Suppression Subtractive Hybridization cDNA Research Articles

Identification of salt-induced transcripts by suppression subtractive hybridization and their expression analysis under the combination of salt and elevated CO2 conditions in Salicornia brachiata

Soil salinity is a major abiotic stress that affects global agricultural productivity. Exploring the mechanisms that halophytes employ to thrive and flourish under saline environments is essential to increase the salt tolerance in sensitive crop species. Of the three halophytes used in this study Salicornia brachiata and Suaeda maritima belong to the same family Chenopodiaceae, while Sesuvium portulacastrum, a mangrove-associated halophyte, belongs to the family Aizoaceae. Assuming that halophytes of same family share similar salt tolerance mechanisms, we generated a suppression subtractive hybridization (SSH1) cDNA library from salt-treated leaf tissues of S. brachiata as tester and that of S. maritima as driver to identify salt-responsive genes unique to S. brachiata. To elucidate the difference in salt-tolerance mechanisms, and to identify salt-tolerance mechanisms amongst different families of halophytes, SSH2 library was generated from salt-treated leaf tissue of S. brachiata as tester and that of S. portulacastrum as driver. Totally, 87 and 49 EST clones representing unique genes were obtained from SSH1 and SSH2 libraries, respectively. Examination of the expression patterns of 17 (SSH1) and 15 (SSH2) differentially expressed genes using semi-quantitative RT-PCR confirmed up-regulation of these genes in shoots in response to salt treatment and elevated CO2 condition, but to a different extent. This study has provided insights into the molecular responses of S. brachiata to salt stress and elevated CO2 conditions.

Development and mapping of functional expressed sequence tag-derived simple sequence repeat markers in a rubber tree RRIM600 × PB217 population

Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.

Activation of Shikimate, Phenylpropanoid, Oxylipins, and Auxin Pathways in Pectobacterium carotovorum Elicitors-Treated Moss.

Plants have developed complex defense mechanisms to cope with microbial pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are perceived by pattern recognition receptors (PRRs), leading to the activation of defense. While substantial progress has been made in understanding the activation of plant defense by PAMPs and DAMPs recognition in tracheophytes, far less information exists on related processes in early divergent plants like mosses. The aim of this study was to identify genes that were induced in P. patens in response to elicitors of Pectobacterium carotovorum subsp. carotovorum, using a cDNA suppression subtractive hybridization (SSH) method. A total of 239 unigenes were identified, including genes involved in defense responses related to the shikimate, phenylpropanoid, and oxylipin pathways. The expression levels of selected genes related to these pathways were analyzed using quantitative RT-PCR, confirming their rapid induction by P.c. carotovorum derived elicitors. In addition, P. patens induced cell wall reinforcement after elicitor treatment by incorporation of phenolic compounds, callose deposition, and elevated expression of Dirigent-like encoding genes. Small molecule defense markers and phytohormones such as cinnamic acid, 12-oxo-phytodienoic acid, and auxin levels all increased in elicitor-treated moss tissues. In contrast, salicylic acid levels decreased while abscisic acid levels remained unchanged. P. patens reporter lines harboring an auxin-inducible promoter fused to β-glucuronidase revealed GUS activity in protonemal and gametophores tissues treated with elicitors of P.c. carotovorum, consistent with a localized activation of auxin signaling. These results indicate that P. patens activates the shikimate, phenylpropanoid, oxylipins, and auxin pathways upon treatment with P.c. carotovorum derived elicitors.

Molecular Characterization of Differentially Expressed ADP Ribosylation Factor from WSSV Infected Black Tiger Shrimp Penaeus monodon

Suppression subtractive hybridization (SSH) cDNA library was constructed using hepatopancreas of the WSSV infected black tiger shrimp, Penaeus monodon. The differentially expressed ESTs of SSH cDNA library represented different functional categories belonging to energy and metabolism, osmoregulation, cytoskeletal and structural proteins, membrane traffic and organelle structure, immune genes, protein folding, transport and synthesis, ovarian and oocyte development, nucleic acid processing, cell cycle and heat shock protein. The ADP ribosylation factor (Arf) was one of the differentially expressed gene which was identified from the SSH cDNA library. The full length cDNA of P. monodon Arf (pmArf) was cloned by 3′- and 5′-rapid amplification of cDNA ends (3′/5′ RACE). The complete nucleotide sequence of pmArf encoded protein sequence of 180 amino acids revealed the characteristic conserved features of the Arf family proteins represented by the P-loop, the switch regions and interswitch region. The sequence homology indicated that the pmArf belonged to class II of Arf family. The gene expression analysis of pmArf and other immune related genes (penaeidin, anti-lipopolysaccharide factor and QM) in the gills, hepatopancreas and gut tissues of the WSSV infected shrimp was carried out by Real-Time PCR. The pmArf gene was up-regulated with highest gene expression levels at 24 h (1.33-fold) in the gill tissues and at 48 h in the gut tissues (1.44-fold) and hepatopancreas (1.46-fold) of WSSV infected shrimp, indicating its potential role in immune response against WSSV infection.

Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection

Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

Deciphering the molecular responses to methanol-enhanced photosynthesis and stomatal conductance in broad bean

This study showed that foliar spray of 2 % methanol significantly increased stomatal conductance, photosynthesis, and growth of broad bean (Vicia faba L.). To understand the molecular mechanisms behind the methanol-enhanced photosynthesis of broad bean, a forward suppression subtractive hybridization (SSH) cDNA library was constructed with 2 % methanol-sprayed leaves. A total of 145 methanol-induced genes were identified, and 63 of these had a known function. Clustering analysis indicated that the largest proportion (41.3 %) comprised genes associated with photosynthesis. RT-PCR analysis verified that the transcription of photosynthetic genes, including Rubisco and chlorophyll a/b-binding proteins of photosystem I and II, was upregulated in 2 % methanol-sprayed leaves, which might contribute to the molecular mechanism of methanol-enhanced photosynthesis in broad bean. Further analysis suggested that the transcription and expression of 14-3-3 protein (present in the SSH library) and PM H+-ATPase encoding genes, PM H+-ATPase phosphorylation and its interactions with 14-3-3 proteins as well as stomatal aperture increased in leaves after 2 % methanol sprayed. Simultaneously, the leaf H2O2 content decreased in a time-dependent manner. Based on these results, we hypothesize that methanol spray induces the expression of 14-3-3 proteins and PM H+-ATPase and simultaneously reduces the H2O2 content in leaves. Consequently, the interaction between phosphorylated PM H+-ATPase and 14-3-3 proteins is enhanced, and PM H+-ATPase and H+ pump activities elevated; these functions may constitute the molecular mechanism through which methanol increases stomatal aperture and conductance in broad bean leaves.

IDENTIFICATION OF ABA INDUCIBLE GENES FROM COLD-HARDY VITIS AMURENSIS RUPR. 'ZUOSHAN-1' BY SUPPRESSION SUBTRACTIVE HYBRIDIZATION

Vitis amurensis Rupr., distributed in the Northeast of China, is the most cold-hardy grape species. It can tolerate temperatures as low as -40~-50°C when fully cold acclimated. Our study showed that as the temperature drops, the cold hardy ?Zuoshan-1? grapevine accumulates ABA quicker and in higher concentration than less cold-hardy Vitis species. To better understand the molecular mechanisms of ABA and its role in grape cold hardiness, we constructed a suppression subtractive hybridization (SSH) cDNA library to isolate ABA inducible genes of ?Zuoshan-1?. In a total of 214 randomly picked clones from the ABA-induced library, 167 were successfully sequenced, from which 31 contigs were assembled. Blast analysis revealed that 14 contigs were homologous to Vitis or other plant species while the other 17 contigs showed high homology to Escherichia coli, which apparently resulted from E. coli contamination. Functional analysis indicated that the putative functions of these contigs were associated with photosynthesis, cell defense or detoxification, and signal transduction.

Identification of genes associated with fruit ripening in Ziziphus jujuba using suppression subtractive hybridization approach

Owing to the high nutritional value and extensive medicinal use of its products, Chinese jujube (Ziziphus jujuba Mill) is one of the most important fruit crops in China. However, jujube fruits are highly perishable and thus have a short shelf life, which is a serious hindrance to the industry. Better understanding of the molecular mechanisms underlying jujube fruit softening is fundamental to overcome the problem. Thus, both forward and reverse suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed genes for fruit at half-red ripening stage and complete red stage. As a result of dot blot confirmation, a total of 154 differentially expressed genes were identified. After removed low-quality regions and screened for vector contamination, blasted with the non-redundant NCBI databases, 78.6 % of sequences exhibited high homology to previously identified or putative proteins. All the ESTs were annotated and classified according to the terms of the three main Gene Ontology vocabularies using the Blast2GO software. Furthermore, the quantitative real-time PCR was carried out for 17 genes to validate the genes differentially expressed from the SSH libraries. And the full-length sequences of galactose oxidase and aldehyde dehydrogenase genes were obtained. It is the first step to explore the functional genomics and regulatory networks during the storage period of jujube fruit. The identification of the genes differentially expressed is helpful to understand the ripening and softening of the jujube fruit at the molecular level.

