

Expression of a Cystatin Transgene in Eggplant Provides Resistance to Root-knot Nematode, Meloidogyne incognita

Root-knot nematodes (RKN) cause substantial yield decline in eggplant and sustainable management options to minimize crop damage due to nematodes are still limited. A number of genetic engineering strategies have been developed to disrupt the successful plant–nematode interactions. Among them, delivery of proteinase inhibitors from the plant to perturb nematode development and reproduction is arguably the most effective strategy. In the present study, transgenic eggplant expressing a modified rice cystatin (OC-IΔD86) gene under the control of the root-specific promoter, TUB-1, was generated to evaluate the genetically modified nematode resistance. Five putative transformants were selected through PCR and genomic Southern blot analysis. Expression of the cystatin transgene was confirmed in all the events using western blotting, ELISA and qPCR assay. Upon challenge inoculation, all the transgenic events exhibited a detrimental effect on RKN development and reproduction. The best transgenic line (a single copy event) showed 78.3% inhibition in reproductive success of RKN. Our results suggest that cystatins can play an important role for improving nematode resistance in eggplant and their deployment in gene pyramiding strategies with other proteinase inhibitors could ultimately enhance crop yield.

Rapid selection of XO embryonic stem cells using Y chromosome-linked GFP transgenic mice.

We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9% of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.

Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases

Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine β-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in β-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

Improved production of transgenic Dioscorea zingiberensis (Dioscoreaceae) by Agrobacterium tumefaciens-mediated transformation

The establishment of high-efficiency Agrobacterium-mediated transformation techniques could improve the production of Dioscorea zingiberensis, a medicinal species with a high diosgenin content. We co-cultivated embryogenic calli induced from mature seeds with A. tumefaciens strain EHA105. A binary vector, pCAMBIA1381, which contains the gfp and hpt genes under the control of the ubiquitin promoter and the CaMV 35S promoter, respectively, was used for transformation. Pre-culture, basic medium, acetosyringone, and bacterial density were evaluated to establish the most efficient protocol. The optimal conditions consisted of MS medium without CaCl(2) for pre- and co-cultivation, three days for pre-culture, addition of 200 μM AS, and an OD(600) of 0.5. The transgenic plants grown under selection were confirmed by PCR analysis and Southern blot analysis. This protocol produced transgenic D. zingiberensis plants in seven months, with a transformation efficiency of 6%.

Molecular and phenotypic characterization of Acinetobacter strains able to degrade diesel fuel

Characterization of bacterial communities in oil-contaminated soils and evaluation of their degradation capacities may serve as a guide for improving remediation of such environments. Using physiological and molecular methods, the aim of this work was to characterize 17 Acinetobacter strains (13 species) able to use diesel fuel oil as sole carbon and energy source. The strains were first tested for their ability to grow on different alkanes on minimal medium containing high NaCl concentrations. The envelope hydrophobicity of each strain was assessed by microbial adhesion to the hydrocarbon test (MATH) when grown in LB medium or minimal medium containing succinate or diesel fuel. Most strains were hydrophobic both in LB and minimal medium, except for strain Acinetobacter venetianus VE-C3 that was hydrophobic only in minimal medium. Furthermore, two A. venetianus strains, RAG-1T and LUH 7437, and strain ATCC 17905 (genomic species 13BJ) displayed biosurfactant activity. The alkM gene encoding alkane hydroxylase was detected in the chromosome of the 15 strains by PCR amplification, sequencing and Southern blot analysis. Phenotype microarray analysis performed on the five A. venetianus strains revealed that they differentially used purines as N-source and confirmed that they are unable to use carbohydrates.

Transgenic Tobacco Plants Resistant to Two Viruses via RNA Silencing

RNA silencing is a phenomenon of homologous RNA degradation induced by dsRNA, which is an effective strategy to obtain virus resistant plants so far. By means of this strategy, higher bio-security immune transgenic plants were obtained, avoiding recombining and transcapsidating with other virus genes. In this study, the recombinant plant expression vector pBIN438-MPRep (i/r) consisting the inverted repeat of TMV-ΔMP and CMV-ΔRep fusion fragment was transformed into tobacco cultivar K326 via Agrobacterium-mediated. The transformants were selected in the culture medium with 100 mg L^(-1) Kan. One hundred and ninety-six transgenic plants were obtained, one hundred and twenty-eight of which were positive plants, and the resistance to CMV and TMV was tested at the degree of virus. PCR-Southern blot and RT-PCR analysis of the transgenic plants demonstrated that the exogenous DNA was integrated into the tobacco genomic DNA and was expressed in transcriptional level. Resistance assay indicated that about 20.3% transgenic plants were immune to the co-infection with TMV and CMV. This result will provide to crops an important reference for plant anti-viral breeding and for preventing the viral co-infection.

