Illumina Transcriptome Sequencing Research Articles

Uncovering the molecular mechanisms behind steroidal saponin accumulation in Liriope muscari (Decne.) Baily through transcriptome sequencing and bioinformatics analysis

The leaves and roots of Liriope muscari (Decne.) Baily were subjected to high-throughput Illumina transcriptome sequencing. Bioinformatics analysis was used to investigate the enzyme genes and key transcription factors involved in regulating the accumulation of steroidal saponins, which are the main active ingredient in L. muscari. These analyses aimed to reveal the molecular mechanism behind steroidal saponin accumulation. The sequencing results of L. muscari revealed 31 enzymes, including AACT, CAS, DXS and DXR, that are involved in the synthesis of steroidal saponins. Among these enzymes, 16 were in the synthesis of terpenoid skeleton, 3 were involved in the synthesis of sesquiterpene and triterpene, and 12 were involved in the synthesis of steroidal compound. Differential gene expression identified 15 metabolic enzymes coded by 34 differentially expressed genes (DEGs) in the leaves and roots, which were associated with steroidal saponin synthesis. Further analysis using gene co-expression patterns showed that 14 metabolic enzymes coded by 31 DEGs were co-expressed. In addition, analysis using gene co-expression analysis and PlantTFDB's transcription factor analysis tool predicted the involvement of 8 transcription factors, including GAI, PIF4, PIL6, ERF8, SVP, LHCA4, NF-YB3 and DOF2.4, in regulating 6 metabolic enzymes such as DXS, DXR, HMGR, DHCR7, DHCR24, and CAS. These eight transcription factors were predicted to play important roles in regulating steroidal saponin accumulation in L. muscari. Promoter analysis of these transcription factors indicated that their main regulatory mechanisms involve processes such as abscisic acid response, drought-induction stress response and light response, especially abscisic acid responsive elements (ABRE) response and MYB binding site involved in drought-inducibility (MBS) response pathway. Furthermore, qRT-PCR analysis of these eight key transcription factors demonstrated their specific differences in the leaves and roots.

Gene Expression Profiling Reveals Potential Players of Sex Determination and Asymmetrical Development in Chicken Embryo Gonads.

Despite the notable progress made in recent years, the understanding of the genetic control of gonadal sex differentiation and asymmetrical ovariogenesis in chicken during embryonic development remains incomplete. This study aimed to identify potential key genes and speculate about the mechanisms associated with ovary and testis development via an analysis of the results of PacBio and Illumina transcriptome sequencing of embryonic chicken gonads at the initiation of sexual differentiation (E4.5, E5.5, and E6.5). PacBio sequencing detected 328 and 233 significantly up-regulated transcript isoforms in females and males at E4.5, respectively. Illumina sequencing detected 95, 296 and 445 DEGs at E4.5, E5.5, and E6.5, respectively. Moreover, both sexes showed asymmetrical expression in gonads, and more DEGs were detected on the left side. There were 12 DEGs involved in cell proliferation shared between males and females in the left gonads. GO analysis suggested that coagulation pathways may be involved in the degradation of the right gonad in females and that blood oxygen transport pathways may be involved in preventing the degradation of the right gonad in males. These results provide a comprehensive expression profile of chicken embryo gonads at the initiation of sexual differentiation, which can serve as a theoretical basis for further understanding the mechanism of bird sex determination and its evolutionary process.

Physiological and transcriptomic responses of Pinellia ternata to continuous cropping

