Multiple Sequence Alignment Analysis Research Articles

Identification and expression of adenosine deaminases acting on tRNA (ADAT) during early tail regeneration of the earthworm.

RNA editing is a widespread phenomenon in all metazoans. One of the common RNA editing event is the chemical conversion of adenosine to inosine (A-to-I) catalyzed by adenosine deaminases acting on tRNA (ADAT). During D. melanogaster development, the ADAT1 transcript was found to localize mainly to the central nervous system including brain and ventral nerve cord during brain development. Although an earthworm adenosine deaminases acting on mRNA (ADAR) has been identified and its possible implication in earthworm regeneration has been investigated, there is little accumulated information on ADAT and tRNA editing in the annelid including terrestrial earthworms. This study aimed to investigate the molecular characteristics and the expression pattern of earthworm ADAT during tail regeneration to understand its physiological significance. Nucleotide sequence of Ean-ADAT was retrieved from the genome assembly of Eisenia andrei via Basic Local Alignment Search Tool (BLAST). The genome assembly of Eisenia andrei was downloaded from National Genomics Data Center ( http://bigd.big.ac.cn/gwh/ ). The alignment and phylogenetic relationship of the core deaminase domains of ADATs and ADARs were analyzed. Its temporal expression during early tail regeneration was measured using real-time PCR. The open reading frame of Ean-ADAT consists of 1719 nucleotides encoding 573 amino acids. Domain analysis indicates that Ean-ADAT has a deaminase domain composed of 498 amino acids and a predicted nuclear localization signal at the N-terminal. Its subcellular localization was predicted to be nuclear. The core deaminase region of Ean-ADAT encompasses the three active-site motifs, including zinc-chelating residues and a glutamate residue for catalytic activity. In addition, Ean-ADAT shares highly conserved RNA recognition region flanking the third cysteine of the deaminase motif with other ADAT1s even from the yeast. Multiple sequence alignment and phylogenetic analysis indicate that Ean-ADAT shows greater similarity to vertebrate ADARs than to yeast Tad1p. Ean-ADAT mRNA expression began to remarkably decrease before 12h post-amputation, showing a tendency to gradual decrease until 7 dpa and then it slightly rebounded at 10 dpa. Our results demonstrate that Ean-ADAT belongs to a class of ADAT1s and support the hypothesis of a common evolutionary origin for ADARs and ADATs. The temporal expression of Ean-ADAT could suggest that its activity is unrelated to the molecular mechanisms of dedifferentiation.

Genome-wide identification, evolution, and transcriptome-based expression profiling analysis of suppressors of cytokine signaling (SOCS) in grass carp (Ctenopharyngodon idella)

Suppressors of cytokine signaling (SOCS) are a crucial negative regulation family that control a variety of signal transduction pathways in immunity, growth, and development. However, they have not been systematically studied in the important economic fish grass carp (Ctenopharyngodon idella). The accumulation of genomic and transcriptomic resources makes it feasible to conduct a systematic analysis of these genes. In the present study, comprehensive analyses, including genome-wide identification, phylogeny, conserved domain/motif, gene structure, chromosomal location, subcellular localization, gene duplication, and expression patterns of the SOCS gene family in grass carp, were performed, based on genomic and transcriptomic data. Here we identified and characterized 14 SOCS genes (CISH, SOCS1a, SOCS1b, SOCS2, SOCS3a, SOCS3b, SOCS4, SOCS5a, SOCS5b, SOCS6a, SOCS6b, SOCS7, SOCS8, and SOCS9) in grass carp. Phylogenetic analysis of deduced protein sequences suggested that all SOCS members were divided into two main types (Type I and Type II). Multiple sequence alignment and domain analyses showed that some CiSOCS members possess Pfam-SOCS, KIR, and ESS structures, in addition to the typical conserved SOCS-box and SH2 region. Gene structure analysis revealed the presence of 1–3 exons among the majority of SOCS genes. The motifs of CiSOCS proteins in the same subfamily were highly conserved. Most SOCS members are predicted to be located in the nucleus. Insights into gene duplication analysis revealed the important role of segmental duplications for the expansion of SOCS genes in grass carp. Purifying selection seemed to be operated during the evolution and expansion of paralogous SOCS genes. mRNA expression analyses indicated that many members were differentially expressed in multiple tissues after grass carp reovirus or Aeromonas hydrophila infection. Taken together, the present comprehensive and systematic studies of the SOCS family members lay a solid foundation for further functional researches of SOCS genes in grass carp.

