Full-length Transcriptome Analysis Research Articles

Transcript Complexity and New Insights of Restorer Line in CMS-D8 Cotton Through Full-Length Transcriptomic Analysis.

Hybrid utilization has proficiently increased crop production worldwide. The cytoplasmic male sterility (CMS) system has emerged as an efficient tool for commercial hybrid cotton seed production. The restorer line with dominant Rf2 gene can restore the fertility of the CMS-D8 sterile line. However, the molecular mechanism of fertility restoration remains unclear in CMS-D8 cotton that limits wider utilization of three-line hybrid breeding. In our study, the Pacific Biosciences (PacBio) Iso-Seq technology was applied to understand fertility restoration mechanism of CMS-D8 cotton. In total, 228,106 full-length non-chimeric transcriptome sequences were obtained from anthers of developing flowering buds. The analysis results identified 3,174 novel isoforms, 2,597 novel gene loci, 652 long non-coding RNAs predicted from novel isoforms, 7,234 alternative splicing events, 114 fusion transcripts, and 1,667 genes with alternative polyadenylation. Specially, two novel genes associated with restoration function, Ghir_D05.742.1 and m64033_190821_201011/21103726/ccs were identified and showed significant higher levels of expression in restorer line than sterile and maintainer lines. Our comparative full-length transcriptome analysis provides new insights into the molecular function of Rf2 fertility restorer gene. The results of this study offer a platform for fertility restoration candidate gene discovery in CMS-D8 cotton.

SMRT Sequencing Reveals Candidate Genes and Pathways With Medicinal Value in Cipangopaludina chinensis.

Cipangopaludina chinensis is an economically important aquatic snail with high medicinal value. However, molecular biology research on C. chinensis is limited by the lack of a reference genome, so the analysis of its transcripts is an important step to study the regulatory genes of various substances in C. chinensis. Herein, we conducted the first full-length transcriptome analysis of C. chinensis using PacBio single-molecule real-time (SMRT) sequencing technology. We identified a total of 26,312 unigenes with an average length of 2,572 bp, of which the largest number of zf-c2h2 transcription factor families (120,18.24%) were found, and also observed that the majority of the 8,058 SSRs contained 4-7 repeat units, which provided data for subsequent work on snail genetics Subsequently, 91.86% (24,169) of the genes were successfully annotated to the four major databases, while the highest homology was observed with Pomacea canaliculata. Functional annotation revealed that the majority of transcripts were enriched in metabolism, signal transduction and Immune-related pathways, and several candidate genes involved in drug metabolism and immune response were identified (e.g., CYP1A1, CYP2J, CYP2U1, GST, ,PIK3, PDE3A, PRKAG). This study lays a foundation for future molecular biology research and provides a reference for studying genes associated with the medicinal value of C. chinensis.

Full-length gonad transcriptome analysis of Amur sturgeon Dmrt family genes: identification, characterization, and expression patterns during gonadal differentiation.

The regulatory mechanisms that govern sex differentiation in sturgeon are still poorly understood. The doublesex and Mab-3-related transcription factor (Dmrt) gene family is known for its extensive roles in sex determination and differentiation across vertebrates. This study aimed to identify new members of sturgeon Dmrt family genes and core actors in the gonadal differentiation of Amur sturgeon. A full-length gonad transcriptome database was exploited to identify Dmrt gene orthologs. Analyses of phylogenetic relationships and selection pressure were performed, and tissue expression profiles and spatiotemporal expression patterns in gonads were then analyzed using real-time PCR. In total, five Dmrt family genes were identified from the full-length gonad transcriptome, including Dmrt2, DmrtA1, DmrtA2, DmrtB1a, and DmrtB1b. Phylogenetic analysis showed that these genes were clustered into clades corresponding to the doublesex/Mav-3 (DM) genes of vertebrates. Furthermore, the analysis of evolutionary selective pressure indicated that DmrtB1a and DmrtB1b were subject to positive selection, suggesting the existence of adaptive evolution in sturgeon. The extensive tissue expression profiling of each Dmrt family gene revealed typical characteristics. Remarkably, according to a spatiotemporal expression pattern analysis, in later stages, DmrtB1b expression increased in testes and was significantly higher in testes than in ovaries at 24months after hatching (M) and 36M. This study provides a genetic resource of full-length Dmrt family genes and increases the understanding of Dmrt functions in sex differentiation in sturgeon.

