Suppression Subtractive Hybridization Technique Research Articles

Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver.

Growth hormone (GH) activates multiple signal transduction pathways. To investigate these pathways, we identified novel genes whose transcription was induced by GH in the liver of hypophysectomized (HPX) rats using the suppression subtractive hybridization technique. We found that regulator of calcineurin 1 (Rcan1) mRNA was upregulated by GH administration. RCAN1 regulates the activity of calcineurin, a Ca/calmodulin-dependent phosphatase. Rcan1 encodes two major transcripts, Rcan1-1 and Rcan1-4, resulting from differential promoter use and first exon choice. We found that a single injection of GH increased the levels of Rcan1-4 mRNA and RCAN1-4 protein transiently, but did not increase Rcan1-1 mRNA in HPX rat liver. Then the molecular mechanism of GH to induce Rcan1-4 transcription was examined in rat hepatoma H4IIE cells. Experiments using inhibitors suggested that c-JUN N-terminal kinase was required for the induction of Rcan1-4 mRNA by GH. GH increased the levels of phosphorylated c-JUN protein and c-Jun mRNA in HPX rat liver. The luciferase and electrophoretic mobility shift assays showed that c-JUN upregulated Rcan1-4 mRNA by binding to the cAMP-responsive element in the upstream of Rcan1 exon 4. These results indicate that GH activates c-JUN to affect the activity of calcineurin by the induction of Rcan1-4 in rat liver.

An improved suppression subtractive hybridization technique to develop species-specific repetitive sequences from Erianthus arundinaceus (Saccharum complex)

BackgroundSugarcane has recently attracted increased attention for its potential as a source of bioethanol and methane. However, a narrow genetic base has limited germplasm enhancement of sugarcane. Erianthus arundinaceus is an important wild genetic resource that has many excellent traits for improving cultivated sugarcane via wide hybridization. Species-specific repetitive sequences are useful for identifying genome components and investigating chromosome inheritance in noblization between sugarcane and E. arundinaceus. Here, suppression subtractive hybridization (SSH) targeting E. arundinaceus-specific repetitive sequences was performed. The five critical components of the SSH reaction system, including enzyme digestion of genomic DNA (gDNA), adapters, digested gDNA concentrations, primer concentrations, and LA Taq polymerase concentrations, were improved using a stepwise optimization method to establish a SSH system suitable for obtaining E. arundinaceus-specific gDNA fragments.ResultsSpecificity of up to 85.42% was confirmed for the SSH method as measured by reverse dot blot (RDB) of an E. arundinaceus subtractive library. Furthermore, various repetitive sequences were obtained from the E. arundinaceus subtractive library via fluorescence in situ hybridization (FISH), including subtelomeric and centromeric regions. EaCEN2-166F/R and EaSUB1-127F/R primers were then designed as species-specific markers to accurately validate E. arundinaceus authenticity.ConclusionsThis is the first report that E. arundinaceus-specific repetitive sequences were obtained via an improved SSH method. These results suggested that this novel SSH system could facilitate screening of species-specific repetitive sequences for species identification and provide a basis for development of similar applications for other plant species.

Differentially expressed genes in the midguts of BmNPV-susceptible and resistant silkworm strains determined using suppression subtractive hybridization

Bombyx mori nuclear polyhedrosis virus (BmNPV) has caused great economic losses to sericulture and considerable effort has been made to identify disease resistance genes in B. mori. We constructed differential expression gene libraries of the resistant near-isogenic line (NIL) BC10 and susceptible strain 306 using the suppression subtractive hybridization technique. A total of 23 differentially expressed genes were obtained, of which 17 genes were upregulated in BC10, and 6 genes were upregulated in 306. These differentially expressed genes are involved in cell metabolism, transmembrane transport, cytoskeleton, protease, development and immunity. Six BC10 upregulated genes were verified by real-time quantitative PCR, and the results were consistent with those of subtractive hybridization. The resistance trait of NIL BC10 was inherited from the resistant strain NB, and its genetic background was 99.99% similar to that of recurrent parent 306. The upregulated genes in NIL BC10 may be associated with silkworm BmNPV resistance.

