Rapid Amplification Of cDNA Ends Research Articles

The selective regulation of immune responses by matrix metalloproteinase MMP14 in Ostrinia furnacalis.

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune-related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection-responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune-related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double-stranded RNA and bacterial infection.

Characterization and tissue expression of twelve selenoproteins in yellow catfish Pelteobagrus fulvidraco fed diets varying in oxidized fish oil and selenium levels

BackgroundSelenium (Se) functions through selenoproteins and is essential to growth and metabolism of vertebrates. The present study was conducted to identify twelve selenoproteins genes (selenoe, selenof, selenoh, selneoi, selenom, selenok, selneon, selenoo, selenot, selenos, selenou and msrb1) from yellow catfish. Their mRNA expression patterns, as well as their response to dietary oxidized fish oils and Se addition were explored. MethodsWe use 3′and 5′ RACE PCR to clone full-length cDNA sequence of twelve selenoprotein genes from yellow catfish. Their mRNA expression patterns were assessed via quantitative real-time PCR. Yellow catfish were fed diet adequate Se+ fresh fish oil, adequate Se+ oxidized fish oil, high Se+ fresh fish oil and high Se+ oxidized fish oil, respectively, for 10 weeks. Their kidney, heart, brain and testis were used to assess the mRNA expression of twelve selenoprotein. ResultsTwelve selenoprotein genes had similar domains with mammals and the other fish. Their mRNAs were expressed widely in eleven tissues but varied with the tissues. Dietary oxidized fish oils and Se addition influenced their mRNA abundances of twelve selenoproteins in a tissue-dependent manner. ConclusionOur study demonstrated the characterization and expression of twelve selenoproteins, and elucidated their responses in yellow catfish fed diets varying in oxidized fish oils and Se addition, which increased our knowledge into the biological function and regulatory mechanism of Se and selenoproteins in fish.

Identification and Characterization of a Novel Quanzhou Mulberry Virus from Mulberry (Morus alba).

In this study, we discovered a new virus named Quanzhou mulberry virus (QMV), which was identified from the leaves of an ancient mulberry tree. This tree is over 1300 years old and is located at Fujian Kaiyuan Temple, a renowned cultural heritage site in China. We obtained the complete genome sequence of QMV using RNA sequencing followed by rapid amplification of complementary DNA ends (RACE). The QMV genome is 9256 nucleotides (nt) long and encodes five open reading frames (ORFs). Its virion was made of icosahedral particles. Phylogenetic analysis suggests that it belongs to the unclassified Riboviria. An infectious clone for QMV was generated and agroinfiltrated into Nicotiana benthamiana and mulberry, resulting in no visible disease symptoms. However, systemic movement of the virus was only observed in mulberry seedlings, suggesting that it has a host-specific pattern of movement. Our findings provide a valuable reference for further studies on QMV and related viruses, contributing to the understanding of viral evolution and biodiversity in mulberry.

Identification and Complete Genome Sequence of an Alternavirus from a Pathogenic Fungus, Fusarium nanum, Collected by Air Sampling

In this study, fungal spores were collected from the air, and mycelium was grown in Petri dishes containing potato dextrose agar medium in the lab. Based on the morphological and molecular characteristics, the fungus was identified as Fusarium nanum. A new double-stranded RNA (dsRNA) mycovirus was isolated from the mycelia of F. nanum and tentatively named Fusarium nanum alternavirus 1 (FnAV1). The complete genome sequence of FnAV1 was obtained by Illumina sequencing, followed by Sanger sequencing and the rapid amplification of cDNA ends (RACE) of the 5′ and 3′ ends. FnAV1 is composed of three dsRNA segments: dsRNA1 (3,546 nt) encodes RdRp, dsRNA2 (2,511 nt) encodes a hypothetical protein, and dsRNA3 encodes the coat protein (2,484 nt). The multiple alignments, along with phylogenetic analysis based on the RdRp (dsRNA1) and coat protein (dsRNA3) amino acid sequences, suggest that FnAV1 clusters with other members of the newly proposed family Alternaviridae, has high identities with Fusarium incarnatum alternavirus 1 (FiAV1), and might be a new strain isolated from a different fungal host, F. nanum. This is the first report of a mycovirus from the mycelial culture of F. nanum, which was obtained from spores collected at a 12-m altitude via air sampling. This suggests that the airborne spores could carry the mycovirus for long-distance movement and dissemination to new locations. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genomic characterization of two new viruses infecting Ageratum conyzoides in China.

