RNA Dot Blot Research Articles

“Role of RNA N6‐methyladenosine methylation in endothelial response to flow”

N6‐methyladenosine (m6A) has the most abundant modification among eukaryotic mRNAs, and plays a vital role in cellular responses to many types of physiological stress. Endothelial cells (ECs) are constantly exposed in the shear stress induced by blood flow over a long period of time. Oscillatory shear (OS) can cause inflammatory response and induce altherosclerotic plaque formation. In contrast, pulsatile shear (PS) has anti‐inflammatory effect and is atheroprotective. However, the relationship between RNA modification and the effects of shear stress in endothelial cells is still unclear. In time‐series experiments on endothelial cells subjected to pulsatile and oscillatory shears, we determined m6A modifications in purified mRNA in by RNA dot blot and the expression levels of regulators of m6A by RT‐PCR and western blot. The results show that the changes of m6A modification in endothelial cells in response to different shear stresses are dynamic events and that the m6A modification of mRNA is lesser in response to OS than PS. The expression levels of m6A writers Mettl3 and Mettl14 were up‐regulated by pulsatile shear as compared with oscillatory shear and static state. In summary, this study has shown that different shear stresses can induce distinct changes of m6A modification and that Mettl3 and Mettl14 were induced by pulsatile force and may regulate RNA N6‐methyladenosine methylation in endothelial response to flow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A high-throughput protocol for message RNA quantification using RNA dot-blots

This study develops a method to rapidly measure the relative abundance of mRNA in total RNA samples using a dot-blotting technique and biotin-labeled detection probes that recognize the polyadenylate tail on mRNA. We demonstrate the effectiveness of this technique by determining the relative total amounts of mRNA in three tissues of turtles (Trachemys scripta elegans) exposed to normoxic versus anoxic conditions. The data emphasize the usefulness of the method for the simple and rapid analysis of relative total mRNA levels for a variety of comparison purposes.

An Orphan Histidine Kinase, OhkA, Regulates Both Secondary Metabolism and Morphological Differentiation in Streptomyces coelicolor

We report here the physiological and genetic characterization of an orphan histidine kinase (HK) (OhkA, SCO1596) in Streptomyces coelicolor and its homolog (OhkAsav, SAV_6741) in Streptomyces avermitilis. The physiological analysis showed that the ohkA mutant of S. coelicolor exhibits impaired aerial mycelium formation and sporulation and overproduction of multiple antibiotics on mannitol-soy flour (MS) medium, especially actinorhodin (ACT) and calcium-dependent antibiotic (CDA), and disruption of ohkAsav in S. avermitilis also led to the similar phenotypes of impaired morphological differentiation and significantly increased oligomycin A production. DNA microarray analysis combined with real-time reverse transcription-PCR (RT-PCR) and RNA dot blot assay in the S. coelicolor ohkA deletion mutant confirmed the physiological results by showing the upregulation of genes involved in the biosynthesis of ACT, CDA, undecylprodigiosin (RED), a yellow type I polyketide (CPK, SCO6273-6289), and a sesquiterpene antibiotic, albaflavenone (SCO5222-5223). The results also suggested that the increased production of ACT and RED in the mutant could be partly ascribed to the enhanced precursor malonyl coenzyme A (malonyl-CoA) supply through increased transcription of genes encoding acetyl-CoA carboxylase (ACCase). Interestingly, DNA microarray analysis also showed that deletion of ohkA greatly downregulated the transcription of chpABCDEFGH genes essential for aerial mycelium formation by S. coelicolor on MS medium but significantly increased transcription of ramS/C/R, which is responsible for SapB formation and regulation and is normally absent on MS medium. Moreover, many other genes involved in development, such as bldM/N, whiG/H/I, ssgA/B/E/G/R, and whiE, were also significantly downregulated upon ohkA deletion. The results clearly demonstrated that OhkA is an important global regulator for both morphological differentiation and secondary metabolism in S. coelicolor and S. avermitilis.