Full-Length Transcriptional Analysis of the Same Soybean Genotype With Compatible and Incompatible Reactions to Heterodera glycines Reveals Nematode Infection Activating Plant Defense Response.

Full-length transcriptome sequencing with long reads is a powerful tool to analyze transcriptional and post-transcriptional events; however, it has not been applied on soybean (Glycine max). Here, a comparative full-length transcriptome analysis was performed on soybean genotype 09-138 infected with soybean cyst nematode (SCN, Heterodera glycines) race 4 (SCN4, incompatible reaction) and race 5 (SCN5, compatible reaction) using Oxford Nanopore Technology. Each of 9 full-length samples collected 8 days post inoculation with/without nematodes generated an average of 6.1 GB of clean data and a total of 65,038 transcript sequences. After redundant transcripts were removed, 1,117 novel genes and 41,096 novel transcripts were identified. By analyzing the sequence structure of the novel transcripts, a total of 28,759 complete open reading frame (ORF) sequences, 5,337 transcription factors, 288 long non-coding RNAs, and 40,090 novel transcripts with function annotation were predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of differentially expressed genes (DEGs) revealed that growth hormone, auxin-activated signaling pathway and multidimensional cell growth, and phenylpropanoid biosynthesis pathway were enriched by infection with both nematode races. More DEGs associated with stress response elements, plant-hormone signaling transduction pathway, and plant–pathogen interaction pathway with more upregulation were found in the incompatible reaction with SCN4 infection, and more DEGs with more upregulation involved in cell wall modification and carbohydrate bioprocess were detected in the compatible reaction with SCN5 infection when compared with each other. Among them, overlapping DEGs with a quantitative difference was triggered. The combination of protein–protein interaction with DEGs for the first time indicated that nematode infection activated the interactions between transcription factor WRKY and VQ (valine-glutamine motif) to contribute to soybean defense. The knowledge of the SCN–soybean interaction mechanism as a model will present more understanding of other plant–nematode interactions.

Full-Length Transcriptome Analysis Provides New Insights Into the Diversity of Immune-Related Genes in Portunus trituberculatus.

Generally, invertebrates were thought to solely rely on their non-specific innate immune system to fight against invading microorganisms. However, increasing studies have implied that the innate immune response of invertebrates displayed diversity and specificity owing to the hyper-variable immune molecules in organisms. In order to get an insight into the diversity of immune-related genes in Portunus trituberculatus, a full-length transcriptome analysis of several immune-related tissues (hemocytes, hepatopancreas and gills) in P. trituberculatus was performed and the diversity of several immune-related genes was analyzed. The full-length transcriptome analysis of P. trituberculatus was conducted using a combination of SMRT long-read sequencing and Illumina short-read sequencing. A total of 17,433 nonredundant full-length transcripts with average length of 2,271 bp and N50 length of 2,841 bp were obtained, among which 13,978 (80.18%) transcripts were annotated. Moreover, numerous transcript variants of various immune-related genes were identified, including pattern recognition receptors, antimicrobial peptides, heat shock proteins (HSPs), antioxidant enzymes and vital molecules in prophenoloxidase (proPO)-activating system. Based on the full-length transcriptome analysis, open reading frames (ORFs) of four C-type lectins (CTLs) were cloned, and tissue distributions showed that the four CTLs were ubiquitously expressed in all the tested tissues, and mainly expressed in hepatopancreas and gills. The transcription of the four CTLs significantly increased in several immune-related tissues (hemocytes, hepatopancreas and gills) of P. trituberculatus challenged with Vibrio alginolyticus and displayed different profiles. Moreover, the four CTLs displayed distinct bacterial binding and antibacterial activities. The recombinant protein PtCTL-1 (rPtCTL-1) and rPtCTL-3 displayed bacterial binding and antibacterial activities against all tested bacteria. rPtCTL-2 only showed bacterial binding and antibacterial activities against V. alginolyticus. No obvious bacterial binding or antibacterial activities for PtCTL-4 was observed against the tested bacteria. This study enriches the transcriptomic information on P. trituberculatus and provides new insights into the innate immune system of crustaceans. Additionally, our study provided candidates of antibiotic agents for the prevention and treatment of bacteriosis.

