Digital Gene Expression Profiling Research Articles

Anthocyanin Biosynthesis Regulation in the Fruit of Citrus sinensis cv. Tarocco

The differential expression profiling of the fruit-specific genes between a blood orange (Citrus sinensis cv. Tarocco) and a blonde orange (Citrus sinensis cv. twenty-first century navel orange) was investigated by fruit transcriptome sequencing. The relative expression folds for Cs6g17570 and Cs5g31400 were significantly greater in the “Tarocco” fruits, but Cs9g04810 transcripts had no significant differences between two accessions by digital gene expression profiling. In particular, R2R3-MYB, bHLH-type, and WD40-repeat proteins (encoded by Cs6g17570, Cs5g31400, and Cs9g04810) were transcription factors with each conserved domain and interacted in vitro and in vivo. Moreover, the dihydroflavonol 4-reductase (DFR), a key enzyme involved into anthocyanin biosynthesis pathway, had greater transcription levels in Tarocco fruit and also in the transgenic Arabidopsis thaliana by Cs6g17570 ectopic expression. Furthermore, Cs6g17570 and Cs5g31400 in Tarroco were upregulated by cold induction and then had similar expression profiling during cold storage. In conclusion, Cs6g17570, Cs5g31400, and Cs9g04810 encoding proteins might be involved into anthocyanin regulation in Tarocco by MYB-bHLH-WD40 complex as well as in other fruit trees. Studies on the anthocyanin regulatory mechanism will aid in development of new biotechnological tools for Citrus breeding.

De novo Sequencing of the Leaf Transcriptome Reveals Complex Light-Responsive Regulatory Networks in Camellia sinensis cv. Baijiguan.

Tea plants (Camellia sinensis L.) possess high genetic diversity that is important for breeding. One cultivar, Baijiguan, exhibits a yellow leaf phenotype, reduced chlorophyll (Chl) content, and aberrant chloroplast structures under high light intensity. In contrast, under low light intensity, the flush shoot from Baijiguan becomes green, the Chl content increases significantly, and the chloroplasts exhibit normal structures. To understand the underlying molecular mechanisms for these observations, we performed de novo transcriptome sequencing and digital gene expression (DGE) profiling using Illumina sequencing technology. De novo transcriptome assembly identified 88,788 unigenes, including 1652 transcription factors from 25 families. In total, 1993 and 2576 differentially expressed genes (DEGs) were identified in Baijiguan plants treated with 3 and 6 days of shade, respectively. Gene Ontology (GO) and pathway enrichment analyses indicated that the DEGs are predominantly involved in the ROS scavenging system, chloroplast development, photosynthetic pigment synthesis, secondary metabolism, and circadian systems. The light-responsive gene POR (protochlorophyllide oxidoreductase) and transcription factor HY5 were identified. Quantitative real-time PCR (qRT-PCR) analysis of 20 selected DEGs confirmed the RNA-sequencing (RNA-Seq) results. Overall, these findings suggest that high light intensity inhibits the expression of photosystem II 10-kDa protein (PsbR) in Baijiguan, thus affecting PSII stability, chloroplast development and chlorophyll biosynthesis.

Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies.

Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells.

Dynamic gene expression profiles during postnatal development of porcine subcutaneous adipose.

A better understanding of the control of lipogenesis is of critical importance for both human and animal physiology. This requires a better knowledge of the changes of gene expression during the process of adipose tissue development. Thus, the objective of the current study was to determine the effects of development on subcutaneous adipose tissue gene expression in growing and adult pigs. Here, we present a comprehensive investigation of mRNA transcriptomes in porcine subcutaneous adipose tissue across four developmental stages using digital gene expression profiling. We identified 3,274 differential expressed genes associated with oxidative stress, immune processes, apoptosis, energy metabolism, insulin stimulus, cell cycle, angiogenesis and translation. A set of universally abundant genes (ATP8, COX2, COX3, ND1, ND2, SCD and TUBA1B) was found across all four developmental stages. This set of genes may play important roles in lipogenesis and development. We also identified development-related gene expression patterns that are linked to the different adipose phenotypes. We showed that genes enriched in significantly up-regulated profiles were associated with phosphorylation and angiogenesis. In contrast, genes enriched in significantly down-regulated profiles were related to cell cycle and cytoskeleton organization, suggesting an important role for these biological processes in adipose growth and development. These results provide a resource for studying adipose development and promote the pig as a model organism for researching the development of human obesity, as well as being used in the pig industry.

Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling.

Tagetes erecta is an important commercial plant of Asteraceae family. The male sterile (MS) and male fertile (MF) two-type lines of T. erecta have been utilized in F1 hybrid production for many years, but no report has been made to identify the genes that specify its male sterility that is caused by homeotic conversion of floral organs. In this study, transcriptome assembly and digital gene expression profiling were performed to generate expression profiles of MS and MF plants. A cDNA library was generated from an equal mixture of RNA isolated from MS and MF flower buds (1 mm and 4 mm in diameter). Totally, 87,473,431 clean tags were obtained and assembled into 128,937 transcripts among which 65,857 unigenes were identified with an average length of 1,188 bp. About 52% of unigenes (34,176) were annotated in Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO (KEGG Ortholog database) and/or GO. Taking the above transcriptome as reference, 125 differentially expressed genes were detected in both developmental stages of MS and MF flower buds. MADS-box genes were presumed to be highly related to male sterility in T. erecta based on histological and cytological observations. Twelve MADS-box genes showed significantly different expression levels in flower buds 4 mm in diameter, whereas only one gene expressed significantly different in flower buds 1 mm in diameter between MS and MF plants. This is the first transcriptome analysis in T. erecta and will provide a valuable resource for future genomic studies, especially in flower organ development and/or differentiation.

The role of melanin pathways in extremotolerance and virulence of Fonsecaea revealed by de novo assembly transcriptomics using illumina paired-end sequencing.

Melanisation has been considered to be an important virulence factor of Fonsecaea monophora. However, the biosynthetic mechanisms of melanisation remain unknown. We therefore used next generation sequencing technology to investigate the transcriptome and digital gene expression data, which are valuable resources to better understand the molecular and biological mechanisms regulating melanisation in F. monophora. We performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of parent (CBS 122845) and albino (CBS 125194) strains using the Illumina RNA-seq system. A total of 17 352 annotated unigenes were found by BLAST search of NR, Swiss-Prot, Gene Ontology, Clusters of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) (E-value <1e‒5). A total of 2 283 unigenes were judged to be the differentially expressed between the two genotypes. We identified most of the genes coding for key enzymes involved in melanin biosynthesis pathways, including polyketide synthase (pks), multicopper oxidase (mco), laccase, tyrosinase and homogentisate 1,2-dioxygenase (hmgA). DEG analysis showed extensive down-regulation of key genes in the DHN pathway, while up-regulation was noted in the DOPA pathway of the albino mutant. The transcript levels of partial genes were confirmed by real time RT-PCR, while the crucial role of key enzymes was confirmed by either inhibitor or substrate tests in vitro. Meanwhile, numbers of genes involved in light sensing, cell wall synthesis, morphology and environmental stress were identified in the transcriptome of F. monophora. In addition, 3 353 SSRs (Simple Sequence Repeats) markers were identified from 21 600 consensus sequences. Blocking of the DNH pathway is the most likely reason of melanin deficiency in the albino strain, while the production of pheomelanin and pyomelanin were probably regulated by unknown transcription factors on upstream of both pathways. Most of genes involved in environmental tolerance to oxidants, irradiation and extreme temperatures were also assembled and annotated in transcriptomes of F. monophora. In addition, thousands of identified cSSR (combined SSR) markers will favour further genetic linkage studies. In conclusion, these data will contribute to understanding the regulation of melanin biosynthesis and help to improve the studies of pathogenicity of F. monophora.

Physiological and Transcriptional Analyses Reveal Differential Phytohormone Responses to Boron Deficiency in Brassica napus Genotypes.

