DNA Blot Analysis Research Articles

Transcribed enhancer sequences are required for maize p1 paramutation.

Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that paramutation occurs at the P1-rr (red pericarp and red cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr' state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr'. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr' epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr' sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation.

The Antioxidant Activity of Betanin protects MRC-5 cellsAgainst Cadmium Induced Toxicity.

Cadmium (Cd) can induce both acute and chronic effects in the lungs depending on the time and the exposure route. Betanin is a component derived from the roots of red beets and it is well-known for its antioxidant and anti-apoptosis effects. The current study aimed to survey the protective effects of betanin on cell toxicity induced by Cd. Different concentration of Cd alone and in combination with betanin was assessed in MRC-5 cells. The viability and oxidative stress were measured using resazurin and DCF-DA methods respectively. Apoptotic cells were assessed by PI staining of the fragmented DNA and western blot analysis detected the activation of caspase 3 and PARP proteins. Cd exposure for 24h declined viability and increased ROS production in MRC-5 cells compared to the control group (p < 0.001). Also, Cd (35μM) elevated DNA fragmentation (p < 0.05), and the level of caspase 3-cleaved and cleaved PARP proteins in MRC-5 cells (p < 0.001). Co-treatment of cells with betanin for 24h significantly enhanced viability in concentrations of 1.25 and 2.5μM (p < 0.001) and 5μM (p < 0.05) and declined ROS generation (1.25 and 5μM p < 0.001, and 2.5μM p < 0.01). As well as, betanin reduced DNA fragmentation (p < 0.01), and the markers of apoptosis (p < 0.001) compared to the Cd-treated group. In conclusion, betanin protects lung cells against Cd-induced toxicity through antioxidant activity and inhibition of apoptosis.

Interference inhibition of Plum pox virus, induced by a hairpin-RNA of viral origin, provides long-term resistance to PPV infection in adult plants of the Startovaya (Prunus domestica L.) variety

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum `Startovaya` (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for β-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, Immuno Strip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene

Probing the Central Role of Phosphatidylinositol Synthesis in Lipid Metabolism of Eukaryotic Cells

Phosphatidylinositol (PI) is the precursor of all phosphoinositides (PPIns), a class of regulatory phospholipids that play key roles in cellular physiology. PI is synthesized in the ER by a single enzyme, phosphatidylinositol synthase (PIS). PI may have important roles of its own in membranes where PPIns are not produced such as in the ER and mitochondria. Disruption of PIS in yeast and zebrafish is lethal and PIS has been postulated to be an essential gene in studies of haploid cells. Therefore, it is difficult to evaluate the processes where this enzyme, or its lipid product, PI plays a critical role. To overcome this problem, we generated stable HEK293 cell lines in which a doxycycline (Dox)‐inducible PIS‐GFP transgene was stably integrated in a safe‐harbor site of the genome and the endogenous PIS locus was inactivated via CRISPR/Cas9 genome editing. We verified the knock‐out of the endogenous PIS and the inducible expression of PIS‐GFP by western blot analysis and sequencing of genomic DNA. Using these cells, we found that PIS enzymatic activity assayed in ER membranes prepared from cells after Dox deprivation was indistinguishable from the assay blank. Dox treatment of cells elevated PIS activity in the isolated ER membrane although it was still lower than that found in the membranes prepared from parental HEK293. Surprisingly, in spite of the undetectable low PIS activity, Dox‐deprived cells still contained about 50% of PI as assessed by equilibrium labeling with 3H‐inositol and retained their PPIns distribution as shown by confocal microscopy using GFP‐tagged PPIns binding modules. This disparity between PIS activity and PI availability was observed even under culture conditions that deprived cells of any external PI source. Such cells were subjected to lipidomic analysis to assess the overall lipid changes associated with the reduction of PI synthesis and to identify processes that require PI. This analysis confirmed the ~50% PI loss and also revealed reprogramming of specific lipid metabolic pathways. Current work focuses on the possibility that alternate source(s) of PI exist and on the question of what lipid synthetic pathways show adaptation to the greatly reduced or missing PIS activity. These experiments have the promise of a better understanding of how PI lipids determine the overall lipid landscape and homeostasis of eukaryotic cells and may unravel new molecular targets that can be exploited for therapeutic interventions in human metabolic and lipid disorders.

