Suppression Subtractive Hybridization Data Research Articles

Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing.

BackgroundAsian Palmyra palm, the source of palm-sugar, is dioecious with a long juvenile period requiring at least 12 years to reach its maturity. To date, there is no reliable molecular marker for identifying sexes before the first bloom, limiting crop designs and utilization. We aimed to identify sex-linked markers for this palm using PCR-based DNA fingerprinting, suppression subtractive hybridization (SSH) and transcriptome sequencing.MethodsDNA fingerprints were generated between males and females based on RAPD, AFLP, SCoT, modified SCoT, ILP, and SSR techniques. Large-scale cloning and screening of SSH libraries and de novo transcriptome sequencing of male and female cDNA from inflorescences were performed to identify sex-specific genes for developing sex-linked markers.ResultsThrough extensive screening and re-testing of the DNA fingerprints (up to 1,204 primer pairs) and transcripts from SSH (>10,000 clones) and transcriptome data, however, no sex-linked marker was identified. Although de novo transcriptome sequencing of male and female inflorescences provided ∼32 million reads and 187,083 assembled transcripts, PCR analysis of selected sex-highly represented transcripts did not yield any sex-linked marker. This result may suggest the complexity and small sex-determining region of the Asian Palmyra palm. To this end, we provide the first global transcripts of male and female inflorescences of Asian Palmyra palm. Interestingly, sequence annotation revealed a large proportion of transcripts related to sucrose metabolism, which corresponds to the sucrose-rich sap produced in the inflorescences, and these transcripts will be useful for further understanding of sucrose production in sugar crop plants. Provided lists of sex-specific and differential-expressed transcripts would be beneficial to the further study of sexual development and sex-linked markers in palms and related species.

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 1.5kb. 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.

Characterization of immune-related genes in the yellow catfish Pelteobagrus fulvidraco in response to LPS challenge.

Fish are considered an excellent model for studies in comparative immunology as they are a representative population of lower vertebrates linked to invertebrate evolution. To gain a better understanding of the immune response in fish, we constructed a subtractive cDNA library from the head kidney of lipopolysaccharide-stimulated yellow catfish (Pelteobagrus fulvidraco) using suppression subtractive hybridization (SSH). A total of 300 putative EST clones were identified which contained 95 genes, including 27 immune-related genes, 7 cytoskeleton-related genes, 3 genes involved in the cell cycle and apoptosis, 9 respiration and energy metabolism-related genes, 7 genes related to transport, 24 metabolism-related genes, 10 genes involved in stress responses, seven genes involved in regulation of transcription and translation and 59 unknown genes. Using real-time quantitative reverse transcription PCR, a subset of randomly selected genes involved in the immune response to lipopolysaccharide challenge were investigated to verify the reliability of the SSH data which identified 16 up-regulated genes. The genes identified in this study provide novel insight into the immune response in fish.

Identification of immune response-related genes in the Chinese oak silkworm, Antheraea pernyi by suppression subtractive hybridization

Insects possess an innate immune system that responds to invading microorganisms. In this study, a subtractive cDNA library was constructed to screen for immune response-related genes in the fat bodies of Antheraea pernyi (Lepidoptera: Saturniidae) pupa challenged with Escherichia coli. Four hundred putative EST clones were identified by suppression subtractive hybridization (SSH), including 50 immune response-related genes, three cytoskeleton genes, eight cell cycle and apoptosis genes, five respiration and energy metabolism genes, five transport genes, 40 metabolism genes, ten stress response genes, four transcription and translation regulation genes and 77 unknown genes. To verify the reliability of the SSH data, the transcription of a set of randomly selected immune response-related genes were confirmed by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time quantitative reverse transcription-PCR (qRT-PCR). These identified immune response-related genes provide insight into understanding the innate immunity in A. pernyi.