Pinellia ternata (Thunb.) Breit. is one of the popular medicinal plants and industrial crops in China, but its production has been severely limited by continuous cropping obstacles (CCO). In order to elucidate the reaction of P. ternata to successive cultivation and the potentially uncover the possible causes of CCO, the research group examined P. ternata relevant indexes, including morphological, physio-biochemical, as well as its transcriptome, under continuous cropping (CC) conditions. Based on the results, the influences of CC were multilevel. Specifically, CC led to chlorophyll content in leaves and antioxidant enzymes (peroxidase and superoxide dismutase, i.e. POD & SOD) activities significant reduced, reactive oxygen species (ROS) and malondialdehyde (MDA) in cells increased significantly. And the obvious changes were also observed in osmoregulatory substances such as proline (Pro), soluble protein (SP) and soluble sugars (SS). As a result, the plants growth was hindered and had the appearance of a "sprout tumble" prematurely. By combining SMRT full-length and Illumina transcriptome sequencing, a database of the root and leaf transcriptome of P. ternata at different years of CC years was established, and 77,783 full-length non-redundant transcripts were assembled. 412 differentially expressed genes (DEGs) were identified in the root during the first planting year (FPR) vs. the root after 2 years of Continuous Cropping (CC2R), of which 138 were up-regulated, meanwhile 274 were down-regulated. Moreover, 5020 DEGs were identified in the leaves during the first planting year (FPL) vs. the leaves after 2 years of Continuous Cropping (CC2L), of which 1894 were up-regulated, and 3126 were down-regulated. The analysis of DEGs showed that photosynthesis and chlorophyll biosynthesis, fatty acid and wax biosynthesis, cell wall synthesis and modification, defense response and plant-pathogen interaction, as well as phytohormone signaling were the key genes differentially expressed. Weighted gene correlation network analysis (WGCNA) analysis revealed that the hub genes in the roots were enriched in pathways associated with sugar synthesis and metabolism, phosphate ion transport, jasmonate metabolism, and fungal defense. At the same time, phosphate ion transport, positive regulation of cellular response to phosphate starvation, chlorophyll biosynthetic process, photosynthesis, and stress response pathways were enriched in the leaves. As a result, it was inferred that phosphorus depletion and the alteration of the soil microbiota by the previous planting cycles, which resulted in nutrient element deficiencies, as well as the damage of roots by pathogenic fungi, were the main CCO causes of P. ternata, and the corresponding changes in the physiological and transcriptional homeostasis of the leaves due to a generalized stress response.

Transcriptional variation in Babesia gibsoni (Wuhan isolate) between in vivo and in vitro cultures in blood stage

BackgroundBabesia gibsoni, the causative agent of canine babesiosis, belongs to the phylum Apicomplexa. The development of in vitro culture technology has driven research progress in various kinds of omics studies, including transcriptomic analysis of Plasmodium spp. between in vitro and in vivo environments, which has prompted the observation of diagnostic antigens and vaccine development. Nevertheless, no information on Babesia spp. could be obtained in this respect, which greatly hinders the further understanding of parasite growth and development in the blood stage.MethodsIn this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization.ResultsThrough laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni.ConclusionThis study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.Graphical

Identification of the GDP-L-Galactose Phosphorylase Gene as a Candidate for the Regulation of Ascorbic Acid Content in Fruits of Capsicum annuum L.

Ascorbic acid (AsA) is an antioxidant with significant functions in both plants and animals. Despite its importance, there has been limited research on the molecular basis of AsA production in the fruits of Capsicum annuum L. In this study, we used Illumina transcriptome sequencing (RNA-seq) technology to explore the candidate genes involved in AsA biosynthesis in Capsicum annuum L. A total of 8272 differentially expressed genes (DEGs) were identified by the comparative transcriptome analysis. Weighted gene co-expression network analysis identified two co-expressed modules related to the AsA content (purple and light-cyan modules), and eight interested DEGs related to AsA biosynthesis were selected according to gene annotations in the purple and light-cyan modules. Moreover, we found that the gene GDP-L-galactose phosphorylase (GGP) was related to AsA content, and silencing GGP led to a reduction in the AsA content in fruit. These results demonstrated that GGP is an important gene controlling AsA biosynthesis in the fruit of Capsicum annuum L. In addition, we developed capsanthin/capsorubin synthase as the reporter gene for visual analysis of gene function in mature fruit, enabling us to accurately select silenced tissues and analyze the results of silencing. The findings of this study provide the theoretical basis for future research to elucidate AsA biosynthesis in Capsicum annuum L.

Comparative transcriptome analysis reveals an insight into the candidate genes involved in anthocyanin and scent volatiles biosynthesis in colour changing flowers of Combretum indicum.