Identification and expression patterns of 20-hydroxyecdysone-responsive genes from Procambarus clarkii

Ecdysteroids play an important role in the regulation of molting process in crustaceans. The suppression subtractive hybridization (SSH) technique was employed to search for 20-hydroxyecdysone-responsive genes in the hepatopancreas of the crayfish Procambarus clarkii. Ninety-three putative expressed sequence tags were identified by SSH, including three immune response-related genes, two cell cycle and apoptosis genes, four respiration and energy metabolism genes, four transport-related genes, six metabolism-related genes, two stress response genes, and eight transcription and translation regulation genes. The expression levels of the examined genes were regulated by 20-hydroxyecdysone and varied in the hepatopancreas during the molting stages; data were confirmed by real-time PCR. RNAi of the ecdysteroid receptor had significant effects on the expression levels of 20-hydroxyecdysone-responsive genes. These differentially expressed genes identified by SSH will provide insight into the ecdysteroid signaling pathway in P. clarkii.

Suppressive activity of a viral histone H4 against two host chromatin remodelling factors: lysine demethylase and SWI/SNF.

Histone H4, a nucleosome subunit in eukaryotes, plays crucial roles in DNA package and regulation of gene expression through covalent modification. A viral histone H4 encoded in Cotesia plutellae bracovirus (CpBV), a polydnavirus, is called CpBV-H4. It is highly homologous to other histone H4 proteins excepting 38 extra amino acid residues in the N terminus. CpBV-H4 can form octamer with other histone subunits and alter host gene expression. In this study, CpBV-H4 was transiently expressed in a natural host (Plutella xylostella) and its suppressive activity on host gene expression was evaluated by the suppressive subtractive hybridization (SSH) technique. The SSH targets down-regulated by CpBV-H4 were read with the 454 pyrosequencing platform and annotated using the genome of P. xylostella. The down-regulated genes (610 contigs) were annotated in most functional categories based on gene ontology. Among these SSH targets, 115 genes were functionally distinct, including two chromatin remodelling factors: a lysine-specific demethylase (Px-KDM) and a chromatin remodelling complex [Px-SWI/SNF (SWItch/Sucrose Non-Fermentable)]. Px-KDM was highly expressed in all tested tissues during the entire larval period. Suppression of Px-KDM expression by specific RNA interference (RNAi) significantly (P<0.05) reduced haemocyte nodule formation in response to immune challenge and impaired both larval and pupal development. Px-SWI/SNF was expressed in all developmental stages. Suppression of Px-SWI/SNF expression by RNAi reduced cellular immune response and interfered with adult metamorphosis. These results suggest that CpBV-H4 can alter host gene expression by interfering with chromatin modification and remodelling factors in addition to its direct epigenetic control activity.

Hypoxic stress -responsive genes in air breathing catfish, Clarias magur (Hamilton 1822) and their possible physiological adaptive function

The Indian catfish, Clarias magur (previous name C. batrachus) is an air breathing fish, inhabitant of aquatic bodies characterized by low dissolved oxygen levels. It is exposed to hypoxic conditions in its natural habitat. Thus, it can be useful model to study the mechanism of hypoxia stress tolerance. In C. magur, molecular processes facilitating its adaptation to hypoxia stress remain largely unexplored, in part due to unavailability of genomic resources. The suppression subtractive hybridization technique (SSH) was employed to compare the differential expression of transcripts under experimental hypoxic conditions, to that of normoxic conditions. Twelve subtracted cDNA libraries (six each forward and reverse) were constructed from brain, heart, liver, muscle, spleen and head kidney tissues. A total of 2020 clones were screened and sequenced, resulting into 1805 high quality expressed sequence tags (ESTs). Annotation of these differentially expressed ESTs resulted into the identification of genes involved in vast majority of pathways/processes affecting metabolism, cellular processes, signal transduction and/or immune functions. Additionally, 18 potential novel genes expressed in hypoxia stress exposed fish were also identified. The study had catalogued the differentially expressed genes from hypoxia stress induced C. magur, where most of them are reported for the first time in a hypoxia-tolerant fish species. The results not only provided insights for the hypoxia stress altered cellular functions in C. magur, but also generated a valuable functional genomics resource to assist targeted studies on functional genomics and future genome projects.