Identification of a novel recombinant mutation in Korean patients with Gaucher disease using a long-range PCR approach

Gaucher disease (GD) is an autosomal recessive, lysosomal disorder caused by mutations in the gene for the β-glucocerebrosidase (GBA) enzyme. Presence of the non-functional GBAP pseudogene, which shares high sequence similarity with the functional GBA gene, has made it difficult to carry out molecular analyses of GD, especially recombinant mutations. Using a long-range PCR approach that has been skillfully devised for the easy detection of GBA recombinant mutations, we identified four recombinant mutations including two gene conversion alleles, Rec 1a and Rec 8a, one reciprocal gene fusion allele, Rec 1b, and one reciprocal gene duplication allele, Rec 7b, in Korean patients with GD. Rec 8a, in which the GBAP pseudogene sequence from intron 5 to exon 11 is substituted for the GBA gene is a novel recombinant mutation. All mutations were confirmed by full sequencing of PCR amplicons and/or Southern blot analysis. These results indicate that the usage of long-range PCR may allow the rapid and accurate detection of GBA recombinant mutations and contribute to the improvement of genotyping efficiency in GD patients.

Fruit-specific Over-expression of LeEXP1 Gene in Tomato Alters Fruit Texture

Expansins are cellular proteins with diverse physiological functions. Expression of fruit-specific expansin gene in tomato is associated with fruit softening — a desirable trait from the processing point of view. In the present study, an expansin gene LeEXP1 was introduced via Agrobacterium tumefaciens in sense orientation under the control of a fruit-specific promoter LeACS4 with nptII gene as selection marker in Indian tomato cv Pusa Uphar. PCR detection and Southern blot analysis confirmed the integration of the transgene in the transformed tomato plants. RT-PCR and northern blot analysis using total RNA isolated from leaves and fruits confirmed over-expression of the LeEXP1 gene in transgenic fruits as compared to the wild type plants. Apart from the visual change in increased red colouration of fruits at different stages of ripening, overexpression of the LeEXP1 gene resulted in enhanced fruit softening, as determined by force required to rupture the fruit pericarp, in the transgenic fruits from breaker stage onwards as compared to the non-transformed wild type fruits. The results thus suggest an improvement in texture of the LeEXP1 over-expressing fruits, which might be useful for tomato processing industry.

Transgenic tomato cv. Pusa Uphar expressing a bacterial mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance

A bacterial mannitol-1-phosphate dehydrogenase (mtlD) gene driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter was transferred into tomato plants using an Agrobacterium tumefaciens-mediated transformation protocol in an attempt to improve abiotic stress tolerance in the transformed plants. Transgene integration was confirmed by PCR analysis and Southern blot analysis, and transgene expression was confirmed by reverse transcription (RT)-PCR and direct mtlD (EC 1.1.1.17) activity. Upon exposure to low temperature stress (4°C) in a cold chamber, transgenic plants survived up to 48 h, while non-transformed plants were unable to survive and gradually died. Transgenic plants subjected to the chilling stress showed a significant decrease in electrolyte leakage and increased lipid peroxidation, as assessed by measuring malondialdehyde (MDA) content. Under the cold condition, transgenic plants also showed a significant increase in the activities of antioxidant enzymes (superoxide dismutase and catalase) and in relative water content (RWC) in comparison to non-transformed plants. Drought (polyethylene glycol in medium) and salinity (sodium chloride in medium) tolerance tests revealed that transgenic lines exhibited a higher tolerance for abiotic stresses than non-transformed plants. These findings indicate that the introduction of a bacterial mtlD gene into tomato conferred tolerance to abiotic stresses to the transformed tomato plants.

CD44 expression during tumor progression of follicular lymphoma

Follicular lymphoma often progresses to diffuse type lymphoma. To elucidate the mechanisms of the diffuse evolution of follicular lymphoma, we investigated the expression pattern of CD44 in 28 cases of follicular lymphomas (FLs) using an immunohistochemical method and semi-quantitative PCR-Southern blot analysis. The FLs were divided into four groups: i) intrafollicular (IF); ii) infiltrative (INF); iii) partially follicular (PF); and iv) minimally follicular (MF), according to the histological classification by Lukes and Collins. Immunohistochemical analysis of CD44 using antibodies against CD44 common (CD44C) epitopes showed that CD44 was expressed in the diffuse area in the INF (0/8 cases), PF (12/12 cases), and MF (2/2 cases) lymphomas, whereas CD44 was not expressed in the lymphoma cells within the area of follicular growth of IF (0/6 cases) and INF (0/8 cases). Semi-quantitative PCR-Southern blot analysis showed that CD19-selected B cells from the FLs were expressed as a product of 482 base pairs (bp) corresponding to a CD44 standard form (CD44s) (5/5 cases). Additionally, the lymphoma cells from the PF were expressed as products of 600 and 1100 bp and the cells from MF were expressed as products of 600, 900, and 1100 bp with the CD44 exon 10 or 11 probes. The results indicated that the expression of CD44s and CD44 variants containing exon 10 and 11 were up-regulated according to the diffuse evolution of the follicular lymphoma.