Two new RNA viruses were identified in Ageratum conyzoides in China using high-throughput sequencing, and their genome sequences were determined using PCR and rapid amplification of cDNA ends. The new viruses, which have positive-sense, single-stranded RNA genomes, were provisionally named "ageratum virus 1" (AgV1) and "ageratum virus 2" (AgV2). AgV1 has a genome of 3,526 nucleotides with three open reading frames (ORFs) and shares 49.9% nucleotide sequence identity with the complete genome of Ethiopian tobacco bushy top virus (genus Umbravirus, family Tombusviridae). The genome of AgV2 consists of 5,523 nucleotides and contains five ORFs that are commonly observed in members of the genus Enamovirus of the family Solemoviridae. Proteins encoded by AgV2 exhibited the highest amino acid sequence similarity (31.7-75.0% identity) to the corresponding proteins of pepper enamovirus R1 (an unclassified enamovirus) and citrus vein enation virus (genus Enamovirus). Based on their genome organization, sequence, and phylogenetic relationships, AgV1 is proposed to be a new umbra-like virus of the family Tombusviridae, and AgV2 is proposed to be a new member of the genus Enamovirus of the family Solemoviridae.

Integration and viral oncogene expression of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma and gastric cancer.

Persistent high-risk human papillomavirus (HR-HPV) infections cause cervical cancer and a fraction of head and neck cancer. To investigate whether HR-HPV infection might be also involved in the development of gastric cancer (GC), we developed a platform utilizing a rolling circle amplification (RCA)-based nested L1 polymerase chain reactionwith Sanger sequencing to genotype the HPV DNA in cancer tissues of 361 GC and 89 oropharyngeal squamous cell carcinomas (OPSCC). HPV transcriptional activity was determined by E6/E7 mRNA expression and a 3' rapid amplification of cDNA ends was performed to identify HPV integration and expression of virus-host fusion transcripts. Ten of 361 GC, 2of 89 OPSCC, and 1of 22 normal adjacent tissues were HPV L1 DNA-positive. Five of the 10 HPV-positive GC were genotyped as HPV16 by sequencing and 1of 2 GC with RCA/nested HPV16 E6/E7 DNA detection exhibited HPV16 E6/E7 mRNA. Two OPSCC displayed HPV16 L1 DNA and E6/E7 mRNA, of which 1OPSCC tissue showed virus-host RNA fusion transcripts from an intron region of KIAA0825 gene. Together, our data reveal viral oncogene expression and/or integration in GC and OPSCC and a possible etiology role of HPV infections in gastric carcinogenesis.

Cloning of the RNA m6A Methyltransferase 3 and Its Impact on the Proliferation and Differentiation of Quail Myoblasts

Methyltransferase 3 (METTL3), which has been demonstrated to play a crucial role in a variety of biological processes, is the key enzyme for catalyzing m6A modification in RNA. However, the complete protein sequence of METTL3 in quail has not been annotated, and its function in skeletal muscle of quails remains unknown. In the current study, the full-length coding sequence of the quail METTL3 was obtained through the 3' rapid amplification of cDNA ends (3' RACE) and its homology with that of other species was predicted based on a generated phylogenetic tree. A Cell Counting Kit-8 assay and flow cytometry in a quail myoblast cell line (QM7) demonstrated that METTL3 promotes myoblast proliferation. The overexpression of METTL3 in QM7 cells significantly increased the expression levels of the myoblast differentiation markers myogenin (MYOG), myogenic differentiation 1 (MYOD1), and myocyte enhancer factor 2C (MEF2C), further demonstrating that METTL3 promotes myoblast differentiation. Additionally, transcriptome sequencing following METTL3 overexpression revealed that METTL3 controls the expression of various genes involved in RNA splicing and the regulation of gene expression, as well as pathways such as the MAPK signaling pathway. Taken together, our findings demonstrated that METTL3 plays a vital function in quail myoblast proliferation and differentiation and that the METTL3-mediated RNA m6A modification represents an important epigenetic regulatory mechanism in poultry skeletal muscle development.