Characterization of the novel protein P9TLDR (temporal lobe down-regulated) with a brain-site-specific gene expression modality in Alzheimer’s disease brain

Alzheimer’s disease (AD) is an aging-related neurodegenerative disorder characterized by irreversible loss of higher cognitive functions. The disease is characterized by the presence of amyloid plaques and neurofibrillary tangles (NFT). In the current study we isolated from an intra-cerebral brain-site-specific (AD temporal lobe vs. AD occipital lobe) polymerase chain reaction (PCR)-select cDNA suppression subtractive hybridization (PCR-cDNA-SSH) expression analysis the novel gene P9TLDR, potentially a microtubule-associated protein involved in neuronal migration, with an altered expression pattern: down-regulated in the temporal lobe cortex of early stage AD brains. In an in vitro AD-related cell model, amyloid-β peptide (Aβ)-treated neurons, reduced P9TLDR expression correlated with increased tau protein phosphorylation. In conclusion, interference with the P9TLDR signalling pathways might be a therapeutic strategy for the treatment of AD.

Differentially expressed genes in dicamba‐resistant and dicamba‐susceptible biotypes

In an ongoing effort to investigate the mechanism of auxinic herbicide resistance in Kochia scoparia (kochia), polymerase chain reaction‐based cDNA suppression subtractive hybridization was used to identify genes that are differentially expressed between dicamba‐resistant (HRd) and dicamba‐susceptible (S1) kochia biotypes in response to herbicide treatment. Both the HRd and S1 adaptor‐ligated cDNAs were used in separate hybridizations in order to generate biotype‐specific clones. A total of 710 cDNAs, representing putative differentially expressed mRNAs, were isolated and subjected to further screening. The false‐positive cDNAs were removed by conducting two colony hybridizations and at least one Northern hybridization. Differential or biotype‐specific expression was confirmed for six clones each from the HRd and S1 plants. The S1‐related genes were constitutively expressed at higher levels than in the HRd plants, but none had significant sequence similarity to known genes. Among the HRd‐related genes, HRd‐88 had 42% amino acid sequence identity to a conserved domain within thiol peptidases, which might be involved in auxin‐regulated gene expression. The constitutively expressed and inducible (by the dicamba treatment) HRd‐39 had 40% identity and 60% similarity to a domain from the Fe(II)/α‐ketoglutarate‐dependent hydroxylase superfamily. The HRd‐39 gene product had the characteristics of an enzyme that is able to detoxify dicamba via oxidative hydroxylation and thus its overexpression might confer the dicamba resistance phenotype.

Efficacy of QCDCR formulated CpG ODN 2007 in Nile tilapia against Streptococcus iniae and identification of upregulated genes

The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P<0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P<0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity.

Isolation and characterization of genes expressed differently in mature fruits of ‘redfield’ and ‘greensleeves’ apples

Anthocyanins are responsible for most of the bright red and blue colors found in higher plants. Anthocyanin accumulation is regulated by MYB transcription factors. Some varieties of apple indicate anthocyanin accumulation with a red color in the skin and, at times, even in the cortex core, but other varieties without anthocyanin production have green-colored skin indicative of chlorophyll accumulation. To obtain gene expression profiles from the apple cultivars with different flesh and skin colors, 2 apple cultivars, the red-colored ‘Redfield’ and the green-colored ‘Greensleeves’, were used. A cDNA suppression subtractive hybridization library was established and analyzed. Six genes encoding methallothionein-like protein, chalcone isomerase, dirigent-like protein, brassinosteroid-6-oxidase, an unnamed protein product, and some unknown proteins were selected, and their expression was confirmed in the ‘Redfield’ cultivar through virtual northern blot analysis. In the ‘Greensleeves’ cultivar, 5 genes encoding gibberellins-regulated protein, a hypothetical 23.5 KD protein, anthranilate N-hydroxycinnamoyl/benzoyltransferase, and isoflavone reductase were chosen, and their upregulated transcriptional levels were identified. These selected genes were differently expressed in each apple cultivar, suggesting that these genes directly or indirectly regulate anthocyanin accumulation.