T-cell factor-4 and MHC upregulation in pigs receiving a live attenuated classical swine fever virus (CSFV) vaccine strain with interferon-gamma adjuvant

The effect of co-administration of interferon (IFN)-γ in pigs undergoing vaccination with an attenuated strain (LPC) of classical swine fever virus (CSFV) was investigated.Unvaccinated pigs demonstrated pyrexia and died 7–9 days after challenge with virulent CSFV. Pigs receiving the attenuated vaccine remained healthy after virus challenge, except for mild, transient pyrexia, whereas pigs receiving IFN-γ simultaneously with the vaccine demonstrated normal body temperatures after virus challenge. Examination by nested RT-PCR revealed greater viral load in the spleens of the pigs vaccinated with the attenuated CSFV, compared with those that had additionally received IFN-γ. Expression of major histocompatibility complex (MHC) class I and MHC class II molecules was upregulated in the spleens of the IFN-γ treated vaccinated pigs, demonstrated by immunohistochemistry. Based on Western blot analysis, anti-CSFV IgG2 antibodies were elevated in vaccinated pigs by co-administration of IFN-γ (IFN-γHi: P < 0.01; IFN-γLo: P < 0.05). By employing the suppression subtractive hybridization technique, RT-PCR, in situ hybridization, and immunohistochemistry, T-cell factor-4 (Tcf-4) mRNA and protein expression were found to be upregulated in the spleens of vaccinated pigs that had received IFN-γ. This study suggests involvement of Tcf-4 in IFN-γ-mediated immune regulation following CSFV vaccination.

FKBP-type peptidyl-prolyl cis-trans isomerase from thermophilic microalga, Scenedesmus sp.: molecular characterisation and demonstration of acquired salinity and thermotolerance in E. coli by recombinant expression

A novel complementary DNA (cDNA) encoding temperature and salt stress-responsive FK506-binding protein 12 (FKBP12), a chaperone with peptidyl-prolyl cis-trans isomerase (PPIase) activity was isolated using suppression subtractive hybridization (SSH) technique from a thermophilic chlorophycean microalga, Scenedesmus sp., isolated from the Manikaran hot springs at Himachal Pradesh, India. To our best knowledge, this is the first report of temperature and salt-responsive FKBP12 isolated from a microalgae. The Scenedesmus FKBP12 (Sce.FKBP12) nucleotide sequence revealed the presence of an open reading frame of 327 bp, encoding 108 amino acid protein containing a single FKBP-like domain with a predicted molecular weight of 12 KDa. Amino acid sequence comparison of Sce.FKBP12 with its ortholog from other eukaryotic organisms showed structural conservation among each other. Expression profile of Sce.FKBP12 gene using quantitative real-time PCR showed significant up-regulated expression under temperature and salinity stress. The recombinant Sce.FKBP12 was over-expressed in Escherichia coli using pET28b expression vector. The recombinant expression of Sce.FKBP12 in E. coli cells imparted temperature and salinity tolerance to the host cells, implying a significant role of Sce.FKBP12 in stress tolerance mechanisms. Sce.FKBP12 could be a promising transgene candidate for imparting abiotic stress tolerance in crop plants, and they need to be further evaluated using plant models like Arabidopsis or Tobacco for confirming its potential as a transgene candidate in development of temperature and salinity-tolerant transgenic crops for improved and sustained productions in adverse agro-climatic conditions.

Downregulation of SWI5 and CTC1 genes: hepatitis B virus DNA polymerase transactivated protein 1-mediated inhibition of DNA repair.