A systematic experimental evaluation of microRNA markers of human bladder cancer

Background: MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. They are aberrantly expressed in many human cancers and are potential therapeutic targets and molecular biomarkers.Methods: In this study, we for the first time validated the reported data on the entire set of published differential miRNAs (102 in total) through a series of transcriptome-wide experiments. We have conducted genome-wide miRNA profiling in 17 urothelial carcinoma bladder tissues and in nine normal urothelial mucosa samples using three methods: (1) An Illumina HT-12 microarray hybridization (MA) analysis (2) a suppression-subtractive hybridization (SSH) assay followed by deep sequencing (DS) and (3) DS alone.Results: We show that DS data correlate with previously published information in 87% of cases, whereas MA and SSH data have far smaller correlations with the published information (6 and 9% of cases, respectively). qRT-PCR tests confirmed reliability of the DS data.Conclusions: Based on our data, MA and SSH data appear to be inadequate for studying differential miRNA expression in the bladder.Impact: We report the first comprehensive validated database of miRNA markers of human bladder cancer.

Physiological and transcriptional analysis of the effects of aluminum stress on Cryptococcus humicola

Aluminum (Al) toxicity is a major factor that limits crop productivity in acid soils. The toxic effects of Al exposure on Al-tolerant Cryptococcus humicola were analyzed at the physiological level. The exposure to 20 mM Al led to a clear increase in malondialdehyde content and a significant decrease in the levels of protein carbonyls, suggesting that Al stress caused oxidative damage to membrane lipids but not to proteins. Suppression subtractive hybridization (SSH) results showed that when C. humicola was exposed to 20 mM Al, a total of 141 ESTs were differentially expressed. Of them, 27 had unknown functions and 48 were newly identified in this study. The genes with known functions were categorized into seven groups. The largest group was related to metabolism and energy, followed by protein synthesis and processing, cell structure, signal transduction and transcription, transporters, stress and defense. Reverse transcription (RT)-PCR analysis of certain genes was performed to confirm the reliability of the SSH data. Nine selected genes were upregulated by Al in a time-dependent manner. The potential functions of some genes involved in Al-tolerance were predicted and are discussed. The diversity of the putative functions of these genes indicates that Al stress results in a complex response in C. humicola.

Transcriptional and physiological changes of alfalfa in response to aluminium stress

SUMMARYMedicago sativais an excellent pasture legume, but it is very sensitive to aluminium (Al) toxicity. To better understand the mechanism ofM. sativasensitivity to Al, a forward suppression subtractive hybridization (SSH) cDNA library for an Al-sensitive cultivar,M. sativaL. cv. Yumu No. 1 (YM1), under 5 μmAl stress over a 24 h period was constructed to analyse changes in its gene expression in response to Al stress. Sequence analysis for the SSH cDNA library generated 291 high-quantity expression sequence tags (ESTs). Of these, 229 were known as functional ESTs, 137 of which have already been reported as Al response genes, whereas the other 92 were potentially novel Al-associated genes. The up-regulation of known Al resistance-associated genes encoding the transcription factor sensitive to proton rhizotoxicity 1 (STOP1) and malate transporterMsALMT1(Al-activated malate transporter) as well as genes for antioxidant enzymes was observed. Reverse transcription polymerase chain reaction analysis validated the reliability of the SSH data and confirmed the up-regulated expression ofSTOP1andMsALMT1under 5 μmAl stress. The analysis of physiological changes indicated that hydrogen peroxide (H2O2) and malondialdehyde levels were elevated rapidly under 5 μmAl stress, suggesting that severe oxidative stress occurred in the YM1 roots. The up-regulation of antioxidant-related genes might be an important protective mechanism for YM1 in response to the oxidative stress induced by 5 μmAl toxicity. Al-induced malate exudation was increased drastically during the early period after Al treatment, which might have been due to the up-regulation and function ofMsALMTandSTOP1. However, malate exudation from the YM1 roots declined quickly during the subsequent period, and a gradual decrease in malate content was simultaneously observed in the YM1 roots. This result is in agreement with the observation that organic acid metabolism-associated enzymes such as phosphoenolpyruvate carboxylase, citrate synthase and malate dehydrogenase were not present in the SSH library. This might be a major reason for the YM1 sensitivity to Al.

