Full-length Open Reading Frame Research Articles

Isolation, genetic analysis of the first Akabane virus from goat in China.

Akabane virus (AKAV) is an important insect-borne virus belonging to the genus Orthobunyavirus, the Peribunyaviridae family. An AKAV defined as GXDH 01 here, was isolated for the first time from blood from a sentinel goat in China in 2016, and its full-length open reading frames (ORFs) were sequenced in this study. Sequence analysis suggested that the isolate GXDH 01 probably had undergone a reassortment incident and acquired L segments from other strain originating from an attenuated vaccine, such as OBE-1. This study aims to provide more understanding as to the origin and epidemiology of AKAV in China.

Inheritance of 5'-Truncated Copies of R2 Retrotransposon in a Series of Generations of German Cockroach, Blattella germanica

Inheritance of 5'-truncated copies of R2 retrotransposon of German cockroach, Blattella germanica, was analyzed in a series of generations. It was shown that copies of certain length are linked and form different patterns associated with X chromosomes where R2 retrotransposons are normally localized within clusters of ribosomal genes and, unexpectedly with autosomes. There was no recombination between certain copies of different length, and new variants of 5'-truncated copies were not found. Taking into account the stable inheritance of particular patterns of various R2 5'-truncated copies, we suggest using them as convenient molecular markers in population genetics studies of German cockroach, Blattella germanica. It was also shown that only short degenerate DNA sequences of R2 retrotransposon were present outside the cluster of ribosomal genes; apparently, DNA sequences corresponding to native full-length copies and/or open reading frames of R2 retrotransposon are present only within the cluster.

Genetic polymorphism of MHC class IIα alleles and its association with resistance/susceptibility to viral nervous necrosis virus (VNNV) in golden pompano (Trachinotus ovatus)

The major histocompatibility complex (MHC) plays an important role in the vertebrate immune system by binding and presenting foreign peptides to T cell receptors. MHC genes encode cellular proteins that regulate the acquired immune response of vertebrates and affect the development of disease in animals. In the present study, we obtained 36 different full-length open reading frame sequences of Trov-MHC IIα from 20 adult golden pompano individuals. High levels of polymorphisms were detected among the 36 sequences. An excess of non-synonymous substitutions (dN) relative to synonymous substitutions (dS) was presented in the peptide-binding regions (PBRs), which suggested that the positive balancing selection driven by pathogens may maintain and promote the high diversity of the PBR domain. Bayesian inference revealed evidence for positive selection of specific codons in Trov-MHC IIα, and most of the positive selection sites were identified in the α1 domain. Furthermore, 26 exon 2 sequences of Trov-MHC IIα were identified from 70 individuals and exhibited high levels of diversity. Six alleles were selected to explore the correlations between the alleles and resistance/susceptibility to viral nervous necrosis virus (VNNV) in golden pompano. The frequency distribution showed that the Trov-DAA*6304 allele in golden pompano was associated with resistance to VNNV, while the Trov-DAA*7701 allele was associated with susceptibility to VNNV. These results confirmed the association between alleles of MHC class IIα and resistance to viral infection in golden pompano and they may provide valuable strategies to enhance the species' resistance to disease caused by viral infections and to help in marker-assisted selection breeding programmes for golden pompano.

Molecular identification and expression analysis of TAB1 from orange-spotted grouper (Epinephelus coioides)

Transforming growth factor-β activated kinase 1 (TAK1) is a crucial signal transducer in multiple signaling pathways. TAK1 binds TAB1, TAB2, and TAB3, which act as activators and adaptors that specifically regulate the activation of TAK1. To date, the role of TABs is largely unknown in fish. In the present study, a TAB1 cDNA sequence was identified in grouper (Epinephelus coioides), and designated EcTAB1. The full-length open reading frame of EcTAB1 is 1, 521 bp; it encodes 506 amino acids that contains an N-terminal PP2C domain. Many important functional sites in mammalian TAB1 were conserved in TAB1 from grouper and from other fish. Multiple sequence alignment showed that EcTAB1 protein shared high sequence identity with TAB1 of other fish, especially with Stegastes partitus (95% identity). TAB1 was clustered into the same subgroup with other fish TAB1 in the phylogenetic tree. Tissue expression analysis indicated that TAB1 was widely distributed in different tissues. After infection with Cryptocaryon irritans, EcTAB1 expression was up-regulated in the infection site (gills). Besides, EcTAB1 was expressed throughout the grouper spleen (GS) cells and significantly enhanced the activation of NF-κB.