Phytohormones play pivotal roles in the response of plants to various biotic and abiotic stresses. Boron (B) is an essential microelement for plants, and Brassica napus (B. napus) is hypersensitive to B deficiency. However, how auxin responds to B deficiency remained a dilemma for many years and little is known about how other phytohormones respond to B deficiency. The identification of B-efficient/inefficient B. napus indicates that breeding might overcome these constraints in the agriculture production. Here, we seek to identify phytohormone-related processes underlying B-deficiency tolerance in B. napus at the physiological and gene expression levels. Our study indicated low-B reduced indole-3-acetic acid (IAA) concentration in both the shoots and roots of B. napus, and affected the expression of the auxin biosynthesis gene BnNIT1 and the efflux gene BnPIN1 in a time-dependent manner. Low-B increased the jasmonates (JAs) and abscisic acid (ABA) concentrations and induced the expression of the ABA biosynthesis gene BnNCED3 and the ABA sensor gene BnPYL4 in the shoot. In two contrasting genotypes, the auxin concentration decreased more drastically in the B-inefficient genotype ‘W10,’ and together the expression of BnNIT1 and BnPIN1 also decreased more significantly in ‘W10’ under long-term B deficiency. While the JAs concentration was considerably higher in this genotype, and the ABA concentration was induced in ‘W10’ compared with the B-efficient genotype ‘QY10.’ Digital gene expression (DGE) profiling confirmed the differential expression of the phytohormone-related genes, indicating more other phyohormone differences involving in gene regulation between ‘QY10’ and ‘W10’ under low-B stress. Additionally, the activity of DR5:GFP was reduced in the root under low-B in Arabidopsis, and the application of exogenous IAA could partly restore the B-defective phenotype in ‘W10.’ Overall, our data suggested that low-B disturbed phytohormone homeostasis in B. napus, which originated from the change of transcriptional regulation of phytohormones-related genes, and the differences between genotypes may partly account for their difference in tolerance (B-efficiency) to low-B.

Identification and expression analysis of genes related to calyx persistence in Korla fragrant pear

BackgroundThe objective of this study was to increase understanding about genetic mechanisms affecting calyx persistence in Korla fragrant pear (Pyrus brestschneideri Rehd). Flowers were collected at early bloom, full bloom, and late bloom. The RNA was extracted from the flowers and then combined according to calyx type. Transcriptome and digital gene expression (DGE) profiles of flowers, ovaries, and sepals with persistent calyx (SC_hua, SC_ep, and SC_zf, respectively) were compared with those of flowers, ovaries, and sepals with deciduous calyx (TL_hua, TL_ep, and TL_zf, respectively). Temporal changes in the expression of selected genes in floral organs with either persistent or deciduous calyx were compared using real-time quantitative PCR (qRT-PCR).ResultsComparison of the transcriptome sequences for SC_hua and TL_hua indicated 26 differentially expressed genes (DEGs) with known relationship to abscission and 10 DEGs with unknown function. We identified 98 MYB and 21 SPL genes from the assembled unigenes. From SC_zf vs TL_zf, we identified 21 DEGs with known relationship to abscission and 18 DEGs with unknown function. From SC_ep vs TL_ep, 12 DEGs with known relationship to abscission were identified along with 11 DEGs with unknown function. Ten DEGs were identified by both transcriptome sequencing and DGE sequencing.ConclusionsMore than 50 DEGs were observed that were related to calyx persistence in Korla fragrant pear. Some of the genes were related to cell wall degradation, plant hormone signal transduction, and stress response. Other DEGs were identified as zinc finger protein genes and lipid transfer protein genes. Further analysis showed that calyx persistence in Korla fragment pear was a metabolic process regulated by many genes related to cell wall degradation and plant hormones.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2470-3) contains supplementary material, which is available to authorized users.

Transcriptomic Analysis for Different Sex Types of Ricinus communis L. during Development from Apical Buds to Inflorescences by Digital Gene Expression Profiling.

The castor plant (Ricinus communis L.) is a versatile industrial oilseed crop with a diversity of sex patterns, its hybrid breeding for improving yield and high purity is still hampered by genetic instability of female and poor knowledge of sex expression mechanisms. To obtain some hints involved in sex expression and provide the basis for further insight into the molecular mechanisms of castor plant sex determination, we performed DGE analysis to investigate differences between the transcriptomes of apices and racemes derived from female (JXBM0705P) and monoecious (JXBM0705M) lines. A total of 18 DGE libraries were constructed from the apices and racemes of a wild monoecious line and its isogenic female derivative at three stages of apex development, in triplicate. Approximately 5.7 million clean tags per library were generated and mapped to the reference castor genome. Transcriptomic analysis showed that identical dynamic changes of gene expression were indicated in monoecious and female apical bud during its development from vegetation to reproduction, with more genes expressed at the raceme formation and infant raceme stages compare to the early leaf bud stage. More than 3000 of differentially expressed genes (DEGs) were detected in Ricinus apices at three developmental stages between two different sex types. A number of DEGs involved in hormone response and biosynthesis, such as auxin response and transport, transcription factors, signal transduction, histone demethylation/methylation, programmed cell death, and pollination, putatively associated with sex expression and reproduction were discovered, and the selected DEGs showed consistent expression between qRT-PCR validation and the DGE patterns. Most of those DEGs were suppressed at the early leaf stage in buds of the mutant, but then activated at the following transition stage (5-7-leaf stage) of buds in the mutant, and ultimately, the number of up-regulated DEGs was equal to that of down-regulation in the small raceme of the mutant. In this study, a large number of DEGs and some suggestions involved in sex expression and reproduction were discovered using DGE analysis, which provides large information and valuable hints for next insights into the molecular mechanism of sex determination. It is useful for other further studies in Ricinus.