Long-Term Oral Administration of Salidroside Alleviates Diabetic Retinopathy in db/db Mice.

Diabetic retinopathy (DR), a microvascular complication of diabetes mellitus, is the leading cause of vision loss in the working-age population worldwide. Unfortunately, current clinical treatments cannot completely prevent the occurrence and development of DR. Salidroside (Sal) is a medicinal supplement that has antioxidative and cytoprotective properties. This study aimed to investigate the therapeutic effect of Sal on DR. Briefly, Sal treatment was applied to wide-type mice and db/db mice (a widely used diabetic mice) at 25 mg/kg by oral gavage once daily from 8 weeks to 20 weeks. Mice’s bodyweight, blood glucose, total cholesterol, triglyceride, high density lipoprotein and low density lipoprotein were recorded and analyzed. Retinal trypsin digestion and evans blue dye assay were used to detect retinal microvessel changes and function. Retinal glutathione and malondialdehyde content measurements were applied to assess retinal oxidative stress. Full-length transcriptome analysis was performed to explore the underlying mechanisms of Sal protection. Our results found that Sal treatment could successfully relieve blood glucose and blood lipid abnormalities, and reduce retinal oxidative stress level in diabetic mice. Also, Sal treatment repaired the abnormal transcriptome caused by diabetes, alleviated the microvascular lesion of the fundus in diabetic mice, and protected retinal normal barrier function. This study enriches the indications of Sal in the treatment of diabetic diseases, providing practical research ideas for the comprehensive preventions and treatments of DR.

Full-length transcriptome analysis of multiple organs and identification of adaptive genes and pathways in Mikania micrantha

Mikania micrantha is a notorious invasive weed that has caused huge economic loss and negative ecological consequences in invaded areas. This species can adapt well to invasive environments with various stress factors. The identification of gene families and functional pathways related to environmental adaptability is lack in M. micrantha at the multi-organ full-length transcriptome level. In this study, we sequenced the transcriptomes of five M. micrantha organs using PacBio single-molecule real-time sequencing and Illumina RNA sequencing technologies. Based on the transcriptome data, full-length transcripts were captured and gene expression patterns among the five organs were analyzed. KEGG enrichment analysis of genes with higher expression indicated their special roles in environmental stress response and adversity adaptation in the various five organs. The gene families and pathways related to biotic and abiotic factors, including terpene synthases, glutathione S-transferases, antioxidant defense system, and terpenoid biosynthesis pathway, were characterized. The expression levels of most differentially expressed genes in the antioxidant defense system and terpenoid biosynthesis pathway were higher in root, stem, and leaf than in the other two organs, suggesting that root, stem, and leaf have strong ability to respond to adverse stresses and form the important organs of terpenoid synthesis and accumulation. Additionally, a large number of transcription factors and alternative splicing events were predicted. This study provides a comprehensive transcriptome resource for M. micrantha, and our findings facilitate further research on the adaptive evolution and functional genomics of this species.

SMRT sequencing of full-length transcriptome and gene expression analysis in two chemical types of Pogostemon cablin (Blanco) Benth.