Combretum indicum is a widely cultivated ornamental species displaying the distinct phenomenon of floral colour change. Flowers display a gradual colour change from white to red, attributed to increased cyanidin 3-O glucoside in petal tissues. The differently coloured flowers also emanate a complex blend of VOCs with trans-linalool oxide (furanoid) as the major compound in the emission profile. To understand molecular mechanisms regulating floral colour shifts and scent biosynthesis, we performed Illumina transcriptome sequencing, including de novo assembly and functional annotation, for the two stages of floral maturation (white and red). Homology analysis with functional classification identified 84 and 42 candidate genes associated with pigment and scent biosynthesis, respectively. Genes encoding transcription factors, such as MYB, ERF, WD40, WRKY, NAC, bHLH and bZIP, that play critical roles in regulating specialized metabolism were also identified in the transcriptome data. Differences in expression of genes were consistent with accumulation patterns of anthocyanins in the two different flower colours. A clear upregulation of flavonoid biosynthesis genes in red flower tissue is associated with increased pigment content. RT-qPCR-based expression analyses gave results consistent with the RNA-Seq data, suggesting the sequencing data are consistent and reliable. This study presents the first report of genetic information for C. indicum. Gene sequences generated from RNA-Seq, along with candidate genes identified by pathway mapping and their expression profiles, provide a valuable resource for subsequent studies towards molecular understanding of specialized metabolism in C. indicum flowers.

Transcriptomics analysis and candidate genes associated with Xinjiang jujube fruits in response to Alternaria alternata infection

Black spot disease, caused by Alternaria alternata, is one of the most destructive diseases of jujube worldwide. To better understand the resistance mechanisms of jujube to A. alternata infection, two different cultivars, Zizyphus jujuba Mill. var. Junzao (susceptible) and Zizyphus jujuba Mill. var. Huizao (resistant) were tested. In this study, we identified 2235 differentially expressed genes (DEGs) in the disease-resistant cultivar and 4958 in the susceptible cultivar. Most were associated with plant phytohormone synthesis and signal transduction, flavonoid synthesis, and glutathione metabolism. The expression of six DEGs associated with disease resistance was detected by real-time quantitative polymerase chain reaction (RT-qPCR), consistent with the results of Illumina transcriptome sequencing. Moreover, the distinct expression level of the six DEGs in Jun jujube and Hui jujube, verified that these are defense response factors. The present study identified several candidate resistance genes and signal transduction pathways that may contribute to black spot disease resistance in jujube, which will assist the investigation of resistance mechanisms in the response of jujube to A. alternata infection.

Evolution and Diversity of Bat and Rodent Paramyxoviruses from North America.

Paramyxoviruses are a diverse group of negative-sense, single-stranded RNA viruses of which several species cause significant mortality and morbidity. In recent years the collection of paramyxovirus sequences detected in wild mammals has substantially grown; however, little is known about paramyxovirus diversity in North American mammals. To better understand natural paramyxovirus diversity, host range, and host specificity, we sought to comprehensively characterize paramyxoviruses across a range of diverse cooccurring wild small mammals in southern Arizona. We used highly degenerate primers to screen fecal and urine samples and obtained a total of 55 paramyxovirus sequences from 12 rodent species and 6 bat species. We also performed Illumina transcriptome sequencing (RNA-seq) and de novo assembly on 14 of the positive samples to recover a total of 5 near-full-length viral genomes. We show there are at least two clades of rodent-borne paramyxoviruses in Arizona, while bat-associated paramyxoviruses formed a putative single clade. Using structural homology modeling of the viral attachment protein, we infer that three of the five novel viruses likely bind sialic acid in a manner similar to other respiroviruses, while the other two viruses from heteromyid rodents likely bind a novel host receptor. We find no evidence for cross-species transmission, even among closely related sympatric host species. Taken together, these data suggest paramyxoviruses are a common viral infection in some bat and rodent species present in North America and illuminate the evolution of these viruses. IMPORTANCE There are a number of viral lineages that are potential zoonotic threats to humans. One of these, paramyxoviruses have jumped into humans multiple times from wild and domestic animals. We conducted one of the largest viral surveys of wild mammals in the United States to better understand paramyxovirus diversity and evolution.

Organelle Genomes and Transcriptomes of Nymphaea Reveal the Interplay between Intron Splicing and RNA Editing.

