Digital Gene Expression Profiling Analysis Research Articles

Intelectin enhances the phagocytosis of macrophages via CDC42-WASF2-ARPC2 signaling axis in Megalobrama amblycephala

Intelectin has been identified in various vertebrates and plays an important role in the host immune system. In our previous studies, recombinant Megalobrama amblycephala intelectin (rMaINTL) protein with excellent bacterial binding and agglutination activities enhances the phagocytic and killing activities of macrophages in M. amblycephala; however, the underlying regulatory mechanisms remain unclear. The present study showed that treatment with Aeromonas hydrophila and LPS induced the expression of rMaINTL in macrophages, and its level and distribution in macrophages or kidney tissue markedly increased after incubation or injection with rMaINTL. The cellular structure of macrophages was significantly affected after incubation with rMaINTL, resulting in an increased surface area and pseudopodia extension, which might contribute to enhancing the phagocytic ability of macrophages. Then, digital gene expression profiling analysis of the kidneys from rMaINTL-treated juvenile M. amblycephala identified some phagocytosis-related signaling factors that were enriched in pathways involved in the regulation of the actin cytoskeleton. In addition, qRT-PCR and western blotting verified that rMaINTL upregulated the expression of CDC42, WASF2, and ARPC2 in vitro and in vivo; however, the expression of these proteins was inhibited by a CDC42 inhibitor in macrophages. Moreover, CDC42 mediated the promotion of rMaINTL on actin polymerization by increasing the F-actin/G-actin ratio, which led to the extension of pseudopodia and remodeling of the macrophage cytoskeleton. Furthermore, the enhancement of macrophage phagocytosis by rMaINTL was blocked by the CDC42 inhibitor. These results suggested that rMaINTL induced the expression of CDC42 as well as the downstream signaling molecules WASF2 and ARPC2, thereby facilitating actin polymerization to promote cytoskeletal remodeling and phagocytosis. Overall, MaINTL enhanced the phagocytosis activity of macrophages in M. amblycephala via activation of the CDC42-WASF2-ARPC2 signaling axis.

Integrated transcriptome analysis of immune-related mRNAs and microRNAs in Macrobrachium rosenbergii infected with Spiroplasma eriocheiris

Macrobrachium rosenbergii (M. rosenbergii), is a major aquaculture species in China and Southeast Asia. However, infection with Spiroplasma eriocheiris (S. eriocheiris) has caused huge economic losses to the cultivation of M. rosenbergii. Currently, there are few reports on the immune response mechanism of M. rosenbergii that are infected with S. eriocheiris. To clarify the immune response mechanism of M. rosenbergii infected with S. eriocheiris, the key immune genes which respond to the infection with the pathogen and the regulation of related microRNAs (miRNAs) on them were identified. In this study, the mRNA and miRNA transcriptome of hepatopancreas of M. rosenbergii at different infection stages were analyzed using high-throughput sequencing and qRT-PCR. In the mRNA transcriptome, 27,703 and 33,402 genes were expressed in healthy and susceptible M. rosenbergii, respectively. By digital gene-expression profiling analysis, 23,929 and 24,325 genes were expressed, and 223 and 373 genes were significantly up-regulated and down-regulated, respectively. A total of 145 key genes related to Toll, IMD, JAK/STAT and MAPK were excavated from the transcriptome. In the miRNA transcriptome, 549 miRNAs (Conserved: 41, PN-type: 83, PC-type: 425) were sequenced, of which 87 were significantly up-regulated and 23 were significantly down-regulated. Among the related immune pathways, there are 259 miRNAs involved in the regulation of target genes in the Toll and IMD pathways, 231 JAK/STAT pathways and 122 MAPK pathways. qRT-PCR differential detection of immune-related miRNAs and mRNAs showed that 22 miRNAs with significant differences (P < 0.05) such as mro-miR-100, PC-mro-3p-27 and PN-mro-miR-316 had corresponding regulatory relationships with 22 important immune genes such as TLR2, TLR3, TLR4, TLR5, MyD88, Pelle and Relish in different stages after infection. In this study, the immune genes and related regulatory miRNAs of M. rosenbergii in response to S. eriocheiris infection were obtained. The results can provide basic data to further reveal the immune defense mechanism of M. rosenbergii against S. eriocheiris infection.