BackgroundPogostemon cablin (Blanco) Benth. also called patchouli, is a traditional medicinal and aromatic plant that grows mainly in Southeast Asia and China. In China, P. cablin is divided into two chemical types: the patchouliol-type and the pogostone-type. Patchouliol-type patchouli usually grow taller, with thicker stems and bigger leaves, and produce more aromatic oil.MethodsTo better understand the genetic differences between the two chemical types that contribute to their differences in morphology and biosynthetic capabilities, we constructed de novo transcriptomes from both chemical types using the Pacific Biosciences (PacBio) Sequel platform and performed differential expression analysis of multiple tissues using Illumina short reads.ResultsIn this study, using single-molecule real-time (SMRT) long-read sequencing, we obtained 22.07 GB of clean data and 134,647 nonredundant transcripts from two chemical types. Additionally, we identified 126,576 open reading frames (ORFs), 100,638 coding sequences (CDSs), 4,106 long noncoding RNAs (lncRNAs) and 6,829 transcription factors (TFs) from two chemical types of P. cablin. We adopted PacBio and Illumina sequencing to identify differentially expressed transcripts (DEGs) in three tissues of the two chemical types. More DEGs were observed in comparisons of different tissues collected from the same chemical type relative to comparisons of the same tissue collected from different chemical types. Furthormore, using KEGG enrichment analysis of DEGs, we found that the most enriched biosynthetic pathways of secondary metabolites of the two chemical types were “terpenoid backbone biosynthesis”, “phenylpropanoid biosynthesis”, “plant hormone signal transduction”, “sesquiterpenoid and triterpenoid biosynthesis”, “ubiquinone and other terpenoid-quinone biosynthesis”, “flavonoid biosynthesis”, and “flavone and flavonol biosynthesis”. However, the main pathways of the patchouliol-type also included “diterpene biosynthesis” and “monoterpene biosynthesis”. Additionally, by comparing the expression levels of the three tissues verified by qRT-PCR, more DEGs in the roots were upregulated in the mevalonate (MVA) pathway in the cytoplasm, but more DEGs in the leaves were upregulated in the methylerythritol phosphate (MEP) pathway in the plastid, both of which are important pathways for terpenoids biosynthesis. These findings promote the study of further genome annotation and transcriptome research in P. cablin.

The dynamic immune responses of Mandarin fish (Siniperca chuatsi) to ISKNV in early infection based on full-length transcriptome analysis and weighted gene co-expression network analysis

Mandarin fish (Siniperca chuatsi) been seriously harmed by infectious spleen and kidney necrosis virus (ISKNV) in recent years, but the early immune response mechanism of infection is still unknown. Here, we performed RNA sequencing on the spleens of mandarin fish infected with ISKNV at 0, 12, 24, 48, and 72 h post-infection (hpi) using short-read Illumina RNA sequencing and long-read Pacific Biosciences isoform sequencing to generate a full-length transcriptome. The immune responses of mandarin fish infected with ISKNV at the molecular level were characterized by RNA-seq analysis and weighted gene co-expression network analysis (WGCNA). A total of 26,528 full-length transcript sequences were obtained. There were 2,729 (1,680 up-regulated and 1,112 down-regulated), 1,874 (1,136 up-regulated and 738 down-regulated), 2,032 (1,158 up-regulated and 847 down-regulated), and 4,176 (2,233 up-regulated and 1,943 down-regulated) differentially expressed genes (DEGs) in mandarin fish at 12, 24, 48, and 72 hpi, compared with uninfected fish, respectively. A total of four modules of co-expressed DEGs identified by WGCNA were significantly positively correlated to the four time points after infection, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the immune-related DEGs in all these modules were mainly enriched in Phagosome, Endocytosis, Herpes simplex infection, and Cytokine-cytokine receptor interaction pathways. Further analysis showed that oher signaling pathways, including CAMs, NOD-like receptor and ER protein processing, Intestinal immune network for IgA production, TLR pathway, and Apoptosis significantly enriched in four modules corresponding to 12, 24, 48, and 72 hpi respectively, had specifically participated in the immune response. Hub genes identified based on the high-degree nodes in the WGCN, including CAM3, IL-8, CCL21, STING, SNX1, PFR and TBK1, and some DEGs such as MHCI, MHCII, TfR, STING, TNF α, TBK1, IRF1, and NF-kB, BCR, IgA and Bcl-XL had involved in dynamic molecular response of mandarin fish to ISKNV infection. In sum, this study provides a set of full-length transcriptome of the spleen tissue of mandarin fish for the first time and revealed a group of immune genes and pathways involved in different temporal responses to ISKNV infection, which has implications for resource conservation and aiding the development of strategies to prevent virus early infection for mandarin fish.

