Full-length Transcriptome Data Research Articles

Nanopore-based full-length transcriptome sequencing for understanding the underlying molecular mechanisms of rapid and slow progression of diabetes nephropathy.

Diabetic nephropathy (DN) has been a major factor in the outbreak of end-stage renal disease for decades. As the underlying mechanisms of DN development remains unclear, there is no ideal methods for the diagnosis and therapy. We aimed to explore the key genes and pathways that affect the rate progression of DN. Nanopore-based full-length transcriptome sequencing was performed with serum samples from DN patients with slow progression (DNSP, n = 5) and rapid progression (DNRP, n = 6). Here, transcriptome proclaimed 22,682 novel transcripts and obtained 45,808 simple sequence repeats, 1,815 transcription factors, 5,993 complete open reading frames, and 1,050 novel lncRNA from the novel transcripts. Moreover, a total of 341 differentially expressed transcripts (DETs) and 456 differentially expressed genes (DEGs) between the DNSP and DNRP groups were identified. Functional analyses showed that DETs mainly involved in ferroptosis-related pathways such as oxidative phosphorylation, iron ion binding, and mitophagy. Moreover, Functional analyses revealed that DEGs mainly involved in oxidative phosphorylation, lipid metabolism, ferroptosis, autophagy/mitophagy, apoptosis/necroptosis pathway. Collectively, our study provided a full-length transcriptome data source for the future DN research, and facilitate a deeper understanding of the molecular mechanisms underlying the differences in fast and slow progression of DN.

SMRT sequencing of a full-length transcriptome reveals cold induced alternative splicing in Vitis amurensis root

Alternative splicing enhances diversity at the transcriptional and protein levels that widely involved in plant response to biotic and abiotic stresses. V. amurensis is an extremely cold-tolerant wild grape variety, however, studies on alternative splicing (AS) in amur grape at low temperatures are currently poorly understood. In this study, we analyzed full-length transcriptome and RNA seq data at 0, 2, and 24 h after cold stress in V. amurensis roots. Following quality control and correction, 221,170 high-quality full-length non-concatemer (FLNC) reads were identified. A total of 16,181 loci and 30,733 isoforms were identified. These included 22,868 novel isoforms from annotated genes and 2815 isoforms from 2389 novel genes. Among the distinguished novel isoforms, 673 Long non-coding RNAs (LncRNAs) and 18,164 novel isoforms open reading frame (ORF) region were found. A total of 2958 genes produced 8797 AS events, of which 189 genes were involved in the low-temperature response. Twelve transcription factors show AS during cold treatment and VaMYB108 was selected for initial exploration. Two transcripts, Chr05.63.1 (VaMYB108short) and Chr05.63.2 (VaMYB108normal) of VaMYB108, display up-regulated expression after cold treatment in amur grape roots and are both localized in the nucleus. Only VaMYB108normal exhibits transcriptional activation activity. Overexpression of either VaMYB108short or VaMYB108normal in grape roots leads to increased expression of the other transcript and both increased chilling resistance of amur grape roots. The results improve and supplement the genome annotations and provide insights for further investigation into AS mechanisms during cold stress in V. amurensis.

Full-length transcriptome analysis of Ophioglossum vulgatum: effects of experimentally identified chloroplast gene clusters on expression and evolutionary patterns.

Genes with similar or related functions in chloroplasts are often arranged in close proximity, forming clusters on chromosomes. These clusters are transcribed coordinated to facilitate the expression of genes with specific function. Our previous study revealed a significant negative correlation between the chloroplast gene expression level of the rare medicinal fern Ophioglossum vulgatum and its evolutionary rates as well as selection pressure. Therefore, in this study, we employed a combination of SMRT and Illumina sequencing technology to analyze the full-length transcriptome sequencing of O. vulgatum for the first time. In particular, we experimentally identified gene clusters based on transcriptome data and investigated the effects of chloroplast gene clustering on expression and evolutionary patterns. The results revealed that the total sequenced data volume of the full-length transcriptome of O. vulgatum amounted to 71,950,652,163bp, and 110 chloroplast genes received transcript coverage. Nine different types of gene clusters were experimentally identified in their transcripts. The chloroplast cluster genes may cause a decrease in non-synonymous substitution rate and selection pressure, as well as a reduction in transversion rate, transition rate, and their ratio. While expression levels of chloroplast cluster genes in leaf, sporangium, and stem would be relatively elevated. The Mann-Whitney U test indicated statistically significant in the selection pressure, sporangia and leaves groups (P < 0.05). We have contributed novel full-length transcriptome data resources for ferns, presenting new evidence on the effects of chloroplast gene clustering on expression land evolutionary patterns, and offering new theoretical support for transgenic research through gene clustering.