Expression of the Streptococcus mutans essential two-component regulatory system VicRK is pH and growth-phase dependent and controlled by the LiaFSR three-component regulatory system

As an inhabitant of the human oral cavity, Streptococcus mutans faces frequent environmental changes. Two-component regulatory systems (TCSs) play a critical role in responding to these changes. Recently, an essential TCS, VicRKX, has been identified. The objective of this study was to identify the environmental signal and bacterial factors regulating the expression of the vicRKX operon. The promoter of the vicRKX operon was fused to a promoterless lacZ reporter gene and introduced into S. mutans UA159. LacZ plate assay identified pH, vancomycin, ampicillin, penicillin G and polymyxin B, but not carbohydrates, as factors affecting expression. Using RNA dot-blotting, high levels of vicR transcript were observed in cells at the mid- and late-exponential phase of growth and in cells grown in media buffered at pH 7.8. Given that vicR expression was pH-dependent, the genes encoding a putative pH-sensing three-component regulatory system (LiaFSR) were deleted. The liaS mutant exhibited upregulation of vicR regardless of the growth condition. The role of VicK, VicX, and the competence-signal peptide (CSP) was also investigated; the results showed that vicR expression was not autoregulated and was downregulated by the CSP in a ComX-independent manner. In conclusion, the expression of vicRKX is influenced by culture pH, growth phase and antibiotic stress, and is regulated by LiaFRS.

Molecular and biological characterization ofgtfregulation-associated genes inStreptococcus mutans

Surface protein antigen (PAc) and glucosyltransferases (GTF) are major adhesive molecules of Streptococcus mutans, though the mechanism of their regulation has not been fully elucidated. To investigate the regulation mechanism, we determined a nucleotide sequence in the upstream region of the pac locus in S. mutans and identified two open reading frames (ORF), designated as orf1 and orf2. Each ORF was inactivated and functional analyses were performed. Western blot analyses revealed that the expression level of PAc was unaffected, while that of cell-associated GTF was diminished in both mutant strains. Furthermore, they showed higher hydrophobicity levels and an impaired sucrose-dependent adherence to smooth surfaces. RNA dot blot analysis demonstrated that transcriptions of the gtfB and the gtfC genes, which encode GTF-I and GTF-SI, respectively, were downregulated, while that of pac was comparable to the wild-type strain. In addition, the GTF activities of the mutant strains were significantly lower than those of the wild-type, though a greater amount of total glucan produced by the mutants was noted in culture supernatants. These findings suggest that orf1 and orf2 are associated with positive regulation of the gtfB and gtfC genes.

Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath

A series of integrative and versatile broad-host-range promoter-probe vectors carrying reporter genes encoding green fluorescent protein (GFP), catechol 2,3-dioxygenase (XylE) or beta-galactosidase (LacZ) were constructed for use in methanotrophs. These vectors facilitated the measurement of in vivo promoter activity in methanotrophs under defined growth conditions. They were tested by constructing transcriptional fusions between the soluble methane monooxygenase (sMMO) sigma(54) promoter or particulate methane monooxygenase (pMMO) sigma(70) promoter from Methylococcus capsulatus and the reporter genes. Reporter gene activity was measured under high- and low-copper growth conditions and the data obtained closely reflected transcriptional regulation of the sMMO or pMMO operon, thus demonstrating the suitability of these vectors for assessing promoter activity in methanotrophs. When beta-galactosidase expression was coupled with the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide it yielded a sensitive and powerful screening system for detecting cells expressing this reporter gene. These data were substantiated with independent experiments using RT-PCR and RNA dot-blot analysis.

Component and protein domain exchange analysis of a thermoresponsive, two-component regulatory system of Pseudomonas syringae

Two closely related phytopathogenic bacterial strains, Pseudomonas syringae pv. glycinea PG4180 and P. syringae pv. tomato DC3000, produce the chlorosis-inducing phytotoxin coronatine (COR) in a remarkably divergent manner. PG4180 produces COR at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C. In contrast, temperature has no effect on COR synthesis in DC3000. A modified two-component system consisting of the histidine protein kinase (HPK), CorS, the response regulator (RR), CorR, and a third component, CorP, governs COR biosynthesis in both strains. A plasmid-based component and domain swapping approach was used to introduce different combinations of RRs, HPKs and hybrid HPKs into corS mutants of both strains. Subsequently, expression levels of the COR biosynthetic cma operon were determined using RNA dot-blot analysis, suggesting that CorRSP of PG4180 mediates a thermoresponsive phenotype dependent on the genomic background of each strain. The reciprocal experiment demonstrated a loss of temperature dependence in the corS mutant of PG4180. The presence of corR from PG4180 led to more pronounced cma expression in DC3000 and was associated with thermoresponsiveness, while corS of PG4180 did not mediate a temperature-dependent phenotype in the DC3000 mutant containing native corR and corP. These findings were substantiated by RT-PCR experiments. The C-terminal domain of CorS of PG4180 mediated thermosensing, while the N terminus did not respond to temperature changes, suggesting cytosolic perception of the temperature signal.

