Genome-Wide Characterization and Expression Analysis of KH Family Genes Response to ABA and SA in Arabidopsis thaliana

K-homologous (KH) family is a type of nucleic acid-binding protein containing the KH domain and has been found to affect splicing and transcriptional regulation. However, KH family genes haven’t been investigated in plant species systematically. In this study, we identified 30 genes that belonged to the KH family based on HMM of the KH domain in Arabidopsis thaliana. Phylogenetic tree analysis showed that the KH family is grouped into three subgroups. Synteny analysis showed that AtKH9 and AtKH29 have the conserved synteny relationship between A. thaliana and the other five species. The AtKH9 and AtKH29 were located in the cytoplasm and nucleus. The seed germination rates of the mutants atkh9 and atkh29 were higher than wild-type after abscisic acid (ABA) and salicylic acid (SA) treatments. In addition, the expression of ABA-related genes, such as ABRE-binding factor 2 (ABF2), ABRE-binding factor 4 (ABF4), and delta 1-pyrroline-5-carboxylate synthase (P5CS), and an SA-related gene pathogenesis-related proteins b (PR1b) were downregulated after ABA and SA treatments, respectively. These results suggested that atkh9 and atkh29 mutants inhibit the effect of ABA and SA on seed germination. In conclusion, our results provide valuable information for further exploration of the function of KH family genes and propose directions and ideas for the identification and characterization of KH family genes in other plants.

Cloning, Functional Characterization and Response to Cadmium Stress of the Thioredoxin-like Protein 1 Gene from Phascolosoma esculenta

Cadmium (Cd) is a heavy metal toxicant and is widely distributed in aquatic environments. It can cause excessive production of reactive oxygen species (ROS) in the organism, which in turn leads to a series of oxidative damages. Thioredoxin (Trx), a highly conserved disulfide reductase, plays an important role in maintaining the intracellular redox homeostasis in eukaryotes and prokaryotes. Phascolosoma esculenta is an edible marine worm, an invertebrate that is extensively found on the mudflats of coastal China. To explore the molecular response of Trx in mudflat organisms under Cd stress, we identified a new Trx isoform (Trx-like protein 1 gene) from P. esculenta for the first time, designated as PeTrxl. Molecular and structural characterization, as well as multiple sequence and phylogenetic tree analysis, demonstrated that PeTrxl belongs to the Trx superfamily. PeTrxl transcripts were found to be ubiquitous in all tissues, and the highest expression level occurred in the coelomic fluid. Exposure to three sublethal concentrations of Cd resulted in the upregulation and then downregulation of PeTrxl expression levels over time in coelomic fluid of P. esculenta. The significant elevation of PeTrxl expression after 12 and 24 h of Cd exposure at 6 and 96 mg/L, respectively, might reflect its important role in the resistance to Cd stress. Recombinant PeTrxl (rPeTrxl) showed prominent dose-dependent insulin-reducing and ABTS free radical-scavenging abilities. After exposure to 96 mg/L Cd for 24 h, the ROS level increased significantly in the coelomic fluid, suggesting that Cd induced oxidative stress in P. esculenta. Furthermore, the injection of rPeTrxl during Cd exposure significantly reduced the ROS in the coelomic fluid. Our data suggest that PeTrxl has significant antioxidant capacity and can protect P. esculenta from Cd-induced oxidative stress.

Isolation and Molecular Characteristics of a Novel Recombinant Avian Orthoreovirus From Chickens in China

In recent years, the emergence of avian orthoreovirus (ARV) has caused significant losses to the poultry industry worldwide. In this study, a novel ARV isolate, designated as AHZJ19, was isolated and identified from domestic chicken with viral arthritis syndrome in China. AHZJ19 can cause typical syncytial cytopathic effect in the chicken hepatocellular carcinoma cell line, LMH. High-throughput sequencing using Illumina technology revealed that the genome size of AHZJ19 is about 23,230 bp, which codes 12 major proteins. Phylogenetic tree analysis found that AHZJ19 was possibly originated from a recombination among Hungarian strains, North American strains, and Chinese strains based on the sequences of the 12 proteins. Notably, the σC protein of AHZJ19 shared only about 50% homology with that of the vaccine strains S1133 and 1733, which also significantly differed from other reported Chinese ARV strains. The isolation and molecular characteristics of AHZJ19 provided novel insights into the molecular epidemiology of ARV and laid the foundation for developing efficient strategies for control of ARV in China.

