scholarly journals Single-Molecule Real-Time and Illumina Sequencing to Analyze Transcriptional Regulation of Flavonoid Synthesis in Blueberry

2021 ◽  
Vol 12 ◽  
Author(s):  
Qi Tang ◽  
Fu-Mei Chi ◽  
Hong-Di Liu ◽  
Hong-Jun Zhang ◽  
Yang Song
Keyword(s):  
Transcriptional Regulation ◽  
Real Time ◽  
Transient Expression ◽  
Single Molecule ◽  
Illumina Sequencing ◽  
Molecular Mechanisms ◽  
Vaccinium Corymbosum ◽  
Background Information ◽  
Anthocyanin Synthesis ◽  
Flavonoid Synthesis

Blueberries (Vaccinium corymbosum) contain large amounts of flavonoids, which play important roles in the plant’s ability to resist stress and can also have beneficial effects on human health when the fruits are eaten. However, the molecular mechanisms that regulate flavonoid synthesis in blueberries are still unclear. In this study, we combined two different transcriptome sequencing platforms, single-molecule real-time (SMRT) and Illumina sequencing, to elucidate the flavonoid synthetic pathways in blueberries. We analyzed transcript quantity, length, and the number of annotated genes. We mined genes associated with flavonoid synthesis (such as anthocyanins, flavonols, and proanthocyanidins) and employed fluorescence quantitative PCR to analyze the expression of these genes and their correlation with flavonoid synthesis. We discovered one R2R3 MYB transcription factor from the sequencing library, VcMYB1, that can positively regulate anthocyanin synthesis in blueberries. VcMYB1 is mainly expressed in colored (mature) fruits. Experiments showed that overexpression and transient expression of VcMYB1 promoted anthocyanin synthesis in Arabidopsis, tobacco (Nicotiana benthamiana) plants and green blueberry fruits. Yeast one-hybrid (Y1H) assay, electrophoretic mobility shift assay, and transient expression experiments showed that VcMYB1 binds to the MYB binding site on the promoter of the structural gene for anthocyanin synthesis, VcMYB1 to positively regulate the transcription of VcDFR, thereby promoting anthocyanin synthesis. We also performed an in-depth investigation of transcriptional regulation of anthocyanin synthesis. This study provides background information and data for studying the synthetic pathways of flavonoids and other secondary metabolites in blueberries.

scholarly journals Chromosome End Repair and Genome Stability in Plasmodium falciparum

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Susannah F. Calhoun ◽  
Jake Reed ◽  
Noah Alexander ◽  
Christopher E. Mason ◽  
Kirk W. Deitsch ◽  
...  
Keyword(s):  
Dna Repair ◽  
Plasmodium Falciparum ◽  
Homologous Recombination ◽  
Real Time ◽  
Single Molecule ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Transmitted Disease ◽  
Subtelomeric Regions

ABSTRACT The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs) is thought to rely almost exclusively on homologous recombination (HR), due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called “telomere healing,” and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity. IMPORTANCE Malaria is a major global health threat, causing approximately 430,000 deaths annually. This mosquito-transmitted disease is caused by Plasmodium parasites, with infection with the species Plasmodium falciparum being the most lethal. Mechanisms underlying DNA repair and maintenance of genome integrity in P. falciparum are not well understood and represent a gap in our understanding of how parasites survive the hostile environment of their vertebrate and insect hosts. Our work examines DNA repair in real time by using single-molecule real-time (SMRT) sequencing focused on the subtelomeric regions of the genome that harbor the multicopy gene families important for virulence and the maintenance of infection. We show that parasites utilize two competing molecular mechanisms to repair double-strand breaks, homologous recombination and de novo telomere addition, with the pathway used being determined by the surrounding DNA sequence. In combination, these two pathways balance the need to maintain genome stability with the selective advantage of generating antigenic diversity. IMPORTANCE Malaria is a major global health threat, causing approximately 430,000 deaths annually. This mosquito-transmitted disease is caused by Plasmodium parasites, with infection with the species Plasmodium falciparum being the most lethal. Mechanisms underlying DNA repair and maintenance of genome integrity in P. falciparum are not well understood and represent a gap in our understanding of how parasites survive the hostile environment of their vertebrate and insect hosts. Our work examines DNA repair in real time by using single-molecule real-time (SMRT) sequencing focused on the subtelomeric regions of the genome that harbor the multicopy gene families important for virulence and the maintenance of infection. We show that parasites utilize two competing molecular mechanisms to repair double-strand breaks, homologous recombination and de novo telomere addition, with the pathway used being determined by the surrounding DNA sequence. In combination, these two pathways balance the need to maintain genome stability with the selective advantage of generating antigenic diversity.

scholarly journals SMRT- and Illumina‑based RNA‑seq Analyses Unveil Triterpenoid Saponin Biosynthesis and Related Key Genes in Platycodon Grandiflorus (Jacq.) A. DC.