Hepatitis B virus (HBV) DNA polymerase transactivated protein 1 (HBVDNAPTP1) is a novel protein upregulated by HBV DNA polymerase, which has been screened by suppression subtractive hybridization technique (SSH) (GenBank Acc. No. AY450389). A vector pcDNA3.1 (-)/myc-His A-HBVDNAPTP1 was constructed and used to transfect acute monocytic leukemia cell line THP-1. HBVDNAPTP1 expression was detected by Western blot analysis in the cells. A cDNA library of genes downregulated by HBVDNAPTP1 in THP-1 cells was made in pGEM-T Easy using SSH. The cDNAs were sequenced and analyzed with BLAST search against the sequences in GenBank. Some sequences, such as DNA repair protein SWI5 homolog (SWI5) and CTS telomere maintenance complex component 1 (CTC1), might be involved in DNA repair. Protein expression of SWI5 and CTC1 was identified by Western blot in THP-1 cells. HBVDNAPTP1 could downregulate the expression of SWI5 and CTC1 at translation level.

DNA Subtraction of In Vivo Selected Phage Repertoires for Efficient Peptide Pathology Biomarker Identification in Neuroinflammation Multiple Sclerosis Model.

To streamline in vivo biomarker discovery, we developed a suppression subtractive DNA hybridization technique adapted for phage-displayed combinatorial libraries of 12 amino acid peptides (PhiSSH). Physical DNA subtraction is performed in a one-tube-all-reactions format by sequential addition of reagents, producing the enrichment of specific clones of one repertoire. High-complexity phage repertoires produced by in vivo selections in the multiple sclerosis rat model (experimental autoimmune encephalomyelitis, EAE) and matched healthy control rats were used to evaluate the technique. The healthy repertoire served as a physical DNA subtractor from the EAE repertoire to produce the subtraction repertoire. Full next-generation sequencing (NGS) of the three repertoires was performed to evaluate the efficiency of the subtraction technique. More than 96% of the clones common to the EAE and healthy repertoires were absent from the subtraction repertoire, increasing the probability of randomly selecting various specific peptides for EAE pathology to about 70%. Histopathology experiments were performed to confirm the quality of the subtraction repertoire clones, producing distinct labeling of the blood–brain barrier (BBB) affected by inflammation among healthy nervous tissue or the preferential binding to IL1-challenged vs. resting human BBB model. Combining PhiSSH with NGS will be useful for controlled in vivo screening of small peptide combinatorial libraries to discover biomarkers of specific molecular alterations interspersed within healthy tissues.

Identification and Characterization of Differentially Expressed Genes in Inferior and Superior Spikelets of Rice Cultivars with Contrasting Panicle-Compactness and Grain-Filling Properties.

Breeding programs for increasing spikelet number in rice have resulted in compactness of the panicle, accompanied by poor grain filling in inferior spikelets. Although the inefficient utilization of assimilate has been indicated as responsible for this poor grain filling, the underlying cause remains elusive. The current study utilized the suppression subtractive hybridization technique to identify 57 and 79 genes that overexpressed in the superior and inferior spikelets (with respect to each other), respectively, of the compact-panicle rice cultivar Mahalaxmi. Functional categorization of these differentially expressed genes revealed a marked metabolic difference between the spikelets according to their spatial location on the panicle. The expression of genes encoding seed storage proteins was dominant in inferior spikelets, whereas genes encoding regulatory proteins, such as serine-threonine kinase, zinc finger protein and E3 ligase, were highly expressed in superior spikelets. The expression patterns of these genes in the inferior and superior spikelets of Mahalaxmi were similar to those observed in another compact-panicle cultivar, OR-1918, but differed from those obtained in two lax-panicle cultivars, Upahar and Lalat. The results first suggest that the regulatory proteins abundantly expressed in the superior spikelets of compact-panicle cultivars and in both the superior and inferior spikelets of lax-panicle cultivars but poorly expressed in the inferior spikelets of compact-panicle cultivars promote grain filling. Second, the high expression of seed-storage proteins observed in the inferior spikelets of compact-panicle cultivars appears to inhibit the grain filling process. Third, the low expression of enzymes of the Krebs cycle in inferior spikelets compared with superior spikelets of compact-panicle cultivars is bound to lead to poor ATP generation in the former and consequently limit starch biosynthesis, an ATP-consuming process, resulting in poor grain filling.

