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The study focuses on an in vivo GST- omega homologue (pCRT7/TPxII intB4) over-expression, purification and characterization. Experiments purport to characterize the antioxidant activity of the LeTPx1, the interacting glutathione S-transferases BI-GST/GPx, LeGST-T1, T2, T3, T4, T5 and the mammalian inhibitor of apoptosis Bcl-2. Upon specific expression, the proteins exerted, differential protective effects in yeast cells treated with lethal doses of the prooxidants hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide. The antioxidant activity of LeTPx1 was highest against the cumene hydroperoxide. The overexpressing GST (omega) homologue TPxintB4 (Baier and Dietz, 1999) which share a considerable homology of the mammalian GST-omega1. In conclusion, the work shows that yeast parental strains are extremely sensitive to very low concentrations (0.2mM) of Cumenehydroperoxide. However, after applying the different antioxidants; it appears that the smallest concentrations t to be tolerated.

Low temperatures limit crop growth, yield, and quality in cold areas, and cold tolerance is the only strategy for plants to survive in those areas. However, mulberry’s (Morus alba L.) molecular regulatory mechanisms in response to cold remain poorly understood. In this report, four cDNA libraries were built from two groups, the cold-treated group and the control group, and evaluated by RNA-Seq analysis. A total of 3593 differential expression genes (DEGs) were identified from 20,279 unigenes. Among these, 2337 up-regulated and 1256 down-regulated DEGs were identified under this standard (corrected p value 0.0). Most of these DEGs are involved in plant hormone signal transduction, MAPK signaling pathway, ubiquinone, and other terpenoid-quinone biosynthesis, nitrogen metabolism, and biosynthesis of secondary metabolites. A series of candidate genes related to cold stress were screened out and discussed based on these results. Furthermore, the expression changes of 8 genes under cold stress from RNA-Seq analysis were further validated by real-time quantitative polymerase chain reaction. This research reflects Morus alba L. transcriptome data and may help elucidate the cold response mechanisms of mulberry trees and would be useful in breeding cold-tolerant mulberry plants.

There is an increasing demand for elucidating the biosynthetic pathway of medicinal plants, which are capable of producing several metabolites with great potentials for industrial drug production. Digitalis species are important medicinal plants for the production of cardenolide compounds. Advancement on culture techniques is strictly related to our understanding of the genomic background of species. There are a limited number of genomic studies on Digitalis species. The goal of this study is to contribute to the genomic data of Digitalis ferruginea subsp. schischkinii by presenting transcriptome annotation. Digitalis ferruginea subsp. schischkinii has a limited distribution in Turkey and Transcaucasia, and has a high level of lanatoside C, an important cardenolide. In the study, we sequenced the cDNA library prepared from RNA pools of D. ferruginea subsp. schischkinii tissues treated with various stress conditions. Comprehensive bioinformatics approaches were used for de novo assembly and functional annotation of D. ferruginea subsp. schischkinii transcriptome sequence data along with TF families predictions and phylogenetic analysis. In the study, 58,369 unigenes were predicted and unigenes were annotated by analyzing the sequence data in the non-redundant (NR) protein database, the non-redundant nucleotide (NT) database, Gene Orthology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), SwissProt, and InterPro databases. This study is the first transcriptome data for D. ferruginea subsp. schischkinii.

Long-chain non-coding RNA HOXA11 antisense RNA (HOXA11-AS) is a kind of lncRNA discovered in recent years. Long-chain non-coding RNA (LncRNA) is an important regulatory factor of protein-coding genes, especially the disorder of LncRNA in more and more diseases which are found, including cancer. HOXA11-AS was first discovered in mouse embryo cDNA library using probes, and then it was discovered by scholars and played an important role in human cervical cancer, gastric cancer, glioma and other malignant tumor cells. Overexpression of HOXA11-AS has been found to promote cell proliferation, migration and tumor invasion, and has a carcinogenic effect. HOXA11-AS can promote tumor proliferation, metastasis and other malignant biological behaviors by interacting with miRNA and EZH2 protein, and is considered to be carcinogenic lncRNA. The discovery of HOXA11-AS provides new ideas for tumor prevention and treatment.

