Identification of differentially expressed genes associated with precocious puberty by suppression subtractive hybridization in goat pituitary tissues.

The aim of this study was to identify genes related to precocious puberty expressed in the pituitary of goats at different growth stages by suppression subtractive hybridization (SSH). The pituitary glands from Jining Gray (JG) goats (early puberty) and Liaoning Cashmere (LC) goats (late puberty) at 30, 90, and 180 days were used in this study. To identify differentially expressed genes (DEGs) in the pituitary glands, mRNA was extracted from these tissues, and SSH libraries were constructed and divided into the following groups: juvenile group (30-JG vs. 30-LC, API), puberty group (90-JG vs. 180-LC, BPI), and control group (90-JG vs. 90-LC, EPI). A total of 60, 49, and 58 DEGs were annotated by 222 Gene Ontology (GO) terms and 75 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Most of the DEGs were significantly enriched in GO terms related to 'structural constituent of ribosome', 'translation' and 'GTP binding', and numerous DEGs were also significantly enriched in KEGG terms related to the Jak-STAT signaling and oocyte meiosis pathways. Candidate genes associated with precocious puberty and sexual development were screened from the SSH libraries. These genes were analyzed to determine if they were expressed in the pituitary tissues of the goats at different growth stages and to identify genes that may influence the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we found precocious puberty-related genes (such as PRLP0, EIF5A, and YWHAH) that may be interesting from an evolutionary perspective and that could be investigated for use in future goat breeding programs. Our results provide a valuable dataset that will facilitate further research into the reproductive biology of goats.

Identification of Therapeutic Drug Target of Stenotrophomonas maltophilia Through Subtractive Genomic Approach and in-silico Screening Based on 2D Similarity Filtration and Molecular Dynamic Simulation.

BACKGROUND: Stenotrophomonas maltophilia is a multi-drug resistant, gram-negative bacterium that causes opportunistic infections and is associated with high morbidity and mortality in severely immunocompromised individuals. AIM: The study aimed to find out the drug target and a novel inhibitor for Stenotrophomonas maltophilia. OBJECTIVES: The current study focused on identifying specific drug targets by subtractive genomes analysis to determine the novel inhibitor for the specified target protein by virtual screening, molecular docking, and molecular simulation approach. MATERIALS AND METHODS: In this study, we performed a subtractive genomics approach to identify the novel drug target for S.maltophilia. After obtaining the specific target, the next step was to identify inhibitors that include calculating 2D similarity search, molecular docking, and molecular simulation for drug development for S.maltophilia. RESULTS: With an efficient subtractive genomic approach, out of 4386 proteins, five unique targets were found, in which UDP-D-acetylmuramic (murF) was the most remarkable target. Further virtual screening, docking, and dynamics analyses resulted in the identification of seven novel inhibitors. CONCLUSION: Further, in vitro and in vivo bioassay of the identified novel inhibitors could facilitate effective drug use against S.maltophilia.

Subtractive genomic approach toward introduction of novel immunogenic targets against Clostridioides difficile: Thinking out of the box.

Clostridioides difficile is one of the major causatives of nosocomial infections worldwide. Antibiotic-associated diarrhea, pseudomembranous colitis, and toxic megacolon are the most common forms of C. difficile infection (CDI). Considering the high antibiotic resistance of C. difficile isolates and the low efficacy of immunization with toxin-related vaccines, we suggested that surface-exposed and secreted proteins could be considered as potential immunogenic targets against CDI. Various immuninformatics databases were used to predict antigenicity, allergenicity, B-cell epitopes, MHC-II binding sites, conserved domains, prevalence and conservation of proteins among the most common sequence types, molecular docking, and immunosimulation of immunogenic targets. Finally, 16 proteins belonging to three functional groups were identified, including proteins involved in the cell wall and peptidoglycan layer (nine proteins), flagellar assembly (five proteins), spore germination (one protein), and a protein with unknown function. Molecular docking results showed that among all the mentioned proteins, WP_009892971.1 (Acd) and WP_009890599.1 (a C40 family peptidase) had the strongest interactions with human Toll-like receptor 2 (TLR-2) and TLR-4. This study proposes a combination of C. difficile toxoid (Tcd) and surface-exposed proteins such as Acd as a promising vaccine formulation for protection against circulating clinical strains of C. difficile.