Transcriptional fates of human-specific segmental duplications in brain.

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their high sequence identity (average >99%) and overlap with human-specific segmental duplications (SDs). We characterized the transcriptional differences between related paralogs to better understand the birth–death process of duplicate genes and particularly how the process leads to gene innovation. In 48% of the cases, we find that the expressed duplicates have changed substantially from their ancestral models due to novel sites of transcription initiation, splicing, and polyadenylation, as well as fusion transcripts that connect duplication-derived exons with neighboring genes. We detect unannotated open reading frames in genes currently annotated as pseudogenes, while relegating other duplicates to nonfunctional status. Our method significantly improves gene annotation, specifically defining full-length transcripts, isoforms, and open reading frames for new genes in highly identical SDs. The approach will be more broadly applicable to genes in structurally complex regions of other genomes where the duplication process creates novel genes important for adaptive traits.

Molecular and genetic characterization of peste des petits ruminants virus Kurdistan 2011 strain based on the haemagglutinin and fusion protein genes sequences

Severe outbreaks of peste des petits ruminants virus (PPRV) occurred during 2011 in wild goats in the Kurdistan region in Iraq. Based on a partial sequence of the nucleoprotein (N) gene the causative virus, named PPRV Kurdistan 2011, was classified as PPRV lineage IV genotype. For further characterization, the open reading frames (ORFs) coding for the haemagglutinin (H) and fusion (F) protein of PPRV Kurdistan 2011 were amplified from infected-cell RNA by RT-PCR. Sequencing of the ORFs revealed sizes of 1830 nucleotides (nt) for the H-ORF and 1641 nt for the F-ORF. This study represents the first report describing the full length ORFs of H and F for a PPRV strain from Kurdistan, Iraq.Comparative sequence analysis performed for the entire H- and F- ORFs of Kurdistan 2011 with respective sequences of PPRV isolates showed the highest homology with PPRV lineage IV isolates which is also reflected by comparative amino acid analysis. Phylogenetic analysis performed based on the partial and complete nucleotide sequences of both H- and F-ORFs clustered Kurdistan 2011 within PPRV lineage IV genotype. H-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Turkey/TR/AFYON/2011-35H and Turkey/2000 strains whereas F-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Qatar/37/10-02, Kurdistan/2012 and Egypt/2009 strains.

Molecular characterization and expression analysis of tumor necrosis factor receptor-associated factors 3 and 6 in large yellow croaker (Larimichthys crocea)

The large yellow croaker (Larimichthys crocea) has a well-developed innate immune system. To gain a better understanding of the defense mechanisms involved in this system, we studied tumor necrosis factor receptor-associated factors (TRAFs), which play important roles in the Toll-like receptor (TLR) pathway. We characterized the full-length open reading frames and protein structures of TRAF3 and TRAF6 to determine their identities, and conducted phylogenetic analysis to determine their evolutionary relationships. To assess the roles of TRAFs in innate immune responses in the large yellow croaker, we performed quantitative reverse-transcription PCR (qRT-PCR) to characterize expression profiles in a range of tissues at different stages after challenge with polyinosinic polycytidylic acid (poly I:C) and Vibrio anguillarum. Following poly I:C challenge, the expression levels of TRAF3 and TRAF6 were highest in the kidneys and lowest in the spleen, whereas after infection with V. anguillarum, TRAF6 expression was the highest in the kidneys and lowest in the liver.