NBS-LRR-type disease resistance gene-like cDNA, induced by salicylic acid (SA) was cloned from rye Secalecereale L. (2n = 14RR) var. Petkus, which has rust resistance genes such as Lr26, Sr31 andRr9. We designed primers based on the NBS region and performed PCR using Petkus genomic DNA as a template. Next, we TA-cloned a 532-bp DNA fragment containing five homologous amino acid sequences in the NBS region. The SA-treated rye showed strong expression of a transcript of approximately 3.5 knt in the Northern blots probed with the NBS fragment; however, no transcripts were observed with the untreated rye. We constructed a cDNA library of rye var. Petkus treated with SA, and then screened the cDNA library using the TA-cloned NBS fragments as a probe. The entire nucleotide sequence of a full length of rye NBS-LRR-containing class cDNA 3,446 bp was determined.

Sweetpotato [Ipomoea batatas (L.) Lam] is an economically important crop for fresh and processed consumption and is widely cultivated worldwide, especially in China. Various sweetpotato cultivars with different storage root colors are presently available. The purple-fleshed sweetpotato obtains its color from anthocyanin accumulation in the storage roots, which is beneficial for both plant and human health. To date, the molecular mechanism of this anthocyanin accumulation has not been studied in detail. In our study, three cDNA libraries generated from 'Xuzi8' with dark-purple flesh, 'Xuzi6' with light-purple flesh, and 'Xu28' with white flesh were sequenced utilizing an Illumina HiSeq™ 2500 platform. Corresponding totals of 28,093,466, 29,239,729 and 27,217,440 raw reads were obtained from the three libraries and assembled into 137,625 unigenes with an average length of 481 bp. Moreover, 79,203 unigenes (57.55%) were found to be annotated in several public databases, and 1285 unigenes were differentially expressed among the Xu28 vs Xuzi8, Xu28 vs Xuzi6, and Xuzi6 vs Xuzi8 libraries. After functional category enrichment analysis of differential expression genes (DEGs), 25 genes were selected as the candidate genes related to anthocyanin accumulation. Furthermore, the expression patterns of some selected DEGs were verified by quantitative real-time PCR (qRT-PCR), and the correlation between expression levels of relevant genes involved in anthocyanin biosynthesis and anthocyanin content was determined. Taken together, the results compose a transcriptomic analysis to investigate the differences in purple flesh formation in the storage roots among different sweetpotato varieties, with the notable outcome that several key genes can now be closely linked to anthocyanin biosynthesis.

Salinity and drought stress are major abiotic stresses negatively affecting the growth and productivity of alfalfa (Medicago sativa L.). Exploration of genes exhibiting superior tolerance to salinity and drought stress in alfalfa will help aid target molecular breeding and developing tolerant forages. In this study, we adopted a high-efficient yeast FOX hunting system for the identification of salinity and drought tolerant genes in alfalfa. Based on the Gateway-compatible vector system, a high-quality expression library was constructed, containing 1.3 × 107 clones with an average size of 1.44 kb. Through heterologous transformation of mixed library plasmid into salt or drought sensitive yeast mutants, monoclonal resistant strains were screened and tolerant genes were captured. Eighteen salinity-tolerance genes were obtained which were involved in several pathways, containing GRAS and zinc finger transcriptional factors, PP2A interaction module, ERVT vesicle transporter and LETM transmembrane protein. Twelve drought tolerance genes were separated, including ERF and SCL transcriptional factors, CIPK and BSK protein kinases, TGL-type ligase, cPGM and cPDL protease. The mRNA transcription levels of these tolerant genes were inducible or suppressible for response to salt or drought stress conditions following quantitative PCR detection, respectively. Furthermore, heterologous transformation of ERVT and CIPK11 can improve the salt and drought stress tolerance in Arabidopsis, which indicates the conservative function of the screening gene in yeast and Arabidopsis. Obtaining these candidate genes can provide new insights for future research with respect to plant salt and drought tolerance.

The herbaceous peony (Paeonia lactiflora Pall.) is considered to be a highly valued cut flower plant. It has large flower with rich colors. However, there has been little or no research into the genes related to its flower development. In this study, we used the Illumina HiSeq platform to analyze the RNA-Seq comparative transcriptome of the P. lactiflora 'Dafugui' in three different flowering periods. Nine cDNA libraries were established, from which 92.53 Gb data with 81,788 unigenes were obtained. We screened the genes related to P. lactiflora flowering, isolated and cloned the PlFT gene related to flowering. The total length of the PlFT gene was 592 bp, which had a complete open reading frame of 522 bp and encoded 173 amino acids. The accession number of the PlFT gene is MT249229. To test the role of PlFT, we constructed an expression vector for genetic transformation. Its expression in Arabidposis mutant indicated that PlFT was involved in the flowering of P. lactiflora. This is the first transcriptome analysis of flower development in P. lactiflora. Our results provide some fundamental information for further analyzing the molecular mechanism underlying flower development of P. lactiflora.