Molecular characterization and tissue distribution of cholecystokinin and its receptor in Yangtze sturgeon (Acipenser dabryanus) and their response to different feeding conditions

Yangtze sturgeon (Acipenser dabryanus) is a species endemic to Yangtze River drainage in China and is listed as a critically endangered species on the IUCN Red List. In the present study, cholecystokinin (CCK), one of the most important neuroregulatory digestive genes, and its receptor (CCKr) were identified from the full-length transcriptome analysis of A. dabryanus. The deduced amino acid sequences of CCK and CCKr from A. dabryanus showed structural features common to those in other vertebrates. Gene expression profile analysis showed that CCK and CCKr were universally expressed in different tissues, and both had the highest expression in the brain. Starvation and refeeding significantly regulated the expression levels of CCK and CCKr in the brain, suggesting that CCK and CCKr were involved in feed intake regulation in A. dabryanus as in mammals. In addition, the expression levels of CCK and CCKr under different feeding frequencies were studied. Compared with the control group (fed two times a day), the expression levels of CCK and CCKr in the intestine and brain did not change significantly in the other groups after 8 weeks of rearing, indicating that the feeding frequency might not influence the appetite of A. dabryanus. The present work provides a basis for further investigation into the regulation of feeding in A. dabryanus.

Comparative full-length transcriptome analysis provides novel insights into the regulatory mechanism of natural rubber biosynthesis in Taraxacum kok-saghyz Rodin roots

Taraxacum kok-saghyz Rodin (TKS) is a promising alternative rubber-producing plant due to the high-quality natural rubber (NR) synthesized in its root. Nevertheless, the comprehensive molecular mechanisms of NR biosynthesis in TKS roots remains segmented and elusive. In the current study, to elucidate the regulatory mechanism of NR synthesis at the transcriptional level, high-quality full-length root transcriptomic data of the TKS were generated based on Oxford Nanopore Technology (ONT). The transcripts with alternative splicing events, polyadenylation sites, and simple sequence repeats (SSRs) were screened. Then, comparative transcriptome analysis was conducted between the lines containing high rubber (HR) and low rubber (LR) contents, and a total of 3134 differentially expressed genes (DEGs) were identified. Among the genes, 1157 DEGs were upregulated, and 1977 DEGs were downregulated in the HR lines vs. the LR lines. The genes involved in NR synthesis were upregulated in HR lines. In contrast, the key genes involved in the synthesis of squalene, phenylpropanoid, fatty acids and ketone, which require the same substrates (isopentenyl pyrophosphate or acetyl-CoA) as NR biosynthesis, were downregulated in HR lines. The results indicated that the expression levels of NR synthesis-related genes were proportional to the NR production, and competition between NR and some metabolites synthesis existed when common substrates were used. Hence, this work provides novel insights into the regulatory mechanism of NR biosynthesis in TKS. Furthermore, the genes or pathways related to NR synthesis identified in this study will be important targets for genetic modification and molecular breeding in further.

SMRT Sequencing of the Full-Length Transcriptome of the Coelomactra antiquata.