The effect of heat stress on gene expression, synthesis of steroids, and apoptosis in bovine granulosa cells.

Summer heat stress (HS) is a major contributing factor in low fertility in lactating dairy cows in hot environments. Heat stress inhibits ovarian follicular development leading to diminished reproductive efficiency of dairy cows during summer. Ovarian follicle development is a complex process. During follicle development, granulosa cells (GCs) replicate, secrete hormones, and support the growth of the oocyte. To obtain an overview of the effects of heat stress on GCs, digital gene expression profiling was employed to screen and identify differentially expressed genes (DEGs; false discovery rate (FDR) ≤ 0.001, fold change ≥2) of cultured GCs during heat stress. A total of 1211 DEGs including 175 upregulated and 1036 downregulated ones were identified, of which DEGs can be classified into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results suggested that heat stress triggers a dramatic and complex program of altered gene expression in GCs. We hypothesized that heat stress could induce the apoptosis and dysfunction of GCs. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression of steroidogenic genes (steroidogenic acute regulatory protein (Star), cytochrome P-450 (CYP11A1), CYP19A1, and steroidogenic factor 1 (SF-1)) and apoptosis-related genes (caspase-3, BCL-2, and BAX). Radio immunoassay (RIA) was used to analyze the level of 17β-estradiol (E2) and progesterone (P4). We also assessed the apoptosis of GCs by flow cytometry. Our data suggested that heat stress induced GC apoptosis through the BAX/BCL-2 pathway and reduced the steroidogenic gene messenger RNA (mRNA) expression and E2 synthesis. These results suggest that the decreased function of GCs may cause ovarian dysfunction and offer an improved understanding of the molecular mechanism responsible for the low fertility in cattle in summer.

The nuclear protein GmbZIP110 has transcription activation activity and plays important roles in the response to salinity stress in soybean.

Plant basic-leucine zipper (bZIP) transcription factors play important roles in many biological processes and are involved in the regulation of salt stress tolerance. Previously, our lab generated digital gene expression profiling (DGEP) data to identify differentially expressed genes in a salt-tolerant genotype of Glycine soja (STGoGS) and a salt-sensitive genotype of Glycine max (SSGoGM). This DGEP data revealed that the expression (log2 ratio) of GmbZIP110 was up-regulated 2.76-fold and 3.38-fold in SSGoGM and STGoGS, respectively. In the present study, the salt inducible gene GmbZIP110 was cloned and characterized through phylogenetic analysis, subcellular localization and in silico transcript abundance analysis in different tissues. The functional role of this gene in salt tolerance was studied through transactivation analysis, DNA binding ability, expression in soybean composite seedlings and transgenic Arabidopsis, and the effect of GmbZIP110 on the expression of stress-related genes in transgenic Arabidopsis was investigated. We found that GmbZIP110 could bind to the ACGT motif, impact the expression of many stress-related genes and the accumulation of proline, Na+ and K+, and enhanced the salt tolerance of composite seedlings and transgenic Arabidopsis. Integrating all these results, we propose that GmbZIP110 plays a critical role in the response to salinity stress in soybean and has high potential usefulness in crop improvement.