Posttranscriptional modifications, including intron splicing and RNA editing, are common processes during regulation of gene expression in plant organelle genomes. However, the intermediate products of intron-splicing, and the interplay between intron-splicing and RNA-editing were not well studied. Most organelle transcriptome analyses were based on the Illumina short reads which were unable to capture the full spectrum of transcript intermediates within an organelle. To fully investigate the intermediates during intron splicing and the underlying relationships with RNA editing, we used PacBio DNA-seq and Iso-seq, together with Illumina short reads genome and transcriptome sequencing data to assemble the chloroplast and mitochondrial genomes of Nymphaea ‘Joey Tomocik’ and analyze their posttranscriptional features. With the direct evidence from Iso-seq, multiple intermediates partially or fully intron-spliced were observed, and we also found that both cis- and trans-splicing introns were spliced randomly. Moreover, by using rRNA-depleted and non-Oligo(dT)-enrichment strand-specific RNA-seq data and combining direct SNP-calling and transcript-mapping methods, we identified 98 and 865 RNA-editing sites in the plastome and mitogenome of N. ‘Joey Tomocik’, respectively. The target codon preference, the tendency of increasing protein hydrophobicity, and the bias distribution of editing sites are similar in both organelles, suggesting their common evolutionary origin and shared editing machinery. The distribution of RNA editing sites also implies that the RNA editing sites in the intron and exon regions may splice synchronously, except those exonic sites adjacent to intron which could only be edited after being intron-spliced. Our study provides solid evidence for the multiple intermediates co-existing during intron-splicing and their interplay with RNA editing in organelle genomes of a basal angiosperm.

The bile acid-activated retinoic acid response in dendritic cells is involved in food allergen sensitization.

Alteration of commensal microbiota is highly correlated with the prevalence of allergic reactions to food in the gastrointestinal tract. The mechanisms by which microbiota modulate food allergen sensitization in the mucosal site are not fully understood. We generate DCs specific knockout of retinoic acid receptor α (Rara) gene mice (DC KO Rara) to evaluate food sensitization. The bile acid-activated retinoic acid response was evaluated by flow cytometry, real-time RT-PCR and Illumina transcriptome sequencing. The global effect of Abx treatment on BA profiles in the mucosal lymph tissue mLN in mice was examined by UPLC-MS analysis. In this study, we demonstrate that depletion of commensal gut bacteria leads to enhanced retinoic acid (RA) signaling in mucosal dendritic cells (DCs). RA signaling in DCs is required for the production of food allergen-specific IgE and IgG1. Antibiotics induced an enlarged bile acid (BA) pool, and dysregulated BA profiles contributed to enhanced RA signaling in mucosal DCs. BA-activated RA signaling promoted DC upregulation of interferon I signature, RA signature, OX40L, and PDL2, which may lead to T helper 2 differentiation of CD4+ T cells. BA-activated RA signaling involved the farnesoid X receptor and RA receptor α (RARa) interaction. Depletion of bile acid reduces food allergen specific IgE and IgG1 levels in mice. Our research unveils a mechanism of food sensitization modulated by BA-RA signaling in DCs, which suggests a potential new approach for the intervention of food allergic reactions.

GA3 is superior to GA4 in promoting bud endodormancy release in tree peony (Paeonia suffruticosa) and their potential working mechanism

BackgroundSufficient low temperature accumulation is the key strategy to break bud dormancy and promote subsequent flowering in tree peony anti-season culturing production. Exogenous gibberellins (GAs) could partially replace chilling to accelerate dormancy release, and different kinds of GAs showed inconsistent effects in various plants. To understand the effects of exogenous GA3 and GA4 on dormancy release and subsequent growth, the morphological changes were observed after exogenous GAs applications, the differentially expressed genes (DEGs) were identified, and the contents of endogenous phytohormones, starch and sugar were measured, respectively.ResultsMorphological observation and photosynthesis measurements indicated that both GA3 and GA4 applications accelerated bud dormancy release, but GA3 feeding induced faster bud burst, higher shoot and more flowers per plant. Full-length transcriptome of dormant bud was used as the reference genome. Totally 124 110 459, 124 015 148 and 126 239 836 reads by illumina transcriptome sequencing were obtained in mock, GA3 and GA4 groups, respectively. Compared with the mock, there were 879 DEGs and 2 595 DEGs in GA3 and GA4 group, 1 179 DEGs in GA3 vs GA4, and 849 DEGs were common in these comparison groups. The significant enrichment KEGG pathways of 849 DEGs highlighted plant hormone signal transduction, starch and sucrose metabolism, cell cycle, DNA replication, etc. Interestingly, the contents of endogenous GA1, GA3, GA4, GA7 and IAA significantly increased, ABA decreased after GA3 and GA4 treatments by LC–MS/MS. Additionally, the soluble glucose, fructose and trehalose increased after exogenous GAs applications. Compared to GA4 treatment, GA3 induced higher GA1, GA3 and IAA level, more starch degradation to generate more monosaccharide for use, and promoted cell cycle and photosynthesis. Higher expression levels of dormancy-related genes, TFL, FT, EBB1, EBB3 and CYCD, and lower of SVP by GA3 treatment implied more efficiency of GA3.ConclusionsExogenous GA3 and GA4 significantly accelerated bud dormancy release and subsequent growth by increasing the contents of endogenous bioactive GAs, IAA, and soluble glucose such as fructose and trehalose, and accelerated cell cycle process, accompanied by decreasing ABA contents. GA3 was superior to GA4 in tree peony forcing culture, which might because tree peony was more sensitive to GA3 than GA4, and GA3 had a more effective ability to induce cell division and starch hydrolysis. These results provided the value data for understanding the mechanism of dormancy release in tree peony.