Subtractive genomics and molecular docking approach to identify drug targets against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a multidrug resistant pathogen associated with high mortality and morbidity in patients having compromised immunity. The efflux systems of S. maltophilia include SmeABC and SmeDEF proteins, which assist in acquisition of multiple-drug-resistance. In this study, proteome based mapping was utilized to find out the potential drug targets for S. maltophilia strain k279a. Various tools of computational biology were applied to remove the human-specific homologous and pathogen-specific paralogous sequences from the bacterial proteome. The CD-HIT analysis selected 4315 proteins from total proteome count of 4365 proteins. Geptop identified 407 essential proteins, while the BlastP revealed approximately 85 non-homologous proteins in the human genome. Moreover, metabolic pathway and subcellular location analysis were performed for essential bacterial genes, to describe their role in various cellular processes. Only two essential proteins (Acyl-[acyl-carrier-protein]-UDP-N acetyl glucosamine O-acyltransferase and D-alanine-D-alanine ligase) as candidate for potent targets were found in proteome of the pathogen, in order to design new drugs. An online tool, Swiss model was employed to model the 3D structures of both target proteins. A library of 5000 phytochemicals was docked against those proteins through the molecular operating environment (MOE). That resulted in to eight inhibitors for both proteins i.e. enterodiol, aloin, ononin and rhinacanthinF for the Acyl-[acyl-carrier-protein]-UDP-N acetyl glucosamine O-acyltransferase, and rhazin, alkannin beta, aloesin and ancistrocladine for the D-alanine-D-alanine ligase. Finally the ADMET was done through ADMETsar. This study supported the development of natural as well as cost-effective drugs against S. maltophilia. These inhibitors displayed the effective binding interactions and safe drug profiles. However, further in vivo and in vitro validation experiment might be performed to check their drug effectiveness, biocompatibility and their role as effective inhibitors.

Ecotoxicogenomic analysis of zebrafish embryos exposed to triclosan and mixture triclosan and methyl triclosan using suppression subtractive hybridization and next-generation sequencing.

Triclosan (TCS) and methyl-triclosan (MTCS), an environmental transformation product of biocide of TCS, have been detected in water, sediment, fish, and invertebrates. In this study, the key pathway perturbation in zebrafish (Danio rerio) embryos exposed to TCS (300 µg/L) and TCS/MTCS mixture (300 µg/L TCS + 30 µg/L MTCS) was assessed by integrating the metabolomic and transcriptomic dysregulation. The differential expressed genes (DEGs) were obtained from the subtracted cDNA libraries by using the suppression subtractive hybridization and next-generation sequencing approach. The dysregulation of twenty-eight GO terms and four KEGG pathways, including oxidative phosphorylation and cardiac muscle contraction, were shown in the TCS treatment group, indicating that TCS could disrupt the mitochondrial inner membrane function by downshifting the electrochemical gradient. Meanwhile, the addition of MTCS in the exposure would cause fourteen additional significant KEGG pathway changes, demonstrating the different effects between two exposure. A pathway-based analysis using the identified DEGs and the altered metabolites in zebrafish embryos treated with TCS and TCS/MTCS mixture, collectively, has been applied. This study demonstrated that the integration of SSH-NGS and metabolomics could reveal toxic effects and potential diseases associated with the exposures of TCS and MTCS in aquatic environments.

Novel ASR isolated from drought stress responsive SSH library in pearl millet confers multiple abiotic stress tolerance in PgASR3 transgenic Arabidopsis.