Transcriptomic profiling of Poa pratensis L. under treatment of various phytohormones.

Poa pratensis L. (Poaceae) is a valuable grass across the north hemisphere, inhabiting diverse environments with wide altitudinal span, where ubiquitous various kinds of stresses. Phytohormones would be helpful to improve tolerance to abiotic and biotic stresses, but the responses of transcriptome regulation of P. pratensis to exogenous phytohormones application remain unclear. In this study, we explored the alteration of plant physiological responses by the application of phytohormones. Aiming to achieve this knowledge, we got full-length transcriptome data 42.76 Gb, of which 74.9% of transcripts were completed. Then used 27 samples representing four treatments conducted at two time points (1 h and 6 h after application) to generate RNA-seq data. 371 and 907 common DEGs were identified in response to four phytohormones application, respectively, these DEGs were involved in "plant hormone signal transduction", "carbon metabolism" and "plant-pathogen interaction". Finally, P. pratensis basic research can gain valuable information regarding the responses to exogenous application of phytohormones in physiological indicators and transcriptional regulations in order to facilitate the development of new cultivars.

Comparative transcriptome analysis and identification of candidate R2R3-MYB genes involved in anthraquinone biosynthesis in Rheum palmatum L.

BackgroundRheum palmatum L. has important medicinal value because it contains biologically active anthraquinones. However, the key genes and TFs involved in anthraquinone biosynthesis and regulation in R. palmatum remain unclear.MethodsBased on full length transcriptome data, in this study, we screened the differentially expressed genes in the anthraquinone biosynthesis pathway. The R2R3-MYB family genes of R. palmatum were systematically identified based on full-length transcriptome sequencing followed by bioinformatics analyses. The correlation analysis was carried out by using co-expression analysis, protein interaction analysis, and real-time fluorescence quantitative analysis after MeJA treatment. The RpMYB81 and RpMYB98 genes were amplified by RT-PCR, and their subcellular localization and self-activation characteristics were analyzed.ResultsComparative transcriptome analysis results revealed a total of 3525 upregulated and 6043 downregulated DEGs in the CK versus MeJA group; 28 DEGs were involved in the anthraquinone pathway. Eleven CHS genes that belonged to the PKS family were differentially expressed and involved in anthraquinone biosynthesis. Twelve differentially expressed MYBs genes were found to be co-expressed and interact with CHS genes. Furthermore, 52 MYB genes were identified as positive regulators of anthraquinone biosynthesis and were further characterized. Three MYB genes including RpMYB81, RpMYB98, and RpMYB100 responded to MeJA treatment in R. palmatum, and the levels of these genes were verified by qRT-PCR. RpMYB81 was related to anthraquinone biosynthesis. RpMYB98 had an interaction with genes in the anthraquinone biosynthesis pathway. RpMYB81 and RpMYB98 were mainly localized in the nucleus. RpMYB81 had self-activation activity, while RpMYB98 had no self-activation activity.ConclusionRpMYB81, RpMYB98, and RpMYB100 were significantly induced by MeJA treatment. RpMYB81 and RpMYB98 are located in the nucleus, and RpMYB81 has transcriptional activity, suggesting that it might be involved in the transcriptional regulation of anthraquinone biosynthesis in R. palmatum.

Functional requirement of alternative splicing in epithelial-mesenchymal transition of pancreatic circulating tumor

Circulating tumor cells (CTCs) that undergo epithelial-to-mesenchymal transition (EMT) can provide valuable information regarding metastasis and potential therapies. However, the current studies on the EMT overlook the alternative splicing. Here, we used the single-cell full-length transcriptome data and mRNA-sequencing of CTCs to identify stage-specific alternative splicing of partial EMT and mesenchymal states during pancreatic cancer metastasis. We classified definitive tumor and normal epithelial cells via genetic aberrations and demonstrated dynamic changes in the epithelial-mesenchymal continuum in both epithelial cancer cells and CTCs. We provided the landscape of alternative splicing in CTCs at different stages of EMT, uncovering cell-type-specific splicing patterns and splicing events in cell-surface proteins suitable for therapies. We showed that MBNL1 governs cell fate through alternative splicing independently of changes in gene expression and affects the splicing pattern during EMT. We found a high frequency of events that contained multiple premature termination codons and were enriched with C and G nucleotides in close proximity, which influence the likelihood of stop codon readthrough and expand the range of potential therapeutic targets. Our study provides insights into the EMT transcriptome's dynamic changes and identifies potential diagnostic and therapeutic targets in pancreatic cancer.