Molecular Detection of blaOXA-type Carbapenemase Genes and Antimicrobial Resistance Patterns among Clinical Isolates of Acinetobacter baumannii

Acinetobacter baumannii is a bacterium found in most places, especially in clinics and hospitals, and an important agent of nosocomial infections. The presence of class D enzymes such as OXA-type carbapenemases in A. baumannii is proven to have a key function in resistance to carbapenem. The aim of the current study is to determine the blaOXA-type carbapenemase genes and antimicrobial resistance among clinically isolated samples of A. baumannii. We assessed 100 clinically isolated specimens of A. baumannii from patients in intensive care units of educational hospitals of Hamadan, West of Iran. The A. baumannii isolates' susceptibility to antibiotics was performed employing disk diffusion method. Multiplex polymerase chain reaction was used to identify the blaOXA-24-like , blaOXA-23-like , blaOXA-58-like , and blaOXA-51-like genes. The blaOXA-23-like , blaOXA-24-like , and blaOXA-58-like genes' prevalence were found to be 84, 58, and 3%, respectively. The highest coexistence of the genes was for blaOXA-51/23 (84%) followed by blaOXA-51/24-like (58%). The blaOXA-51/23- like pattern of genes is a sort of dominant gene in resistance in A. baumannii from Hamadan hospitals. The highest resistance to piperacillin (83%) and ciprofloxacin (81%) has been observed in positive isolates of blaOXA-23-like . The A. baumannii isolates with blaOXA-58-like genes did not show much resistance to antibiotics. Based on the results of the phylogenetic tree analysis, all isolates have shown a high degree of similarity. This study showed the high frequency of OXA-type carbapenemase genes among A. baumannii isolates from Hamadan hospitals, Iran. Thus, applying an appropriate strategy to limit the spreading of these strains and also performing new treatment regimens are necessary.

Genome-wide characterization of bZIP gene family identifies potential members involved in flavonoids biosynthesis in Ginkgo biloba L.

Ginkgo biloba L. is an ancient relict plant with rich pharmacological activity and nutritional value, and its main physiologically active components are flavonoids and terpene lactones. The bZIP gene family is one of the largest gene families in plants and regulates many processes including pathogen defense, secondary metabolism, stress response, seed maturation, and flower development. In this study, genome-wide distribution of the bZIP transcription factors was screened from G. biloba database in silico analysis. A total of 40 bZIP genes were identified in G. biloba and were divided into 10 subclasses. GbbZIP members in the same group share a similar gene structure, number of introns and exons, and motif distribution. Analysis of tissue expression pattern based on transcriptome indicated that GbbZIP08 and GbbZIP15 were most highly expressed in mature leaf. And the expression level of GbbZIP13 was high in all eight tissues. Correlation analysis and phylogenetic tree analysis suggested that GbbZIP08 and GbbZIP15 might be involved in the flavonoid biosynthesis. The transcriptional levels of 20 GbbZIP genes after SA, MeJA, and low temperature treatment were analyzed by qRT-PCR. The expression level of GbbZIP08 was significantly upregulated under 4°C. Protein–protein interaction network analysis indicated that GbbZIP09 might participate in seed germination by interacting with GbbZIP32. Based on transcriptome and degradome data, we found that 32 out of 117 miRNAs were annotated to 17 miRNA families. The results of this study may provide a theoretical foundation for the functional validation of GbbZIP genes in the future.

Comparative Genome Analysis of Streptococcus suis Serotype 9 Isolates from China, The Netherland, and the U.K.