Complete nucleotide sequence of hackberry virus A, a tentative member of the genus Waikavirus

The complete genomic sequence of a waikavirus from Chinese hackberry in Zhejiang province, China, named "hackberry virus A" (HVA), was determined using high-throughput sequencing (HTS) combined with reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The bicistronic genomic RNA of HVA was found to consist of 12,691 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and to encode a large polyprotein of 3783 amino acids (aa) and an additional 10.3-kDa protein. The aa sequences of the Pro-Pol and the CP regions of this virus share 39.8-44.2% and 25.5-36.4% identity, respectively, with currently known waikaviruses. These values are significantly below the current species demarcation threshold (< 75% and < 80% aa identity for the CP and Pro-Pol region, respectively) for the family Secoviridae, indicating that HVA represents a new species in the genus Waikavirus. This is the first report of a virus infecting Chinese hackberry.

Cloning and Expression Analysis of Onion (Allium cepa L.) MADS-Box Genes and Regulation Mechanism of Cytoplasmic Male Sterility.

Flower organ development is one of the most important processes in plant life. However, onion CMS (cytoplasmic male sterility) shows an abnormal development of floral organs. The regulation of MADS-box transcription factors is important for flower development. To further understand the role of MADS-box transcription factors in the regulation of cytoplasmic male sterility onions. We cloned the full-length cDNA of five MADS-box transcription factors from the flowers of onion using RACE (rapid amplification of cDNA ends) technology. We used bioinformatics methods for sequence analysis and phylogenetic analysis. Real-time quantitative PCR was used to detect the expression patterns of these genes in different onion organs. The relative expression levels of five flower development genes were compared in CMS onions and wild onions. The results showed that the full-length cDNA sequences of the cloned MADS-box genes AcFUL, AcDEF, AcPI, AcAG, and AcSEP3 belonged to A, B, C, and E MADS-box genes, respectively. A phylogenetic tree construction analysis was performed on its sequence. Analysis of MADS-box gene expression in wild onion and CMS onion showed that the formation of CMS onion was caused by down-regulation of AcDEF, AcPI, and AcAG gene expression, up-regulation of AcSEP3 gene expression, and no correlation with AcFUL gene expression. This work laid the foundation for further study of the molecular mechanism of onion flower development and the molecular mechanism of CMS onion male sterility.

The microRNA ppe-miR393 mediates auxin-induced peach fruit softening by promoting ethylene production.

Auxin can inhibit or promote fruit ripening, depending on the species. Melting flesh (MF) peach fruit (Prunus persica L. Batsch) cultivars produce high levels of ethylene caused by high concentrations of indole-3-acetic acid (IAA), which leads to rapid fruit softening at the late stage of development. In contrast, due to the low concentrations of IAA, the fruit of stony hard (SH) peach cultivars does not soften and produces little ethylene. Auxin seems necessary to trigger the biosynthesis of ethylene in peach fruit; however, the mechanism is not well understood. In this study, we identified miRNA gene family members ppe-miR393a and ppe-miR393b that are differentially expressed in SH and MF fruits. RNA ligase-mediated 5' rapid amplification of cDNA ends and transient transformation of Nicotiana benthamiana revealed TRANSPORT INHIBITOR RESPONSE 1 (PpTIR1), part of the auxin perception and response system, as a target of ppe-miR393a and b. Yeast 2-hybrid assay and bimolecular fluorescence complementation assay revealed that PpTIR1 physically interacts with an Aux/IAA protein PpIAA13. The results of yeast 1-hybrid assay, electrophoretic mobility shift assay, and dual-luciferase assay indicated that PpIAA13 could directly bind to and trans-activate the promoter of 1-aminocyclopropane-1-carboxylic acid synthase 1 (PpACS1), required for ethylene biosynthesis. Transient overexpression and suppression of ppe-miR393a and PpIAA13 in peach fruit induced and repressed the expression of PpACS1, confirming their regulatory role in ethylene synthesis. Gene expression analysis in developing MF and SH fruits, combined with postharvest α-naphthalene acetic acid (NAA) treatment, supports a role for a ppe-miR393-PpTIR1-PpIAA13-PpACS1 module in regulating auxin-related differences in ethylene production and softening extent in different types of peach.