Enhanced expression of the tumour necrosis factor/cachectin gene in peripheral blood mononuclear cells from patients with systemic vasculitis

The expression of tumour necrosis factor-alpha (TNF-alpha) gene in peripheral blood mononuclear cells from patients with systemic vasculitis, periarteritis nodosa and Wegener's granulomatosis, has been studied by RNA dot blot and Northern blot assays. We further examined the transcriptional level of TNF-alpha gene in peripheral blood mononuclear cells from these patients by performing nuclear run on transcription assay. We demonstrate enhanced TNF-alpha gene expression in mononuclear cells from these patients compared with healthy subjects and patients with bronchial asthma. These findings suggest that the enhanced transcription of TNF-alpha gene in peripheral blood mononuclear cells from patients with systemic vasculitis may be involved in the pathophysiology/pathogenesis of these diseases by cytokine dysregulation.

MRNA Distribution and Heterologous Expression of Orphan Cytochrome P450 20A1

Cytochrome P450 (P450) 20A1 is one of the so-called "orphan" P450s without assigned biological function. mRNA expression was detected in human liver, and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3- to 4-fold higher in brain and heart than in liver. In situ hybridization of rat whole brain sections showed an mRNA expression pattern similar to that observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200-280 nmol of P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function.

Identification and Characterization of Duck Enteritis Virus dUTPase Gene

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous and important enzyme that hydrolyzes dUTP to dUMP. Many viruses encode virus-specific dUTPase, which plays an essential role in maintaining the integrity of the viral DNA both by reducing the dUTP levels and by providing the substrate for the thymidylate synthase. A 1344-bp gene of duck enteritis virus (DEV) homologous to herpesviral dUTPase was first reported in this paper. The gene encodes a protein of 477 amino acids, with a predicted molecular mass of 49.7 kDa. Multiple sequence alignment suggested that DEV dUTPase was quite similar to other identified herpesviral dUTPase and functioned as a homotrimer. The five conserved motifs of DEV dUTPase with 3-1-2-4-5 arrangement have been recognized, and the phylogenetic analysis showed that DEV dUTPase was genetically close to the avian herpesvirus. Furthermore, RNA dot blot, western blot, and immunofluorescence analysis indicated that the enzyme was expressed at early and late stages after infection. Immunofluorescence also confirmed that DEV dUTPase localized in the cytoplasm of DEV-infected duck embryo fibroblasts as early as 4 hr postinfection (hpi). Later, the enzyme transferred from cytoplasm to nucleus at 8 hpi, and then reached its expression peak at 12 hpi, both in the cytoplasm and nucleus. The results suggested that the DEV dUTPase gene might be an early viral gene in DEV vitro infection and contribute to ensuring the fidelity of genome replication.

Characterization of the RND-Type Multidrug Efflux Pump MexAB-OprM of the Plant Pathogen Pseudomonas syringae

In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB-oprM-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.

A comparison of in vivo targeted gene expression during fungal colonization of DED‐susceptible Ulmus americana

SummaryThis study examined in vivo gene expression associated with the colonization of Dutch elm disease‐susceptible Ulmus americana by H175, an aggressive strain of Ophiostoma novo‐ulmi Brasier. Stress‐related genes encoding phenylalanine ammonia‐lyase (PAL), chitinase (CHT) and polygalacturonase inhibiting protein (PGIP) were used to observe gene expression changes during U. americana colonization by H175. A novel non‐invasive method employing leaf midribs was used to observe plant gene expression, fungal colonization and mansonone F accumulation. RNA dot blots determined that all transcripts probed were induced during colonization compared with unchallenged controls. PAL and PGIP expression were increased in leaf midrib tissue prior to fungal colonization, suggesting a remote signal induction. CHT expression was increased locally with the presence of fungus, suggesting a local signal induction. Mansonone F accumulation was detected prior to fungal colonization suggesting a remote signal induction. The in vivo method used will provide a useful tool for in vivo disease research, particularly in trees, where chemically complex tissue can impede RNA isolation and downstream analysis.