The BES1/BZR1 transcription factors regulate the expression of genes responsive to brassinosteroids and play pivotal roles in plant development, but their role in regulating kernel development in maize remains unclear. In this study, we found that ZmBES1/BZR1-5 positively regulates kernel size. Association analysis of candidate genes in 513 diverse maize inbred lines indicated that three SNPs related to ZmBES1/BZR1-5 were significantly associated with kernel width and whilst four SNPs were related to 100-kernel weight. Overexpression of ZmBES1/BZR1-5 in Arabidopsis and rice both significantly increased seed size and weight, and smaller kernels were produced in maize Mu transposon insertion and EMS mutants. The ZmBES1/BZR1-5 protein locates in the nucleus, contains bHLH and BAM domains, and shows no transcriptional activity as a monomer but forms a homodimer through the BAM domain. ChIP-sequencing analysis, and yeast one-hybrid and dual-luciferase assays demonstrated that the protein binds to the promoters of AP2/EREBP genes (Zm00001d010676 and Zm00001d032077) and inhibits their transcription. cDNA library screening showed that ZmBES1/BZR1-5 interacts with casein kinase II subunit β4 (ZmCKIIβ4) and ferredoxin 2 (ZmFdx2) in vitro and in vivo, respectively. Taken together, our study suggests that ZmBES1/BZR1-5 positively regulates kernel size, and provides new insights into understanding the mechanisms of kernel development in maize.

Background: Prmt5 plays major role in regulation of gene expression, RNA processing, cell growth and differentiation, signal transduction, germ cell development, etc., in mammals. Prmt5 is also related to cancer. Knowing the proteins interacting with Prmt5 is important to understand Prmt5’s function in cells. Although there have been reports on proteins binding with Prmt5 in mammals, the partner proteins of Prmt5 in fish are still unclear. Objectives: The objective was to obtain proteins that bind with Prmt5 in medaka, a model fish. Methods: Yeast two hybridization was adopted to achieve the objective. Medaka Prmt5 was used as a bait to fish the prey, binding proteins in a cDNA library of medaka. Co-immunoprecipitation and in silicon analysis were performed to study the interaction of medaka Mep50 and Prmt5. Results: Eight proteins were identified to bind with Prmt5 from 69 preliminary positive colonies. The binding proteins are methylosome protein 50 (Mep50), apolipoprotein A-I-like (Apo-AI), PR domain containing protein 1a with zinc fingers (Prdm1a), Prdm1b, T-cell immunoglobulin mucin family member 3 (Tim-3), phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (Paics), NADH dehydrogenase subunit 4 (ND4) and sciellin (Scl). Co-immunoprecipitation confirmed the interaction of medaka Prmt5 and Mep50. Predicted structures of medaka Prtm5 and Mep50 are similar to that of human PRMT5 and MEP50. Conclusion: Medaka Mep50, Prdm1a, Prdm1b, Apo-AI, Tim-3, Paics, ND4, and Scl bind with Prmt5.

Background: Coconut tissues consist of a complex network of polysaccharides, proteins, polyphenols, and lipids that can bind to nucleic acids and pose difficulty in isolation. Certainly, a vigorous method is required to isolate high quality and quantity of RNA from such tissues for the purpose of downstream experiments. In this paper, we discuss a newly developed method for the Isolation of RNA from Complex Matrices (IRCM) method from coconut tissues. Results: The method is robust, cheap, and efficient for the extraction of quality RNA in high quantities from the solid endosperm of stored and fresh coconut (150 μg/g FW with A260/280 = 1.89 and 247.5 μg/g FW with A260/280 = 1.91), coconut apple (263.8 μg/g FW with A260/280 = 1.97), and coconut bud (1052.5 μg/g FW with A260/280 = 2.00). The other well established methods, such as Method of RNA Isolation from Palm (MRIP), Cetyl Trimethyl Ammonium Bromide (CTAB), TRIZOL, and RNA plant kit failed to isolate quality RNA in appreciable quantities from the coconut tissues. Furthermore, the resultant RNA performed well in the downstream experiment, that is, RT-PCR for the production and amplification of cDNA. Conclusions: From the study, we concluded that the present method will play a vital role in the extraction of high quality RNA from complex matrices in a short time. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Anti-viral RNA therapy is on high demand nowadays due to the emergence of several new viral infections. The small non-coding regulatory RNAs (dsRNA) from the microbial sources are not yet explored for anti-viral activity. In this study, we assessed the anti-HIV activity of the small dsRNA produced by 12 different microbial species isolated from naturally fermented foods of North-East India. For this, we selectively extracted the dsRNA from the microbial culture, confirmed its double-stranded nature by immunoblotting, and deep sequenced the cDNA library using Illumina platform. Further, we used conventional algorithms to predict the potential targets of the dsRNA sequences within the 3′-UTR region of HIV-1. A small dsRNA fragment with 34 bases in size with a sequence of 3′-UUGGUACACGAGAUGGUUCGACUCGAUGAAGGGC-5′ produced abundantly (9.17% of the total dsRNA fraction) by Bacillus subtilis MTCC5480 showed a much higher base complementarity values than previously reported miRNAs analysed against HIV-1. We separated the dsRNA fraction and validated the anti-HIV activity against human peripheral blood mononuclear cells (PBMC) infected with JRCSF strain of HIV-1 virus and the EC50 value ranges from 0.2–0.3 μM. This small dsRNA abundantly produced by B. subtilis could be studied further for its application as an anti-viral therapeutic agent.