A genomic resource of drought stress responsive genes/ESTs was generated using Suppression Subtractive Hybridization (SSH) approach in a drought stress tolerant Pennisetum glaucum genotype 841B. Fifty five days old plants were subjected to drought stress after withholding water for different time intervals (10 days, 15 days, 20 days and 25 days). A forward subtractive cDNA library was prepared from isolated RNA of leaf tissue. Differential gene expression under drought stress was validated for selected nine contigs by RT-qPCR. A transcript homologous to Setaria italica ASR3 upregulated under drought stress was isolated from genotype 841B and characterized. Heterologous expression of PgASR3 was validated in Arabidopsis and confirmed under multiple abiotic stress conditions. A total of four independent transgenic lines overexpressing gene PgASR3 were analyzed by Southern blot at T1 stage. For drought stress tolerance, three independent lines (T2 stage) were analyzed by biochemical and physiological assays at seedling stage. The growth rate (shoot and root length) of transgenic seedlings improved as compared to WT seedling under differenct abiotic stress conditions. The three transgenic lines were also validated for drought stress tolerance and RT-qPCR analysis, at maturity stage. Under drought stress conditions, the mature transgenic lines showed higher levels of RWC, chlorophyll and proline but lower levels of MDA as compared to WT plants. PgASR3 gene isolated and validated in this study can be utilized for developing abiotic stress tolerant crops.

Screening of immune-related differentially expressed genes from primary lymphatic organs of broilers fed with probiotic bacillus cereus PAS38 based on suppression subtractive hybridization.

To explore the molecular mechanism of the effect of Bacillus cereus PAS38 on the immunity of broilers, sixty 7-day-old broilers were divided into two groups with three replicates. The control group was fed with basal diet, and the treatment group was fed with basal diet containing Bacillus cereus PAS38 1×106 CFU/g. Thymus and bursa of fabricius were taken from two groups of broilers at the age of 42 days, total RNA was extracted, differential gene library was constructed by SSH technology, and immune-related differential genes were screened. Then, we used siRNA to interfere with the expression of some differential genes in the original generation lymphocytes of broiler blood to detect the change of cytokines mRNA expression level. A total of 42 immune-related differentially expressed genes were screened, including 22 up-regulated genes and 20 down-regulated genes. When 7 differentially up-regulated genes associated with enhanced immune function were interfered with in lymphocytes, some immune-promoting cytokines were down-regulated. These results showed that Bacillus cereus PAS38 might up-regulate the expression of JCHAIN, PRDX1, CD3E, CDK6 and other genes in immune organs of broilers, thereby affecting the development of immune organs, the expression of various cytokines and the transduction of immune signals, improving the immune capacity of broilers.

Identification of Vibrio alginolyticus virulent strain-specific DNA regions by suppression subtractive hybridization and PCR.

AIMS: Vibrio alginolyticus was frequently isolated from diseased farmed fish in the coaster waters of Hainan Island over the past two decades. In this study, we attempted to identify candidates of virulent strain-specific DNA regions for this pathogen. METHODS AND RESULTS: Suppression subtractive hybridization (SSH) and PCR were successively performed between the typical virulent strain and avirulent strain of V. alginolyticus, in which they shared 99·54% homology of 16S rDNAs. Out of 2873 subtracted clones, nine clones were finally indicated to harbour virulent strain-specific DNA fragments. The receivable functions of the major fragments in the nine clones were believed to encode methyl-accepting chemotaxis protein (n = 1), type VI secretion system-associated FHA domain protein TagH (n = 1), diguanylate cyclase (n = 1), AraC family transcriptional regulator (n = 1), ABC-type uncharacterized transport system permease component (n = 1) and hypothetical proteins (n = 4). Two hypothetical proteins contain several disordered regions. CONCLUSIONS: Some specific DNA regions existed in the virulent strain of V. alginolyticus, and the SSH assay could be a highly sensitive method for identifying virulent regions in pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This report is the first to describe the identification of virulent strain-specific DNA regions in the V. alginolyticus genome, which is helpful in developing virulent strain-specific rapid detection methods and is a pivotal precondition for clarifying the molecular virulence mechanism of V. alginolyticus.