Differential expression of genes involved in Bengal macrothrombocytopenia (BMTCP)

Bengal macrothrombocytopenia (BMTCP) is a giant platelet disorder with mild to moderate thrombocytopenia, clinically characterized by mild bleeding symptoms to totally asymptomatic condition. The pathophysiological mechanism of this condition is not fully understood yet. In the present study, 5 subjects (P1–P5) with BMTCP whose platelet counts ranged between 36140X109/l and mean platelet volume (MPV)13.5–16.1fl were analyzed for differential gene expression of platelets by suppressive subtractive hybridization (SSH) technique. Four genes i.e. myotubularin related protein 9 (MTMR9), iron responsive element binding protein 2 (IREB2), alpha tubulin(TUBA) and tyrosine kinase ligand (TKL) were found to be differentially expressed in patient platelets as compared to that of normal healthy controls which was further confirmed by quantitative RT PCR analysis. The study highlights a multi-factorial etiology for BMTCP which is widely prevalent in the northeastern region of the Indian subcontinent.

Cadmium induces cadmium-tolerant gene expression in the filamentous fungus Trichoderma harzianum

The filamentous fungus Trichoderma harzianum, strain IMI 393899, was able to grow in the presence of the heavy metals cadmium and mercury. The main objective of this research was to study the molecular mechanisms underlying the tolerance of the fungus T. harzianum to cadmium. The suppression subtractive hybridization (SSH) method was used for the characterization of the genes of T. harzianum implicated in cadmium tolerance compared with those expressed in the response to the stress induced by mercury. Finally, the effects of cadmium exposure were also validated by measuring the expression levels of the putative genes coding for a glucose transporter, a plasma membrane ATPase, a Cd(2+)/Zn(2+) transporter protein and a two-component system sensor histidine kinase YcbA, by real-time-PCR. By using the aforementioned SSH strategy, it was possible to identify 108 differentially expressed genes of the strain IMI 393899 of T. harzianum grown in a mineral substrate with the addition of cadmium. The expressed sequence tags identified by SSH technique were encoding different genes that may be involved in different biological processes, including those associated to primary and secondary metabolism, intracellular transport, transcription factors, cell defence, signal transduction, DNA metabolism, cell growth and protein synthesis. Finally, the results show that in the mechanism of tolerance to cadmium a possible signal transduction pathway could activate a Cd(2+)/Zn(2+) transporter protein and/or a plasma membrane ATPase that could be involved in the compartmentalization of cadmium inside the cell.

Genetic differences between two Leishmania major-like strains revealed by suppression subtractive hybridization

Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of β-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.

Identification of genes associated with blood feeding in the cat flea, Ctenocephalides felis.

BackgroundThe cat flea (Ctenocephalides felis) is a blood-feeding ectoparasitic insect and particular nuisance pest of companion animals worldwide. Identification of genes that are differentially expressed in response to feeding is important for understanding flea biology and discovering targets for their control.MethodsC. felis fleas were maintained and fed for 24 h using an artificial rearing system. The technique of suppression subtractive hybridization was employed to screen for mRNAs specifically expressed in fed fleas.ResultsWe characterized nine distinct full-length flea transcripts that exhibited modulated or de novo expression during feeding. Among the predicted protein sequences were two serine proteases, a serine protease inhibitor, two mucin-like molecules, a DNA topoisomerase, an enzyme associated with GPI-mediated cell membrane attachment of proteins and a component of the insect innate immune response.ConclusionsOur results provide a molecular insight into the physiology of flea feeding. The protein products of the genes identified may play important roles during flea feeding in terms of blood meal digestion, cellular growth/repair and protection from feeding-associated stresses.

Transcript dynamics at early stages of molecular interactions of MYMIV with resistant and susceptible genotypes of the leguminous host, Vigna mungo.