The bioaccumulation of cadmium (Cd) in crop and the subsequent food chain has aroused extensive concerns. However, the underlying molecular mechanisms of plant Cd tolerance remain to be clarified from the viewpoint of novel candidate genes. Here we described a highly efficient approach for preliminary identifying rice Cd-tolerant genes through the yeast-based cDNA library survival screening combined with high-throughput sequencing strategy. About 690 gene isoforms were identified as being Cd-tolerant candidates using this shotgun approach. Among the Cd-tolerant genes identified, several categories of genes such as BAX inhibitor (BI), NAC transcription factors and Rapid ALkalinization Factors (RALFs) were of particular interest, and their function of Cd tolerance was further validated via heterologous expression, which suggested that SNAC1, RALF12, OsBI-1 can confer Cd tolerance in yeast and tobacco leaves. Regarding the genes involved in ion transport, the validated Cd-tolerant heavy metal-associated domain (HMAD) isoprenylated protein HIPP42 was particularly noteworthy. Further elucidation of these genes associated with Cd tolerance in rice will benefit agricultural activities.

Ligation-independent cloning (LIC), such as Gibson Assembly, tends to produce clones without an insert, depending on the sequences present at the ends of linearized vectors. We used a nicking enzyme-mediated LIC (NE-LIC) method to construct a cDNA library in a binary vector pER8. Prior to constructing the cDNA library, pilot experiments were carried out, in which the GUS coding sequence was cloned into pER8 using NE-LIC. Approximately 12% of input vector DNAs were converted to plasmids carrying a GUS insert, and no plasmids without an insert were detected, indicating that this strategy is highly effective for cloning with the binary vector pER8. Therefore, NE-LIC was adopted to construct a cDNA library in pER8, by using cDNA that was PCR-amplified from a library constructed in another vector. As a result, a cDNA library in pER8 was successfully constructed. During library construction, it is important to exclude plasmids without an insert, since contamination from plasmids without inserts decreases the efficiency of screening. Therefore, NE-LIC is useful for the construction of cDNA libraries.

SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER− breast cancer patients.

Cytoplasmic male sterility is a well-proven mechanism for cotton hybrid production. Long non-coding RNAs belong to a class of transcriptional regulators that function in multiple biological processes. The cDNA libraries from the flower buds of the cotton CGMS, it's restorer (Rf) and maintainer lines were sequenced using high throughput NGS technique. A total of 1531 lncRNAs showed significant differential expression patterns between these three lines. Functional analysis of the co-expression network of lncRNA-mRNA using gene ontology vouchsafes that, lncRNAs play a crucial role in cytoplasmic male sterility and fertility restoration through pollen development, INO80 complex, development of anther wall tapetum, chromatin remodeling, and histone modification. Additionally, 94 lncRNAs were identified as putative precursors of 49 miRNAs. qRT-PCR affirms the concordance of expression pattern to RNA-seq data. These findings divulge the lncRNA driven miRNA-mediated regulation of gene expression profiling superintended for a better understanding of the CMS mechanisms of cotton.