Cloning, Characterization, and Expression Pattern Analysis of the BBM Gene in Tree Peony (Paeonia ostii)

BABY BOOM (BBM) is one of the members of the plant-specific APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor superfamily. It acts as a key regulator of plant cell pluripotency, playing a significant role in promoting somatic embryogenesis. In this study, a BBM gene named PoBBM was screened, cloned, and identified from the third-generation full-length transcriptome data of Paeonia ostii. Its open reading frame was 2136 bp, encoding 711 amino acids. Sequence feature analysis revealed that it possessed two AP2 conserved domains and eight motifs, including bbm-1. The phylogenetic tree indicated that PoBBM clusters with AtBBM in the euANT group of the Arabidopsis AP2 family, which is most closely related to grape VvBBM and may have the same ancestry as grape. Subcellular localization demonstrated that the PoBBM protein was localized in the nucleus. Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the PoBBM transcript levels during ten developmental stages of somatic embryos and in five tissue types of peonies. The results indicate that PoBBM was highly expressed in the early stages of peony somatic embryo development. The expression on 0–15 d was the highest and decreased gradually with somatic embryogenesis. The gene is almost not expressed after 40 d since somatic embryo formation. PoBBM was expressed in roots, stems, leaves, seeds, and calli, with the highest levels in seeds, followed by leaves and calli. The PoBBM protein displayed transcriptional self-activation activity, which may facilitate further research on its relationships with other proteins. The above results provide a key gene PoBBM for somatic embryogenesis in peonies, which is significant for advancing the establishment of a stable and efficient regeneration and genetic transformation system for peonies.

Single-Molecule Real-Time Sequencing for Identifying Sexual-Dimorphism-Related Transcriptomes and Genes in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)’s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-β-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-β pathway. Female-biased genes such as Smad4, Wif1, and 17-β-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis.

Integrative analyses of transcriptomes and metabolomes provide insight into salinity adaption in Bangia (Rhodaphyta)

The salinity of the external environment poses a serious threat to most land plants. Although seaweeds can adapt to this, intertidal species are subject to wide fluctuations in salinity, including hypo- and hyper-saline conditions. The red algal genus Bangiales is a typical example; it is one of the oldest eukaryotes with sexual reproduction and has successfully adapted to both marine and freshwater environments. However, there is a dearth of research focused on elucidating the mechanism by which marine Bangia (Bangia fuscopurpurea) adapts to hypo-salinity, as well as the mechanism by which freshwater Bangia (Bangia atropurpurea) adapts to hyper-salinity. The objective of this study is to employ third-generation full-length transcriptome data and untargeted metabolome data, to provide insights into the salinity adaptation mechanism of as well as the evolutionary relationship between both Bangia species. B. fuscopurpurea and B. atropurpurea exhibited 9112 and 8772 differentially expressed genes (DEGs), respectively, during various periods of hyper-saline condition. These genes were primarily enriched in secondary metabolites and energy-related metabolic pathways. Additionally, B. fuscopurpurea displayed 16,285 DEGs during different periods of hypo-saline condition, which were mainly enriched in metabolic pathways related to ion transport and membrane proteins. In the hyper- and hypo-saline adapt response processes of B. fuscopurpurea, a total of 303 transcription factors were identified, which belonged to 26 families. Among these, 85 and 142 differential transcription factors were identified, respectively, mainly belonging to the C2H2 and MYB family. Similarly, in the response process of B. atropurpurea to hyper-saline condition, a total of 317 transcription factors were identified, mainly belonging to 17 families. Among these, 121 differential transcription factors were identified, mainly belonging to the C2H2 and bZIP family. Furthermore, a correlation analysis was conducted to examine the relationship between the transcriptional and metabolic levels of both species under saline adaptation. The findings demonstrated that Bangia exhibits intricate adaptations to salinity, which involve swift regulation of its photosynthetic processes, alternations in membrane contents, and a robust anti-oxidation system to mitigate the effects of excess redox energy during exposure to varying salinity. Notably, the unsaturated fat and glutathione metabolic pathways were found to be significantly enriched in this context.