Abstract 176: Detecting neoepitopes from tumor RNA sequencing datasets

Abstract Epitopes are peptides that present on the surface of the cell and can be recognized by immune cells to initiate the immune response. Identification of neoepitopes – tumor-specific, MHC-bound epitopes recognized specifically by T-cells – is valuable for predicting response to immunotherapies, including checkpoint blockade therapies. Tumors with more neoepitopes tend to be more responsive to immune checkpoint therapies compared to tumors with fewer neoepitopes. ProTECT is a previously published computational method that uses Illumina whole genome and transcriptome sequencing data from tumor and matched normal tissues to identify neoepitopes. Tumor and normal whole genome sequencing data are used to infer a patient's HLA haplotypes, as well as annotate variants as either somatic or germline. While whole genome sequencing is comprehensive, it is quite costly and not available for many samples. Here we adapt ProTECT to use only tumor RNA sequencing data and HLA haplotype information available to the clinician to identify neoepitopes in a tumor sample. Prior to running ProTECT, we use the computational tools Opossum and Platypus for variant calling instead of Radia (which is designed for variant calling using both RNA and DNA sequencing data as input). To determine which variants are somatic and therefore could represent tumor neoepitopes, variants found in RNA are compared to a panel of normals, for example the Genome Aggregation Database (gnomAD; containing variants from 125,748 exome sequences and 15,708 whole-genome sequences). With the resulting somatic variants and the HLA type, ProTECT proceeds as usual, with translation of variants into proteins, MHC:Peptide binding predictions and neoepitope ranking. We find that high quality neoepitopes are identifiable using an RNA-only approach, when genomic data is absent. Future work will validate the sensitivity of our method by benchmarking it against the original ProTECT predictions in the TCGA Prostate Adenocarcinoma cohort. Citation Format: Drew Thompson, Olena M. Vaske, Arjun Rao, Holly C. Beale. Detecting neoepitopes from tumor RNA sequencing datasets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 176.

Transcriptomic analysis reveals the parallel transcriptional regulation of UV-B-induced artemisinin and flavonoid accumulation in Artemisia annua L.

UV-B radiation is a pivotal photomorphogenic signal and positively regulates plant growth and metabolite biosynthesis. In order to elucidate the transcriptional regulation mechanism underlying UV-B-induced artemisinin and flavonoid biosynthesis in Artemisia annua, the transcriptional responses of A. annua L. leaves to UV-B radiation were analyzed using the Illumina transcriptome sequencing. A total of 10705 differentially expressed genes (DEGs) including 533 transcription factors (TFs), were identified. Based on the expression trends of the differentially expressed TFs as well as artemisinin and flavonoid biosynthesis genes, we speculated that TFs belonging to 6 clusters were most likely to be involved in the regulation of artemisinin and/or flavonoid biosynthesis. The regulatory relationship between TFs and artemisinin/flavonoid biosynthetic genes was further studied. Dual-LUC assays results showed that AaMYB6 is a positive regulator of AaLDOX which belongs to flavonoid biosynthesis pathway. In addition, we identified an R2R3 MYB TF, AaMYB4 which potentially mediated both artemisinin and flavonoid biosynthesis pathways by activating the expression of AaADS and AaDBR2 in artemisinin biosynthesis pathway and AaUFGT in flavonoid biosynthesis pathway. Overall, our findings would provide an insight into the elucidation of the parallel transcriptional regulation of artemisinin and flavonoid biosynthesis in A. annua L. under UV-B radiation.

Characterization of the development dynamics within the linear growth bamboo leaf.