Non‑invasive prenatal diagnosis of thalassemia through multiplex PCR, target capture and next‑generation sequencing

Prenatal clinical detection of thalassemia involves gap-PCR and reverse dot blot (RDB) analysis of fetal DNA acquired through invasive methods. The present study aimed to develop a non-invasive prenatal diagnostic method for thalassemia based on next-generation sequencing (NGS). A total of eight families with proband children with thalassemia were recruited for the study during a subsequent pregnancy. The sequence of the thalassemia genes of the parents and proband were determined using NGS, based on a thalassemia AmpliSeq panel. Cell-free plasma DNA from pregnant women related to the aforementioned proband was analyzed using an NGS panel, based on thalassemia-associated capture probes. Heterozygous single nucleotide polymorphisms within the 10 kb regions flanking exons of the targeted thalassemia genes were acquired using probes or AmpliSeq and employed for parental haplotype construction using Trio-based panel sequencing. The fetal haplotype was deduced from the parental haplotypes and relative haplotype dosage, and subsequently validated using gap-PCR and RDB, based on invasively sampled amniotic fluid. A non-invasive prenatal diagnosis procedure from maternal plasma fetal DNA was successfully developed based on haplotype analysis. The deduced haplotypes of eight fetuses were identical to the results of invasive prenatal diagnosis procedures, with an accuracy rate of 100%. Taken together, the present study demonstrated the potential for non-invasive prenatal diagnosis of α- and β-thalassemia using NGS and haplotype-assisted analysis.

Research Note: Longitudinal monitoring of chicken houses in a commercial layer farm for antimicrobial resistance in Escherichia coli with special reference to plasmid-mediated quinolone resistance

Plasmid-mediated quinolone resistance (PMQR) genes located on conjugative plasmids can be transferred to other bacteria in the absence of antimicrobial selective pressure. To elucidate the prevalence of resistance, including PMQR in an egg-producing commercial layer farm in western Japan where no antimicrobials were used, minimum inhibitory concentrations (MIC) for a total of 375 Escherichia coli isolates obtained from chicken houses in the farm between 2012 and 2017 were determined using the agar dilution methods. Eighty-seven isolates resistant to oxytetracycline (OTC) accounted for 23.0% of the tested isolates, followed by isolates resistant to dihydrostreptomycin (DSM) (18.4%), sulfisoxazole (18.1%), ampicillin (AMP) (14.4%), trimethoprim (TMP) (14.4%), and nalidixic acid (10.1%). The prevalence rate of multidrug-resistant (MDR) isolates—which are resistant to 3 or more antimicrobial classes, including β-lactams, aminoglycosides, quinolones, folate pathway inhibitors, tetracyclines, and phenicols—was inversely related to the age of chickens at the time of bacterial examination. Probably, the prevalence of MDR isolates in layer chickens may have decreased with age owing to the absence of selective pressure. Furthermore, 45 isolates exhibiting enrofloxacin MICs of more than 0.25 μg/mL were examined for PMQR genes. The transfer of PMQR genes was tested by conjugation analysis. Southern blot analysis of genomic DNA revealed that the qnrS1 (5 isolates), qnrS2 (1 isolate), and qnrS13 genes (1 isolate) were located on plasmids with sizes ranging from approximately 60 to 120 kpb. In 1 of the 5 qnrS1-positive isolates and in an isolate with qnrS13, the qnrS genes were transferred to recipient strains. The plasmid harboring the qnrS1 gene was typed as IncF by PCR-based replicon typing. On this plasmid, the blaTEM, aadA, tetA, and dfrA1 genes responsible for resistance to AMP, DSM, OTC, and TMP, respectively, were detected. The tetA gene was detected in the plasmid harboring the qnrS13 gene, which was typed as IncI1. These results suggest that despite the low prevalence of quinolone resistance in this farm, various PMQR genes, located on diverse plasmids, exist.

Plasmid-mediated quinolone resistance in Escherichia coli isolates from commercial broiler chickens and selection of fluoroquinolone-resistant mutants