High-quality wild barley genome assemblies and annotation with Nanopore long reads and Hi-C sequencing data

Wild barley, from “Evolution Canyon (EC)” in Mount Carmel, Israel, are ideal models for cereal chromosome evolution studies. Here, the wild barley EC_S1 is from the south slope with higher daily temperatures and drought, while EC_N1 is from the north slope with a cooler climate and higher relative humidity, which results in a differentiated selection due to contrasting environments. We assembled a 5.03 Gb genome with contig N50 of 3.53 Mb for wild barley EC_S1 and a 5.05 Gb genome with contig N50 of 3.45 Mb for EC_N1 using 145 Gb and 160.0 Gb Illumina sequencing data, 295.6 Gb and 285.35 Gb Nanopore sequencing data and 555.1 Gb and 514.5 Gb Hi-C sequencing data, respectively. BUSCOs and CEGMA evaluation suggested highly complete assemblies. Using full-length transcriptome data, we predicted 39,179 and 38,373 high-confidence genes in EC_S1 and EC_N1, in which 93.6% and 95.2% were functionally annotated, respectively. We annotated repetitive elements and non-coding RNAs. These two wild barley genome assemblies will provide a rich gene pool for domesticated barley.

PacBio full-length transcriptome analysis provides new insights into transcription of chloroplast genomes

ABSTRACT Chloroplast and mitochondrial DNA (cpDNA and mtDNA) are apart from nuclear DNA (nuDNA) in a eukaryotic cell. The transcription system of chloroplasts differs from those of mitochondria and eukaryotes. In contrast to nuDNA and animal mtDNA, the transcription of cpDNA is still not well understood, primarily due to the unresolved identification of transcription initiation sites (TISs) and transcription termination sites (TTSs) on the genome scale. In the present study, we characterized the transcription of chloroplast (cp) genes with greater accuracy and comprehensive information using PacBio full-length transcriptome data from Arabidopsis thaliana. The major findings included the discovery of four types of artifacts, the validation and correction of cp gene annotations, the exact identification of TISs that start with G, and the discovery of polyA-like sites as TTSs. Notably, we proposed a new model to explain cp transcription initiation and termination at the whole-genome level. Four types of artifacts, degraded RNAs and splicing intermediates deserve the attention from researchers working with PacBio full-length transcriptome data, as these contaminant sequences can lead to incorrect downstream analysis. Cp transcription initiates at multiple promoters and terminates at polyA-like sites. Our study provides new insights into cp transcription and new clues to study the evolution of promoters, TISs, TTSs and polyA tails of eukaryotic genes.

Comprehensive Analysis of the NF-YB Gene Family and Expression under Abiotic Stress and Hormone Treatment in Larix kaempferi.

NF-YB, a subfamily of Nuclear Factor Y (NF-Y) transcription factor, play crucial role in many biological processes of plant growth and development and abiotic stress responses, and they can therefore be good candidate factors for breeding stress-resistant plants. However, the NF-YB proteins have not yet been explored in Larix kaempferi, a tree species with high economic and ecological values in northeast China and other regions, limiting the breeding of anti-stress L. kaempferi. In order to explore the roles of NF-YB transcription factors in L. kaempferi, we identified 20 LkNF-YB family genes from L. kaempferi full-length transcriptome data and carried out preliminary characterization of them through series of analyses on their phylogenetic relationships, conserved motif structure, subcellular localization prediction, GO annotation, promoter cis-acting elements as well as expression profiles under treatment of phytohormones (ABA, SA, MeJA) and abiotic stresses (salt and drought). The LkNF-YB genes were classified into three clades through phylogenetic analysis and belong to non-LEC1 type NF-YB transcription factors. They have 10 conserved motifs; all genes contain a common motif, and their promoters have various phytohormones and abiotic stress related cis-acting elements. Quantitative real time reverse transcription PCR (RT-qPCR) analysis showed that the sensitivity of the LkNF-YB genes to drought and salt stresses was higher in leaves than roots. The sensitivity of LKNF-YB genes to ABA, MeJA, SA stresses was much lower than that to abiotic stress. Among the LkNF-YBs, LkNF-YB3 showed the strongest responses to drought and ABA treatments. Further protein interaction prediction analysis for LkNF-YB3 revealed that LkNF-YB3 interacts with various factors associated with stress responses and epigenetic regulation as well as NF-YA/NF-YC factors. Taken together, these results unveiled novel L. kaempferi NF-YB family genes and their characteristics, providing the basic knowledge for further in-depth studies on their roles in abiotic stress responses of L. kaempferi.