Transcriptomic analysis of the succulent xerophyte Zygophyllum xanthoxylum in response to salt treatment and osmotic stress

Accumulating a great quantity of Na+, maintaining the stability of the concentration of important nutrition elements, increasing the activities of enzymes related to ROS-scavenging are crucial strategies for the xerophyte Zygophyllum xanthoxylum surviving under adverse saline and drought environments; besides, actively regulating the photosynthesis is also a main reason for Z. xanthoxylum to adapt to mild salt conditions. However, the possible molecular basis of above physiological mechanisms is poorly understood. By performing Illumina sequencing combined with a digital gene expression profiling technique, differentially expressed genes in leaves and roots of Z. xanthoxylum under 50 mM NaCl and −0.5 MPa osmotic stress for 6 and 24 h were identified, respectively, mainly focused on genes related to ion transport, ROS-scavenging system and photosynthesis. Under 50 mM NaCl and −0.5 MPa osmotic stress, the transcripts of genes encoding transporters/channels for Na+, K+, Ca2+, Mg2+, nitrogen, phosphate and important micro-elements significantly increased, which is conducive to enhance the uptake and transport of nutrient elements in Z. xanthoxylum; and more importantly, besides Na+, genes related to vacuolar compartmentalization of K+, Ca2+, NO3 − in leaves plays vital roles in the adaptation to mild salt condition. Meanwhile, NaCl treatment and osmotic stress significantly increased the transcripts of a number of genes related to ROS-scavenging system, which is beneficial to accelerate the ROS-scavenging under 50 mM NaCl and mitigate the damage of ROS to cell biomembrane system under osmotic stress. In addition, in contrast to osmotic stress, 50 mM NaCl significantly induced the expression of genes encoding proteins participated in photosynthetic electron transport and carbon fixation, while inhibited the expression of genes related to chlorophyll catabolism. The present study identified potential genes underling the principal physiological mechanisms of salt and drought tolerance in Z. xanthoxylum. The results provided abundant genetic resources from desert xerophyte for genetic improvement of stress-resistance of important forage and crop species in arid area.

The Protein Elicitor PevD1 Enhances Resistance to Pathogens and Promotes Growth in Arabidopsis

The protein elicitor PevD1, isolated from Verticillium dahlia, could enhance resistance to TMV in tobacco and Verticillium wilt in cotton. Here, the pevd1 gene was over-expressed in wild type (WT) Arabidopsis, and its biological functions were investigated. Our results showed that the transgenic lines were more resistant to Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 than the WT line was. In transgenic plants, both the germination time and bolting time required were significantly shorter and fresh weights and plant heights were significantly higher than those in the WT line. A transcriptomics study using digital gene expression profiling (DGE) was performed in transgenic and WT Arabidopsis. One hundred and thirty-six differentially expressed genes were identified. In transgenic Arabidopsis, three critical regulators of JA biosynthesis were up-regulated and JA levels were slightly increased. Three important repressors of the ABA-responsive pathway were up-regulated, indicating that ABA signal transduction may be suppressed. One CML and two WRKY TFs involved in Ca2+-responsive pathways were up-regulated, indicating that this pathway may have been triggered. In conclusion, we show that PevD1 is involved in regulating several plant endogenous signal transduction pathways and regulatory networks to enhance resistance and promote growth and development in Arabidopsis.

Genome-wide identification and expression analysis along the leaf developmental gradient of the sigma factor gene family in foxtail millet (Setaria italica)

Sigma factors are necessary for the initiation of transcription by RNA polymerase in bacteria and plastids of plants. In plants, a small family of nuclear genes is responsible for encoding the sigma factor proteins. In this study, a genome-wide identification and expression analysis of leaf gradient in millet (Setaria italica) were performed to characterize sigma factor genes and their proteins. By applying several bioinformatics tools, we identified chromosome locations of seven sigma factor genes in millet and their protein 3D structures. All these proteins contained three conserved domains of σ-70 family. These Sigma factor genes have a closer phylogenetic relationship with their orthologs in maize than that in rice. The digital gene expression (DGE) analysis along the millet leaf developmental gradient indicated that Sisig1, Sisig5, Sisig6 showed extremely high expression levels in leaf middle and tip regions. Combining the conservation analysis of residues of each sigma factor protein with the DGE profiles of these proteins, it reveals that Sisig5 plays the housekeeping role compared with other Sisig proteins. Our study will facilitate the future research on crop evolution and the functional studies of sigma factor genes in millet.