Lack of Transmission of Foot-and-Mouth Disease Virus From Persistently Infected Cattle to Naïve Cattle Under Field Conditions in Vietnam.

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV; Aphthovirus, Picornaviridae), is a highly contagious and economically important disease of cloven-hoofed domestic livestock and wildlife species worldwide. Subsequent to the clinical phase of FMD, a large proportion of FMDV-infected ruminants become persistently infected carriers, defined by detection of FMDV in oropharyngeal fluid (OPF) samples 28 days or more post-infection. The goal of this prospective study was to characterize the FMD carrier state in cattle subsequent to natural infection under typical husbandry practices in Vietnam. Ten persistently infected cattle on eight farms in the Long An province in southern Vietnam were monitored by monthly screening of serum and oropharyngeal fluid samples for 12 months. To assess transmission from FMDV carriers, 16 naïve cattle were intentionally brought into direct contact with the persistently infected animals for 6 months, and were monitored by clinical and laboratory methods. The restricted mean duration of the FMD carrier state was 27.7 months, and the rate of decrease of the proportion of carrier animals was 0.03 per month. There was no evidence of transmission to naïve animals throughout the study period. Additionally, there was no detection of FMDV infection or seroconversion in three calves born to carrier animals during the study. The force of infection for carrier-to-contact transmission was 0 per month, with upper 95% confidence limit of 0.064 per month. Phylogenetic analysis of viral protein 1 (VP1) coding sequences obtained from carriers indicated that all viruses recovered in this study belonged to the O/ME-SA/PanAsia lineage, and grouped phylogenetically with temporally and geographically related viruses. Analysis of within-host evolution of FMDV, based upon full-length open reading frame sequences recovered from consecutive samples from one animal, indicated that most of the non-synonymous changes occurred in Lpro, VP2, and VP3 protein coding regions. This study suggests that the duration of FMDV persistent infection in cattle may be longer than previously recognized, but the risk of transmission is low. Additional novel insights are provided into within-host viral evolution under natural conditions in an endemic setting.

Genome-Wide Characterization and Expression Analyses of Pleurotus ostreatus MYB Transcription Factors during Developmental Stages and under Heat Stress Based on de novo Sequenced Genome.

Pleurotus ostreatus is a commercially grown mushroom species in China. However, studies on the mechanisms of the fruiting body development and stress response of P. ostreatus are still at a primary stage. In this study, we report the entire genome sequence of P. ostreatus CCMSSC03989. Then, we performed comprehensive genome-wide characterization and expression analysis of the MYB transcription factor family during a series of developmental stages and under the condition of heat stress. A 34.76 Mb genome was obtained through next-generation sequencing (NGS) and Bionano optical mapping approaches. The genome has a scaffold N50 of 1.1 Mb and contains 10.11% repeats, and 10,936 gene models were predicted. A total of 20 MYB genes (PoMYB) were identified across the genome, and the full-length open reading frames were isolated. The PoMYBs were classified into 1 repeat (1R), 2R, and 3R-MYB groups according to their MYB domain repeat numbers, and 3R-MYBs possessed relatively more introns than 1R and 2R-MYBs. Based on phylogenetic analysis, the PoMYBs were divided into four groups and showed close relationships with the MYB genes of plants and fungi. RNA-sequencing (RNA-Seq) and quantitative PCR (qPCR) analyses revealed that PoMYB expression showed stage-specific patterns in reproductive stages and could be induced by heat stress. The P. ostreatus draft genome will promote genome-wide analysis, and our study of PoMYBs will promote further functional analysis of MYB genes in mushrooms.