Full-length transcriptome sequencing and transgenic tobacco revealed the key genes in the chlorogenic acid synthesis pathway of Sambucus chinensis L.

Chlorogenic acid is a key chemical in antioxidation and antisepsis. Sambucus chinensis L. is a herbaceous plant rich in chlorogenic acid and a potential genetic resource for breeding high-chlorogenic acid plants. However, there are few studies on the synthesis pathway of chlorogenic acid in S. chinensis. Our study found chlorogenic acid accumulation in S. chinensis to be organ-specific, higher in leaves and buds but lower in roots, stems and fruits. A total number of 546,844 CCS (Circular Consensus Sequence), including 402,767 full-length non-chimeric (FLNC) and 39 annotated sequences related to the synthesis of chlorogenic acid, was obtained by single-molecule real-time sequencing technology (SMRT). qRT-PCR showed that a number of key genes involved in chlorogenic acid synthesis were differentially expressed in various tissues of S. chinensis. Transgenic tobacco revealed that ectopic expression of the HCT homologous gene HCT-45178 increased the content of chlorogenic acid. Our results should be the first report of full-length transcriptome data of S. chinensis, which help to understand the basis of chlorogenic acid synthesis and provide a novel strategy for breeding tobacco cultivars with higher levels of chlorogenic acid. This article is protected by copyright. All rights reserved.

Abstract 6781: Spatial omics using spatially-resolved laser-activated cell sorting for cancer biomarker discovery

Abstract Cancer is spatially heterogeneous in terms of genetic molecules. Revealing genomic, epigenomic, transcriptomic, and epitranscriptomic features that are unique to the malignant cell populations within cancer lead to cancer biomarker discoveries that can be translated into diagnostic or therapeutic tools. Here, we introduce spatially-resolved laser-activated cell sorting (SLACS) technology coupled to next generation sequencing and mass spectrometry that analyzes genetic molecules from regions of interest within spatial context. Specifically, we demonstrate application of SLACS in various cancers such as breast cancer, glioblastoma, glioma, meningioma, multiple myeloma, and leukemia. Examples of discovering cancer biomarker and their mechanism of action within spatial context are also provided. Specifically, we describe Select-seq which isolates the regions of interest as small as single cells from the immunofluorescence stained tissue and obtains the full-length transcriptome data. We demonstrated Select-seq on tumor tissue section from triple negative breast cancer patient who have received neoadjuvant chemotherapy. Select-seq produced full-length spatial transcriptome data, with which we analyzed the transcriptomic and epitranscriptomic features including alternative splicing variant, complementarity-determining region analysis of B cells, and even adenosine-to-inosine base editing. We were also able to analyze the immune- and stem-cell-like microniches, in which the complex mechanisms behind ferroptosis inhibition might suggest therapeutic options for the TNBC patients. Bridging spatial technologies to omics technologies, the discovery of specific markers within spatial context will provide insights into the next generation diagnostics and cancer therapeutics such as cancer vaccines. Citation Format: Amos C. Lee, Sumin Lee, Sunghoon Kwon. Spatial omics using spatially-resolved laser-activated cell sorting for cancer biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6781.