Review article: nitric oxide from dysbiotic bacterial respiration of nitrate in the pathogenesis and as a target for therapy of ulcerative colitis

Factors initiating human ulcerative colitis (UC) are unknown. Dysbiosis of bacteria has been hypothesized to initiate UC but, to date, neither the nature of the dysbiosis nor mucosal breakdown has been explained. To assess whether a dysbiosis of anaerobic nitrate respiration could explain the microscopic, biochemical and functional changes observed in colonocytes of UC. Published results in the gastroenterological, biochemical and microbiological literature were reviewed concerning colonocytes, nitrate respiration and nitric oxide in the colon in health and UC. A best-fit explanation of results was made regarding the pathogenesis and new treatments of UC. Anaerobic nitrate respiration yields nitrite, nitric oxide (NO) and nitrous oxide. Colonic bacteria produce NO and UC in remission has a higher lumenal NO level than control cases. NO with sulphide, but not NO alone, impairs beta-oxidation, lipid and protein synthesis explaining the membrane, tight junctional and ion channel changes observed in colonocytes of UC. The observations complement therapeutic mechanisms of those probiotics, prebiotics and antibiotics useful in treating UC. The prolonged production of bacterial NO with sulphide can explain the initiation and barrier breakdown, which is central to the pathogenesis of UC. Therapies to alter bacterial nitrate respiration and NO production need to evolve. The production of NO by colonic bacteria and that of the mucosa need to be separated to pinpoint the sequential nature of NO damage in UC.

Physiological characterization and identification of genes differentially expressed in response to drought induced by PEG 6000 in Populus canadensis leaves

We report here about the physiological and molecular responses of Populus canadensis (clone Dorskamp) to drought. The stress was applied to young rooted cuttings by PEG 6000 application over 30 days. This stress induces a decrease in predawn leaf water potential. After 10 days of stress, there was a decrease in stomatal conductance and a slight retardation of leaf growth, but the osmotic potential remained constant. Using the differential display technique, we searched for genes differentially expressed in response to drought at this date. Thirty-six differentially expressed leaf cDNAs were detected between stressed and control conditions. Thirty-four cDNAs clones were successfully cloned and 23 were found to share high identity with Arabidopsis thaliana and Populus trichocarpa genes. The transcriptional regulation of 21 genes was examined by reverse RNA dot blot, confirming an increase in expression for 16 of them after 10 days of treatment. Among these 16 genes, most of them are involved in a different cellular metabolic pathway. These differentially expressed genes are also involved and/or regulated by other treatments such as salt, withholding water or auxin application. The maintenance of growth observed during the first 10 days of the stress period could be due to the regulation of these genes and can be a common response between herbaceous plants and trees.

RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1α during Hypoxia

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.

Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds

To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21-47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm(-3). Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B.

ENOS Deficient Mice Develop Progressive Cardiac Hypertrophy with Altered Cytokine and Calcium Handling Protein Expression

Although studies have shown that endothelial nitric oxide synthase (eNOS) homozygous knockout mice (eNOS-/-) develop left ventricular (LV) hypertrophy, well compensated at least to 24 wks, uncertainty still exists as to the cardiac functional and molecular mechanistic consequences of eNOS deficiency at later time-points. To bridge the gap in existent data, we examined whole hearts from eNOS-/- and age-matched wild-type (WT) control mice ranging in age from 18 to 52 wks for macroscopic and microscopic histopathology, LV mRNA and protein expression using RNA Dot blots and Western blots, respectively, and LV function using isolated perfused work-performing heart preparations. Heart weight to body weight (HW/BW in mg/g) ratio increased significantly as eNOS-/- mice aged (82.2%, P < 0.001). Multi-focal replacement fibrosis and myocyte degeneration/death were first apparent in eNOS-/- mouse hearts at 40 wks. Progressive increases in LV atrial natriuretic factor (ANF) and alpha-skeletal actin mRNA levels both correlated significantly with increasing HW/BW ratio in aged eNOS-/- mice (r = 0.722 and r = 0.648, respectively; P < 0.001). At 52 wks eNOS-/- mouse hearts exhibited basal LV hypercontractility yet blunted beta adrenergic receptor (betaAR) responsiveness that coincided with a significant reduction in the LV ratio of phospholamban to sarcoplasmic reticulum Ca2+-ATPase-2a protein levels and was preceded by a significant upregulation in LV steady-state mRNA and protein levels of the 28 kDa membrane-bound form of tumor necrosis factor-alpha. We conclude that absence of eNOS in eNOS-/- mice results in a progressive concentric hypertrophic cardiac phenotype that is functionally compensated with decreased betaAR responsiveness, and is associated with a potential cytokine-mediated alteration of calcium handling protein expression.