A new WRKY transcription factor gene was isolated by ESTs screening from a cDNA library of suspension cultured cells of Sweet potato (Ipomoea batatas). The 2,285 bp cDNA fragment, IbWRKY, was sequenced, from which a 505 amino acid residue protein was deduced. A search of the protein BLAST database identified significant similarity to other plant WRKY31 protein sequences. RT-PCR analysis showed expression patterns of IbWRKY31 in various intact tissues and suspension cultured cells of Sweet potato, and in leaves exposed to different stresses. The IbWRKY31 gene was highly expressed in suspension cultured cells. In leaf tissues, IbWRKY31 showed strong expression during salicylic acid and methyl jasmonate treatments. Expression of IbWRKY31 was also induced under various abiotic stress and pathogen infection conditions, such as wounding, H2O2, MV, PEG, NaCl, and bacterial pathogen infection. These results suggest that IbWRKY31 is involved in plant responses to various stress conditions, such as abiotic stresses and pathogen infection through a defense signaling pathway.

Hami melon (Cucumis melo L. var. inodorus Jacq) is an important horticultural crop but susceptible to chilling injury during low temperature storage. To characterize the gene expression profiles of Gold Queen Hami melon under the cold stress and discover the key cold stress-induced genes, five cDNA libraries, C0 (control), C12 (control for 12 d), C24 (control for 24 d), L12 (cold treatment at 0.5°C for 12 d) and L24 (cold treatment at 0.5°C for 24 d), were constructed for RNA sequencing (RNA-Seq) and gene expression profiling. A total of 6479 and 7914 differentially expressed genes (DEGs) in L12 and L24 compared with C0 were respectively detected. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified the relevant biological functions and metabolic pathways of these differentially expressed genes. In addition, a diverse range of cold response candidate genes were mainly obtained from starch and sucrose metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis, such as genes encoding sucrose-phosphate synthase, α-amylase, β-amylase, sucrose synthase and glucose-6-phosphate dehydrogenase. Various transcription factors with divergent expression patterns were identified. Finally, quantitative real time polymerase chain reaction (qRT-PCR) was conducted to validate the RNA-Seq and gene differentially expression results. This study provides valuable information for further studies on cold response mechanisms and genetic improvement of cold tolerance in Gold Queen Hami melons.

There is now abundant information on genes involved in molluscan oogenesis and their associations with ovarian development. However, few studies have investigated the ovarian transcriptome of Pacific abalone (Haliotis discus hannai).The objective of this study was to identify genes related to ovarian development and maturation in Pacific abalone utilizing RNA-sequencing (RNA-seq) and to verify the genes most relevant to different stages of maturation.RNA samples from the ovarian tissues of sexually immature and mature abalone were used to construct cDNA libraries, which were paired-end sequenced on an Illumina HiSeq 2500 platform. Reads from individual samples (unigenes) were aligned to reference transcriptome databases for identification of differentially expressed genes (DEGs) between immature and mature ovarian libraries. Reverse transcription-quantitative polymerase chain reaction was used to verify the RNA-seq data.A total of 8779 unigenes were obtained from the ovaries of immature and mature abalone, with a total length of 3323,279 bp and an average length of 379 bp per gene. Gene ontology analysis assigned 5860 unigenes to biological processes, 855 to cellular components, and 1352 to molecular functions. Overall, 470 DEGs were identified, including 213 and 257 genes down-regulated and up-regulated in mature abalone, respectively. Among these, 13 relevant transcripts, including VTG1 and FZD7, were significantly highly expressed in the ovaries of mature abalone (p 0.05, fold change 2).This H. discus hannai ovary transcriptome provides molecular targets to better understand ovarian development, oogenesis, and sexual maturation, and to enhance Pacific abalone production.

Recently, single-cell RNA-seq technologies have been rapidly updated, leading to a revolution in biology. We previously developed Microwell-seq, a cost-effective and high-throughput single cell RNA sequencing(scRNA-seq) method with a very simple device. Most cDNA libraries are sequenced using an expensive Illumina platform. Here, we present the first report showing combined Microwell-seq and BGI MGISEQ2000, a less expensive sequencing platform, to profile the whole transcriptome of 11,883 individual mouse adult adrenal gland cells and identify 18 transcriptionally distinct clusters. Moreover, we performed a single-cell comparative analysis of human and mouse adult adrenal glands to reveal the conserved genetic networks in these mammalian systems. These results provide new insights into the sophisticated adrenal gland hierarchy and provide a benchmark, low-cost strategy for high-throughput single-cell RNA study.