Characterization of antigenic variant infectious bursal disease virus strains identified in South Korea

ABSTRACT Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases of young chickens, causing considerable economic losses to the poultry industry. More than 30 years ago, an antigenic variant (av) pathotype of the IBD virus (IBDV) was reported to originate in, and subsequently spread among, poultry farms in the USA. Recently, a novel avIBDV lineage was identified in China and was shown to exhibit clear differences in its pathogenicity as well as molecular characteristics compared with the previously isolated variant strains. In this study, we conducted a passive surveillance of chicken carcasses submitted to our research division from June–December 2019, and detected the IBDV strains by reverse transcription PCR. Five avIBDV strains were isolated, and their pathogenicity was determined by necropsy and molecular analysis. Additionally, a coinfection field case involving an avIBDV strain and a very virulent IBDV (vvIBDV) strain was identified. Multiple sequence alignment and phylogenetic analysis of partial viral protein 1 (VP1) and hypervariable region (hv) VP2 genes revealed that those strains originated from two different avIBDV lineages. The co-occurrence of two sub-groups of avIBDVs in South Korea confirms for the first time the evolution of antigenic variant IBDV strains, and highlights the urgency for the development of new strategies for IBDV intervention in South Korea. RESEARCH HIGHLIGHTS Five avIBDV strains were identified in South Korea by passive surveillance test in 2019. A coinfection between two IBDV strains from different genogroups was reported in a field case. By phylogenetic analysis, Korean avIBDVs belonged to two distinct lineages of antigenic variant genogroup.

Cloning and expression analysis of protein kinase C gene from Whitmania pigra

Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

Understanding how Mutations in Non-Muscle Myosin 2A Drive Human Disease

Non-muscle myosin 2A (NM2A) is a ubiquitously expressed myosin important for organizing actin filaments in areas of cell protrusion, cell adhesion, cell migration and cytokinesis and it is essential for early embryogenesis. Dephosphorylation of its regulatory light chain promotes formation of a compact inactive, ‘shutdown’ state in which the filament-forming tail is folded onto one of the two heads (known as the “10S” molecule). In this state, molecules cannot form filaments and the motors are inactivated. Over 80 mutations in the gene that encodes the heavy chain for NM2A (MYH9) have been described, most of which are single amino acid substitutions. Collectively these cause an autosomal-dominant disorder known as MYH9 disease, which result in a combination of symptoms from clotting disorders in which platelets are enlarged and bleeding time prolonged, aggregation of NM2A in neutrophils, cataracts, deafness and glomerulosclerosis. Here, using the cryo-electron microscopy structure we recently solved of shutdown smooth muscle myosin, we map known MYH9 mutations onto a homology model of NM2A. Multiple sequence alignment analysis reveals a high degree of conservation for residues involved in 10S-stabilising ionic interactions as well as many known mutation sites. Light meromyosin constructs (LMM is the region of the coiled-coil that assembles to form filaments) have been expressed, purified, and the effects of selected mutations have been assessed via negative stain electron microscopy. We suspect that these mutations may perturb the equilibrium between filamentous and 10S inactive molecules by actively destabilising the 10S conformation or by disrupting natural filament packing. With these results, we provide new structural insight on how mutations result in NM2A dysregulation and its implication in disease.

Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains

BackgroundLumpy skin disease (LSD) is a contagious viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD has recently spread in Asia following outbreaks in the Middle East and Europe. The disease emerged in Bangladesh in July 2019 in the Chattogram district, then rapidly spread throughout the entire country. We investigated six LSD outbreaks in Bangladesh to record the clinical signs and collect samples for diagnostic confirmation. Furthermore, we performed the molecular characterization of Bangladesh isolates, analyzing the full RPO30 and GPCR genes and the partial EEV glycoprotein gene.ResultsClinical observations revealed common LSD clinical signs in the affected cattle. PCR and real-time PCR, showed the presence of the LSDV genome in samples from all six districts. Phylogenetic analysis and detailed inspection of multiple sequence alignments revealed that Bangladesh isolates differ from common LSDV field isolates encountered in Africa, the Middle East, and Europe, as well as newly emerged LSDV variants in Russia and China. Instead, they were closely related to LSDV KSGP-0240, LSDV NI2490, and LSDV Kenya.ConclusionsThese results show the importance of continuous monitoring and characterization of circulating strains and the need to continually refine the strategies for differentiating vaccine strains from field viruses.