Initial phases of the MYMIV- Vigna mungo interaction is crucial in determining the infection phenotype upon challenging with the virus. During incompatible interaction, the plant deploys multiple stratagems that include extensive transcriptional alterations defying the virulence factors of the pathogen. Such molecular events are not frequently addressed by genomic tools. In order to obtain a critical insight to unravel how V. mungo respond to Mungbean yellow mosaic India virus (MYMIV), we have employed the PCR based suppression subtractive hybridization technique to identify genes that exhibit altered expressions. Dynamics of 345 candidate genes are illustrated that differentially expressed either in compatible or incompatible reactions and their possible biological and cellular functions are predicted. The MYMIV-induced physiological aspects of the resistant host include reactive oxygen species generation, induction of Ca2+ mediated signaling, enhanced expression of transcripts involved in phenylpropanoid and ubiquitin-proteasomal pathways; all these together confer resistance against the invader. Elicitation of genes implicated in salicylic acid (SA) pathway suggests that immune response is under the regulation of SA signaling. A significant fraction of modulated transcripts are of unknown function indicating participation of novel candidate genes in restricting this viral pathogen. Susceptibility on the other hand, as exhibited by V. mungo Cv. T9 is perhaps due to the poor execution of these transcript modulation exhibiting remarkable repression of photosynthesis related genes resulting in chlorosis of leaves followed by penalty in crop yield. Thus, the present findings revealed an insight on the molecular warfare during host-virus interaction suggesting plausible signaling mechanisms and key biochemical pathways overriding MYMIV invasion in resistant genotype of V. mungo. In addition to inflate the existing knowledge base, the genomic resources identified in this orphan crop would be useful for integrating MYMIV-tolerance trait in susceptible cultivars of V. mungo.

IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY EXPRESSED GENES ASSOCIATED WITH CITRUS BLIGHT (Citrus spp.)

Brazil is the largest citrus producer in the world, being responsible for more than 20% of its production, which is, however still low due to phytosanitary issues such as citrus blight. Citrus blight is an anomaly whose causes still have not yet been determined, therefore there are no efficient control measures to minimize the production losses with the use of resistant varieties being considered the most appropriate method. However, little is known about the genes involved in the defense response of the plants to this anomaly. Considering that many physiological alterations associated with plant stress responses are controlled at a transcriptional level, in this study we sought the identification and characterization of the gene expression products differentially expressed in the response to the citrus blight. Through the suppressive subtractive hybridization technique, expressed cDNA libraries were built using mRNAs isolated from "Cravo" lemon tree roots (Citrus limonia L. Osbeck) under "Pera" orange (Citrus sinensis L. Osbeck) of healthy and sick plants. 129 clones were obtained by subtraction and their sequences were compared in databases. 34 of them linked to proteins associated to stress processes, while the others were similar to sequences of unknown functions or did not present similarity with sequences deposited in the databases. 3 genes were selected and their expressions were studied by RT - qPCR in real-time. Plants with citrus blight presented an increase of the expression level in two of those genes, suggesting that these can be directly involved with this anomaly.

Identification of Differentially Expressed Genes in Sugarcane During Pathogenesis of Colletotrichum falcatum by Suppression Subtractive Hybridization (SSH)

A suppression subtractive hybridization technique was used to identify differentially expressed genes in sugarcane in response to Colletotrichum falcatum, the fungal pathogen causing red rot in sugarcane. Messenger RNA (mRNA) samples collected from red rot resistant cv. Co 93009 at 6, 12, 24 and 48 h after pathogen challenge were pooled together and used as tester population. mRNA samples collected from red rot susceptible cv. CoC 671 at 6, 12, 24 and 48 h after pathogen challenge and control of samples of cv. Co 93009 and cv. CoC 671 were pooled together and used as the driver population. A forward subtraction enriched for differentially expressed ESTs in the resistant cultivar was carried out. At the end of subtraction, cloning and sequencing, 136 EST sequences were assembled into ten clusters/contigs. Based on TIGR homology search, the clusters were found to be involved in reactive oxygen species signaling, defense and the secretory pathway of plant innate immunity associated with hypersensitive response—mediated programmed cell death. Temporal gene expression pattern of the EST clusters by quantitative real time PCR revealed that, resistant cultivar had a higher level of gene expression than the susceptible cultivar in all the clusters till 48 h after pathogen inoculation. In conclusion, this study identified for the first time a set of differentially expressed EST clusters in red rot resistant sugarcane cultivar in response to C. falcatum infection.