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections

To understand the molecular mechanisms involved in response of Nile tilapia ( Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6 h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly ( p < 0.05) upregulated in Nile tilapia at 6 h post infection with A. hydrophila at an injection dose of 10 5 CFU per fish (∼20% mortality). Of the nine upregulated genes, four were also significantly ( p < 0.05) induced in Nile tilapia at 6 h post infection with A. hydrophila at an injection dose of 10 6 CFU per fish (∼60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly ( p < 0.05) upregulated in Nile tilapia at 6 h post infection with Streptococcus iniae at doses of 10 6 and at 10 5 CFU per fish (∼70% and ∼30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.

The Mining of Pearl Formation Genes in Pearl Oyster Pinctada fucata by cDNA Suppression Subtractive Hybridization

Recent researches revealed the regional preference of biomineralization gene transcription in the pearl oyster Pinctada fucata: it transcribed mainly the genes responsible for nacre secretion in mantle pallial, whereas the ones regulating calcite shells expressed in mantle edge. This study took use of this character and constructed the forward and reverse suppression subtractive hybridization (SSH) cDNA libraries. A total of 669 cDNA clones were sequenced and 360 expressed sequence tags (ESTs) greater than 100bp were generated. Functional annotation associated 95 ESTs with specific functions, and 79 among them were identified from P. fucata at the first time. In the forward SSH cDNA library, it recognized mass amount of nacre protein genes, biomineralization genes dominantly expressed in the mantle pallial, calcium-ion-binding genes, and other biomineralization-related genes important for pearl formation. Real-time PCR showed that all the examined genes were distributed in oyster mantle tissues with a consistence to the SSH design. The detection of their RNA transcripts in pearl sac confirmed that the identified genes were certainly involved in pearl formation. Therefore, the data from this work will initiate a new round of pearl formation gene study and shed new insights into molluscan biomineralization.

Transcriptional Effects of Dietary Exposure of Oil-Contaminated Calanus finmarchicus in Atlantic Herring (Clupea harengus)

Suppression subtractive hybridization (SSH) cDNA library construction and characterization was used to identify differentially regulated transcripts from oil exposure in liver of male Atlantic herring (Clupea harengus) fed a diet containing 900 mg crude oil/kg for 2 mo. In total, 439 expressed sequence tags (EST) were sequenced, 223 from the forward subtracted library (enriched for genes putatively upregulated by oil exposure) and 216 from the reverse subtracted library (enriched for genes putatively downregulated by oil exposure). Follow-up reverse-transcription (RT) quantitative polymerase chain reaction (qPCR) analyses of gene transcription were conducted on additional herring exposed to food containing 9 (low), 90 (medium), and 900 (high) mg crude oil/kg feed for 2 mo. Chronic exposure of Atlantic herring to an oil-contaminated diet mediated upregulation of transcripts encoding antifreeze proteins, proteins in the classical complement pathway (innate immunity), and iron-metabolism proteins. Gene ontology (GO) analysis showed that “cellular response to stress,” “regulation to biological quality,” “response to abiotic stimuli,” and “temperature homeostasis” were the most affected go at the biological processes level, and “carbohydrate binding,” “water binding,” and “ion binding” at the molecular function level. Of the genes examined with RT-qPCR, CYP1A, antifreeze protein, retinol binding protein 1, deleted in malignant brain tumor 1, and ovary-specific C1q-like factor demonstrated a significant upregulation. Myeloid protein 1, microfibrillar-associated protein 4, WAP65, and pentraxin were downregulated in liver of fish from the high exposure group. In conclusion, this study suggests that 2 mo of oil exposure affected genes encoding proteins involved in temperature homeostasis and possible membrane stability in addition to immune-responsive proteins in Atlantic herring.

Identification of Cold-Temperature-Regulated Genes in Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of F. psychrophilum. Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no F. psychrophilum gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of F. psychrophilum.