Coelomactra antiquata is an important aquatic economic shellfish with high medicinal value. However, because C. antiquata has no reference genome, a lot of molecular biology research cannot be carried out, so the analysis of its transcripts is an important step to study the regulatory genes of various substances in C. antiquata. In the present study, we conducted the first full-length transcriptome analysis of C. antiquata by using PacBio single-molecule real-time (SMRT) sequencing technology. The results identified a total of 39,209 unigenes with an average length of 2,732 bp, 23,338 CDSs, 251 AS events, 9,881 lncRNAs, 20,106 SSRs, and 2,316 TFs. Subsequently, 59.22% (23,220) of the unigenes were successfully annotated, of which 23,164, 18,711, 15,840, 13,534, and 13,474 unigenes could be annotated using NR, Swiss-prot, KOG, GO, and KEGG databases, respectively. This study lays the foundation for the follow-up research of molecular biology and provides a reference for studying the more medicinal value of C. antiquata.

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

BackgroundEimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix.MethodsSecond-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied.ResultsSMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2.ConclusionsWe obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control.Graphical

Characterization of two lipid metabolism-associated genes and their expression profiles under different feeding conditions in Acipenser dabryanus

Acipenser dabryanus is an endemic species restricted to the upper reaches of the Yangtze River and is listed as a national first-class protected animal in China. In the present study, two lipid metabolism-associated genes, namely, lipoprotein lipase (LPL) and apolipoprotein E (apoE), were identified from the full-length transcriptome analysis of A. dabryanus. The full-length genes encoded polypeptides with typical conserved motifs and functional sites shared with their homologues in other fish. Gene expression profile analysis showed that the LPL and apoE genes were constitutively expressed in A. dabryanus. The highest expression level of LPL was found in the muscle, while apoE was predominantly expressed in the liver. In the fasting and refeeding experiment, one hundred twenty fish were randomly assigned to four groups (fasting 0, 3, 7 or 14 d and then refed for 14 d). Fasting and refeeding significantly regulated the expression level of LPL with various expression patterns in different tissues, whereas starvation showed little effect on apoE expression. In addition, the expression levels of LPL and apoE were significantly affected by feeding frequency. The fish were bred for eight weeks at six different feeding frequencies groups (once a day, two times a day, three times a day, four times a day, five times a day and six times a day). Both LPL and apoE expression levels were significantly upregulated in the liver in the highest feeding frequency group. Our study suggests potential roles for LPL and apoE in lipid metabolism in A. dabryanus and will be useful for further study of nutrition regulation in fish.

Full-Length Transcriptome Analysis Reveals Candidate Genes Involved in Terpenoid Biosynthesis in Artemisia argyi.

Artemisia argyi is an important medicinal plant widely utilized for moxibustion heat therapy in China. The terpenoid biosynthesis process in A. argyi is speculated to play a key role in conferring its medicinal value. However, the molecular mechanism underlying terpenoid biosynthesis remains unclear, in part because the reference genome of A. argyi is unavailable. Moreover, the full-length transcriptome of A. argyi has not yet been sequenced. Therefore, in this study, de novo transcriptome sequencing of A. argyi's root, stem, and leaf tissues was performed to obtain those candidate genes related to terpenoid biosynthesis, by combining the PacBio single-molecule real-time (SMRT) and Illumina sequencing NGS platforms. And more than 55.4 Gb of sequencing data and 108,846 full-length reads (non-chimeric) were generated by the Illumina and PacBio platform, respectively. Then, 53,043 consensus isoforms were clustered and used to represent 36,820 non-redundant transcripts, of which 34,839 (94.62%) were annotated in public databases. In the comparison sets of leaves vs roots, and leaves vs stems, 13,850 (7,566 up-regulated, 6,284 down-regulated) and 9,502 (5,284 up-regulated, 4,218 down-regulated) differentially expressed transcripts (DETs) were obtained, respectively. Specifically, the expression profile and KEGG functional enrichment analysis of these DETs indicated that they were significantly enriched in the biosynthesis of amino acids, carotenoids, diterpenoids and flavonoids, as well as the metabolism processes of glycine, serine and threonine. Moreover, multiple genes encoding significant enzymes or transcription factors related to diterpenoid biosynthesis were highly expressed in the A. argyi leaves. Additionally, several transcription factor families, such as RLK-Pelle_LRR-L-1 and RLK-Pelle_DLSV, were also identified. In conclusion, this study offers a valuable resource for transcriptome information, and provides a functional genomic foundation for further research on molecular mechanisms underlying the medicinal use of A. argyi leaves.