Direct identification of pneumococcal serotypes in blood cultures by a PCR-reverse-hybridisation technique / Identificación directa de serotipos de neumococo en frascos de hemocultivo mediante una técnica de PCR seguida de hibridación reversa

INTRODUCTION: The objective of this study was to assess the performance of a technique (S. PneumoStrip test) based on PCR followed by reverse strip hybridisation for the detection of Streptococcus pneumoniae serotypes directly in blood culture vials. METHODS: One hundred and ten (110) pairs of isolated strains and their corresponding original blood cultures vials were studied in parallel. Pure isolated strains were conventionally serotyped using latex agglutination and the Quellung reaction. The S. PneumoStrip test was carried out directly in the original blood culture samples. RESULTS: In 102 cases (92.7%), results of the serotype obtained by Quellung coincided with their corresponding original blood cultures typed by S. PneumoStrip. CONCLUSIONS: S. PneumoStrip test is a good alternative technique for direct pneumococcal serotyping in blood culture clinical simples

INTRODUCCIÓN: El objetivo de este estudio fue evaluar el rendimiento de una técnica de PCR seguida de hibridación reversa (S. PneumoStrip test) para su aplicación directa en muestras de frasco de hemocultivo. MÉTODOS: Se estudiaron 110 cepas aisladas en sangre y sus correspondientes muestras de frasco de hemocultivo. Las cepas fueron serotipadas convencionalmente mediante aglutinación por látex y reacción de Quellung. El test de S. PneumoStrip se realizó directamente en las muestras originales de hemocultivo. RESULTADOS: En 102 casos (92,7%) el serotipado por Quellung coincidió con el obtenido directamente en su original frasco de hemocultivo. CONCLUSIONES: S. PneumoStrip constituye una buena alternativa para el serotipado directo en muestras de frasco de hemocultivo

Regulation of vitellogenin gene expression under the negative modulator, gonad-inhibiting hormone in Penaeus monodon.

Vitellogenesis is a principal process during ovarian maturation in crustaceans. This process is negatively regulated by gonad-inhibiting hormone (GIH), a neuronal peptide hormone from eyestalks. However, the detailed mechanism through which GIH regulates Vg expression is still ambiguous. In this study, suppression subtractive hybridization (SSH) under specific GIH-knockdown condition was utilized to determine the expression of genes in the ovary that may act downstream of GIH to control vitellogenin synthesis in Penaeus monodon. The total of 102 and 82 positive clones of up-regulated and down-regulated genes in GIH- knockdown shrimp were identified from the forward and reverse SSH libraries, respectively. Determination of the expression profiles of these reproduction-related genes during ovarian development revealed that the expression of calreticulin (CALR) was significantly reduced in vitellogenic ovary suggesting its role in vitellogenesis. Suppression of CALR by specific dsRNA showed elevated vitellogenin (Vg) transcript level in the ovary at day 7 post-dsRNA injection. Since CALR can bind to steroid hormone receptors and prevents the binding of the receptor to its responsive element to regulate gene expression, it is possible that CALR is an inhibitory mediator of vitellogenin synthesis via steroidal pathway. Our results posted a possible novel pathway of GIH signaling that might interfere the steroid signaling cascade to mediate Vg synthesis in the shrimp.

Screening of differentially expressed immune-related genes from spleen of broilers fed with probiotic Bacillus cereus PAS38 based on suppression subtractive hybridization.