Sequencing and phylogenetic analysis of mitochondrial cox1 and nad1 genes in Toxocara canis and Toxascaris leonina isolates from Iran

Toxocara canis and Toxascaris leonina are the most important ascaridoid nematodes of the family Toxocaridae. The present study was aimed to characterisation and analysis of genetic variation within and among T. canis and T. leonina isolates obtained from Iran by sequencing partial mitochondrial cytochrome c oxidase subunit 1 (pcox1) and partial NADH dehydrogenase subunit 1 (pnad1) genes. A total number of 134 adult nematodes belonging to Toxocaridae family were collected from stray dogs in Alborz province, Iran during 2015 and 2016. Polymerase chain reaction (PCR) was performed and products were sequenced. Sequences of two mitochondrial cox1 and nad1 genes were compared with other sequences in the GenBank, while multiple sequences alignment analysis was performed using the Bioedit and MEGA6 software and phylogenetic tree was plotted. For all isolates, amplicons of about 450 and 350 base pairs (bp) were successfully produced by PCR for cox1 and nad1, respectively. All sequences of T. canis isolates from present study were 100% homologous across the nad1 gene but not in the cox1 gene. The results indicate that the PCR method based on sequence of cox1 and nad1 genes is a suitable technique for the differentiation of T. canis and T. leonina species and that mtDNA regions could be used as genetic markers for the identification and differentiation of Toxocara species.

Molecular cloning and characterization of a lipoxygenase gene (CsLOX1) from cucumber

Lipoxygenases (LOXs) are non-heme iron enzymes that play crucial roles in many developmental processes during plant life, and defense responses against biotic and abiotic stresses. In this study, a lipoxygenase gene (CsLOX1) was cloned and characterized from cucumber (Cucumis sativus). The coding sequence (CDS) of CsLOX1 was 741 bp, and encoded an 878 amino-acid residue protein, which was predicted to be located in the cytoplasm. CsLOX1 contained the conserved LH2/PLAT and lipoxygenase domains, as well as the representative 38 amino acids motif [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His]. Multiple sequence alignment and phylogenetic analysis indicated that CsLOX1 was closely related to other dicot 9-LOXs and posesess the essential conserved residues involved in the binding of the iron atom. Promoter analysis suggested that several development-, stress-, and hormone-related cis-acting regulatory elements were present in the promoter region of CsLOX1. The function of CsLOX1 was assessed by overexpression it in Arabidopsis, and the transgenic plants were male sterile and displayed obviously increased floral shoots. These results provide some clues for revealing the biological roles of CsLOX1 in cucumber.

Characterization, expression and function analysis of the TLR3 gene in golden pompano (Trachinotus ovatus)

Toll-like receptors (TLRs)are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many fishes, Toll-like receptors subfamily contain a large number of members with different functions that need to research in deep. In the present study, the full-length cDNA of TLR3 from the golden pompano, Trachinotus ovatus, was cloned and characterized. The full length of ToTLR3 cDNA was 3710 bp including an open reading frame of 2760 bp encoding a peptide of 919 amino acids. The derived amino acids sequence comprised of 14 leucine-rich repeats (LRR), capped with LRRCT followed by transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). Multiple sequence alignment and phylogenetic analysis revealed that ToTLR3 shared the highest similarity to the teleost fish and suggested ToTLR3 is fairly conservative in evolution process. Tissues distribution analysis indicated that ToTLR3 showed a tissue-specific variation with high expression in blood and liver. After the fish were stimulated by poly(I:C), flagellin and LPS, ToTLR3 expression in the liver, intestine, blood, kidney, skin and muscle was significantly upregulated in a time-depended manner, especially in immune related tissues such as liver, blood and kidney. Binding assay revealed the specificity of rToTLR3 for pathogen-associated molecular patterns (PAMPs) and bacteria that included Vibrio harveyi, V. vulnificus, V. anguillarum, Photobacterium damselae, Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus and PolyI:C, LPS, Flagellin, and PGN. In addition, a luciferase reporter assay showed that overexpression ToTLR3 significantly increased NF-κB activity. Collectively, our results suggested that ToTLR3 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, and transmiss the danger signal to downstream signaling pathways.