Abstract IA09: Single cell RNA-sequencing of circulating tumor cells

Abstract Isolation of circulating tumor cells (CTCs) in the blood allows noninvasive tumor sampling from patients with cancer. Molecular analysis of single CTCs may uncover heterogeneous cellular pathways that underlie disease progression and resistance to therapy. Using a microfluidic device, we isolated individual CTCs from patients with metastatic prostate cancer. Single candidate CTCs were micromanipulated after cell surface staining for epithelial (EpCAM) and mesenchymal (CDH11) markers, and then subjected to single cell RNA-sequencing. Digital gene expression profiles of lineage-confirmed CTCs were compared with each other, with primary prostate tumors, and with annotated markers of cellular signaling pathways. Single prostate CTCs displayed considerable heterogeneity in transcriptional profiles, but clustered according to patient of origin, indicating higher diversity in CTCs across different individuals (mean correlation 0.10 for CTCs within patients vs. 0.0014 for CTCs across patients, P=2.0x10E-11). Compared to primary tumors, CTCs were significantly enriched in 37 molecular pathways (FDR &amp;lt;0.1), with the majority implicated in growth factor, cell adhesion, and hormone signaling. Gene mutations and alternative splice variants of the Androgen Receptor (AR) gene were rare in primary prostate tumors and CTCs from untreated patients, but were prevalent in patients with castration-resistant prostate cancer. Distinct AR variants, including AR-V7, were present within different cells of individual patients, as well as within individual CTCs. Together, single cell molecular analysis of human CTCs points to multiple mechanisms of drug resistance in advanced prostate cancer, and suggests the role of heterogeneous signaling pathways that cooperate with co-existing abnormalities in AR in mediating disease progression. Citation Format: David T. Miyamoto, Yu Zheng, Ben S. Wittner, Richard J. Lee, Huili Zhu, Katherine T. Broderick, Rushil Desai, Brian W. Brannigan, Kshitij S. Arora, Douglas M. Dahl, Lecia V. Sequist, Matthew R. Smith, Ravi Kapur, Chin-Lee Wu, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Single cell RNA-sequencing of circulating tumor cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA09.

Genome-wide expression profiles of Pyropia haitanensis in response to osmotic stress by using deep sequencing technology.

BackgroundPyropia haitanensis is an economically important marine crop grown in harsh intertidal habitats of southern China; it is also an excellent model system for studying mechanisms of stress tolerance. To understand the molecular mechanisms underlying osmotic tolerance and adaptation to intertidal environments, a comprehensive analysis of genome-wide gene expression profiles in response to dehydration and rehydration in Py. haitanensis was undertaken using digital gene expression profile (DGE) approaches combined with de novo transcriptome sequencing.ResultsRNA-sequencing of the pooled RNA samples from different developmental phases and stress treatments was performed, which generated a total of 47.7 million clean reads. These reads were de novo assembled into 28,536 unigenes (≥200 bp), of which 18,217 unigenes (63.83 %) were annotated in at least one reference database. DGE analysis was performed on four treatments (two biological replicates per treatment), which included moderate dehydration, severe dehydration, rehydration, and normal conditions. The number of raw reads per sample ranged from 12.47 to 15.79 million, with an average of 14.69 million reads per sample. After quality filtering, the number of clean reads per sample ranged from 11.83 to 15.04 million. All distinct sequencing reads were annotated using the transcriptome of Py. haitanensis as reference. A total of 1,681 unigenes showed significant differential expression between moderate dehydration and normal conditions, in which 977 genes were upregulated, and 704 genes were downregulated. Between severe dehydration and normal conditions, 1,993 unigenes showed significantly altered expression, which included both upregulated (1,219) and downregulated genes (774). In addition, 1,086 differentially expressed genes were detected between rehydration and normal conditions, of which 720 genes were upregulated and 366 unigenes were downregulated. Most gene expression patterns in response to dehydration differed from that of rehydration, except for the synthesis of unsaturated fatty acids, several transcription factor families, and molecular chaperones that have been collectively implicated in the processes of dehydration and rehydration in Py. haitanensis.ConclusionsTaken together, these data provide a global high-resolution analysis of gene expression changes during osmotic stress that could potentially serve as a key resource for understanding the biology of osmotic acclimation in intertidal red seaweed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2226-5) contains supplementary material, which is available to authorized users.