Plasmid-mediated quinolone resistance (PMQR) is a potential concern for animal husbandry and public health. Escherichia coli isolates from a total of 109 fecal samples collected from 6 commercial broiler farms between 2007 and 2011 were examined for PMQR genes, and transfer of these genes was tested by conjugation analysis to elucidate the prevalence and spread of PMQR in broiler chickens. Two isolates from 2 farms harbored the aac(6')-Ib-cr gene that was not detected in plasmids using Southern blot analysis of S1 nuclease-digested genomic DNA separated by pulsed-field gel electrophoresis. In these 2 isolates, nucleotide mutations in the gyrA and parC genes that result in amino acid substitutions were detected. Additionally, a total of 6 isolates originating from 6 chickens from the 2 farms were positive for the qnrS1 gene. In 2 of the 6 isolates, the qnrS1 gene was transferred to a recipient strain. Two transconjugants harboring the qnrS1 gene were cultured on media supplemented with successively higher concentrations of enrofloxacin (ERFX). After a 5-time subcultivation, the ERFX MICs reached 8 and 16 μg/mL, and no nucleotide mutations were detected in the gyrA, gyrB, parC, and parE genes. Our results suggest that the prevalence of PMQR was relatively low in broiler chickens and that exposure of bacteria carrying PMQR genes to the selective pressure of fluoroquinolones can result in resistance to fluoroquinolone, which is not caused by mutations in genes encoding topoisomerases.

Agrobacterium-Mediated Transformation of Russian Commercial Plum cv. "Startovaya" (Prunus domestica L.) With Virus-Derived Hairpin RNA Construct Confers Durable Resistance to PPV Infection in Mature Plants.

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum “Startovaya” (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for β-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, ImmunoStrip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene construct encoding intron-spliced hairpin RNAs provided a highly effective protection of plum trees against permanent viral infection. At the same time, this observation indicates the lack of the systemic spread of resistance from GM tissues to an infected plum graft even after years of joint growth.

Production of microbial mutan polysaccharide by expression of a mutansucrase gene (gtfI) in sugarcane

Due to its high productivity and sucrose content, sugarcane (Saccharum officinarum) is becoming the source of high-value bioproducts. Expression of bacterial extracellular polysaccharide genes in non-biopolymer accumulating plants is an excellent resource for production of added-value products. To this end, an expression cassette containing a full-length glucosyltransferase (gtfI) gene from Streptococcus downei driven by a CaMV promoter was expressed in a commercial sugarcane cultivar (CP48-103) using a biolistic approach. Copy number was assessed for a number of selected transgenic sugarcane lines by DNA blot analysis, where it was corroborated that each transgenic line contained at least two gtfI copies. The southern blot analysis of gtfI-expressing lines showed that the number of integrated copies ranged from two to four. The expression of gtfI in transgenic sugarcane plants was confirmed by mRNA blot analysis and qRT-PCR analysis. The expression of gtfI in transgenic sugarcane plants resulted in an approximate 30% reduction in sucrose accumulation, suggesting that mutansucrase actively converted sucrose to mutan polymer. In internodal stalk tissues, mutan polymer accumulated up to 55.9 mg/g FW, which apparent through glucan staining. The levels of glucose and fructose increased nearly by twofold, suggesting that mutansucrase may also have hydrolyzing activity.

The vacuolar proton pyrophosphatase gene (SbVPPase) from the Sorghum bicolor confers salt tolerance in transgenic Brahmi [Bacopa monnieri (L.) Pennell

Plants overcome the effect of Na+ toxicity either by excluding Na+ at the plasma membrane or bysequestering them into the vacuoles. Influx of Na+ ions into the plant vacuoles is usually driven by H+ generated by vacuolar-type H+-ATPase as well as vacuolar proton pyrophosphatse (VPPase). In the present study, we have developed Bacopa monnieri transgenics via Agrobacterium tumefaciens containing the recombinant vector pCAMBIA2300-SbVPPase gene. Transformants were produced using nodal explants. Transformants were confirmed by PCR and DNA blot analysis. qPCR analysis showed higher transcript levels of SbVPPase compared to untransformed control (UC). Higher VPPase activity was recorded in transgenics compared to UC. Under 150mM salt stress, transgenic shoots showed enhanced Na+ accumulation with better biomass production, increased glycine betaine content, and total soluble sugar levels than UC. Transgenic shoots showed 2.9-3.8-folds lower levels of malondialdehyde content indicating lesser membrane damage. Increase in antioxidant enzyme activities (1.4-3.2-folds) was observed in transgenics compared to UC. Transgenics also displayed 7.3-9.0-folds enhanced accumulation of the medicinally important compound bacoside A. Increased biomass production, accumulation of Na+, osmolytes (glycine betaine, sugars etc.), and elevated antioxidant enzyme activities indicate better osmotic adjustment in transgenics by compartmentalization of Na+ into the vacuoles under salt stress conditions. Thus, overexpression of SbVPPase in Bacopa alleviated salt stress by sequestering Na+.