The leaf is the main photosynthetic organ in plants, such as bamboo. Leaves from bamboo are used as a food additive. However, according to our investigation, to date there have been no reports concerning the leaf development of bamboo. By measuring over 7500 parenchymal cells, we discovered that the linear leaf growth of Pseudosasa japonica could be divided into three growth sections. The first one is a roughly 1-cm long division zone (DZ), containing about 1580 cells, located at the bottom of the leaf; the second one is an about 3-cm long elongation zone (EZ), with ~1905 cells, located above the DZ; and the last is a mature zone (MZ) in which cell elongation is completed. The cell production rate of the DZ was 25.33-35.81 cells per hour, with an average of 29.73; and the cell division rate was ~0.45 cells per cell every 24 h. PacBio and Illumina transcriptome sequencing found 21 933 unigenes expressed in these zones. Further analysis revealed a dynamic transcriptome, with transcripts for cell division in the DZ changing to transcripts for cell elongation, photosynthetic development, secondary metabolism, stress resistance, and nutrition transport toward the leaf distal. Those transcriptome transformations correlated well with the changes of relative water content, biomass accumulation, and cellulose crystal degree and were supported by quantitative polymerase chain reaction data. These results revealed a developmental gradient of the bamboo linear growth leaf, which offers a foundation to elucidate and engineer leaf development in bamboo, an economically valuable plant.

PacBio full length transcript sequencing and Illumina transcriptome insight into immune defense mechanism of Litopenaeus vannamei under ammonia-N stress

To investigate the immune defense mechanism of Litopenaeus vannamei (L. vannamei) exposed to high concentration of environmental ammonia-N, PacBio full-length transcriptome and Illumina transcriptome sequencing were performed. After ammonia-N exposure, 24,763 and 32,132 full-length transcripts were obtained, while 4465 and 3022 new genes were annotated at 0 and 6 h after compared to L. vannamei whole genome, respectively. Besides, haemocytes transcriptomes, performed at 0, 6 and 48 h, obtained a total of 1017 differentially expressed genes (DEGs), and among them 160 DEGs related to neuroendocrine-immune (NEI) were screened, of which 40 DEGs have alternative splicing form. The following experiments showed that plasma 5-HT and ACh concentrations increased significantly, and then returned to the initial level at 48 h. Moreover, cell membrane receptors (5-HT7R, ACh7R and Toll3), G-protein effectors (AC2 and PLC) and CaM all significantly upregulated, and nuclear transcription factor NF-кB was significantly downregulated, suggesting that 5-HT can act on AC-PKA, PLC - PKC and PLC - CaM pathways through 5-HT7R, and subsequently function on NF-кB together with ACh through ACh7R pathway to affects downstream effector proteins expression. Toll3 pathway is also activated to participate in signal transduction process simultaneously. Furthermore, the expressions of BGBP, PPO3, C-lectin, ALF, LYC and AIF3 were significantly increased, while Syntaxin and Cubilin were significantly decreased. The corresponding immune defense parameters including the THC, PO activity, haemocytes phagocytosis rate, antibacterial and bacteriolytic activities were significantly decreased. All results indicate that after ammonia-N stress, 5-HT, ACh and Toll3 can transfer signal into haemocytes to affect effector proteins and finally reduce immune defense parameters of L. vannamei. This study contributes to better exploration the immune defense mechanism of crustaceans under ammonia-N stress.

De novo Transcriptome Assembly, Gene Annotation and SSR Marker Development in the Moon Seed Genus Menispermum (Menispermaceae).

The moonseed genus Menispermum L. (Menispermaceae) is disjunctly distributed in East Asia and eastern North America. Although Menispermum has important medicinal value, genetic and genomic information is scarce, with very few available molecular markers. In the current study, we used Illumina transcriptome sequencing and de novo assembly of the two Menispermum species to obtain in-depth genetic knowledge. From de novo assembly, 53,712 and 78,921 unigenes were generated for M. canadense and M. dauricum, with 37,527 (69.87%) and 55,211 (69.96%) showing significant similarities against the six functional databases, respectively. Moreover, 521 polymorphic EST-SSRs were identified. Of them, 23 polymorphic EST-SSR markers were selected to investigate the population genetic diversity within the genus. The newly developed EST-SSR markers also revealed high transferability among the three examined Menispermaceae species. Overall, we provide the very first transcriptomic analyses of this important medicinal genus. In addition, the novel microsatellite markers developed here will aid future studies on the population genetics and phylogeographic patterns of Menispermum at the intercontinental geographical scale.