Stable Expression of the Chlorocatechol Dioxygenase Gene fromRalstonia eutrophaNH9 in Hybrid Poplar Cells

The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 under the control of a modified cauliflower mosaic virus 35S promoter was introduced into a hybrid poplar (Populus tremula x P. tremuloides). Integration of the cbnA gene in transgenic poplar was confirmed by PCR and genomic Southern blot analysis. Expression of the cbnA gene was analyzed by Western blot analysis. Transgenic poplar calli efficiently converted 3-chlorocatechol to 2-chloro-cis,cis-muconate.

Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method

Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10(2) transformants/microg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.

Over-Expression of ICE1 Gene in Transgenic Rice Improves Cold Tolerance

ICE1, an Arabidopsis thaliana transcription factor gene, was cloned by RT-PCR and successfully transformed into rice variety Kenjiandao 10 by the Agrobacterium-mediated transformation method. PCR amplification and Southern blot analysis indicated that ICE1 had been integrated into rice genome. Compared with the non-transgenic plants, the transgenic plants exhibited high resistance to hygromycin B and were consistent with the Mendelian inheritance of a single copy of the transgenic ICE1. Under the low temperature stress, the transgenic plants showed the lower mortality rate and the increased proline content. These results suggest that the Arabidopsis ICE1 is functional in rice and the over-expression of ICE1 improves the tolerance to cold stress in rice.

Cloning of HvBADH1 Gene from Hulless Barley and Its Transformation to Tobacco

To reveal the relationship between the expression of betaine aldehyde dehydrogenase (BADH) and the tolerance to salt stress resistance in hulless barley ( Hordeum vulgare L. var . nudum Hook. f), a 1,512 bp cDNA encoding BADH was cloned from hulless barley using the methods of reverse transcription PCR (RT-PCR) and rapid application of cDNA ends (RACE). The cDNA, designated HvBADH1 under the accession number EF492983 in GenBank, encodes a 54.2 kD protein with 232 amino acid residues. Gene HvBADH1 exhibited a homology (98.4%) in amino acid sequence with BBD2 gene encoding an isoenzyme of BADH from barley. It also shared a high homology of 97.0%, 84.7%, and 85.1% with BADH in wheat ( Triticum aestivum L.), maize ( Zea mays L.), and rice ( Oryza sativa L.), respectively. Gene HvBADH1 was inserted into pMAL c2x, and the recombined plasmid was then transferred into Escherichia coli TB1 cells. The recombinant TB1 harboring pMAL c2x- HvBADH1 and the control TB1 harboring empty pMAL c2x cells were induced with IPTG. The results revealed that the recombinant E. coli cells expressed a fusion protein with molecular weight of 96.3 kD. This fusion protein was fused by maltose binding protein (MBP, about 42.1 kD) and the peptide (about 54.2 kD) encoded by HvBADH1. The open reading frame (ORF) of HvBADH1 was inserted between CaMV35S promoter and NOS polyA in T-DNA region of the binary expression vector pCAMBIA1301. The recombinant plasmid, designated pCAM-ba, was transferred into Agrobacterium tumefaciens strain LBA4404. The HvBADH1 gene was transformed to tobacco ( Nicotiana tabacum L,) mediated by Agrobacterium. Two hygromycin B (Hyg)-resistant plants were obtained. PCR detection and Southern blot analysis indicated that all the Hyg-resistant tobacco plants contained the alien BADH gene. RT-PCR analysis showed that HvBADH1 gene was expressed on the mRNA level in the transgenic tobacco plants. This suggested that HvBADH1 gene was related to the salt tolerance in hulless barley, which can be expressed in transgenic plants.