Sprouted Sorghum Extract Elicits Coleoptile Emergence, Enhances Shoot and Root Acclimatization, and Maintains Genetic Fidelity in indica Rice

The high growth-stimulating effect of plant extract has urged the plant biotechnologists to use natural supplements in the culture media instead of synthetic phytohormones. We advocated the effect of sprouted sorghum extract (SSE) on emergence, in vitro acclimatization, and genetic fidelity in coleoptile derived callus of indica rice variety ADT36. The use of SSE with Murashige Skoog medium efficiently acclimatized the root and shoot apical systems. A higher mat and seminal roots (3.4 g biomass) with an efficient shoot primordium elongation were observed with an increase in the concentration of SSE. Seeds treated with SSE medium showed higher germination and earlier coleoptile maturation about 48 h compared to untreated seeds, and there was a higher expression of eEF-1α with an increase in coleoptile length. B5 medium was effective on inducing embryogenic and nodular callus from 3-day-old coleoptile with 3.0 mg/L 2,4-dichlorophenoxyacetic acid and further proliferated effectively with 0.8 mg/L kinetin with a fresh weight of 180 mg. Highly significant regeneration was observed with combination of 2.5 mg/L 6-benzylamino purine and 3.0 mg/L α-naphthaleneacetic acid. The metabolic and genetic profiles of in vitro and directly cultivated plants were the same, examined through Fourier-transform infrared spectroscopy, random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and R-ISSR (combination of RAPD and ISSR) markers, respectively, and thus confirming the significant efficacy of the SSE incorporated medium. Disarmed T-DNA was transformed to coleoptile derived callus through Agrobacterium tumefaciens LBA4404 and confirmed by GUS assay. The T-DNA integration was confirmed by DNA blot analysis using DNA from transient GUS-expressed explants. Thus, SSE can be used as a natural and organic supplement for organogenesis and efficient acclimatizations of shoot and root apical meristems in regenerated plants.

Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment.

Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene (GUS) and neomycin phosphotransferase II gene (npt II) as a reporter and as a selectable marker gene, respectively. The effect of optimized physical parameters on the frequency of transient (79.2±1.52%) and stable (41.9%) expressions has been investigated. The optimized biolistic parameters for petiole segments of Momordica charantia L. were determined as follows: 650 psi helium pressure and 6cm target distance. Using the optimized parameters, transformation of bitter melon was carried out for generation of putative transformants from bombarded tissues on SRM-K medium, with a mean number of 50.3 explants surviving at the end of the final selection (50mgl-1 kanamycin) round. Finally, the transformants produced were subjected to GUS histochemical assay, and integration of the transgenes (GUS and npt II) into the nuclear genome was confirmed by PCR analysis. DNA blot analysis confirmed the transgene integration in the transformed plantlet genomes. The present study may be used for developing transplastomic technology in this valuable medicinal plant for enhanced metabolic engineering pathways and production of biopharmaceuticals.

Overexpression of SbAP37 in rice alleviates concurrent imposition of combination stresses and modulates different sets of leaf protein profiles.

SbAP37 transcription factor contributes to a combination of abiotic stresses when applied simultaneously in rice. It modulates a plethora of proteins that might regulate the downstream pathways to impart salt stress tolerance. APETALA type of transcription factor was isolated from Sorghum bicolor (SbAP37), overexpressed in rice using a salt inducible abscisic acid 2 (ABA2) promoter through Agrobacterium tumefaciens following in planta method. Transgenics were confirmed by PCR amplification of SbAP37, hygromycin phosphotransferase (hptII) marker and ABA2 promoter and DNA blot analysis. Plants were exposed to 150mM NaCl coupled with high day/night 36 ± 2/25 ± 2 °C temperatures and also drought stress by withholding water for 1-week separately at the booting stage. SbAP37 expression was 2.8- to 4.7-folds higher in transgenic leaf under salt, but 1.8- to 3.3-folds higher in roots under drought stress. Native gene expression analysis showed that it is expressed more in stem than in roots and leaves under 150mM NaCl and 6% PEG stress. In the present study, proteomic analysis of transgenics exposed to 150mM NaCl coupled with elevated temperatures was taken up using quadrupole time-of-flight (Q-TOF) mass spectrometry (MS). The leaf proteome revealed 11 down regulated, 26 upregulated, 101 common (shared), 193 newly synthesized proteins in transgenics besides 368 proteins in untransformed plants. Some of these protein sets appeared different and unique to combined stresses. Our results suggest that the SbAP37 has the potential to improve combined stress tolerance without causing undesirable phenotypic characters when used under the influence of ABA2 promoter.

Biolistic transformation of Scoparia dulcis L.

Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0%) transient expression of GUS gene bombarded at a flight distance of 6cm and rupture disc pressure/acceleration pressure of 650psi. Highest stable expression of 52.0% was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.

Double flower formation in Tricyrtis macranthopsis is related to low expression of AGAMOUS ortholog gene

Complex flowers containing large numbers of petals are especially prized as ornamental plants. Double-flowered Tricyrtis macranthopsis, in which stamens and carpels are converted to petaloid organs, is related to AGAMOUS (AG) gene expression. So we isolated AGAMOUS gene from T. macranthopsis, named as TrimAG. Since TrimAG expression was limited to whorls 3 and 4 and ectopic expression of TrimAG in Arabidopsis thaliana caused the same phenotype as overexpression of other AG orthologs in transgenic Arabidopsis, this gene has similar function as its AG orthologs. In the double-flowered T. macranthopsis cultivar, the level of TrimAG expression was reduced in the two inner reproductive whorls (stamens and carpels) and caused reproductive organ replacement with petaloid tepals and floral indeterminacy. Southern blot analysis of genomic DNA indicated that there were no significant differences between the double-flowered cultivar and wild-type plants. Analysis of promoter and AG intron II sequences did not identify any critically mutated sequences in the doubled-flowered cultivar. These results suggest that reduced TrimAG expression in whorls 3 and 4 might be caused by mutation in one of the genes in regulatory network which controls AGAMOUS expression or transcriptional silencing by methylation of TrimAG promoter/intron sequences.

Isolation and Functional Identification of BrDSR, a New Gene Related to Drought Tolerance Derived from Brassica rapa

건조 스트레스는 작물의 생존과 생산성을 결정하는데 매우 중요한 환경요인이다. 본 연구의 목적은 배추에서 신규 건조 스트레스 저항성 유전자를 동정 검정하는 것이다. 건조 스트레스 하에서 생육된 지부('Chiifu') 배추를 이용하여 제작된 KBGP-24K 마이크로어레이 데이터 분석을 통해 738개의 건조 반응 유전자 중 기능은 밝혀져 있지 않지만, 건조 스트레스 하에서 발현량이 6배 이상 크게 증가한 1개의 유전자를 선발하여 BrDSR(B. rapa Drought Stress Resistance)이라 명명하였다. 이의 검정을 위해 내혼계배추('CT001')에서 BrDSR을 동정한 결과 438bp의 오픈리딩프레임과 145개의 아미노산을 가지고 있음을 확인하였고, 동정된 완전장의 cDNA 염기서열은 형질전환용 과발현 vector인 'pSL100' 제작에 이용하였다. BrDSR이 식물체에서 건조 스트레스 저항성을 향상시켜줄 수 있는지 분석하기 위해 담배 형질전환을 수행하였다. PCR과 DNA 블롯 분석으로 선발된 T1 세대 담배 형질전환체들을 대상으로 quantitative real-time RT PCR 분석을 수행한 결과, 형질전환체의 BrDSR 발현량은 비형질 전환체 보다 2.6배까지 증가하였다. 또한 건조처리 10일째 수행한 표현형 분석에서 BrDSR이 발현되는 담배 형질전환체들이 비형질전환체들 보다 우수한 건조 저항성을 보였다. 연구 결과들을 종합하면 BrDSR은 건조 스트레스 하에서 식물의 생장과 생존에 효과적인 저항성 기능을 할 것으로 기대된다. Drought stress is a crucial environmental factor determining crop survival and productivity. The goal of this study was to clearly identify a new drought stress-tolerance gene in Brassica rapa. From KBGP-24K microarray data with the B. rapa ssp. pekinensis inbred line 'Chiifu' under drought stress treatment, a gene which was named BrDSR (B. rapa Drought Stress Resistance) was chosen among 738 drought-responsive unigenes. BrDSR function has yet to be determined, but its expression was induced over 6-fold by drought. To characterize BrDSR, the gene was isolated from B. rapa inbred line 'CT001' and found to contain a 438-bp open reading frame encoding a 145 amino acid protein. The full-length cDNA of BrDSR was used to construct an over-expression vector, 'pSL100'. Tobacco transformation was then conducted to analyze whether the BrDSR gene can increase drought tolerance in plants. The BrDSR expression level in T1 transgenic tobacco plants selected via PCR and DNA blot analyses was up to 2.6-fold higher than non-transgenic tobacco. Analysis of phenotype clearly showed that BrDSR-expressing tobacco plants exhibited more tolerance than wild type under 10 d drought stress. Taking all of these findings together, we expect that BrDSR functions effectively in plant growth and survival of drought stress conditions.

Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

BackgroundIdentifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.ResultsWe used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.ConclusionsThis study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users.

Efficient shoots regeneration and genetic transformation of Bacopa monniera.

Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles. Present report describes efficient in vitro multiplication and transformation method for genetic manipulation of this species. MS medium supplemented with 2mgl(-1) BA and 0.2mgl(-1) IAA was found optimum for maximum shoot regeneration (98.33%) from in vitro leaves with 2-3 longitudinal cuts. Agrobacterium tumefaciens-mediated transformation method was used for generating transgenic B. monniera plants. Putative transformants were confirmed by GUS assay and PCR based confirmation of hptII gene. DNA blot analysis showed single copy insertion of transgene cassette. An average of 87.5% of the regenerated shoots were found PCR positive for hptII gene and GUS activity was detected in leaves of transgenic shoots at a frequency of 82.5% The efficient multiple shoots regeneration system described herein may help in mass production of B. monniera plant. Also, the high frequency transformation protocol described here can be used for genetic engineering of B. monniera for enhancement of its pharmaceutically important metabolites.

Intestine-specific expression of the β-glucanase in mice

Guan, L.-Z., Xi, Q.-Y., Sun, Y.-P., Wang, J.-L., Zhou, J.-Y., Shu, G., Jiang, Q.-Y. and Zhang, Y.-L. 2014. Intestine-specific expression of the β-glucanase in mice. Can. J. Anim. Sci. 94: 287–293. The β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was cloned into a specific expression plasmid (MUC2-GLU-LV). Transgenic mice were prepared by microinjection. Polymerase chain reaction and Southern blot analysis of genomic DNA extracted from the tail tissue of transgenic mice showed that the mice carried the β-glucanase gene. Northern blot analysis indicated that β-glucanase was specifically expressed in the intestine of the transgenic mice. The β-glucanase activity in the intestinal contents was found to be 1.23±0.32 U mL−1. The crude protein, crude fat digestibility of transgenic mice were increased by 9.32 and 5.09% (P&lt;0.05), respectively, compared with that of the non-transgenic mice, while moisture in feces was reduced by 12.16% (P&lt;0.05). These results suggest that the expression of β-glucanase in the intestine of animals offers a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.

Genotyping of Trypanosoma cruzi in a hyper-endemic area of Colombia reveals an overlap among domestic and sylvatic cycles of Chagas disease

BackgroundChagas disease is a neglected illness caused by the Trypanosoma cruzi parasite, which widely affects American communities. This study attempted to identify T. cruzi genotypes circulating in four indigenous communities of the Sierra Nevada of Santa Marta, Colombia, to investigate parasite transmission dynamics in these communities. In addition, some epidemiological variables to determine the risk factors for infection with this parasite, such as the prevalence of T. cruzi infection, the triatomine species, and the domestic and sylvatic mammals that act as vectors and reservoirs of the parasite in the domestic, peridomestic and sylvatic cycles, were examined.MethodsWe developed a prospective study to identify the main risk factors associated with T. cruzi infection in the region. The T. cruzi prevalence was determined by ELISA, IFA and PCR. Triatomines species and both domestic and sylvatic mammals from all communities were captured and sampled. To analyze parasite transmission dynamics in these four communities, eight DNA parasite probes were generated from insect and reservoir samples, and a DNA blot analysis were carried out.ResultsSerological studies revealed 37% prevalence in the four communities, and Kasakumake was the most endemic region, containing approximately 70% seropositives. Moreover, the molecular diagnosis showed a high correlation between the serological data and the T. cruzi circulating in the patients’ blood. A total of 464 triatomine insects were collected in domestic, peridomestic and sylvatic environments, and these insects belonged to five different species; Rhodnius prolixus and Triatoma dimidiata were the two more important species transmitting the parasite. After studying the eco-epidemiological factors in these four communities, the most important risk factors for infection with the parasite were determined. These risk factors are a high infection rate of people and domestic animals, the construction materials of the houses, the presence of infected triatomines inside the human dwellings, the proximity between houses and a sylvatic environment with several triatomine species and wild animals. Finally, the molecular characterization of T. cruzi showed the presence of three haplotypes and complex T. cruzi mixed infections in all reservoirs.ConclusionsActive transmission of T. cruzi is present in four indigenous communities of the Sierra Nevada de Santa Marta with overlap between the domestic and the sylvatic transmission cycles of Chagas disease.