MiR-330 targeting BCO2 is involved in carotenoid metabolism to regulate skin pigmentation in rainbow trout (Oncorhynchus mykiss)

BackgroundMicroRNAs (miRNAs) play a critical role in regulating skin pigmentation. As a key economic trait, skin color directly affects the market value of rainbow trout (Oncorhynchus mykiss), however, the regulatory mechanism of most miRNAs in fish skin color is still unclear.ResultsIn this study, the full-length cDNA sequence of β-carotene oxygenase 2 (BCO2, a key regulator of carotenoid metabolism) from the rainbow trout was obtained using rapid-amplification of cDNA ends (RACE) technology, and qRT-PCR was used to investigate the differential expression of miR-330 and BCO2 in 14 developmental stages and 13 tissues between wild-type rainbow trout (WTrt) and yellow mutant rainbow trout (YMrt). Additionally, the function of miR-330 was verified by overexpression and silencing in vitro and in vivo. The results showed that the complete cDNA sequence of BCO2 was 2057 bp with a 1707 bp ORF, encoding a 568 amino acid protein having a molecular weight of 64.07 kD. Sequence alignment revealed that higher conservation of BCO2 protein amongst fishes than amongst other vertebrates, which was further confirmed by phylogenetic analysis. The analysis of spatial and temporal expression patterns suggested that BCO2 and miR-330 were abundantly expressed from fertilized-stage to multi-cell as well as in the dorsal and ventral skin of WTrt and YMrt, and their expression patterns were opposite in most of the same periods and tissues. In vitro, luciferase reporter assay confirmed that BCO2 was a direct target of miR-330, and transfection of miR-330 mimics into rainbow trout liver cells resulted in a decrease in the expression of BCO2; conversely, miR-330 inhibitor had the opposite effect to the miR-330 mimics. In vivo, miR-330 agomir significantly decreased BCO2 expression in dorsal skin, tail fin, and liver. Furthermore, overexpression of miR-330 could suppress cell proliferation and induce apoptosis.ConclusionOur results showed that miR-330 is involved in the regulation of skin pigmentation in rainbow trout by targeting BCO2 and shows its promise as a potential molecular target to assist the selection of rainbow trout with better skin color patterns.

Long noncoding RNAs are substrates for cytoplasmic capping enzyme.

Cytoplasmic capping returns a cap to specific mRNAs, thus protecting uncapped RNAs from decay. Prior to the identification of cytoplasmic capping, uncapped mRNAs were thought to be degraded. Here, we test whether long noncoding RNAs (lncRNAs) are substrates of the cytoplasmic capping enzyme (cCE). The subcellular localisation of 14 lncRNAs associated with sarcomas were examined in U2OS osteosarcoma cells. We used 5' rapid amplification of cDNA ends (RACE) to assay uncapped forms of these lncRNAs. Inhibiting cytoplasmic capping elevated uncapped forms of selected lncRNAs indicating a plausible role of cCE in targeting them. Analysis of published cap analysis of gene expression (CAGE) data shows increased prevalence of certain 5'-RACE cloned sequences, suggesting that these uncapped lncRNAs are targets of cytoplasmic capping.