The aim of this study was to construct the spleen differential genes library of broilers fed with probiotic Bacillus cereus PAS38 by suppression subtractive hybridization (SSH) and screen the immune-related genes. Sixty seven-day-old broilers were randomly divided into two groups. The control group was fed with basal diet, and the treated group was fed with basal diet containing Bacillus cereus PAS38 1×106 CFU/g. Spleen tissues were taken and extracted its total RNA at 42 days old, then SSH was used to construct differential gene library and screen immune-related genes. A total of 119 differentially expressed sequence tags (ESTs) were isolated by SSH and 9 immune-related genes were screened out by Gene ontology analysis. Nine differentially expressed genes were identified by qRT-PCR. JCHAIN, FTH1, P2RX7, TLR7, IGF1R, SMAD7, and SLC7A6 were found to be significantly up-regulated in the treated group. Which was consistent with the results of SSH. These findings imply that probiotic Bacillus cereus PAS38-induced differentially expressed genes in spleen might play an important role in the improvement of immunity for broilers, which provided useful information for further understanding of the molecular mechanism of probiotics responsible to affect the poultry immunity.

Transcript profiling of chickpea pod wall revealed the expression of floral homeotic gene AGAMOUS-like X2 (CaAGLX2).

The differentially expressed genes in the chickpea pod wall have been identified for the first time using a forward suppression subtractive hybridization (SSH) library. In all, 226 clones of SSH library were sequenced and analyzed. A total of 179 high-quality expressed sequence tags (ESTs) were generated and based on the CAP3 assembly of these ESTs, 126 genes (97 singletons and 29 contigs) were computationally annotated. The mapping of 88.26% ESTs by gene ontology (GO) annotation distributed them into 751 GO terms of three categories, cellular location, molecular function, and biological process. The KEGG pathway analysis revealed 45 ESTs are involved in 49 different biological pathways. Also, 67 ESTs encodes four different classes of enzymes such as oxidoreductases (29), transferase (20), hydrolases (16) and isomerase (2). Six genes were selected and subjected to qPCR analysis, of these, two genes (FHG Floral homeotic AGAMOUS-like isoform X2, MADS1 MADS-box transcription factor) showed significant up-regulation in the pod wall compared to leaves. Surprisingly, one of the MADS1 box gene, FHG (CaAGLX2), responsible for flower development expressed in the pod wall. Therefore, understanding its specific role in the pod wall could be interesting. Thus, the transcript dynamics of the chickpea pod wall revealed differentially expressed genes in the pod wall, which may be participating in the metabolic build-up of both pod wall and seeds.

Karyotyping of circulating tumor cells for predicting chemotherapeutic sensitivity and efficacy in patients with esophageal cancer.

BACKGROUND: Aneuploidy of chromosome 8 in circulating tumor cells (CTCs) has been reported correlates with therapeutic efficacy and prognosis in patients with advanced gastric cancer. However, it is not clear whether it is also appropriate for other cancer. Therefore, in this study, we evaluate the clinical application aneuploidy of CTCs for esophageal cancer. METHODS: Peripheral blood were collected for karyotyping analysis before and after first 4-cycles chemotherapy from seventy nine patients with newly diagnosed esophageal cancer. Karyotyping of chromosome 8 in CTCs detected by SET-iFISH (Subtraction Enrichment-Immunostaining fluorescence in situ hybridizatio) in those patients were grouped into two categories according to CTC number: triploid group and non-triploid group. Pearson Chi-Square were used to compare the association between different aneuploidy type and chemotherapeutic sensitivity and efficacy. RESULTS: Among the 16 patients with triploid of chromosome 8, 4 patients benefit, and of the 63 patients with non-triploid, 54 patients benefit. Chi-square test analysis found that clinical benefit of non-triploid patients was significantly higher than triploid patients, suggesting non-triploid patients were more sensitive to chemotherapy than triploid patients. After 4-cycles chemotherapy, it is found that chemotherapeutic efficacy was positively correlated with non-triploid proportion. These results suggest that non-triploid proportion could be used as a candidate maker for assessing chemotherapeutic efficacy. CONCLUSIONS: Monitoring aneuploidy of chromosome 8 in CTCs before and after chemotherapy may help predict sensitivity and efficacy of chemotherapy in patients with esophageal cancer.

Two-factor ANOVA of SSH and RNA-seq analysis reveal development-associated Pi-starvation genes in oilseed rape.

MAIN CONCLUSION: The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings. Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.