기름으로 오염된 토양에서 작물생육을 위한 계면활성제 생산 Bacteria의 활용에 관한 연구

Bacillus LPO3 (계면활성제와 같이 유화물질을 생산하는)을 hydrocarbon을 감소시키는 bio-control agent로 사용했다. 토양 (시중구입)은 gasoline을 섞어 오염시킨 토양을 사용하였다. 13일 동안 bacteria의 성장, hydrocarbon의 감소, 그리고, Bacillus LP03 의 작물 성장에 미친 요인(식물의 싹과 뿌리의 길이를 포함한)을 관찰하였다. 우리는 이 bacteria의hydrocarbon을 감소 시키는 물질로서 이 균이 생산하는 계면활성제일 것이라는 가능성을 이미 확인 하였으며 이는 식물의 싹과 뿌리의 생장을 촉진하였고, 회색 곰팡이에 대하여항 진균 활성을 보였다. 본 연구에서는 bacteria를 넣지 않은 기름으로 오염된 토양 실험 군에서는 식물이 자라나지 않았을 뿐 아니라 살아남지도 못했다. 실험 결과로 볼 때, gasoline의 hydrocarbon이 시간이 지남에 따라 감소되었고, RNA blotting실험에서는 오염된 토양에서 균의 증식과 더불어 계면활성제관련 유전자 산물의 증가를 확인하였다 결과적으로, 이 bacteria (계면활성제를 생산하는)는 회색 곰팡이에 감염된 토양이나 기름으로 오염된 토양에서 작물이 자랄 수 있게 하기 위한 bio-control agent로서 유효한 균으로 생각된다. Bacillus sp.LPO3 (producing emulsifying substances such as bio-surfactant) was used as a bio-control agent to degrade hydrocarbon (gasoline in oil spilled crop soil). The soil (brought from fertilizer store)was mixed with gasoline-spilled soil (made with Diatomaceous Earth, Sigma.U.S.A). The study was conducted for a period of 13 days, 13 days during which bacterial growth, hydrocarbon degradation and growth parameters of Bacillus sp.LP03 including shoot and root length were studied. We found that the effective of bacterial producing substance might bio-surfactants let the plants survive even more promote the growth of shoot and root length and showed antifungal activity against gray mold. Without the bacteria, they couldn't grow in oil-spilled soil not even survive. According to the results of the above experiments, we can see with following results, hydrocarbon in gasoline was reduced, day by day, then RNA dot blotting was done and it fit the results we had done. Finally, this Bacteria(producing bio-surfactant) were found to have effective bio-control agent for cropping in oil spilled soil and infected by gray mold.

Soybean dwarf virus-resistant transgenic soybeans with the sense coat protein gene

We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.

Identification of two new genes from drought tolerant peanut up-regulated in response to drought

Using differential display of mRNA transcripts, we obtained nine partial cDNA clones that were up-regulated and down-regulated by exposure to drought. Two full length cDNAs of 825 bp and 700 bp, designated as Arachis hypogaea serine rich protein (AhSrp) and Arachis hypogaea leucine rich protein (AhLrp) were obtained using RLM-RACE. Based on the cDNA sequence, the transcriptional start site of AhSrp and AhLrp was assigned. AhSrp is predicted to code for a protein of 233 amino acids containing a signal peptide, three potential transmembrane domains and a sumolyation motif. AhLrp is predicted to code for a protein of 206 amino acids containing a signal peptide. The expression of partial cDNA and full-length cDNA were confirmed by RNA dot blot analysis. Expression of AhSrp was observed between 6 and 10 days of stress and prominent expression of AhLrp was visualized at 10 days of stress by semi quanititative PCR. Our studies have resulted in the identification of two new genes that may play a role in plant response to drought stress in peanut.