Transcriptome-wide survey, gene expression profiling and exogenous chemical-induced transcriptional responses of cytochrome P450 superfamily genes in migratory locust (Locusta migratoria)

Cytochrome P450 monooxygenases (CYPs) belong to a large superfamily of heme-containing enzymes catalyzing at least 60 different types of chemically distinct reactions. Insect CYPs play key roles in biotransformation of insecticides and plant chemicals, and are implicated in insecticide resistance and insect adaptation to their host plants. Insect CYPs are well studied in model insects, but little is known about the CYP superfamily in paurometabolous insects. We employed Illumina sequencing technology to identify 71 partial and 78 full-length open reading frames (ORFs) of LmCYP genes from the migratory locust (Locusta migratoria), one of the most destructive paurometabolous insect pests in the world. Seventy-eight LmCYPs with complete ORFs were formally named and classified into 19 families and 43 subfamilies. The majority of LmCYPs were mainly expressed in nymphal and adult stages, but LmCYP expression varied widely among thirteen different tissues examined. Regulatory elements were predicted in the promoter regions of LmCYP genes, and subsequent exposure of locusts to 12 different exogenous chemicals showed that 2-tridecanone and xanthotoxin were the most effective at increasing LmCYP expression. Our results represent the first transcriptome-wide analysis of the LmCYP superfamily from migratory locust, and provide a foundation for understanding the physiological functions, functional diversity, evolution, and regulatory mechanisms controlling the expression of the CYP gene superfamily in the locust.

Ribavirin-induced mutagenesis across the complete open reading frame of hepatitis C virus genotypes 1a and 3a.

Ribavirin (RBV) has been used for the last 20 years to treat patients with chronic hepatitis C virus (HCV) infection. This pluripotent drug is believed to induce mutagenesis in HCV RNA. However, for cell-cultured HCV (HCVcc) this phenomenon has only been investigated in genotype 2a recombinants. Here we studied the mutations that developed in HCVcc of genotypes 1a and 3a treated with RBV or ribavirin triphosphate (RBV-TP) compared to non-treated controls. Analysis was performed on the amplified full-length open reading frame (ORF) of recovered viruses following next-generation sequencing and clonal analyses. Compared to non-treated controls, the spread of TNcc(1a) and DBN3acc(3a) HCVcc was delayed by RBV and RBV-TP at concentrations of 40 µM or higher. The delay in HCVcc spread was associated with increased new single-nucleotide polymorphisms (SNP). Significantly higher numbers of new SNP were observed in TNcc(1a) viruses treated with RBV or RBV-TP compared to matched non-treated controls. RBV or RBV-TP treatment led to significantly increased proportions of new G-to-A and C-to-U SNP compared to non-treated TNcc(1a). Clonal analyses confirmed a significantly increased mutation rate in RBV-treated TNcc(1a). Synonymous pairwise distances increased in both viruses across the complete ORF under RBV and RBV-TP treatment compared to controls. Consensus-shifts in single samples of RBV- or RBV-TP-treated TNcc(1a) viruses occurred in proteins E1, p7, NS3 and NS4B. No non-synonymous consensus changes were observed in DBN3acc(3a). This study supports a biased G-to-A and C-to-U mutagenic effect of RBV and RBV-TP throughout the entire ORF of HCV genotypes 1a and 3a.

A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus

BackgroundThe current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins. ObjectiveTo develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype. Study designIn total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. ResultsThe method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. ConclusionsThe presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.

Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385, from Rhodopseudomonas palustris PSB-S

Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30–46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l−1 and 0.918 ± 0.025 U·µg−1, respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.

Gene Cloning, Prokaryotic Expression, and Biochemical Characterization of a Soluble Trehalase in Helicoverpa armigera Hübner (Lepidoptera: Noctuidae).