Chinese Sapium sebiferum (L.) Roxb has great potential to emerge as a promising biodiesel resource plant due to abundant oil content in seed. However, the unavailability of transcriptomic and genomic data impedes the progress of application in biodiesel production. Transcriptome analysis will help to further exploit tallow and kernel oil utilization in industry and facilitate molecular-breeding-based genetic improvement of S. sebiferum. Illumina hiseq 2000 sequencing platform was used to perform de novo transcriptome assembly and gene expression analyses of seeds from three strains in three developmental stages. A total of 188,254 unigenes over 200 bp were assembled with average length of 699 bp. These transcriptic unigenes were functionally assigned by GO and KEGG pathways. RPKM was used to identify differentially expressed unigenes of five cDNA libraries. Transcriptic genes endcoding enzymes related to fatty acid metabolism were evaluated. qRT-PCR was employed to confirm five key enzymes involved in fatty acid and triacylglycerol biosynthesis. This study provided comprehensive gene expression information of Chinese S. sebiferum seed at transcriptional level. These transcriptome data will facilitate high-oil-content genetic cultivation of S. sebiferum, which will further exploit tallow and kernel oil utilization in biodiesel industry.

Bone morphogenetic protein 9 (BMP9) (or GDF2) was originally identified from fetal mouse liver cDNA libraries. Emerging evidence indicates BMP9 exerts diverse and pleiotropic functions during postnatal development and in maintaining tissue homeostasis. However, the expression landscape of BMP9 signaling during development and/or in adult tissues remains to be analyzed. Here, we conducted a comprehensive analysis of the expression landscape of BMP9 and its signaling mediators in postnatal mice. By analyzing mouse ENCODE transcriptome datasets we found Bmp9 was highly expressed in the liver and detectable in embryonic brain, adult lungs and adult placenta. We next conducted a comprehensive qPCR analysis of RNAs isolated from major mouse tissues/organs at various ages. We found that Bmp9 was highly expressed in the liver and lung tissues of young adult mice, but decreased in older mice. Interestingly, Bmp9 was only expressed at low to modest levels in developing bones. BMP9-associated TGFβ/BMPR type I receptor Alk1 was highly expressed in the adult lungs. Furthermore, the feedback inhibitor Smads Smad6 and Smad7 were widely expressed in mouse postnatal tissues. However, the BMP signaling antagonist noggin was highly expressed in fat and heart in the older age groups, as well as in kidney, liver and lungs in a biphasic fashion. Thus, our findings indicate that the circulating BMP9 produced in liver and lungs may account for its pleiotropic effects on postnatal tissues/organs although possible roles of BMP9 signaling in liver and lungs remain to be fully understood. Keywords: BMP9/GDF2, Bone morphogenetic proteins (BMPs), Hepatic metabolism, Mesenchymal stem cells, Neurogenesis, Osteogenic differentiation, Pulmonary arterial hypertension, Tumorigenesis

Soil salinity of fields is often non-uniform. To obtain a better understanding of molecular response to non-uniform salt stress, we conducted transcriptomic analysis on the leaves and roots of alfalfa grown under 0/0, 200/200, and 0/200 mM NaCl treatments. A total of 233,742 unigenes were obtained from the assembled cDNA libraries. There were 98 and 710 unigenes identified as significantly differentially expressed genes (DEGs) in the leaves of non-uniform and uniform salt treatment, respectively. Furthermore, there were 5178 DEGs in the roots under uniform salt stress, 273 DEGs in the non-saline side and 4616 in the high-saline side roots under non-uniform salt stress. Alfalfa treated with non-uniform salinity had greater dry weight and less salt damage compared to treatment with uniform salinity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs in roots revealed that both sides of the non-uniform salinity were enriched in pathways related to “phenylpropanoid biosynthesis” and “linoleic acid metabolism”; and “MAPK signaling pathway–plant” was also indicated as a key pathway in the high-saline roots. We also combined a set of important salt-response genes and found that roots from the non-saline side developed more roots with increased water uptake by altering the expression of aquaporins and genes related to growth regulation. Moreover, the hormone signal transduction and the antioxidant pathway probably play important roles in inducing more salt-related genes and increasing resistance to non-uniform salt stress on both sides of the roots.

Lizards of the Helodermatidae (Anguimorpha) family consist of at least two well recognized species: Heloderma horridum horridum and Heloderma suspectum suspectum. They contain specialized glands in their jaws that produce venomous secretions that causes envenoming symptoms to bitten animals. One way to study proteins from such secretions is by RNA-seq; a powerful molecular tool to characterize the transcriptome of such specialized gland, and its protein secretions. The total RNA from venom gland tissues of H. horridum horridum was extracted and a cDNA library was constructed and sequenced. Overall, 114,172 transcripts were found, and 199 were annotated based on sequence similarities to previously described peptides/proteins. Transcripts coding for putative exendins, defensins, natriuretics and serine protease inhibitors were the most highly expressed. Transcripts that code for several putative serine proteases, phospholipases, metalloproteases, lipases, L-amino oxidase and nucleases were also found. Some of the novel identified transcripts were translationally controlled tumor proteins, venom factors, vespryns, waprins, lectins, cystatins and serine protease inhibitors. All these new protein structures may contribute to a better understanding of the venomous secretions of the Helodermatidae family.