2021 ◽  
Author(s):  
Hanwen Yu ◽  
Mengli Liu ◽  
Minzhen Yin ◽  
Tingyu Shan ◽  
Huasheng Peng ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Total Content ◽  
Triterpenoid Saponin ◽  
Triterpenoid Saponins ◽  
Sequencing Technologies ◽  
Saponin Biosynthesis ◽  
Genes Encoding ◽  
Key Genes

Abstract Background: Platycodon grandiflorus, a traditional Chinese medicine, contains considerable triterpene saponins with broad pharmacological activities. To date, information on the molecular mechanism of triterpenoid saponin biosynthesis in P. grandiflorus is limited. Here, single-molecule real-time (SMRT) and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and unveil triterpenoid saponin biosynthesis in P. grandiflorus.Results: We quantified four saponin monomers in P. grandiflorus, and found that the total content of the four saponins was the highest in the roots and the lowest in the stems and leaves. A total of 173,354 non-redundant transcripts generated from the PacBio platform were successfully annotated to seven functional databases, among which 1,765 transcripts were aligned to the "metabolism of terpenoids and polyketides" pathway in the KEGG database. Three full-length transcripts of β-amyrin synthase (β-AS), the key synthase of the β-amyrin, were identified. Furthermore, a total of 132,610 clean reads of BGISEQ sequences were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes (DEGs) involved were selected as candidates. Notably, 9 of the 96 DEGs showed the highest expression in the roots, which were considered key genes for synthesising triterpenoid saponins in P. grandiflorus. Furthermore, 3,469 genes encoding transcription factors (TFs) were identified and classified into 57 TF families, including MYB, bHLH, mTERF, and AP2-EREBP. The expression levels of genes were verified by quantitative real-time PCR.Conclusions: Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide new insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

scholarly journals Complete Genome Sequence of Achromobacter xylosoxidans MN001, a Cystic Fibrosis Airway Isolate

2015 ◽  
Vol 3 (4) ◽  
Author(s):  
Jonathan P. Badalamenti ◽  
Ryan C. Hunter
Keyword(s):  
Cystic Fibrosis ◽  
Cystic Fibrosis Patient ◽  
Real Time ◽  
Genome Sequence ◽  
Single Molecule ◽  
Illumina Sequencing ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Achromobacter Xylosoxidans ◽  
Cystic Fibrosis Airway

The genome of Achromobacter xylosoxidans MN001, a strain isolated from sputum derived from an adult cystic fibrosis patient, was sequenced using combined single-molecule real-time and Illumina sequencing. Assembly of the complete genome resulted in a 5,876,039-bp chromosome, representing the smallest A. xylosoxidans genome sequenced to date.

scholarly journals Complete Genome Sequence of the Clostridium difficile Type Strain DSM 1296 T

2015 ◽  
Vol 3 (5) ◽  
Author(s):  
Thomas Riedel ◽  
Boyke Bunk ◽  
Johannes Wittmann ◽  
Andrea Thürmer ◽  
Cathrin Spröer ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Real Time ◽  
Genome Sequence ◽  
Single Molecule ◽  
Illumina Sequencing ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Type Strain ◽  
Sequencing Technology ◽  
Extrachromosomal Elements

In this study, we sequenced the complete genome of the Clostridium difficile type strain DSM 1296 T . A combination of single-molecule real-time (SMRT) and Illumina sequencing technology revealed the presence of one chromosome and two extrachromosomal elements, the bacteriophage phiCDIF1296T and a putative plasmid-like structure harboring genes of another bacteriophage.

scholarly journals Single-Molecule Real-Time and Illumina-Based RNA Sequencing Data Identified Vernalization-Responsive Candidate Genes in Faba Bean (Vicia faba L.)

2021 ◽  
Vol 12 ◽  
Author(s):  
Xingxing Yuan ◽  
Qiong Wang ◽  
Bin Yan ◽  
Jiong Zhang ◽  
Chenchen Xue ◽  
...  
Keyword(s):  
Real Time ◽  
Candidate Genes ◽  
Vicia Faba ◽  
Single Molecule ◽  
Faba Bean ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Full Length ◽  
Differentially Expressed ◽  
Sequencing Data

Faba bean (Vicia faba L.) is one of the most widely grown cool season legume crops in the world. Winter faba bean normally has a vernalization requirement, which promotes an earlier flowering and pod setting than unvernalized plants. However, the molecular mechanisms of vernalization in faba bean are largely unknown. Discovering vernalization-related candidate genes is of great importance for faba bean breeding. In this study, the whole transcriptome of faba bean buds was profiled by using next-generation sequencing (NGS) and single-molecule, real-time (SMRT) full-length transcriptome sequencing technology. A total of 29,203 high-quality non-redundant transcripts, 21,098 complete coding sequences (CDS), 1,045 long non-coding RNAs (lncRNAs), and 12,939 simple sequence repeats (SSRs) were identified. Furthermore, 4,044 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these differentially expressed transcripts were found to be enriched in binding and transcription factor activity, electron carrier activity, rhythmic process, and receptor activity. Finally, 50 putative vernalization-related genes that played important roles in the vernalization of faba bean were identified; we also found that the levels of vernalization-responsive transcripts showed significantly higher expression levels in cold-treated buds. The expression of VfSOC1, one of the candidate genes, was sensitive to vernalization. Ectopic expression of VfSOC1 in Arabidopsis brought earlier flowering. In conclusion, the abundant vernalization-related transcripts identified in this study will provide a basis for future researches on the vernalization and faba bean breeding and established a reference full-length transcriptome for future studies on faba bean.