Alignment of Biological Sequences with Jalview.

In this chapter, we introduce core functionality of the Jalview interactive platform for the creation, analysis, and publication of multiple sequence alignments. A workflow is described based on Jalview's core functions: from data import to figure generation, including import of alignment reliability scores from T-Coffee and use of Jalview from the command line. The accompanying notes provide background information on the underlying methods and discuss additional options for working with Jalview to perform multiple sequence alignment, functional site analysis, and publication of alignments on the web.

JA-Responsive Transcription Factor SmMYB97 Promotes Phenolic Acid and Tanshinone Accumulation in Salvia miltiorrhiza.

Phenolic acids and tanshinones are active principles in Salvia miltiorrhiza Bunge administered for cardiovascular and cerebrovascular diseases. Jasmonic acid (JA) promotes secondary metabolite accumulation, but the regulatory mechanism is unknown in S. miltiorrhiza. We identified and characterized the JA-responsive gene SmMYB97. Multiple sequence alignment and phylogenetic tree analyses showed that SmMYB97 was clustered with AtMYB11, AtMYB12, and ZmP1 in the subgroup S7 regulating flavonol biosynthesis. SmMYB97 was highly expressed in S. miltiorrhiza leaves and induced by methyl jasmonate (MeJA). SmMYB97 was localized in the nucleus and had strong transcriptional activation activity. SmMYB97 overexpression increased phenolic acid and tanshinone biosynthesis and upregulated the genes implicated in these processes. Yeast one-hybrid and transient transcriptional activity assays disclosed that SmMYB97 binds the PAL1, TAT1, CPS1, and KSL1 promoter regions. SmJAZ8 interacts with SmMYB97 and downregulates the genes that it controls. This study partially clarified the regulatory network of MeJA-mediated secondary metabolite biosynthesis in S. miltiorrhiza.

Modifications of mgrB instigate colistin resistance in poultry gut-isolates in Bangladesh

Background: Acquisition of colistin resistance by a plasmid-mediated gene, mcr-1, was described for Enterobacteriaceae in many countries. We identified mcr-1-carrying colistin-resistant poultry gut-bacteria in Bangladesh and observed a group of colistin-resistant isolates without harboring mcr-1. We sought to investigate the genetic factor conferring colistin-resistance in those mcr-1-naive isolates. Several recent studies have reported inactivation of a chromosomal gene, mgrB, to be responsible for acquired colistin-resistance in absence of mcr-1. In this study, we investigated any potential deletion or mutation in mgrB gene to link the colistin-resistance phenotype. Methods and materials: Different selective culture media were used to isolate various bacteria from chicken dropping. Conventional biochemical procedures were followed by API 20E kit (BioMèrieux, Durham, NC) for identification of bacteria. A part of bacterial identification was validated further by genotyping using 16S rDNA analyses. Disc-diffusion and minimal inhibitory concentration (MIC) measurement were performed to determine the colistin susceptibility of the isolates. mgrB gene was amplified by polymerase chain reactions (PCR). Any alterations of nucleotides and/or amino acids in the mgrB gene clones were checked primarily by single sequence blasting with online database and multiple sequence alignment analysis by ClustalW program. Results: Among 99 isolates 87.5% showed phenotypic colistin-resistant by disc-diffusion assessment. All the resistant isolates showed MIC level, between >8 μg/mL to >256 μg/mL. Among these resistant isolates only 36.2% were harboring mcr-1 gene. The identified mcr-1 gene showed complete harmony to phenotypic colistin-resistance. To determine the mgrB alteration, 15 isolates were analyzed in three groups: group-1 was colistin-resistant carrying mcr-1 gene; group-2 was colistin-resistant without mcr-1 gene; and group-3 was colistin-susceptible isolates. Analyses of nucleotide and amino acid sequences revealed deletion mutation and shortening of MgrB protein in all the colistin-resistant isolates. In addition, a novel single amino acid substitution at 28 position of MgrB from phenylalanine (F) to cysteine (C) was observed associating colistin-resistance. Conclusion: Modulation of mgrB gene expression was the key factor for the acquired resistance to colistin. The deletion or non-synonymous substitution of the mgrB gene was shown to associate colistin resistance in gram negative bacteria. These findings attested the regulatory role of mgrB leading to phenotypic polymyxins-resistance.