Dipeptidase PEPDA Is Required for the Conidiation Pattern Shift in Metarhizium acridum

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involves a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum, which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in ΔpepdA mutant. The defect of MC in ΔpepdA can be rescued when nonpolar amino acids, α-alanine, β-alanine, or proline, were added into sucrose yeast extract agar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum. IMPORTANCE Conidia, as the asexual propagules in many fungi, are the start and end of the fungal life cycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the ΔpepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.

Digital gene expression profiling analysis of A549 cells cultured with PM10 in moxa smoke

Moxibustion is a traditional Chinese medicine therapy to cure diseases by fumigating meridians or affected parts via burning of moxa floss. Moxa smoke (MS) is one of the key factors in moxibustion. In this study, we adopted digital gene expression profiling, a next-generation gene sequencing technology, to investigate the effect of MS, inhalable particulate matter (PM 10 ), on human lung adenocarcinoma A549 cells. The effects of MS PM 10 on A549 cells, over different treatment durations were investigated in different groups: the 4-h group (4-h MS group and 4-h control group) and the 20-h group (20-h MS group and 20-h control group). Samples collected from the four groups were stored at −80 °C for subsequent digital gene expression analysis. The differentially expressed genes (DEGs), identified after PM 10 treatment, were screened, and their expression patterns analyzed by cluster analysis, Gene Ontology term enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Compared with two control groups, 1109 DEGs were identified after 4 h of MS intervention and 3565 DEGs were found after 20 h of MS intervention, respectively. Compared with that after 4-h intervention, 2149 DEGs were identified after 20-h intervention. Cluster analysis demonstrated that PM 10 can significantly inhibit cell cycle process with the prolongation of intervention time. Significant pathway enrichment analysis showed that MS PM 10 can inhibit A549 cell cycle process at all phases. When MS PM 10 exposure time prolongs, the inhibitory effect on cell cycle process becomes more obvious. MS PM 10 has many biological activities, and may cause differential expression of genes involved in various biological processes. Nevertheless, further research on MS is warranted for better understanding of the mechanistic details.

Glutamate-Cysteine Ligase Catalytic Subunit Attenuated Hepatitis C Virus-Related Liver Fibrosis and Suppressed Endoplasmic Reticulum Stress

The study aimed to clarify the role and molecular mechanism of glutamate-cysteine ligase catalytic subunit (GCLC) in modulating Hepatitis C virus (HCV)-related liver fibrosis. Twenty patients with HCV-related liver fibrosis and 15 healthy controls were enrolled. Differentially expressed plasma mRNAs were detected by digital gene expression profile analysis and validated by qRT-PCR. Hepatic histopathology was observed by H&E and Masson stained liver sections. The mRNA and protein expression of GCLC, endoplasmic reticulum (ER) stress markers, and inflammatory and fibrogenic factors were detected in liver tissues from patients with HCV-related hepatic fibrosis and HCV core protein-expressing LX-2. The GCLC-overexpressing LX-2 were established by transfecting puc19-GCLC plasmid. Then, glutathione and reactive oxygen species (ROS) levels were measured respectively by spectrophotometric diagnostic kit and dihydrodichlorofluorescein diacetate kit. GCLC were dramatically down-regulated in HCV-related fibrotic livers and activated HSCs, which companied with up-regulation of ER stress-related genes, including inositol-requiring 1 (IRE1) and glucose-regulated protein 78 (GRP78). Also, the proinflammatory and profibrogenic gene, including nuclear factor kappa B (NF-κB), tumor necrosis factor α (TNFα), and transforming growth factor 1(TGFβ1), was highly upregulated. Overexpression of GCLC in hepatic stellate cells could suppress α-SMA and collagen I expression, produce hepatic GSH and reduce ROS, and down-regulate IRE1, GRP78, NF-κB, TNF-α, and TGFβ1 expression. GCLC was a negative regulatory factor in the development of HCV-related liver fibrosis and might be a potential therapeutic target for liver fibrosis.