Heat stress has a severe impact on potato growth and tuberization process, always resulting in the decrease of tuber yield and quality. Therefore, it is of great significance for potato breeding to illuminate the mechanism of heat stress on potato and explore heat resistant genes. In this study, two cDNA libraries from normal potato leaves (20 °C day/18 °C night) and potato leaves with 3 days of heat treatment (35 °C day/28 °C night) were constructed respectively. Totally, 1420 differentially expressed genes (DEGs) were identified. The expression patterns of 12 randomly selected genes detected using droplet digital PCR agreed with the sequencing data. Gene ontology analysis showed that these DEGs were clustered into 49 different GO types, reflecting the functional diversity of the heat stress response genes. The results of KEGG pathway enrichment showed the potential biological pathways in which the DEGs were involved, indicating that these pathways may be involved in heat tolerance regulation. Most potato heat transcription factors (StHsfs) and heat shock proteins (StHsps) were not expressed efficiently based on expression profile of these DEGs. StHsp26-CP and StHsp70 were markedly increased after 3 days of heat treatment. These data will be useful for further understanding the molecular mechanisms of potato plant tolerance to heat stress and provide a basis for breeding heat-tolerance varieties.

The light–dark cycle is the primarily environmental stimulus responsible for entraining the major circadian clock located within the suprachiasmatic nucleus (SCN), which in turn coordinates various physiological processes in fish, including those in non‐SCN regions of the brain. To better understand differences in gene expression due to the light–dark cycle, we compared the transcriptomes of Pelteobagrus vachellii in non‐SCN regions of the brain in the daytime (12:00) and nighttime (24:00). The two cDNA libraries yielded 217,575,058 and 350,784,004 paired‐end clean reads, and 311,143 unique transcript fragments (unigenes) averaging 600.94 base pairs were assembled. It was possible to annotate ~7,426 (2.39%) unigenes based on homology with all database sequences. Gene expression analysis revealed that 2,406 and 1,665 annotated unigenes were up‐ and downregulated, respectively, with ≥twofold differences between daytime and nighttime. Enrichment analysis of differentially expressed genes predicted the pathways in which they operate, including development, endocrine function, adaptation to environmental changes and immunity. In summary, characterization of daytime and nighttime gene expression patterns in P. vachellii showed that the light–dark cycle coordinates gene expression in non‐SCN regions of the brain.

Members of the C. neoformans/C. gattiii species complex are an important cause of serious humans infections, including meningoencephalitis. We describe here a 45 kDa extracellular cellulase purified from culture supernatants of C. neoformans var. neoformans. The N-terminal sequence obtained from the purified protein was used to isolate a clone containing the full-length coding sequence from a C. neoformans var. neoformans (strain B-3501A) cDNA library. Bioinformatics analysis indicated that this gene is present, with variable homology, in all sequenced genomes of the C. neoformans/C. gattii species complex. The cDNA clone was used to produce a recombinant 45 kDa protein in E. coli that displayed the ability to convert carboxymethyl cellulose and was therefore designated as NG-Case (standing for Neoformans Gattii Cellulase). To explore its potential use as a vaccine candidate, the recombinant protein was used to immunize mice and was found capable of inducing T helper type 1 responses and delayed-type hypersensitivity reactions, but not immune protection against a highly virulent C. neoformans var grubii strain. These data may be useful to better understand the mechanisms underlying the ability C. neoformans/C. gattii to colonize plant habitats and to interact with the human host during infection.