Conserved patterns and interactions in the unfolding transition state across SH3 domain structural homologues.

Proteins with similar structures are generally assumed to arise from similar sequences. However, there are more cases than not where this is not true. The dogma is that sequence determines structure; how, then, can very different sequences fold to the same structure? Here, we employ high temperature unfolding simulations to probe the pathways and specific interactions that direct the folding and unfolding of the SH3 domain. The SH3 metafold in the Dynameomics Database consists of 753 proteins with the same structure, but varied sequences and functions. To investigate the relationship between sequence and structure, we selected 17 targets from the SH3 metafold with high sequence variability. Six unfolding simulations were performed for each target, transition states were identified, revealing two general folding/unfolding pathways at the transition state. Transition states were also expressed as mathematical graphs of connected chemical nodes, and it was found that three positions within the structure, independent of sequence, were consistently more connected within the graph than any other nearby positions in the sequence. These positions represent a hub connecting different portions of the structure. Multiple sequence alignment and covariation analyses also revealed certain positions that were more conserved due to packing constraints and stabilizing long-range contacts. This study demonstrates that members of the SH3 domain with different sequences can unfold through two main pathways, but certain characteristics are conserved regardless of the sequence or unfolding pathway. While sequence determines structure, we show that disparate sequences can provide similar interactions that influence folding and lead to similar structures.

Functional characterization and gene expression profile of perforin-2 in starry flounder (Platichthys stellatus)

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli, and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus).

Computational drug re-purposing targeting the spike glycoprotein of SARS-CoV-2 as an effective strategy to neutralize COVID-19

COVID-19 has intensified into a global pandemic with over a million deaths worldwide. Experimental research analyses have been implemented and executed with the sole rationale to counteract SARS-CoV-2, which has initiated potent therapeutic strategy development in coherence with computational biology validation focusing on the characterized viral drug targets signified by proteomic and genomic data. Spike glycoprotein is one of such potential drug target that promotes viral attachment to the host cellular membrane by binding to its receptor ACE-2 via its Receptor-Binding Domain (RBD). Multiple Sequence alignment and relative phylogenetic analysis revealed significant sequential disparities of SARS-CoV-2 as compared to previously encountered SARS-CoV and MERS-CoV strains. We implemented a drug re-purposing approach wherein the inhibitory efficacy of a cluster of thirty known drug candidates comprising of antivirals, antibiotics and phytochemicals (selection contingent on their present developmental status in underway clinical trials) was elucidated by subjecting them to molecular docking analyses against the spike protein RBD model (developed using homology modelling and validated using SAVES server 5.0) and the composite trimeric structures of spike glycoprotein of SARS-CoV-2. Our results indicated that Camostat, Favipiravir, Tenofovir, Raltegravir and Stavudine showed significant interactions with spike RBD of SARS-CoV-2. Proficient bioavailability coupled with no predicted in silico toxicity rendered them as prospective alternatives for designing and development of novel combinatorial therapy formulations for improving existing treatment regimes to combat COVID-19.

Molecular cloning and functional analysis of type I IFNd of Chinese sea bass (Lateolabrax maculatus) under immune stimulation

Chinese sea bass (Lateolabrax maculatus) is an economically important marine cultured species in China. Interferons (IFNs) play an essential role in innate antiviral immunity. The study on IFN immune system helps prevent and control viral diseases of L. maculatus. We have obtained cloning and characterization of the type I IFNd gene from L. maculatus (LmIFNd) in the present study. The full length of cDNA was 1190 bp, including 5’UTR (untranslated region) of 354 bp, 3’UTR of 278 bp, and an open reading frame (ORF) of 558 bp. It encodes 185 amino acids, and the first 20 amino acids are hypothetical signal peptides. The results of amino acid multiple sequence alignment and phylogenetic tree analysis showed that LmIFNd and mandarin fish (Siniperca chuatsi) IFNd were clustered into one branch, and the gene sequence similarity was as high as 88.9%. The expression of LmIFNd was tissue-specific and highly expressed in the head kidney, spleen, and gill. After infection with Rana grylio virus (RGV), and polyinosinic-polycytidylic acid [Poly(I:C)], the expression of LmIFNd in gill, spleen, and head-kidney was up-regulated significantly. Besides, the expression level of LmIFNd has increased significantly under the stimulation of Vibrio harveyi and Streptococcus iniae. The results show that LmIFNd may play a protective role in both viral and bacterial infections.