沙田柚自交不亲和花柱数字基因表达谱及相关基因分析

沙田柚自交不亲和花柱数字基因表达谱及相关基因分析

Comparative transcriptome analyses reveal candidate genes regulating wood quality in Japanese larch (Larix kaempferi)

We studied the molecular mechanism of the quality traits of wood formation in larch. We used the immature latewood cells of two Japanese larch (Larix kaempferi) clones with significant differences in density and in microfibrillar angle (MFA) as materials to analyze their gene expression profiles. A total of 1735 differentially expressed genes were detected in immature latewood cells of the two clones, among which, 971 were up-regulated and 764 were down-regulated. Digital gene expression profiling analysis revealed that genes encoding transcription factor members NAC66 and R2R3-MYB4, microtubule-associated protein, actin-related protein, cell wall protein members, arabinogalactan protein, Fasciclin-like arabinogalactan protein and glycine-rich protein, and several cell-wall-synthesis genes affected wood density and MFA by regulating latewood formation at transcriptional level. Our study results represent a basis for selection of quality traits and genetic improvement of larch wood.

Digital gene-expression profiling analysis of the fatty liver of Landes geese fed different supplemental oils

The goose liver is an ideal model for deciphering lipogenesis molecular mechanisms. This study was designed to investigate the effect of different lipid sources on hepatic lipogenesis in overfed geese. Sixty Landes geese were fed ad libitum with no fat (control) or overfed diets containing 2% goose fat (GF) or rapeseed oil (RO) for 20 days. We measured fatty acid composition of the liver at day 20 of overfeeding. We performed a transcriptomic comparison of fatty liver between GF and RO-fed geese to gain insights into the molecular and cellular events mediating lipogenesis activity. The results showed that there was no substantial effect on fatty liver performance between GF- and RO-fed geese. Significant differences in fatty acid composition were detected between GF- and RO-fed geese. Total ω−6 PUFAs increased and saturated fatty acid decreased (P < 0.05) with RO supplementation when compared with GF, but ω−3 PUFAs did not differ between the two diets. Concentrations of C16:1, C18:1, C18:2, C20:2, and C22:1 were higher (P < 0.05) in the fatty liver of RO-fed geese compared to those in the GF group. Analysis of transcriptome sequencing showed that there were 124 up-regulated and 129 down-regulated differentially expressed genes in the fatty liver of RO and GF-fed geese. Many of these genes code for proteins involved in the lipid metabolic process, including bile secretion, adipocytokine signalling pathway, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, fatty acid elongation and fatty acid biosynthesis, and fat digestion and absorption. Moreover, genes involved in lipid-related pathways such as peroxisome, steroid biosynthesis, steroid hormone biosynthesis, retinol metabolism, and apoptosis were altered, suggesting that the fatty liver of goose fed different oils undertakes both an oxidation function and hormone-related metabolic function.In conclusions, these data suggest that RO supplementation reduces liver lipid oxidation and improves lipogenesis. These findings provide new insights into the molecular mechanisms involved in fatty liver formation and provide valuable resources for analysing mechanisms underlying the effects of oils from different sources on the goose fatty liver.

Digital gene expression profiling analysis and its application in the identification of genes associated with improved response to neoadjuvant chemotherapy in breast cancer

BackgroundThis study aimed to screen sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer.MethodsIn this study, Illumina digital gene expression sequencing technology was applied and differentially expressed genes (DEGs) between patients presenting pathological complete response (pCR) and non-pathological complete response (NpCR) were identified. Further, gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed. The genes in significant enriched pathways were finally quantified by quantitative real-time PCR (qRT-PCR) to confirm that they were differentially expressed. Additionally, GSE23988 from Gene Expression Omnibus database was used as the validation dataset to confirm the DEGs.ResultsAfter removing the low-quality reads, 715 DEGs were finally detected. After mapping to KEGG pathways, 10 DEGs belonging to the ubiquitin proteasome pathway (HECTD3, PSMB10, UBD, UBE2C, and UBE2S) and cytokine–cytokine receptor interactions (CCL2, CCR1, CXCL10, CXCL11, and IL2RG) were selected for further analysis. These 10 genes were finally quantified by qRT-PCR to confirm that they were differentially expressed (the log2 fold changes of selected genes were − 5.34, 7.81, 6.88, 5.74, 3.11, 19.58, 8.73, 8.88, 7.42, and 34.61 for HECTD3, PSMB10, UBD, UBE2C, UBE2S, CCL2, CCR1, CXCL10, CXCL11, and IL2RG, respectively). Moreover, 53 common genes were confirmed by the validation dataset, including downregulated UBE2C and UBE2S.ConclusionOur results suggested that these 10 genes belonging to these two pathways might be useful as sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer.

Digital Gene Expression Profiling Analysis of Aged Mice under Moxibustion Treatment.