Genomic characterization of a novel torradovirus infecting Arctium lappa L. in China

Burdock (Arctium lappa L.) is not only a popular vegetable crop but also an important medicinal plant. In burdock plants with symptoms of leaf mosaic, a novel torradovirus tentatively named "burdock mosaic virus" (BdMV) was identified by high-throughput sequencing. The complete genomic sequence of BdMV was further determined using RT-PCR and the rapid amplification of cDNA ends (RACE) method. The genome is composed of two positive-sense single-stranded RNAs. RNA1 (6991 nt) encodes a polyprotein of 2186 aa, and RNA2 (4700 nt) encodes a protein of 201 aa and a polyprotein of 1212 aa that is predicted to be processed into one movement protein (MP) and three coat proteins (CPs). The Pro-Pol region of RNA1 and the CP region of RNA2 shared the highest amino acid sequence identity of 74.0% and 70.6%, respectively, with the corresponding sequences of lettuce necrotic leaf curl virus (LNLCV) isolate JG3. Phylogenetic analysis based on the amino acid sequences of the Pro-Pol and CP regions showed that BdMV clustered with other non-tomato-infecting torradoviruses. Taken together, these results suggest that BdMV is a new member of the genus Torradovirus.

Cloning and Functional Analysis of the Root-knot Nematode Resistance Gene NtRk1 in Tobacco.

Tobacco (Nicotiana tabacum L.) is an economically important crop worldwide. Root-knot nematodes (RKNs) are responsible for yield losses in tobacco and other crops, such as tomato, potato, peanut, and soybean. Therefore, screening for resistance genes that can prevent RKN infestation and the associated damage is crucial. However, there is no report of cloning tobacco RKN resistance genes to date. Here, we cloned the tobacco RKN resistance gene NtRk1 from the resistant variety TI706, using rapid amplification of cDNA ends. NtRk1 has high homology with other RKN resistance genes (CaMi in pepper, Mi-1.1, and Mi-1.2 in tomato). Under normal conditions, NtRk1 was barely expressed in the roots; however, following RKN infection, its expression level rapidly increased. Overexpression of NtRk1 in the susceptible cultivar 'Changbohuang' enhanced its resistance to Meloidogyne incognita, while RNA interference of NtRk1 in the resistant cultivar K326 resulted in its susceptibility to M. incognita. Moreover, compared with resistant variety K326, we found the salicylic acid and jasmonic acid contents of RNAi plants decreased after inoculation with M. incognita, and confirmed that the function of NtRk1 is related to these phytohormones. These findings indicate that NtRk1 is an RKN resistance gene, which is abundantly expressed in response to RKN infection and may enhance host defence responses by elevating salicylic acid and jasmonic acid levels.

Only fourteen 3′-end poly(A)s sufficient for rescuing Senecavirus A from its cDNA clone, but inadequate to meet requirement of viral replication

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense, single-strand RNA that has 5′ and 3′ untranslated regions. There is a poly(A) tail at the 3′ end of viral genome. Although the number of poly(A)s is variable, the length of poly(A) tail generally has the minimum nucleotide limit for picornaviral replication. To identify a range limit of poly(A)s for SVA recovery, five SVA cDNA clones, separately containing 25, 20, 15, 10 and 5 poly(A)s, were constructed for rescuing viruses. Replication-competent SVAs could be rescued from the first three cDNA clones, implying the range limit of poly(A)s was (A)15 to (A)10. To recognize the precise limit, four extra cDNA clones, separately containing 14, 13, 12 and 11 poly(A)s, were constructed to rescue SVAs further. The replication-competent SVA was rescued only from the poly(A)14-containing plasmid, indicating that the precise limit was poly(A)14 at the 3′ end of cDNA clone for SVA recovery. The rescued SVA was serially passaged in cells. The passage-5 and -10 progenies were independently subjected to the analysis of 3′-rapid amplification of cDNA ends. Both progenies showed their own poly(A) tails far more than 14 (A)s, implying extra (A)s added to the poly(A)14 sequence during viral passaging. It can be concluded that fourteen (A)s are sufficient for rescuing a replication-competent SVA from its cDNA clone, but inadequate for maintaining viral propagation in cells.