Transcriptome analysis of metabolic pathways associated with oil accumulation in developing seed kernels of Styrax tonkinensis, a woody biodiesel species

BackgroundStyrax tonkinensis (Pierre) Craib ex Hartwich has great potential as a woody biodiesel species having seed kernels with high oil content, excellent fatty acid composition and good fuel properties. However, no transcriptome information is available on the molecular regulatory mechanism of oil accumulation in developing S. tonkinensis kernels.ResultsThe dynamic patterns of oil content and fatty acid composition at 11 time points from 50 to 150 days after flowering (DAF) were analyzed. The percent oil content showed an up-down-up pattern, with yield and degree of unsaturation peaking on or after 140 DAF. Four time points (50, 70, 100, and 130 DAF) were selected for Illumina transcriptome sequencing. Approximately 73 million high quality clean reads were generated, and then assembled into 168,207 unigenes with a mean length of 854 bp. There were 5916 genes that were differentially expressed between different time points. These differentially expressed genes were grouped into 9 clusters based on their expression patterns. Expression patterns of a subset of 12 unigenes were confirmed by qRT-PCR. Based on their functional annotation through the Basic Local Alignment Search Tool and publicly available protein databases, specific unigenes encoding key enzymes, transmembrane transporters, and transcription factors associated with oil accumulation were determined. Three main patterns of expression were evident. Most unigenes peaked at 70 DAF, coincident with a rapid increase in oil content during kernel development. Unigenes with high expression at 50 DAF were associated with plastid formation and earlier stages of oil synthesis, including pyruvate and acetyl-CoA formation. Unigenes associated with triacylglycerol biosynthesis and oil body development peaked at 100 or 130 DAF.ConclusionsTranscriptome changes during oil accumulation show a distinct temporal trend with few abrupt transitions. Expression profiles suggest that acetyl-CoA formation for oil biosynthesis is both directly from pyruvate and indirectly via acetaldehyde, and indicate that the main carbon source for fatty acid biosynthesis is triosephosphate originating from phosphohexose outside the plastid. Different sn-glycerol-3-phosphate acyltransferases are implicated in diacylglycerol biosynthesis at early versus late stages of oil accumulation. Triacylglycerol biosynthesis may be accomplished by both diacylglycerol and by phospholipid:diacylglycerol acyltransferases.

High-throughput identification of heavy metal binding proteins from the byssus of chinese green mussel (Perna viridis) by combination of transcriptome and proteome sequencing.

The Byssus, which is derived from the foot gland of mussels, has been proved to bind heavy metals effectively, but few studies have focused on the molecular mechanisms behind the accumulation of heavy metals by the byssus. In this study, we integrated high-throughput transcriptome and proteome sequencing to construct a comprehensive protein database for the byssus of Chinese green mussel (Perna viridis), aiming at providing novel insights into the molecular mechanisms by which the byssus binds to heavy metals. Illumina transcriptome sequencing generated a total of 55,670,668 reads. After filtration, we obtained 53,047,718 clean reads and subjected them to de novo assembly using Trinity software. Finally, we annotated 73,264 unigenes and predicted a total of 34,298 protein coding sequences. Moreover, byssal samples were analyzed by proteome sequencing, with the translated protein database from the foot transcriptome as the reference for further prediction of byssal proteins. We eventually determined 187 protein sequences in the byssus, of which 181 proteins are reported for the first time. Interestingly, we observed that many of these byssal proteins are rich in histidine or cysteine residues, which may contribute to the byssal accumulation of heavy metals. Finally, we picked one representative protein, Pvfp-5-1, for recombinant protein synthesis and experimental verification of its efficient binding to cadmium (Cd2+) ions.

Comparative transcriptome analysis to identify candidate genes related to chlorogenic acid biosynthesis in Eucommia ulmoides Oliv.