Identification and characterization of drought stress responsive genes in faba bean (Vicia faba L.) by suppression subtractive hybridization

Faba bean (Vicia faba L.) ranks fourth in food legume crop production in the world. However, drought is a potential major constraint to faba bean production and improved faba bean cultivars and development of drought-resistant varieties play a key role in enhancing faba bean crop production. In this study, suppression subtractive hybridization (SSH) technique was used to study differential expression in response to water stress and to identify genes involved in molecular mechanism of drought tolerance. A forward subtractive cDNA library induced by water deficit conditions was constructed used Hara faba bean cultivar grown in pots and treated with either well-watered (WW) or water-stressed (WS). A total of 28 clones were identified as drought stress induced. After sequencing, ten unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis which showed homology to known drought responsive genes including heat shock protein (HSP), late embryogenic abundant (LEA), zinc finger protein transcription factors (ZFP), lipid transfer protein (LTP), chlorophyll a/b-binding protein (ChlBP), thioredoxin h (Trx h), and ATP synthase as well as some functionally unknown transcripts. Their expression was characterized in Leaf, root, flower, cotyledon, and stem tissue. Quantitative RT-PCR analysis revealed that eight genes were consistently up-regulated in Hara compared to Giza3 cultivar, known as drought-tolerant and sensitive respectively under water deficit treatment. The expression of six genes was differentially expressed in different stages of water stress faba bean plant. Drought responsive genes showed changed expression patterns, indicating that they may play important roles in faba bean water stress response. Furthermore, these results indicate that drought-induced genes are related to metabolic pathways and genetic regulation of stress and development and can serve as a foundation for future studies to elucidate drought stress mechanisms of faba bean.

Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs.

BackgroundMosquito-borne diseases threaten over half the world’s human population, making the need for environmentally-safe mosquito population control tools critical. The sterile insect technique (SIT) is a biological control method that can reduce pest insect populations by releasing a large number of sterile males to compete with wild males for female mates to reduce the number of progeny produced. Typically, males are sterilized using radiation, but such methods can reduce their mating competitiveness. The method is also most effective if only males are produced, but this requires the development of effective sex-sorting methods. Recent efforts to use transgenic methods to produce sterile male mosquitoes have increased interest in using SIT to control some of our most serious disease vectors, but the release of genetically modified mosquitoes will undoubtedly encounter considerable delays as regulatory agencies deal with safety issues and public concerns.MethodsTestis genes in the dengue vector Aedes aegypti were identified using a suppression subtractive hybridization technique. Mosquito larvae were fed double-stranded RNAs (dsRNAs) that targeted both the testis genes and a female sex determination gene (doublesex) to induce RNA interference (RNAi) -mediated sterility and inhibition of female development. Fertility and mating competiveness of the treated males were assessed in small-scale mating competition experiments.ResultsFeeding mosquito larvae dsRNAs targeting testis genes produced adult males with greatly reduced fertility; several dsRNAs produced males that were highly effective in competing for mates. RNAi-mediated knockdown of the female-specific isoform of doublesex was also effective in producing a highly male-biased population of mosquitoes, thereby overcoming the need to sex-sort insects before release.ConclusionsThe sequence-specific gene-silencing mechanism of this RNAi technology renders it adaptable for species-specific application across numerous insect species. We envisage its use for traditional large-scale reared releases of mosquitoes and other pest insects, although the technology might also have potential for field-based control of mosquitoes where eggs deposited into a spiked larval site lead to the release of new sterile males.