Aging is closely connected with death, progressive physiological decline, and increased risk of diseases, such as cancer, arteriosclerosis, heart disease, hypertension, and neurodegenerative diseases. It is reported that moxibustion can treat more than 300 kinds of diseases including aging related problems and can improve immune function and physiological functions. The digital gene expression profiling of aged mice with or without moxibustion treatment was investigated and the mechanisms of moxibustion in aged mice were speculated by gene ontology and pathway analysis in the study. Almost 145 million raw reads were obtained by digital gene expression analysis and about 140 million (96.55%) were clean reads. Five differentially expressed genes with an adjusted P value < 0.05 and |log⁡2(fold change)| > 1 were identified between the control and moxibustion groups. They were Gm6563, Gm8116, Rps26-ps1, Nat8f4, and Igkv3-12. Gene ontology analysis was carried out by the GOseq R package and functional annotations of the differentially expressed genes related to translation, mRNA export from nucleus, mRNA transport, nuclear body, acetyltransferase activity, and so on. Kyoto Encyclopedia of Genes and Genomes database was used for pathway analysis and ribosome was the most significantly enriched pathway term.

Identification of Putative Genes Involved in Limonoids Biosynthesis in Citrus by Comparative Transcriptomic Analysis.

Limonoids produced by citrus are a group of highly bioactive secondary metabolites which provide health benefits for humans. Currently there is a lack of information derived from research on the genetic mechanisms controlling the biosynthesis of limonoids, which has limited the improvement of citrus for high production of limonoids. In this study, the transcriptome sequences of leaves, phloems and seeds of pummelo (Citrus grandis (L.) Osbeck) at different development stages with variances in limonoids contents were used for digital gene expression profiling analysis in order to identify the genes corresponding to the biosynthesis of limonoids. Pair-wise comparison of transcriptional profiles between different tissues identified 924 differentially expressed genes commonly shared between them. Expression pattern analysis suggested that 382 genes from three conjunctive groups of K-means clustering could be possibly related to the biosynthesis of limonoids. Correlation analysis with the samples from different genotypes, and different developing tissues of the citrus revealed that the expression of 15 candidate genes were highly correlated with the contents of limonoids. Among them, the cytochrome P450s (CYP450s) and transcriptional factor MYB demonstrated significantly high correlation coefficients, which indicated the importance of those genes on the biosynthesis of limonoids. CiOSC gene encoding the critical enzyme oxidosqualene cyclase (OSC) for biosynthesis of the precursor of triterpene scaffolds was found positively corresponding to the accumulation of limonoids during the development of seeds. Suppressing the expression of CiOSC with VIGS (Virus-induced gene silencing) demonstrated that the level of gene silencing was significantly correlated to the reduction of limonoids contents. The results indicated that the CiOSC gene plays a pivotal role in biosynthesis of limonoids.

Digital gene expression profiling analysis of DNA repair pathways in colon cancer stem population of HT29 cells.

Cancer stem cells (CSCs) contribute to the relapse and development of new neoplasm lesions. While most available clinical approaches, such as chemical and radiation therapies, will kill the majority of cancer cells, they do not kill them all. Some resisting cells, like CSCs, are able to survive due to their excellent self-maintaining capabilities, even in challenging environments. In the present study, we investigated the mRNA level of DNA repair genes of colon CSCs from the HT29 cell line in response to single-strand damage and double-strand breaks, as well as the evident upregulation of key genes in base excision repair, mismatch repair, non-homologous end-joining, and homologous recombination pathways in these cells. Digital gene expression analysis identified upregulated genes in CD44+ HT29 cells that may play important roles in DNA repair. Our results reveal that colon CSCs bear efficient DNA repair abilities, which might explain the survival of colon CSCs after repeated chemical and radiation therapy.