Using digital gene expression profile to detect representational difference of Chlamys farreri genes after laboratory exposure to persistent organic pollutants

Chlamys farreri (C. farreri), as an economic species of marine bivalves, can tolerate manifold anthropogenic stressors including persistent organic pollutants (POPs) stress. A systematic study of transcriptome after POPs exposure may provide insights into the mechanism of acquired pollution tolerance. In this study, we compared C. farreri transcriptome by reanalyzing DGE (digital gene expression) data from previous study. Our results revealed a toxicant-dependent pattern of global transcriptional responses, with 108, 126 and 138 DEGs regulated by BaP, TBBPA and PAHs cocktail (mixed by benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene and chrysene with the ratio of 5:3:1:1) exposure, respectively. Among these DEGs, 219 were commonly regulated by all pollutants, whereas the minority of differences were condition-specific. In addition, we performed the first hierarchical cluster analysis of the common transcripts throughout three types of contaminants stress ulteriorly, leading to new discoveries of genes’ association information with dynamical gene expression data of C. farreri, which goes beyond the focus on individuals. It revealed a cascade of gene expression patterns in the response of C. farreri to different environmental stresses and may be beneficial for further analysis of novel environmental metrics.

Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses.

De novo transcriptome profiling of cold-stressed siliques during pod filling stages in Indian mustard (Brassica juncea L.).

Low temperature is a major abiotic stress that impedes plant growth and development. Brassica juncea is an economically important oil seed crop and is sensitive to freezing stress during pod filling subsequently leading to abortion of seeds. To understand the cold stress mediated global perturbations in gene expression, whole transcriptome of B. juncea siliques that were exposed to sub-optimal temperature was sequenced. Manually self-pollinated siliques at different stages of development were subjected to either short (6 h) or long (12 h) durations of chilling stress followed by construction of RNA-seq libraries and deep sequencing using Illumina's NGS platform. De-novo assembly of B. juncea transcriptome resulted in 133,641 transcripts, whose combined length was 117 Mb and N50 value was 1428 bp. We identified 13,342 differentially regulated transcripts by pair-wise comparison of 18 transcriptome libraries. Hierarchical clustering along with Spearman correlation analysis identified that the differentially expressed genes segregated in two major clusters representing early (5–15 DAP) and late stages (20–30 DAP) of silique development. Further analysis led to the discovery of sub-clusters having similar patterns of gene expression. Two of the sub-clusters (one each from the early and late stages) comprised of genes that were inducible by both the durations of cold stress. Comparison of transcripts from these clusters led to identification of 283 transcripts that were commonly induced by cold stress, and were referred to as “core cold-inducible” transcripts. Additionally, we found that 689 and 100 transcripts were specifically up-regulated by cold stress in early and late stages, respectively. We further explored the expression patterns of gene families encoding for transcription factors (TFs), transcription regulators (TRs) and kinases, and found that cold stress induced protein kinases only during early silique development. We validated the digital gene expression profiles of selected transcripts by qPCR and found a high degree of concordance between the two analyses. To our knowledge this is the first report of transcriptome sequencing of cold-stressed B. juncea siliques. The data generated in this study would be a valuable resource for not only understanding the cold stress signaling pathway but also for introducing cold hardiness in B. juncea.

Genome-wide identification and comparative analysis of grafting-responsive mRNA in watermelon grafted onto bottle gourd and squash rootstocks by high-throughput sequencing.

Grafting is an important agricultural technique widely used to improve plant growth, yield, and adaptation to either biotic or abiotic stresses. However, the molecular mechanisms underlying grafting-induced physiological processes remain unclear. Watermelon (Citrullus lanatus L.) is an important horticultural crop worldwide. Grafting technique is commonly used in watermelon production for improving its tolerance to stresses, especially to the soil-borne fusarium wilt disease. In the present study, we used high-throughput sequencing to perform a genome-wide transcript analysis of scions from watermelon grafted onto bottle gourd and squash rootstocks. Our transcriptome and digital gene expression (DGE) profiling data provided insights into the molecular aspects of gene regulation in grafted watermelon. Compared with self-grafted watermelon, there were 787 and 3485 genes differentially expressed in watermelon grafted onto bottle gourd and squash rootstocks, respectively. These genes were associated with primary and secondary metabolism, hormone signaling, transcription factors, transporters, and response to stimuli. Grafting led to changes in expression of these genes, suggesting that they may play important roles in mediating the physiological processes of grafted seedlings. The potential roles of the grafting-responsive mRNAs in diverse biological and metabolic processes were discussed. Obviously, the data obtained in this study provide an excellent resource for unraveling the mechanisms of candidate genes function in diverse biological processes and in environmental adaptation in a graft system.