Identification of novel differentially expressed zooxanthellal genes from Aiptasia-Symbiodinium endosymbiosis through SDS-based RNA purification

Endosymbiosis between dinoflagellates and cnidarian hosts first occurred more than 200 million years ago; however, symbiosis-specific genes and cellular processes involved in the establishment, maintenance, and breakdown of endosymbiosis remain unclear. Therefore, this study aimed to identify the zooxanthellal genes associated with the aforementioned biological processes during endosymbiosis in Aiptasia-Symbiodinium endosymbionts. Here, zooxanthellae isolates were treated with 0.02% SDS to decrease potential host RNA contamination and to enhance the identification of novel symbiosis/nonsymbiosis-associated differentially expressed zooxanthellal genes through suppressive subtractive hybridization (SSH) and next-generation sequencing (NGS) methods. Consequently, among 214 symbiosis-specific transcripts identified herein that displayed identity to only 5.6% of host-derived transcripts, 64% were well-known functional genes. In the nonsymbiotic stage, 181 differentially expressed transcripts were identified, of which 64.1% belonged to well-known functional genes. BLAST revealed that 8 categories of cellular processes were significantly induced in symbiotic or nonsymbiotic zooxanthellae. Together with the results of quantitative analysis, the results revealed that photosynthesis, flagellate biosynthesis and motility, stress-induced responses, cell wall biosynthesis, starch synthesis and transport, lipid biosynthesis and metabolism, host/symbiont immune response, intercellular communication, cell growth, and cell cycle regulation were the major cellular processes occurring in symbiotic/nonsymbiotic stages. The present results provide insights into the mechanisms involved in regulating the different physiological processes in symbiotic/nonsymbiotic zooxanthellae and may guide future studies.

Screening of Differentially Expressed Microsporidia Genes from Nosema ceranae Infected Honey Bees by Suppression Subtractive Hybridization

The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly; however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

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 gene fragments related to nitrogen deficiency in Eichhornia crassipes (Pontederiaceae)

<p><em>Eichhornia crassipes</em> is an aquatic plant native to the Amazon River Basin. It has become a serious weed in freshwater habitats in rivers, lakes and reservoirs both in tropical and warm temperate areas worldwide. Some research has stated that it can be used for water phytoremediation, due to its strong assimilation of nitrogen and phosphorus, and the accumulation of heavy metals, and its growth and spread may play an important role in environmental ecology. In order to explore the molecular mechanism of <em>E. crassipes</em> to responses to nitrogen deficiency, we constructed forward and reversed subtracted cDNA libraries for <em>E. crassipes</em> roots under nitrogen deficient condition using a suppressive subtractive hybridization (SSH) method. The forward subtraction included 2 100 clones, and the reversed included 2 650 clones. One thousand clones were randomly selected from each library for sequencing. About 737 (527 unigenes) clones from the forward library and 757 (483 unigenes) clones from the reversed library were informative. Sequence BlastX analysis showed that there were more transporters and adenosylhomocysteinase-like proteins in <em>E. crassipes</em> cultured in nitrogen deficient medium; while, those cultured in nitrogen replete medium had more proteins such as UBR4-like e3 ubiquitin-protein ligase and fasciclin-like arabinogalactan protein 8-like, as well as more cytoskeletal proteins, including actin and tubulin. Cluster of Orthologous Group (COG) analysis also demonstrated that in the forward library, the most ESTs were involved in coenzyme transportation and metabolism. In the reversed library, cytoskeletal ESTs were the most abundant. Gene Ontology (GO) analysis categories demonstrated that unigenes involved in binding, cellular process and electron carrier were the most differentially expressed unigenes between the forward and reversed libraries. All these results suggest that <em>E. crassipes</em> can respond to different nitrogen status by efficiently regulating and controlling some transporter gene expressions, certain metabolism processes, specific signal transduction pathways and cytoskeletal construction. </p>

Screening and identification of specific markers for bladder transitional cell carcinoma from urine urothelial cells with suppressive subtractive hybridization and cDNA microarray

Objective: The objective of this study was to screen and identifydifferentially expressed genes in invasive bladder transitional cellcarcinoma (BTCC).Methods: Voided urine samples were collected from consecutivepatients with BTCC and patients under surveillance for bladdercancer recurrence; voided urine samples from patients with nonmalignantdiseases served as control. We identified the differentiallyexpressed genes by comparing urine samples of bladdercarcinoma to that of the control group with suppressive subtractivehybridization (SSH) and cDNA microarray. The differentiallyexpressed genes were verified by quantitative real-time polymerasechain reaction (QPCR).Results: From the 762 white colonies, a total of 449 positive cloneswere obtained in which 112 were found to be upregulated in BTCC.Sequencing and homology analysis were performed for these 112clonies. The detection rates of some known genes (including IGF-1, human telomerase reverse transcriptase [hTERT], bladder cancerspecific nuclear matrix protein 4 [BLCA-4] and homeobox A13[HOXA13]) for BTCC at the Ta, T1 and >T1 stages were 48%, 90%and 100%, respectively, with a specificity of 85%. The test specificitywas 80% for the 30 control patients with urinary tract infections. Thecombination of BLCA-4 and HOXA13 could distinguish betweenlow- and high-grade tumours, with specificity and sensitivity of 80%.Conclusion: We successfully constructed a reliable SSH library ofBTCC and found that combination detection insulin-like growthfactor 1 (IGF-1), hTERT, BLCA-4 and HOXA13 genes could helpto evaluate BTCC at different stages.