RPS24c Isoform Facilitates Tumor Angiogenesis Via Promoting the Stability of MVIH in Colorectal Cancer

2020 ◽  
Vol 20 (5) ◽  
pp. 388-395 ◽  
Author(s):  
Yue Wang ◽  
Youjun Wu ◽  
Kun Xiao ◽  
Yingjie Zhao ◽  
Gang Lv ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Ribosomal Protein ◽  
Real Time ◽  
Tumor Angiogenesis ◽  
Real Time Pcr ◽  
Molecular Mechanisms ◽  
Migration And Invasion ◽  
Binding Interaction ◽  
Tubule Formation ◽  
The Stability

Background: Colorectal cancer (CRC) is the second leading cause of death worldwide, and distant metastasis is responsible for the poor prognosis in patients with advanced-stage CRC. RPS24 (ribosomal protein S24) as a ribosomal protein, multiple transcript variant encoding different isoforms have been found for this gene. Our previous studies have demonstrated that RPS24 is overexpressed in CRC. However, the mechanisms underlying the role of RPS24 in tumor development have not been fully defined. Methods: Expression of RPS24 isoforms and lncRNA MVIH in CRC tissues and cell lines were quantified by real-time PCR or western blotting assay. Endothelial tube formation assay was performed to determine the effect of RPS24 on tumor angiogenesis. The cell viability of HUVEC was determined by MTT assay, and the migration and invasion ability of HUVEC were detected by transwell assay. PGK1 secretion was tested with a specific ELISA kit. Results: Here, we found that RPS24c isoform was a major contributor to tumor angiogenesis, a vital process in tumor growth and metastasis. Real-time PCR revealed that RPS24c isoform was highly expressed in CRC tissues, while other isoforms are present in both normal and CRC tissues with no statistical difference. Moreover the change of RPS24 protein level is mainly due to the fluctuation of RPS24c. Furthermore, we observed that silencing RPS24c could decrease angiogenesis by inhibiting tubule formation, HUVEC cell proliferation and migration. Additionally, we investigated the molecular mechanisms and demonstrated that RPS24c mRNA interacted with lncRNA MVIH, the binding-interaction enhanced the stability of each other, thereby activated angiogenesis by inhibiting the secretion of PGK1. Conclusion: RPS24c facilitates tumor angiogenesis via the RPS24c/MVIH/PGK1 pathway in CRC. RPS24c inhibition may be a novel option for anti-vascular treatment in CRC.

scholarly journals Combined Transcriptome Analysis Reveals the Ovule Abortion Regulatory Mechanisms in the Female Sterile Line of Pinus tabuliformis Carr.

2021 ◽  
Vol 22 (6) ◽  
pp. 3138
Author(s):  
Zaixin Gong ◽  
Rui Han ◽  
Li Xu ◽  
Hailin Hu ◽  
Min Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Regulatory Mechanisms ◽  
Cdna Libraries ◽  
Ovule Development ◽  
Ovule Abortion ◽  
Pinus Tabuliformis ◽  
Wild Type Female ◽  
Fertile Line ◽  
Next Generation Sequencing Ngs

Ovule abortion is a common phenomenon in plants that has an impact on seed production. Previous studies of ovule and female gametophyte (FG) development have mainly focused on angiosperms, especially in Arabidopsis thaliana. However, because it is difficult to acquire information about ovule development in gymnosperms, this remains unclear. Here, we investigated the transcriptomic data of natural ovule abortion mutants (female sterile line, STE) and the wild type (female fertile line, FER) of Pinus tabuliformis Carr. to evaluate the mechanism of ovule abortion during the process of free nuclear mitosis (FNM). Using single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS), 18 cDNA libraries via Illumina and two normalized libraries via PacBio, with a total of almost 400,000 reads, were obtained. Our analysis showed that the numbers of isoforms and alternative splicing (AS) patterns were significantly variable between FER and STE. The functional annotation results demonstrate that genes involved in the auxin response, energy metabolism, signal transduction, cell division, and stress response were differentially expressed in different lines. In particular, AUX/IAA, ARF2, SUS, and CYCB had significantly lower expression in STE, showing that auxin might be insufficient in STE, thus hindering nuclear division and influencing metabolism. Apoptosis in STE might also have affected the expression levels of these genes. To confirm the transcriptomic analysis results, nine pairs were confirmed by quantitative real-time PCR. Taken together, these results provide new insights into ovule abortion in gymnosperms and further reveal the regulatory mechanisms of ovule development.

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