Full-length transcriptome analysis provides new insights into the early bolting occurrence in medicinal Angelica sinensis

Angelica sinensis (Oliv.) Diels root part is an integral component of traditional Chinese medicine, widely prescribed to improve blood circulation and blood stasis. However, early bolting of A. sinensis compromises the quality of the roots and hence is a major limitation for yield of medicinal materials. To date, little information about the molecular mechanisms underlying bolting is available for this important medicinal plant. To identify genes putatively involved in early bolting, we have conducted the transcriptome analysis of the shoot tips of the early-bolting plants and non-bolting (normal) plants of A. sinensis, respectively, using a combination of third-generation sequencing and next-generation sequencing. A total of 43,438 non-redundant transcripts were collected and 475 unique differentially expressed genes (DEGs) were identified. Gene annotation and functional analyses revealed that DEGs were highly involved in plant hormone signaling and biosynthesis pathways, three main flowering pathways, pollen formation, and very-long-chain fatty acids biosynthesis pathways. The levels of endogenous hormones were also changed significantly in the early bolting stage of A. sinensis. This study provided new insights into the transcriptomic control of early bolting in A. sinensis, which could be further applied to enhance the yield of medicinally important raw materials.

The Full-Length Transcriptome Sequencing and Identification of Na+/H+ Antiporter Genes in Halophyte Nitraria tangutorum Bobrov.

Nitraria tangutorum Bobrov is a halophyte that is resistant to salt and alkali and is widely distributed in northwestern China. However, its genome has not been sequenced, thereby limiting studies on this particular species. For species without a reference genome, the full-length transcriptome is a convenient and rapid way to obtain reference gene information. To better study N. tangutorum, we used PacBio single-molecule real-time technology to perform full-length transcriptome analysis of this halophyte. In this study, a total of 21.83 Gb of data were obtained, and 198,300 transcripts, 51,875 SSRs (simple sequence repeats), 55,574 CDS (coding sequence), and 74,913 lncRNAs (long non-coding RNA) were identified. In addition, using this full-length transcriptome, we identified the key Na+/H+ antiporter (NHX) genes that maintain ion balance in plants and found that these are induced to express under salt stress. The results indicate that the full-length transcriptome of N. tangutorum can be used as a database and be utilized in elucidating the salt tolerance mechanism of N. tangutorum.

Mechanisms by Which Fermented Soybean Meal and Soybean Meal Induced Enteritis in Marine Fish Juvenile Pearl Gentian Grouper.