수온 스트레스에 의한 명태의 Heat Shock Factor 1의 분자생물학적 특성과 Heat Shock Protein 70의 단백질 발현 패턴

This study evaluated the levels of mRNA expression of HSF1 and HSP70 expression patterns in walleye pollock, Gasus chalcogrammus according to water temperature. Walleye pollack HSF1, which has not been reported, was used as a Rapid Amplification of cDNA Ends (RACE)-PCR strategy. The HSF1 amino acid sequence of walleye pollack showed 93% identification with the Atlantic cod. On the other hand, it showed relatively low identification with 50% in zebrafish. However, in the phylogenetic analysis, fish HSF1 formed a single clade group. The relative mRNA expression of HSF1 in the liver, intestine, gill, kidney, and heart of juveniles and immatures was investigated. The tissues in juvenile and immature, HSF1 mRNA expression was the highest at 14℃, and HSF1 mRNA levels increased significantly from 11℃ compared to 5 and 8℃. The mRNA levels of HSF1 were particularly high in the gill both juveniles and immatures. The expression of HSP70 was also evident in 11℃ and 14℃. These results indicate that water temperature of 11°C or higher is exposed to stressed-condition in walleye pollock. Therefore, HSF1 and HSP70 can be considered as important indicator for water temperature stress in walleye pollock.

B-Cell Receptor Features and Database Establishment in Recovered COVID-19 Patients by Combining 5'-RACE with PacBio Sequencing.

Antibodies induced by viral infection can not only prevent subsequent virus infection, but can also mediate pathological injury following infection. Therefore, understanding the B-cell receptor (BCR) repertoire of either specific neutralizing or pathological antibodies from patients convalescing from Coronavirus disease 2019 (COVID-19) infection is of benefit for the preparation of therapeutic or preventive antibodies, and may provide insight into the mechanisms of COVID-19 pathological injury. In this study, we used a molecular approach of combining 5' Rapid Amplification of cDNA Ends (5'-RACE) with PacBio sequencing to analyze the BCR repertoire of all 5 IgH and 2 IgL genes in B-cells harvested from 35 convalescent patients after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We observed numerous BCR clonotypes within most COVID-19 patients, but not in healthy controls, which validates the association of the disease with a prototypical immune response. In addition, many clonotypes were found to be frequently shared between different patients or different classes of antibodies. These convergent clonotypes provide a resource to identify potential therapeutic/prophylactic antibodies, or identify antibodies associated with pathological effects following infection with SARS-CoV-2.

Enhanced Expression of a Novel Lamin A/C Splice Variant in Idiopathic Pulmonary Fibrosis Lung.

In idiopathic pulmonary fibrosis (IPF), the normal delicate lung architecture is replaced with rigid extracellular matrix (ECM) as a result of the accumulation of activated myofibroblasts and excessive deposition of ECM. Lamins have a role in fostering mechanosignaling from the ECM to the nucleus. Although there is a growing number of studies on lamins and associated diseases, there are no prior reports linking aberrations in lamins with pulmonary fibrosis. Here, we discovered, through analysis of RNA sequencing data, a novel isoform of lamin A/C that is more highly expressed in IPF compared with control lung. This novel LMNA (lamin A/C) splice variant includes retained introns 10 and 11 and exons 11 and 12 as documented by rapid amplification of cDNA ends. We found that this novel isoform is induced by stiff ECM. To better clarify the specific effects of this novel isoform of lamin A/C and how it may contribute to the pathogenesis of IPF, we transduced the lamin transcript into primary lung fibroblasts and alveolar epithelial cells and found that it impacts several biological effects, including cell proliferation, senescence, cell contraction, and the transition of fibroblasts to myofibroblasts. We also observed that type II epithelial cells and myofibroblasts in the IPF lung exhibited wrinkled nuclei, and this is notable because this has not been previously described and is consistent with laminopathy-mediated cellular effects.

Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort.

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.