Further confirmation of second- and third-generation Eimeria necatrix merozoite DEGs using suppression subtractive hybridization.

In our previous study, we obtained a large number of differentially expressed genes (DEGs) between second-generation merozoites (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix using RNA sequencing (RNA-seq). Here, we report two subtractive cDNA libraries for MZ2 (forward library) and MZ3 (reverse library) that were constructed using suppression subtractive hybridization (SSH). PCR amplification revealed that the MZ2 and MZ3 libraries contained approximately 96.7% and 95% recombinant clones, respectively, and the length of the inserted fragments ranged from 0.5 to 1.5 kb. A total of 106 and 111 unique sequences were obtained from the MZ2 and MZ3 libraries, respectively, and were assembled into 13 specific consensus sequences (contigs or genes) (5 from MZ2 and 8 from MZ3). The qRT-PCR results revealed that 11 out of 13 genes were differentially expressed between MZ-2 and MZ-3. Of 13 genes, 11 genes were found in both SSH and our RNA-seq data and displayed a similar expression trend between SSH and RNA-seq data, and the remaining 2 genes have not been reported in both E. necatrix genome and our RNA-seq data. Among the 11 genes, the expression trends of 8 genes were highly consistent between SSH and our RNA-seq data. These DEGs may provide specialized functions related to the life-cycle transitions of Eimeria species.

PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust.

BACKGROUND: Protein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the reliable identification of the full range of genes involved. The present work reports the technique of protein kinase (PK)-profiling for the identification of the PK genes induced in the partially resistant oats line MN841801-1 following exposure to the fungus Puccinia coronata. This is the first time this technique has been used with cDNA (complementary DNA) from a suppression subtractive hybridization library obtained after the hybridization of cDNAs from inoculated and mock-inoculated plants. RESULTS: Six degenerate primers based on the conserved domains of protein kinases were used in a PK-profiling assay including cDNA from mock-inoculated leaves and subtracted cDNA. Of the 75.7% of sequences cloned and sequenced that showed significant similarity to resistance genes, 76% were found to code for PKs. Translation and ClustalW2 alignment of each sequence cloned with the complete sequences of the most similar B. distachyon PKs allowed those of the partially resistant oat line to be deduced and characterized. Further, a phylogenetic study carried out after alignment of these B. distachyon PK sequences with the most similar protein sequences of related species also allowed to deduce different functions for the PK cloned. RT-qPCR (Reverse Transcription-quantitative PCR) was analyzed on nine representative sequences to validate the reliability of the employed PK-profiling method as a tool for identifying the expression of resistance-associated genes. CONCLUSIONS: PK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata, and perhaps other pathogens. Most of the PKs studied are related to receptor-like protein kinases expressed shortly after infection. This is in agreement with previous studies indicating a close relationship between partial resistance and the first layer of defense against pathogen used by plants.

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

BACKGROUND: Sugarcane 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. RESULTS: Specificity 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. CONCLUSIONS: This 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.

Identification of up-regulated transcripts during Pleurotus ostreatus primordium stage and characterization of PoALDH1.

In order to isolate the differentially expressed genes in the primordium stage of Pleurotus ostreatus, the SSH cDNA library was constructed using the cDNA from dikaryotic mycelium stage as a driver and the cDNA from primordium stage as a tester. There were 423 significantly differently expressed clones among 2055 positive clones after three times of reverse Northern blot differential screening. After the repeated sequences being removed, 46 genes were identified which were putatively involved in cell rescue and defense, energy metabolism, transcription and protein regulation, membrane proteins, and signal transduction; 18 genes encoding hypothetical proteins with unknown function; 5 genes without any homology. PoALDH1 and its full-length cDNA sequence were cloned using the Aldehyde dehydrogenase EST isolated from the library. The amino acid sequence of PoALDH1 contains conservative glutamic acid and cysteine residues active sites of aldehyde dehydrogenase family. When exposed to different concentrations of sodium chloride, the mycelium growth was inhibited and the expression level of PoALDH1 was significantly higher than that of the control one, which indicated that PoALDH1 may have the ability to relieve salt stress. The results of this study will provide useful information for isolating growth and development related genes of P. ostreatus.