Full-Length Transcriptome Sequencing and Identification of Hsf Genes in Cunninghamia lanceolata (Lamb.) Hook

Cunninghamia lanceolata (Lamb.) Hook. (Chinese fir) is an important timber species that is widely cultivated in southern China. However, the shallow root system and weak drought resistance of Chinese fir are not enough to cope with high temperature and drought. In recent years, molecular biology has been used to modify plants to make them more resilient. Therefore, improving heat and drought resistance of Chinese fir by molecular biology technology is one of the best choices, whereas fewer genetic information resources for C. lanceolata limit more comprehensive molecular studies. In this study, single-molecule full-length transcriptome (SMRT) sequencing technology was used to obtain full-length transcriptome data on Chinese fir. A total of 21,331 transcripts were obtained via co-assembly, and 11,094 gene sets were obtained via further de-redundancy. In addition, gene function annotation and gene structure analysis were performed. We also used these data to identify nine heat shock transcription factors (Hsfs) in Chinese fir, and heat stress transcriptome and real-time quantitative polymerase chain reaction (PCR) analyses revealed expression changes in response to heat stress, indicating that these may play roles in heat resistance. These studies have enriched the genetic information resources of Chinese fir, which may be utilized for further species promotion, improvement, and application.

Full-length transcriptome sequencing reveals extreme incomplete annotation of the goat genome.

Despite recent advances in generating high-quality reference genome assemblies, the genome sequences for most livestock species, including goats, are still poorly annotated. Single-molecule long-read sequencing has greatly facilitated gene annotation by obtaining full-length transcripts. In this study, we generated full-length transcriptome data for samples from abomasum (n=2) and testicle (n=1), using PacBio Iso-Seq technology. We further combined these data with published data from abomasum (5ZY, SRR8618141) to evaluate and improve the gene annotation of the goat genome. We identified 14.5-16.3% of novel genes per sample from the four Iso-Seq datasets. At the transcript level, 40.6% of them were novel, including 29.7% novel transcripts from known genes and 10.9% from novel genes. We further verified the expression of novel genes in four additional RNA-seq data and found that the expression level of novel genes was significantly lower than that of known genes, indicating that the lowly expressed genes tend to be missed in the current genome annotation. This study shows the superiority of full-length transcriptome data in gene annotation, and more such data are required to improve the gene annotation for goat genome and other species.

Enhancement of Pathogen Toxicity by Feeding Reticulitermes chinensis Snyder Sonicated Bacteria Expressing Double-Stranded RNA That Interferes with Olfaction

Reticulitermes chinensis Snyder is a serious pest in China, and the odorant receptor co-receptor gene RcOrco plays a crucial role in olfaction. However, the function of RcOrco in the resistance of termites to entomopathogens has not been reported. We constructed dsRcOrco-HT115 engineered bacteria based on the RcOrco sequence from the full-length transcriptome data of R. chinensis. The engineered bacteria expressed dsRNA of RcOrco. Sonication was used to inactivate the dsRNA-HT115 strain and obtain a large amount of dsRcOrco. The dsRcOrco produced using this method overcame the problem that genetically engineered bacteria could not be applied directly and improved its effectiveness against termites. Bioassays using the dsRcOrco generated using this method showed that dsRcOrco significantly increased the toxicity of the bacterial and fungal pathogens to R. chinensis. The present study showed, for the first time, the function of Orco in termite resistance to pathogens, and the results provide a theoretical basis for the development and application of termite RNA biopesticides.

Full-Length Transcriptome Characterization and Functional Analysis of Pathogenesis-Related Proteins in Lilium Oriental Hybrid 'Sorbonne' Infected with Botrytis elliptica.

Gray mold (Botrytis elliptica) causes a deleterious fungal disease that decreases the ornamental value and yield of lilies. Lilium oriental hybrid 'Sorbonne' is a variety that is resistant to gray mold. Understanding the mechanism of resistance against B. elliptica infection in 'Sorbonne' can provide a basis for the genetic improvement in lily plants. In this study, a PacBio Sequel II system was used to sequence the full-length transcriptome of Lilium 'Sorbonne' after inoculation with B. elliptica. A total of 46.64 Gb subreads and 19,102 isoforms with an average length of 1598 bp were obtained. A prediction analysis revealed 263 lncRNAs, and 805 transcription factors, 4478 simple sequence repeats, and 17,752 coding sequences were identified. Pathogenesis-related proteins (PR), which may play important roles in resistance against B. elliptica infection, were identified based on the full-length transcriptome data and previously obtained second-generation transcriptome data. Nine non-redundant potential LhSorPR proteins were identified and assigned to two groups that were composed of two LhSorPR4 and seven LhSorPR10 proteins based on their genetic relatedness. The real-time quantitative reverse transcription PCR (qRT-PCR) results showed that the patterns of expression of nine differentially expressed PR genes under B. elliptica stress were basically consistent with the results of transcriptome sequencing. The pattern of expression of LhSorPR4s and LhSorPR10s genes in different tissues was analyzed, and the expression of each gene varied. Furthermore, we verified the function of LhSorPR4-2 gene in Lilium. The expression of LhSorPR4-2 was induced by phytohormones such as methyl jasmonate, salicylic acid, and ethephon. Moreover, the promoter region of LhSorPR4-2 was characterized by several functional domains associated with phytohormones and stress response. The overexpression of LhSorPR4-2 gene in 'Sorbonne' increased the resistance of the lily plant to B. elliptica and correlated with high chitinase activity. This study provides a full-length transcript database and functionally analyzed the resistance of PR gene to B. elliptica in Lilium, thereby introducing the candidate gene LhSorPR4-2 to breed resistance in Lilium.