Trehalase is an indispensable component of insect hemolymph that plays important role in energy metabolism and stress resistance. In this study, we cloned and expressed the gene encoding soluble trehalase (HaTreh-1) of Helicoverpa armigera (cotton bollworm) and characterized the enzyme. HaTreh-1 had a full-length open reading frame encoding a protein of 571 amino acids. Sequence comparison indicated that HaTreh-1 was similar to some known insect trehalases. Two essential active sites (D321 and E519) and three essential residues (R168, R221, and R286) were conserved in HaTreh-1. The recombinant trehalase was expressed in Escherichia coli and purified by nickel exchange chromatography. Molecular weight of the recombinant protein was about 71 kDa, and the optimum HaTreh-1 enzyme activity is at 55°C with pH 6.0. Enzymatic assays showed a Km value of 72.8 mmol/liter and a Vmax value of 0.608 mmol/(liter·min). Inhibition assays in vitro indicated that castanospermine, a polyhydroxylated alkaloid, was an effective competitive inhibitor of trehalase with a Ki value of 6.7 μmol/liter. The inhibitor action of castanospermine was linked to its modification effect on trehalase structure. The circular dichroism spectrum showed that the percentage of α-helix increased under the presence of castanospermine. Results of our study will aid in developing effective trehalase inhibitors for controlling H. armigera in the future.

Molecular cloning of a plasma membrane aquaporin in Stipa purpurea, and exploration of its role in drought stress tolerance

Stipa purpurea is widely distributed on the Tibetan Plateau, and has high drought resistance. Plasma membrane intrinsic proteins are a type of aquaporin. They regulate the movement of water and are associated with plant protective reactions to biotic and abiotic stresses. We characterized a plasma membrane intrinsic protein from S. purpurea (SpPIP1) and elucidated its role in molecular aspects of the plant's response to drought stress. The full-length open reading frame of SpPIP1 was 870 bp and encoded 289 amino acids. The transcript level of SpPIP1 was higher in the root of S. purpurea than in the flower, leaf and stem. The level of SpPIP1 transcript increased significantly when treated with drought treatment. Subcellular localization result showed that SpPIP1 was localized in the plasma membrane. Ectopic expression of SpPIP1 in Arabidopsis thaliana resulted in plants with higher tolerance to drought treatment. SpPIP1 of S. purpurea may mediate plant response to arid environments.

The root-knot nematode Meloidogyne incognita produces a functional mimic of the Arabidopsis INFLORESCENCE DEFICIENT IN ABSCISSION signaling peptide

INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is a signaling peptide that regulates cell separation in Arabidopsis including floral organ abscission and lateral root emergence. IDA is highly conserved in dicotyledonous flowering plant genomes. IDA-like sequences were also found in the genomic sequences of root-knot nematodes, Meloidogyne spp., which are globally deleterious pathogens of agriculturally important plants, but the role of these genes is unknown. Exogenous treatment of the Arabidopsis ida mutant with synthetic peptide identical to the M. incognita IDA-like 1 (MiIDL1) protein sequence minus its N-terminal signal peptide recovered both the abscission and root architecture defects. Constitutive expression of the full-length MiIDL1 open reading frame in the ida mutant substantially recovered the delayed floral organ abscission phenotype whereas transformants expressing a construct missing the MiIDL1 signal peptide retained the delayed abscission phenotype. Importantly, wild-type Arabidopsis plants harboring an MiIDL1-RNAi construct and infected with nematodes had approximately 40% fewer galls per root than control plants. Thus, the MiIDL1 gene produces a functional IDA mimic that appears to play a role in successful gall development on Arabidopsis roots.

Genetic and Pathologic Characterization of a Novel Recombinant TC07-2-Type Avian Infectious Bronchitis Virus.

Avian infectious bronchitis viruses (IBVs) with the TC07-2 genotype have spread rapidly in East Asia since they were first reported in China in 2007. In 2015, an IBV with the TC07-2 genotype (designated KrD1515) was isolated from layer chickens with severe respiratory symptoms in Korea. In the present study, the full-length open reading frames of the spike (S) and nucleocapsid (N) genes of the virus were sequenced and analyzed. S1 gene phylogenetic analysis revealed that the KrD1515 virus clustered with viruses with the TC07-2 genotype, whereas N gene phylogenetic analysis revealed that the KrD1515 virus clustered with Korean IBVs, but not with Chinese TC07-2 IBV. When 7-day-old specific-pathogen-free chickens were inoculated with the KrD1515 virus, they developed severe respiratory symptoms and tracheal lesions. However, there were no other clinical symptoms or pathologic lesions in other tissues. The virus was shed from the trachea for at least a week and from the cloaca for only a day. Our findings suggest that the KrD1515 virus is a recombinant virus between a Chinese TC07-2 IBV and a non-TC07-2 Korean IBV and engages in respiratory tropism in chickens.

Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis.

An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

A novel GATA-like zinc finger transcription factor involving in hematopoiesis of Eriocheir sinensis

GATA transcription factor is a family of DNA-binding proteins that can recognize and bind to sequence of (A/T) GATA (A/G). In the present study, a GATA-like protein (named as EsGLP) was characterized from Eriocheir sinensis, including an 834 bp full length open reading frame of EsGLP, encoding a polypeptide of 277 amino acids. The deduced amino acid sequence of EsGLP contained one conserved GATA-type zinc finger of the form Cys-X2-Cys-X17-Cys-X2-Cys, with four cysteine sites. The EsGLP mRNA transcripts were mainly detected in the hematopoietic tissue, hepatopancreas and gonad. The recombinant EsGLP protein was prepared for the antibody production. The EsGLP protein was mainly distributed in the edge of lobules in the HPT and the cytoplasm of hemocytes. The mRNA transcripts of EsGLP in hemocytes were significantly decreased at 24 h (0.39-fold and 0.27-fold, p < .05) and 48 h (0.35-fold and 0.16-fold, p < .05) after LPS and Aeromonas hydrophila stimulation, respectively. However, one peak of EsGLP mRNA transcripts were recorded at 24 h (8.71-fold, p < .05) in HPT after A. hydrophila stimulation. The expression level of EsGLP mRNA in HPT was significantly up-regulated at 2 h, 2.5 h and 9 h (41.74-fold, 45.38-fold and 26.07-fold, p < .05) after exsanguination stimulation. When EsGLP gene expression was inhibited by the injection of double-stranded RNA, both the total hemocytes counts and the rate of EdU-positive hemocytes were significantly decreased (0.32-fold and 0.56-fold compared to that in control group, p < .05). All these results suggested that EsGLP was an important regulatory factor in E. sinensis which involved in the hemocytes generation and the immune response against invading pathogens.

Bioinformatics and expression analysis of NAC transcription factor genes in Scutellaria baicalensis

Background: NAC, as a unique transcription factor to plants, plays important roles in multiple biological functions, such as regulation of plant growth and development, hormone levels, and responses to various kinds of stresses. However, there is a lack of research of NAC genes in Chinese herbs. Objective: The study aimed to evaluate the potential functions of NAC genes in Scutellaria baicalensis by bioinformatics and expression analysis, and provide evidence of the molecular regulation mechanism involved in flavonoid biosynthesis in S. baicalensis. Methods: The genes of NAC transcription factors in S. baicalensis were obtained from cDNA library and their functions were explored using bioinformatic methods. The NAC genes were screened from the cDNA library of S. baicalensis using BLAST comparison software. Then, the open reading frame (ORF) finder online tool was used to predict the full-length ORFs of NAC genes and their protein characteristics were explored by bioinformatic methods. The expression of NAC genes was then detected by quantitative polymerase chain reaction in different parts of S. baicalensis and different leaves treated by gibberellin GA3 treatment. Results: Six genes of NAC transcription factors were cloned, two of which had complete ORFs. NAC genes cloned in this study were mainly expressed in the flowers of S. baicalensis. The expression levels of NAC2, NAC3, NAC4, NAC5, NAC6 were increased firstly and then decreased gradually after 100 μM GA3 treatment. Meanwhile, some NACs and PAL2 in S. baicalensis showed strong correlation. Conclusion: This study suggested that NACs cloned in this study were mainly regulated the flavonoid biosynthesis in the flowers of S. baicalensis; NAC6 in S. baicalensis might be involved in the regulation of PAL2 transcription and affected the accumulation of flavonoids in the root of S. baicalensis. Our results provided a basis for further understanding the molecular regulation mechanism of flavonoid biosynthesis in S. baicalensis.