Establishment of genetic transformation system and obtaining transgenic sugarcane (var. badila) transformed with RS gene

Resveratrol Synthase (RS) is one of the key enzymes in resveratrol biosynthesis, which catalyzes one molecule of coumaroyl CoA and three molecules of malonyl CoA to form one molecule of resveratrol. In this study, the genetic transformation system for badila, a chewing cane variety, was established. The optimized media for regeneration were as follows: induction medium was MS+2,4-D 2mg.L−1; differentiation medium was MS+NAA 0.2 mg.L−1+BA 2 mg.L−1; rooting medium was 1/2 MS+NAA 2 mg.L−1. The addition of 0.5 % activated carbon in the differentiation and rooting medium could decrease the phenol poison and promote rooting. At the same time, the optimal concentration of G418 in subculture and differentiation period was determined as: 30 mgL−1 for differentiation and 25 mgL−1 for rooting. RS gene was transferred into badila via gene gun bombardment through the constructed plant expression vector pBIL-RS. PCR detection and southern blot analysis showed that seven putative transgenic plants had been obtained. Therefore, the genetic transformation system of badila had been elementarily established and proved to be efficient.

Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum

Methanotrophic bacteria constitute a ubiquitous group of microorganisms playing an important role in the biogeochemical carbon cycle and in control of global warming through natural reduction of methane emission. These bacteria share the unique ability of using methane as a sole carbon and energy source and have been found in a great variety of habitats. Phylogenetically, known methanotrophs constitute a rather limited group and have so far only been affiliated with the Proteobacteria. Here, we report the isolation and initial characterization of a nonproteobacterial obligately methanotrophic bacterium. The isolate, designated Kam1, was recovered from an acidic hot spring in Kamchatka, Russia, and is more thermoacidophilic than any other known methanotroph, with optimal growth at approximately 55 degrees C and pH 3.5. Kam1 is only distantly related to all previously known methanotrophs and belongs to the Verrucomicrobia lineage of evolution. Genes for methane monooxygenases, essential for initiation of methane oxidation, could not be detected by using standard primers in PCR amplification and Southern blot analysis, suggesting the presence of a different methane oxidation enzyme. Kam1 also lacks the well developed intracellular membrane systems typical for other methanotrophs. The isolate represents a previously unrecognized biological methane sink, and, due to its unusual phylogenetic affiliation, it will shed important light on the origin, evolution, and diversity of biological methane oxidation and on the adaptation of this process to extreme habitats. Furthermore, Kam1 will add to our knowledge of the metabolic traits and biogeochemical roles of the widespread but poorly understood Verrucomicrobia phylum.

Wolbachiagenome integrated in an insect chromosome: Evolution and fate of laterally transferred endosymbiont genes

Recent accumulation of microbial genome data has demonstrated that lateral gene transfers constitute an important and universal evolutionary process in prokaryotes, while those in multicellular eukaryotes are still regarded as unusual, except for endosymbiotic gene transfers from mitochondria and plastids. Here we thoroughly investigated the bacterial genes derived from a Wolbachia endosymbiont on the nuclear genome of the beetle Callosobruchus chinensis. Exhaustive PCR detection and Southern blot analysis suggested that approximately 30% of Wolbachia genes, in terms of the gene repertoire of wMel, are present on the insect nuclear genome. Fluorescent in situ hybridization located the transferred genes on the proximal region of the basal short arm of the X chromosome. Molecular evolutionary and other lines of evidence indicated that the transferred genes are probably derived from a single lateral transfer event. The transferred genes were, for the length examined, structurally disrupted, freed from functional constraints, and transcriptionally inactive. Hence, most, if not all, of the transferred genes have been pseudogenized. Notwithstanding this, the transferred genes were ubiquitously detected from Japanese and Taiwanese populations of C. chinensis, while the number of the transferred genes detected differed between the populations. The transferred genes were not detected from congenic beetle species, indicating that the transfer event occurred after speciation of C. chinensis, which was estimated to be one or several million years ago. These features of the laterally transferred endosymbiont genes are compared with the evolutionary patterns of mitochondrial and plastid genome fragments acquired by nuclear genomes through recent endosymbiotic gene transfers.

Pollen‐mediated transformation of Sorghum bicolor plants

Pollen from sorghum (Sorghum bicolor L. Moench) were transformed by a novel genetic transformation approach using mild ultrasonication. The plasmid pBI121 contained the NPT II gene encoding NPT II (neomycin phosphotransferase II) and a GUS (beta-glucuronidase) reporter gene. Plasmid DNA and pollen from sorghum A(2)V4B were submerged in a 0.3 mol/l sucrose solution and then subjected to ultrasonication. The treated pollen was then used for pollination of stigmas from the male sterile line A(2)V4A. Detection of transient and stable GUS activity confirmed that the GUS gene was expressed in transgenic plants. PCR amplification and Southern-blot analysis of transgenic plants confirmed that the NPT II gene had been integrated into the sorghum genome. These results indicate that direct gene transfer to pollen can be mediated by mild ultrasonication.