Transcriptome sequencing of the spleen of the Yangtze sturgeon (Acipenser dabryanus) under Edwardsiella tarda and poly(I:C) treatments

Acipenser dabryanus is an ancient living fossil and a flagship species of the Yangtze River. However, A. dabryanus was listed as a critically endangered species due to the dramatic decrease in its population, and disease outbreaks occurring during captive breeding have hindered the protection of this species. Here, we investigated the effect of the pathogenic bacterium Edwardsiella tarda and poly(I:C) on A. dabryanus at the histopathological and transcriptomic levels. After 48 h of infection with E. tarda, A. dabryanus exhibited marked pathological changes in the intestine and liver. We then conducted full-length transcriptome sequencing of the spleen by SMRT sequencing technology. Using the full-length transcriptome data as a reference, transcriptomic results obtained through Illumina sequencing technology revealed that a total of 894 and 963 unigenes were differentially expressed between the PBS and bacterial infection groups and between the PBS and poly(I:C) challenge groups, respectively. E3 ubiquitin ligases family members, T-cell receptor and clusters of differentiation were significantly upregulated by bacteria or poly(I:C) treatment, while the complement was significantly downregulated, suggesting that they might participate in multiple immune pathways in the response to pathogen invasion. Pathogen infection also induced changes in some key rate-limiting enzymes involved in energy metabolism. Our study can provide important information on the molecular mechanisms underlying the immune response of fish to pathogenic microbial infections and help explore the antibacterial and antiviral immunity of A. dabryanus.

Analysis of Rac/Rop Small GTPase Family Expression in Santalum album L. and Their Potential Roles in Drought Stress and Hormone Treatments.

Plant-specific Rac/Rop small GTPases, also known as Rop, belong to the Rho subfamily. Rac proteins can be divided into two types according to their C-terminal motifs: Type I Rac proteins have a typical CaaL motif at the C-terminal, whereas type II Rac proteins lack this motif but retain a cysteine-containing element for membrane anchoring. The Rac gene family participates in diverse signal transduction events, cytoskeleton morphogenesis, reactive oxygen species (ROS) production and hormone responses in plants as molecular switches. S. album is a popular semiparasitic plant that absorbs nutrients from the host plant through the haustoria to meet its own growth and development needs. Because the whole plant has a high use value, due to the high production value of its perfume oils, it is known as the "tree of gold". Based on the full-length transcriptome data of S. album, nine Rac gene members were named SaRac1-9, and we analyzed their physicochemical properties. Evolutionary analysis showed that SaRac1-7, AtRac1-6, AtRac9 and AtRac11 and OsRac5, OsRacB and OsRacD belong to the typical plant type I Rac/Rop protein, while SaRac8-9, AtRac7, AtRac8, AtRac10 and OsRac1-4 belong to the type II Rac/ROP protein. Tissue-specific expression analysis showed that nine genes were expressed in roots, stems, leaves and haustoria, and SaRac7/8/9 expression in stems, haustoria and roots was significantly higher than that in leaves. The expression levels of SaRac1, SaRac4 and SaRac6 in stems were very low, and the expression levels of SaRac2 and SaRac5 in roots and SaRac2/3/7 in haustoria were very high, which indicated that these genes were closely related to the formation of S. album haustoria. To further analyze the function of SaRac, nine Rac genes in sandalwood were subjected to drought stress and hormone treatments. These results establish a preliminary foundation for the regulation of growth and development in S. album by SaRac.