In order to identify genes involved in the pathogenesis of clear cell carcinoma of the ovary (CCC), functional screening using a cDNA expression library was performed. We extracted mRNA from a CCC cell line (RMG-1), established a cDNA library using a retroviral vector, transfected that library into mouse NIH3T3 cells and sequenced the resultant foci. The tissue-type specific expression of isolated genes and their transforming activities were evaluated. Seven genes were isolated. Of these genes, the mRNA expression of SEC61B and DVL1 is significantly stronger in CCC than in other histological types (

The basic helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors. Recent research has shown that the bHLH transcription factor DevR is involved in both sexual and asexual development as well as conidial melanin production in Aspergillus species. Our previous research also found that DevR significantly influences polysaccharide metabolism in Aspergillus oryzae. In this study, to further explore the function of DevR, its interaction proteins were screened by a yeast two-hybrid assay. An A. oryzae cDNA library was transformed into the Y187 strain by using the SMART technique and the homologous recombination method, and then hybridized with a constructed DevR bait plasmid introducing strain to obtain positive clones. Through sequencing analysis, the potential interaction proteins of DevR were determined. Among them, an AO090701000363 gene-encoding protein (named DipA), which was predicted to be a basic leucine zipper (bZIP) transcription factor, was a possible candidate. Phenotypic analysis indicated that overexpression of the AodipA may significantly suppress growth of the strain. Additionally, although no obvious change in the growth rate was found, the deletion of AodipA resulted in thicker hyphae morphology relative to the control. Comparative proteomic analysis further indicated that DipA was potentially involved in the regulation of cell wall integrity, carbon utilization, acetate catabolic process and other biological processes. Partial similarity of the phenotype to that of DevR suggested a correlation between them and implied that the DipA has a function partially similar to that of DevR.

Sogatella furcifera is an important rice pest. In order to understand the molecular basis of the sex determination in this pest, we performed de novo transcriptome sequencing of six cDNA libraries (three biological replicates) of female and male fifth-instar nymphs. Total 65,199 unigenes were obtained, with an average length of 971.5 bp and N50 length of 1708 bp. 20,287 open reading frames (ORFs) were predicted and annotated. Total 1019 differentially expressed genes with 873 upregulated and 146 downregulated were found in male compared to female. Total 164 sex-determining genes were identified, including the key sex-determining genes in fruit flies, such as Sxl, tra, dsx, etc. It implied that the sex determination mechanisms of S. furcifera may be the same as that of fruit flies. This study provided transcriptome resource as a fundamental support for future functional studies to elucidate the sex determination regulatory networks governing sexual dimorphism of S. furcifera.

Background Sequencing of miRNAs isolated from exosomes has great potential to identify novel disease biomarkers, but exosomes have low amount of RNA, hindering adequate analysis and quantification. Here, we have assessed several steps in developing an optimized small RNA (sRNA) library preparation protocol for next-generation sequencing (NGS) miRNA analysis from urinary exosomes. Methods A total of 24 urinary exosome samples from donors were included in this study. RNA was extracted by column-based methods. The quality of extracted RNA was assessed by spectrophotometric quantification and Bioanalyzer software analysis. All libraries were prepared using the CleanTag small RNA library preparation protocol and the effect of our additional modifications on adapter-dimer presence, sequencing data and tagged small RNA library population was also analyzed. Results Our results show that good quality sequencing libraries can be prepared following our optimized small RNA library preparation protocol from urinary exosomes. When the size selection by gel purification step was included within the workflow, adapter-dimer was totally removed from cDNA libraries. Furthermore, the inclusion of this modification step within small RNA library protocol augmented the small RNA mapped reads, with an especially significant 37% increase in miRNA reads, and the gel purification step made no difference to the tagged miRNA population. Conclusions This study provides researchers with an optimized small RNA library preparation workflow for next generation sequencing based exosome-associated miRNA analysis that yields a high amount of miRNA mapped reads without skewing the tagged miRNA population significantly.

Although the function and regulation of SnRK1 have been studied in various plants, its molecular mechanisms in response to abiotic stresses are still elusive. In this work, we identified an AP2/ERF domain-containing protein (designated GsERF7) interacting with GsSnRK1 from a wild soybean cDNA library. GsERF7 gene expressed dominantly in wild soybean roots and was responsive to ethylene, salt, and alkaline. GsERF7 bound GCC cis-acting element and could be phosphorylated on S36 by GsSnRK1. GsERF7 phosphorylation facilitated its translocation from cytoplasm to nucleus and enhanced its transactivation activity. When coexpressed in the hairy roots of soybean seedlings, GsSnRK1(wt) and GsERF7(wt) promoted plants to generate higher tolerance to salt and alkaline stresses than their mutated species, suggesting that GsSnRK1 may function as a biochemical and genetic upstream kinase of GsERF7 to regulate plant adaptation to environmental stresses. Furthermore, the altered expression patterns of representative abiotic stress-responsive and hormone-synthetic genes were determined in transgenic soybean hairy roots after stress treatments. These results will aid our understanding of molecular mechanism of how SnRK1 kinase plays a cardinal role in regulating plant stress resistances through activating the biological functions of downstream factors.