RNA-seq approach to discriminate gene expression profiles in RIXI overexpressing transgenic rice

To investigate whether RIXI-overexpressing transgenic plants influence the expression of other genes, we analyzed transcriptomic changes between R7 (RIXI-overexpression transgenic line 7) plants and WT (wild-type) plants using deep RNA sequencing combined with digital gene expression profile analysis. The differentially expressed genes between the WT and R7 libraries were identified by DEG-Seq (differentially expressed genes from RNA-seq) software. The profiling analysis revealed that the overexpression of RIXI in rice resulted in numerous changes in gene expressions, including upregulation of 391 genes and downregulation of 905 genes. These differentially expressed genes were categorized into 30 groups with broad functions using gene ontology (GO) assignments. Among the 30 groups, five groups (single-organism metabolic process, biological regulation, anion binding, small molecule binding and nucleotide binding) had more differentially expressed genes. Biological pathways affected by RIXI overexpression were mapped using the detected genes to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). The differentially expressed genes were assigned to 98 KEGG pathways, and four enriched pathways were identified: metabolic, biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone signal transduction. The measurement of agronomic traits of R7 showed that the overexpression of RIXI did not influence the growth and development of rice. Thus, we conclude that the xylanase inhibitor gene RIXI may play a role in activation of a complex signal transduction network in response to various biotic and abiotic stresses, but does not have a negative influence on growth and development of rice plants.

The Salinity Responsive Mechanism of a Hydroxyproline-Tolerant Mutant of Peanut Based on Digital Gene Expression Profiling Analysis.

Soil salinity seriously limits plant growth and yield. Strategies have been developed for plants to cope with various environmental stresses during evolution. To screen for the broad-spectrum genes and the molecular mechanism about a hydroxyproline-tolerant mutant of peanut with enhanced salinity resistance under salinity stress, digital gene expression (DGE) sequencing was performed in the leaves of salinity-resistant mutant (S2) and Huayu20 as control (S4) under salt stress. The results indicate that major transcription factor families linked to salinity stress responses (NAC, bHLH, WRKY, AP2/ERF) are differentially expressed in the leaves of peanut under salinity stress. In addition, genes related to cell wall loosening and stiffening (xyloglucan endotransglucosylase/hydrolases, peroxidases, lipid transfer protein, expansin, extension), late embryogenesis abundant protein family, fatty acid biosynthesis and metabolism (13-lipoxygenase omega-6 fatty acid desaturase, omega-3 fatty acid desaturase) and some previously reported stress-related genes encoding proteins such as defensin, universal stress protein, metallothionein, peroxidase etc, and some other known or unknown function stress related genes, have been identified. The information from this study will be useful for further research on the mechanism of salinity resistance and will provide a useful genomic resource for the breeding of salinity resistance variety in peanut.

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.

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.

A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

Identification of genes involved in biosynthesis of mannan polysaccharides in Dendrobium officinale by RNA-seq analysis

Dendrobium officinale is a traditional Chinese medicinal plant. The stems of D. officinale contain mannan polysaccharides, which are promising bioactive polysaccharides for use as drugs. However, the genes involved in the biosynthesis of mannan polysaccharides in D. officinale have not yet been identified. In this study, four digital gene expression profiling analyses were performed on developing stems of greenhouse-grown D. officinale to identify such genes. Based on the accumulation of mannose and on gene expression levels, eight CELLULOSE SYNTHASE-LIKE A genes (CSLA), which are highly likely to be related to the biosynthesis of bioactive mannan polysaccharides, were identified from the differentially expressed genes database. In order to further analyze these DoCSLA genes, a full-length cDNA of each was obtained by RACE. The eight genes, belonging to the CSLA family of the CesA superfamily, contain conserved domains of the CesA superfamily. Most of the genes, which were highly expressed in the stems of D. officinale, were related to abiotic stress. Our results suggest that the CSLA family genes from D. officinale are involved in the biosynthesis of bioactive mannan polysaccharides.

Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii.

Mogrosides, the major bioactive components isolated from the fruits of Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as high-potency sweeteners and possess a variety of notable pharmacological activities. Mogrosides are synthesized from 2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase (CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However, the relevant genes have not been characterized to date. In this study, we report successful identification of SgCbQ and UGT74AC1, which were previously predicted via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis of the fruits of S. grosvenorii. SgCbQ was functionally characterized by expression in the lanosterol synthase-deficient yeast strain GIL77 and was found to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously expressed in Escherichia coli as a His-tag protein and it showed specificity for mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE by in vitro enzymatic activity assays. This study reports the identification of CbQ and glycosyltransferase from S. grosvenorii for the first time. The results also suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene saponins.

De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-alzheimer’s properties

BackgroundThe major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid β protein (a pathological marker of Alzheimer’s disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown.ResultsIn this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis.ConclusionA combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria, and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-676) contains supplementary material, which is available to authorized users.