Abstract B58: hnRNP H1, a novel coactivator of the androgen receptor implicated in progression and hormone resistance of prostate cancer in African Americans

Abstract The causes of ethnic disparity in the clinical manifestation and outcome of prostate cancer (CaP) are not well understood. African Americans (AA) have twice the incidence and mortality of CaP than Caucasian Americans (CA). We identified by a novel functional genomics approach, encompassing laser capture microdissection (LCM), suppressive subtractive hybridization (SSH) and custom race-based CaPspecific cDNA array chips, selective expression of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) in prostate tumor cells of AA in comparison to CA men. hnRNP H1 expression correlates with disease progression and score and poor prognosis in AA men. Subsequent functional studies demonstrate that hnRNP H1 up-regulates transcription, and physically interacts with, and confers hormone-dependent and independent transactivation of androgen receptor (AR) in CaP cells. In addition, hnRNP H1 enhances AR binding to its cognate DNA element on promoter and enhancer element of PSA gene and selective exonic domains of the AR gene. These findings support a model in which hnRNP H1 is an auxiliary coactivator for ligand-dependent and independent transactivation of AR in tumor cells. Furthermore, siRNA silencing of hnRNP H1 elicited growth arrest and enhanced cytotoxicity of antiandrogen bicalutamide in CaP cells. Our data support a new role for hnRNP H1 in ethnic disparity of CaP and further demonstrate a previously uncharacterized mechanism for AR-hnRNP H1 axis in disease progression and development of hormone refractory in AA men. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B58.

Expression analysis of sugarcane shaggy-like kinase (SuSK) gene identified through cDNA subtractive hybridization in sugarcane (Saccharum officinarum L.)

Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e(-108)), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may indicate involvement in stress signaling.

Expression‐based identification of genetic determinants of the bacterial symbiosis ‘Chlorochromatium aggregatum’

The phototrophic consortium 'Chlorochromatium aggregatum' is a highly structured association of green sulfur bacterial epibionts surrounding a central, motile bacterium and is the most specific symbiosis currently known between two phylogenetically distinct bacterial species. Genes and gene products potentially involved in the symbiotic interaction were identified on the genomic, transcriptomic and proteomic level. As compared with the 11 available genomes of free-living relatives, only 186 open reading frames were found to be unique to the epibiont genome. 2-D differential gel electrophoresis (2-D DIGE) of the soluble proteomes recovered 1612 protein spots of which 54 were detected exclusively in consortia but not in pure epibiont cultures. Using mass spectrometry analyses, the 13 most intense of the 54 spots could be attributed to the epibiont. Analyses of the membrane proteins of consortia, of consortia treated with cross-linkers and of pure cultures indicated that a branched chain amino acid ABC-transporter binding protein is only expressed in the symbiotic state of the epibiont. Furthermore, analyses of chlorosomes revealed that an uncharacterized 11 kDa epibiont protein is only expressed during symbiosis. This protein may be involved in the intracellular sorting of chlorosomes. Application of a novel prokaryotic cDNA suppression subtractive hybridization technique led to identification of 14 differentially regulated genes, and comparison of the transcriptomes of symbiotic and free-living epibionts indicated that 328 genes were differentially transcribed. The three approaches were mostly complementary and thereby yielded a first inventory of 352 genes that are likely to be involved in the bacterial interaction in 'C. aggregatum'. Notably, most of the regulated genes encoded components of central metabolic pathways whereas only very few (7.5%) of the unique 'symbiosis genes' turned out to be regulated under the experimental conditions tested. This pronounced regulation of central metabolic pathways may serve to fine-tune the symbiotic interaction in 'C. aggregatum' in response to environmental conditions.

Identification of Water Deficit Stress Upregulated Genes in Sugarcane

Sugarcane represents an important renewable source among biofuel crops with high capability to assimilate carbon among the C4 plants. Limited availability of freshwater renders this crop uneconomical, warranting the necessity for the development of varieties with higher water use efficiency and tolerant to water deficiency stress. Sugarcane variety cv. Co740 was subjected to varied levels of water deficiency stress to isolate transcripts differentially expressed to the imposed stress. The leaf relative water content was used as a measure to estimate the stress response. PCR-based cDNA suppression subtractive hybridization technique was applied to construct forward subtracted library for differentially expressed genes under stress. Dot blot-selected 158 clones showing elevated response were sequenced, of which 62% resembled similarity with known functional genes, 12% with hypothetical proteins of plant origin, while 26% represented new unknown sequences. Annotation of these differentially expressed sequence tags (ESTs) in the moderately water deficit stress-tolerant cultivar predicted that most of them encoded proteins involved in cellular organization, protein metabolism, signal transduction, and transcription. Further, semi-quantitative reverse transcriptase PCR carried out for five genes projected the involvement of these ESTs in stress alleviation/tolerance. Results from this study may help in targeting useful genes for improving drought tolerance in sugarcane and other grasses.