Transcriptional analysis identifies major pathways as response components to Sporisorium scitamineum stress in sugarcane.

BACKGROUND: Sugarcane smut, which is caused by Sporisorium scitamineum, is a severe fungal disease affecting sugarcane. However, the major pathways involved in the interaction between sugarcane and S. scitamineum remains unclear. RESULTS: In the present study, suppression subtractive hybridization (SSH) library construction, together with reverse northern blotting, was conducted on the most prevalent sugarcane genotype ROC22 challenged with S. scitamineum. After alignment and homologous expressed sequence tag (EST) assembly, a total of 155 differentially expressed unigenes were identified from SSH libraries. Totally, 26 of 155 differentially expressed unigenes were analyzed by qRT-PCR in sugarcane smut-resistant genotype YC05-179 and susceptible genotype ROC22. Genes encoded two unknown protein (Q1 and Q11), serine/threonine kinase (Q2), fiber protein (Q3), eukaryotic translation initiation factor 5A (Q23), and Sc14-3-3-like protein (Q24) were induced in sugarcane smut-resistant genotype YC05-179 but inhibited in susceptible genotype ROC22. Based on the differential expression data achieved from SSH libraries and qRT-PCR, we found that, serine/threonine kinases, Ca2+ sensors, mitogen-activated protein genes and some NBS-LRR genes may involve in the signal recognition and transduction of smut fungus infection in sugarcane. While in the plant hormone signaling pathways, the genes related to auxin, abscisic acid, salicylic acid and ethylene were more apparently in response to smut fungus invasion. The hypersensitive response, protein metabolism, polyamine synthesis, and cell wall formation may play an important role in sugarcane defense against smut fungus colonization. Additionally, the Sc14-3-3 might serve as a molecular modulator in sugarcane being immune to smut disease by interacting with proteins like ScGAPN (Q10), which have been further verified by BiFC assay. CONCLUSIONS: The findings of the present study could provide a general view about gene pathways involving in sugarcane defense against smut disease and facilitate a better understanding of the molecular mechanism underlying sugarcane-S. scitamineum interaction.

Gene expression and metabolite accumulation during strawberry (Fragaria × ananassa) fruit development and ripening.

MAIN CONCLUSION: A coordinated regulation of different metabolic pathways was highlighted leading to the accumulation of important compounds that may contribute to the final quality of strawberry fruit. Strawberry fruit development and ripening involve complex physiological and biochemical changes, ranging from sugar accumulation to the production of important volatiles compounds that contribute to the final fruit flavor. To better understand the mechanisms controlling fruit growth and ripening in cultivated strawberry (Fragaria × ananassa), we applied a molecular approach combining suppression subtractive hybridization and next generation sequencing to identify genes regulating developmental stages going from fruit set to full ripening. The results clearly indicated coordinated regulation of several metabolic processes such as the biosynthesis of flavonoid, phenylpropanoid and branched-chain amino acids, together with glycerolipid metabolism and pentose and glucuronate interconversion. In particular, genes belonging to the flavonoid pathway were activated in two distinct phases, the first one at the very early stages of fruit development and the second during ripening. The combination of expression analysis with metabolomic data revealed that the functional meaning of these two inductions is different, as during the early stages gene activation of flavonoid pathway leads to the production of proanthocyanidins and ellagic acid-derived tannins, while during ripening anthocyanins are the main product of flavonoid pathway activation. Moreover, the subtractive approach allowed the identification of different members of the same gene family coding for the same or very similar enzymes that in some cases showed opposite regulation during strawberry fruit development. Such regulation is an important trait that can help to understand how plants specifically channel metabolic intermediates towards separate branches of a biosynthetic pathway or use different isoforms of the same enzyme in different organs or developmental stages.