We performed a comparative transcriptome between two different concentrations of CGA from leaves in E. ulmoides. The key genes in the biosynthesis of CGA were identified by differential expression analysis. Eucommia ulmoides Oliv. is native to China and famous for its extensive medicinal value, such as anti-fatigue, anti-aging, and enhanced immunity, etc. Chlorogenic acid (CGA) is the main medicinal ingredient of E. ulmoides. In E. ulmoides, the gene functions related to CGA biosynthesis are not fully understood. In this study, we conducted Illumina transcriptome sequencing and digital gene expression (DGE) for E. ulmoides with high content of CGA (H01, H02, and H03) and low content of CGA (L01, L02, and L03) to explore the potential genes involved in CGA biosynthesis. A total of 82,065 unigenes were generated. Of these unigenes, 28 were involved in CGA biosynthesis pathways and 116 regulated the CGA biosynthesis. Furthermore, by gene expression profiling and differential expression analysis, three functional genes including two PALs (c133625.graph_c5 and c133625.graph_c7), one 4CL (c139078.graph_c0), and three transcription factors (TFs), including one ERF2 (c139016.graph_c3) and two WRKY (c139022.graph_c3 and c139022.graph_c0) were different expressed, these genes may play key roles in CGA biosynthesis. This study will provide useful genomic resources for the investigation of CGA biosynthesis and genetic improvements of E. ulmoides.

Multiple Mechanisms Driving Alternative Polyadenylation of Cyclin D1 (CCND1) pre‐mRNA Processing

Eukaryotic 3′end formation involves binding of 3′end processing factors to the pre‐mRNA, cleavage downstream of a polyadenylation signal (PAS), followed by polyadenylation. The existence of multiple PASs in ~70% of human genes has led to the increased recognition of alternative polyadenylation (APA) as a new paradigm of global gene regulation involving the 3′end. Transcripts resulting from APA of the same terminal exon share the same open reading frame but have 3′UTRs of different length which affect the stability, localization and translatability of the mRNA transcript. One of the genes that undergoes APA is cyclin D1 (CCND1), which is rate limiting for G1‐S phase cell cycle transition. Cyclin D1 expression is tightly regulated and is normally transiently expressed in response to mitogenic stimuli. Both the normal protein and full length CCND1 mRNA transcript have a short half–life (~30 minutes). It is therefore not surprising that cyclin D1 expression is deregulated in several pathological states including cancer. In the B‐cell malignancy mantle cell lymphoma (MCL), the initiating lesion is a t(11; 14) (q13; q32) chromosomal translocation event that juxtaposes the CCND1 gene downstream of the IgH locus, resulting in constitutive expression of cyclin D1. Some MCL patients express CCND1 transcripts with truncated 3′UTRs and this is associated with a decrease in survival of almost two years. The truncated CCND1 transcript eliminates ~ 3kb from the 3′UTR leaving a 3′UTR <0.5kb in length. Our goal is to determine the mechanisms involved in APA as well as develop tools to identify and detect changes in the 3′UTR of genes. We developed different qRT‐PCR primers that can detect CCND1 transcripts with long/short 3′UTRs as well as nested primers for 3′RACE to help map the 3′ends of different CCND1 transcripts. In addition, used Illumina HiSeq and Pacific Biosciences (PacBio) next generation sequencing platforms to detect alternative transcripts. Reporter plasmids containing 3′UTRs were used to determine effects of RNAi. We found that highly proliferative cell lines were associated with higher levels of truncated CCND1 3′UTRs. One cell line had a mutation that resulted in the generation of a canonical PAS from a non‐optimal PAS proximal to the stop codon causing a switch from using a more distal PAS to the mutated PAS proximal to the stop codon. Illumina transcriptome sequencing after knockdown of a 3′end processing factor, CFIm25, resulted in increased shortening of the 3′UTR of CCND1 and over 1,400 other genes. Hence APA of CCND1 may be regulated by altered expression/function of genes involved in 3′end formation. When we mapped the 3′ends of CCND1 transcripts from multiple cell lines using 3′RACE, we identified a novel CCND1 transcript with a chimeric 3′UTR consisting of sequences from CCND1 fused to another gene, MCRK. This transcript containing the chimeric CCND1‐MRCK sequence was only detected in some MCL cell lines and patient DNA. Full length CCND1 3′UTR has over 60 potential miRNA target sites and reporter assays showed that the CCND1‐MRCK 3′UTR was able to evade miRNA regulation. Preliminary full length transcript PacBio sequencing resulted in the de novo identification of the CCND1‐MRCK fusion transcript. In conclusion, there are multiple ways in which CCND1 optimizes its expression in cells at the mRNA level and these mechanisms may be used by other genes under different physiological states.Support or Funding InformationDoD W81XWH‐12‐1‐0218This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.