Identification of Genes Differentially Expressed by Metarhizium anisopliae Growing on Locusta migratoria Wings Using Suppression Subtractive Hybridization

Insect-pathogenic fungi penetrate their hosts directly through the cuticle. To better understand this process, we identified genes that were up-regulated by Metarhizium anisopliae germinating and differentiating on Locusta migratoria wings using suppression subtractive hybridization (SSH). A total of 78 unique expressed sequence tags (ESTs) up-regulated more than twofold during fungal growth on locust wings were identified. Among these 78 ESTs, 30 (38.5%) shared significant similarity with NCBI annotated hypothetical proteins, 16 (20.5%) shared low similarity to known or predicted genes, might represent novel genes, and 32 (41.0%) shared significant similarity with known proteins that are involved in various cell and molecular processes such as cell metabolism, protein metabolism, stress response and defense, and cell structure and function. Semi-quantitative RT-PCR analysis of six randomly selected genes confirmed the SSH results, verifying the fidelity of the SSH data. The results of this study provide novel information on genes expressed during early stages of infection with M. anisopliae, and improve current understanding of fungal pathogenesis.

Identification and characterization of genes associated with tapping panel dryness from Hevea brasiliensis latex using suppression subtractive hybridization

BackgroundTapping panel dryness (TPD) is one of the most serious threats to natural rubber production. Although a great deal of effort has been made to study TPD in rubber tree, the molecular mechanisms underlying TPD remain poorly understood. Identification and systematical analyses of the genes associated with TPD are the prerequisites for elucidating the molecular mechanisms involved in TPD. The present study is undertaken to generate information about the genes related to TPD in rubber tree.ResultsTo identify the genes related to TPD in rubber tree, forward and reverse cDNA libraries from the latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. Among the 1106 clones obtained from the two cDNA libraries, 822 clones showed differential expression in two libraries by reverse Northern blot analyses. Sequence analyses indicated that the 822 clones represented 237 unique genes; and most of them have not been reported to be associated with TPD in rubber tree. The expression patterns of 20 differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and real-time PCR analysis. According to the Gene Ontology convention, 237 unique genes were classified into 10 functional groups, such as stress/defense response, protein metabolism, transcription and post-transcription, rubber biosynthesis, etc. Among the genes with known function, the genes preferentially expressed were associated with stress/defense response in the reverse library, whereas metabolism and energy in the forward one.ConclusionsThe genes associated with TPD were identified by SSH method in this research. Systematic analyses of the genes related to TPD suggest that the production and scavenging of reactive oxygen species (ROS), ubiquitin proteasome pathway, programmed cell death and rubber biosynthesis might play important roles in TPD. Therefore, our results not only enrich information about the genes related to TPD, but also provide new insights into understanding the TPD process in rubber tree.

Characterization of genes associated with induced resistance against Penicillium expansum in apple fruit treated with quercetin

Penicillium expansum causes blue mould, a serious postharvest disease of apples, and is the main producer of the mycotoxin patulin. Since control by synthetic fungicides is less accepted by consumers, the demand for alternative means is pressing. In a recent study, the flavonoid quercetin, although scarcely effective in in vitro assays against P. expansum growth, significantly reduced blue mould rots on ‘Golden Delicious’ apples, suggesting an enhancement of host disease resistance. To confirm or reject this hypothesis, genes differentially expressed in quercetin-treated ‘Golden Delicious’ apples were identified by suppression subtractive hybridization (SSH). A pool of 88 unique gene transcripts was obtained. Several sequences revealed high similarities with different classes of pathogenesis-related proteins (RNase-like PR10 and PR8), or with proteins expressed under stress conditions. Other transcripts had high similarity to genes of unknown function or genes coding for proteins having a role in pathogen recognition and in signalling pathways. SSH data were validated by analysing the expression of 14 genes by quantitative real-time PCR (qPCR). Eleven genes proved to be up-regulated at a medium-high level in freshly harvested apples. Among these, 5 genes selected for temporal expression profiling revealed the existence of a combined effect, particularly at 24 or 48 h, between wounding and phenolic treatment. These results provide evidence that quercetin induces resistance to P. expansum in apples, by acting on the transcription level of genes involved in several distinct metabolic processes.

Transcriptional Analysis of Normal Human Fibroblast Responses to Microgravity Stress

To understand the molecular mechanism(s) of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the space-flown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization (SSH) to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several G1-phase cell cycle traverse genes. Other genes showing up-regulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the “hub” for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.