Soy meals can cause intestinal inflammation and even injury in animals, especially infants and juvenile individuals. This study investigated the effects of fermented soybean meal (FSBM) on the growth and intestinal homeostasis of juvenile pearl gentian grouper and examined the mechanisms by which FSBM and soybean meal (SBM) induced enteritis in fish, using “3+2” full-length transcriptome sequencing. We randomly assigned 720 female juvenile groupers into three treatment groups: FM control group, 20% FSBM group (FSBM20), and FSBM40 group (n = 4). Three iso-nitrogenous (50% protein) and iso-lipidic (10% lipid) diets were prepared and fed to fish for 10 weeks. The water volume in each barrel was about 1 m3, using natural light and temperature. Results showed that dietary FSBM, at experimental level, significantly affected fish growth and intestinal structure negatively and significantly increased enteritis indices. The degree of intestinal injury and inflammation was determined by the enzyme activities of trypsin and lysozyme, and the contents of IgM, C3, C4, and malondialdehyde, and the expressions of pro-inflammatory genes (IL1β, IL8, IL17, and TNFα) and anti-inflammatory genes (IL4, IL10, and TGFβ1). Full-length transcriptome analysis identified 2,305 and 3,462 differentially expressed genes (DEGs) in SBM40 and FSBM40 groups, respectively. However, only 18.98% (920/5,445) of DEGs had similar expression patterns, indicating that high levels of SBM40 and FSBM40 have different metabolic strategies. KEGG enrichment analysis indicated that among the significant pathways, ~45% were related to immune diseases/systems, infectious diseases, and signal transduction in both SBM and FSBM groups. Based on PacBio SMRT sequencing, nine toll-like receptor (TLR) members, including TLR1, TLR2, TLR3, TLR5, TLR8, TLR9, TLR13, TLR21, and TLR22, were detected in intestinal tissues of pearl gentian grouper. TLR-MyD88-NF-κB signaling pathway played an important role in the development of FSBM- and SBM-induced enteritis in pearl gentian grouper; however, TLR receptors used in SBM and FSBM groups were different. TLR1, TLR8, TLR13, and TLR22 were the main receptors used in FSBM group, while TLR5, TLR8, TLR9, TLR21, and TLR22 were the main receptors used in SBM group. Present study provides valuable theoretical references for further research on soy protein-induced enteritis in fish.

De novo full length transcriptome analysis of Arachis glabrata provides insights into gene expression dynamics in response to biotic and abiotic stresses

The perennial ornamental peanut Arachis glabrata represents one of the most adaptable wild Arachis species. This study used PacBio combined with BGISEQ-500 RNA-seq technology to study the transcriptome and gene expression dynamics of A. glabrata. Of the total 109,747 unique transcripts obtained, >90,566 transcripts showed significant homology to known proteins and contained the complete coding sequence (CDS). RNA-seq revealed that 1229, 1039, 1671, 3923, 1521 and 1799 transcripts expressed specifically in the root, stem, leaf, flower, peg and pod, respectively. We also identified thousands of differentially expressed transcripts in response to drought, salt, cold and leaf spot disease. Furthermore, we identified 30 polyphenol oxidase encoding genes associated with the quality of forage, making A. glabrata suitable as a forage crop. Our findings presented the first transcriptome study of A. glabrata which will facilitate genetic and genomics studies and lays the groundwork for a deeper understanding of the A. glabrata genome.

Full-length transcriptome sequencing and identification and immune response of TRIM genes in Dabry's sturgeon (Acipenser dabryanus)

Acipenser dabryanus, also called Yangtze sturgeon or Dabry's sturgeon, is an endemic species restricted to the upper reaches of the Yangtze River in China. In the present study, we conducted the first full-length transcriptome analysis of A. dabryanus by the use of SMRT sequencing technology. Transcriptome sequencing generated a total of 155,348 unigenes with a mean length of 2996 bp. A total of 131,711 (84.78%) unigenes were annotated in various databases and comparative analysis was performed between A. dabryanus and its the related species. Based on the sequence information, three members in the tripartite motif (TRIM) family, namely, TRIM32, TRIM36 and TRIM71, were identified. The full-length TRIM genes encoded polypeptides with a typical TRIM structure: a RING domain, one or two B-box domains, and variable domains at the C-terminus. Gene expression profile analysis showed that the three TRIM genes were constitutively expressed in A. dabryanus, and the highest expression levels of TRIM32 and TRIM36 were found in the brain, while TRIM71 was predominantly expressed in the muscle. Upon challenge with the bacterial pathogen Edwardsiella tarda, significant upregulation of TRIMs was observed in both mucosal and systemic-associated immune tissues. Our data revealed the possible functional role of TRIMs in both mucosal and systemic defence of A. dabryanus in the immune response against bacterial infection.