Streptococcus suis (S. suis) is an important swine pathogen and an emerging zoonotic agent worldwide. Serotype 9 is the most prevalent serotype in several European countries but it is relatively rare in China. In this study, through the investigation of the serotypes of 279 S. suis strains isolated from China from 2015 to 2017, it was found that serotype 9 is the second most prevalent serotype (43 out of 279), behind serotype 2 (83 out of 279). Next, the 43 serotype 9 isolates were sequenced and compared with those from the Netherland (28) and the U.K. (eight). For the purpose of comparison, the strain D12 (GCA_000231905), which has completed genome sequences, was also incorporated. Phylogenetic tree analysis showed that the strains from China and the U.K. were heterogeneous. In contrast, all but one from the Netherland belonged to the same clade. The dominant clades of Chinese strains (33) and strains from the Netherland (27) were very similar. Both of them may have originated from the same strain about 70 years ago. Then, the distributions of virulence-associated genes and antibiotic resistance genes among different clades and sources were analyzed. By comparison, strains from the Netherland carried more virulence-associated genes and those from the U.K. had more antibiotic resistance genes. Additionally, some virulence-associated genes (salK and salR) and antibiotic resistance genes (lincomycin and spectinomycin) existed only in several Chinese strains. In conclusion, our data displayed the population characteristics and differences of S. suis serotype 9 between China and Europe, suggesting that they have taken different evolutionary paths.

Analysis and Sequence Alignment of Peste des Petits Ruminants Virus ChinaSX2020

The peste des petits ruminants virus (PPRV) mainly infects goats and sheep and causes a highly contagious disease, PPR. Recently, a PPRV strain named ChinaSX2020 was isolated and confirmed following an indirect immunofluorescence assay and PCR using PPRV-specific antibody and primers, respectively. A sequencing of the ChinaSX2020 strain showed a genome length of 15,954 nucleotides. A phylogenetic tree analysis showed that the ChinaSX2020 genome was classified into lineage IV of the PRRV genotypes. The genome of the ChinaSX2020 strain was found to be closely related to PPRVs isolated in China between 2013 and 2014. These findings revealed that not a variety of PRRVs but similar PPRVs were continuously spreading and causing sporadic outbreaks in China.

Molecular Cloning and Identification of NADPH Cytochrome P450 Reductase from Panax ginseng

Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology.

Genome-wide investigation of the GRAS transcription factor family in foxtail millet (Setaria italica L.)

Background GRAS transcription factors perform indispensable functions in various biological processes, such as plant growth, fruit development, and biotic and abiotic stress responses. The development of whole-genome sequencing has allowed the GRAS gene family to be identified and characterized in many species. However, thorough in-depth identification or systematic analysis of GRAS family genes in foxtail millet has not been conducted. Results In this study, 57 GRAS genes of foxtail millet (SiGRASs) were identified and renamed according to the chromosomal distribution of the SiGRAS genes. Based on the number of conserved domains and gene structure, the SiGRAS genes were divided into 13 subfamilies via phylogenetic tree analysis. The GRAS genes were unevenly distributed on nine chromosomes, and members of the same subfamily had similar gene structures and motif compositions. Genetic structure analysis showed that most SiGRAS genes lacked introns. Some SiGRAS genes were derived from gene duplication events, and segmental duplications may have contributed more to GRAS gene family expansion than tandem duplications. Quantitative polymerase chain reaction showed significant differences in the expression of SiGRAS genes in different tissues and stages of fruits development, which indicated the complexity of the physiological functions of SiGRAS. In addition, exogenous paclobutrazol treatment significantly altered the transcription levels of DELLA subfamily members, downregulated the gibberellin content, and decreased the plant height of foxtail millet, while it increased the fruit weight. In addition, SiGRAS13 and SiGRAS25 may have the potential for genetic improvement and functional gene research in foxtail millet. Conclusions Collectively, this study will be helpful for further analysing the biological function of SiGRAS. Our results may contribute to improving the genetic breeding of foxtail millet.

Genetic Characterization and Pathogenesis of Three Novel Reassortant H5N2 Viruses in South Korea, 2018

The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia–Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China’s H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.