Genome-wide analysis of Toll-like receptors in zebrafish and the effect of rearing temperature on the receptors in response to stimulated pathogen infection.

Water temperature has a major influence on the host innate immune defence and the infectivity of pathogens in ectothermic teleosts. Toll-like receptors (TLRs) are the first and well-characterized innate immune receptors that are conserved in vertebrates. However, little is known about the effect of temperature variation on TLRs in fish species. In this study, we used adult zebrafish as a research model to investigate the effect of water temperature on TLRs. Whole genome searches identified 20 TLR homologue genes in zebrafish. Multiple sequence alignment and protein structure analysis revealed the conserved domains for these TLR proteins. To identify TLR genes related to temperature variation, TLR family genes from 12 species with different body temperatures were assigned to conduct phylogenetic analyses. Based on the phylogenetic relationships, TLR3, TLR4, TLR5 and TLR20~21 were selected as candidate genes. Immunostimulation data indicated that TLR3, TLR5, and TLR21 were more sensitive to temperature variation and their expression levels were affected in response to pathogen stimulation. Taken together, our results provide a new opportunity to understand the roles of temperature on host innate immune response in fishes and have broader implications for disease prevention in aquaculture.

A comparative analysis of GH18 chitinases and their isoforms from Beauveria bassiana: An in-silico approach

Fifteen different chitinases from various Beauveria bassiana strains were selected and their physicochemical characteristics, secondary structure evaluation and functional analyses were conducted using multiple bioinformatics tools. The molecular mass of chitinases varied from 34.25 to 49.27 kDa, and theoretical pI from 4.81 to 7.94 respectively. Most of the chitinases were hydrophilic, thermostable and negatively charged with good in vivo half-life. Nearly all the chitinases were extracellular with half of them having the standard secretory peptide, and chitinase family 18 active site motif. Phylogeny, multiple sequence alignment and indel analysis confirmed the presence of highly conserved active site residues in seven sequences. Furthermore, a three-dimensional model of chitinase (AIT18869.1) was constructed using the SWISS-MODEL server and validated by ERRAT, Verify 3D and RAMPAGE. The presence of 98.1 % of its residues in the Ramachandran plot’s favoured region further established the quality of the model. CASTp and MetaPocket 2.0 analysis followed by protein-ligand docking using allosamidin and chitotriose thiazoline, suggested Asp-208, Gln-242, Gln-265 and Asn-268 as the most conserved active residues for the enzyme. The information gathered through the in-silico approach would be beneficial in unravelling the properties of B. bassiana chitinases in vitro, which could be subsequently exploited for various industrial applications.

Molecular characterization of Rab5 and Rab6, and their involvement in innate immunity in red swamp crayfish Procambarus clarkii

Rab5 and Rab6 are members of the Rab GTPases protein family, and they play important roles in regulating trafficking organelles, especially in phagosome formation. In this study, full-length cDNA sequences of Rab5 and Rab6 were cloned from crayfish Procambarus clarkii, which were designated as PcRab5 and PcRab6, respectively. The full-length cDNA of PcRab5 is 1367 bp and contains an open reading frame (ORF) of 639 bp encoding 212 amino acids with an estimated molecular weight of 23.0 kDa. The full-length cDNA of PcRab6 is 1097 bp, and ORF is 636 bp encoding 211 amino acids with 23.7 kDa. The deduced amino acid sequence of PcRab5 displayed high identity to that of Penaeus vannamei (97.17%), and PcRab6 shared high similarity to that of Macrobrachium rosenbergii (99.05%). According to the results of multiple sequence alignment and phylogenetic analysis, the guanine-base binding motif (G), phosphate/magnesium-binding motif (PM) and Rab family motifs (RabF) of PcRab5 and PcRab6 are high conserved among different species, however, the N- and C-terminal regions were hypervariable. Besides, the mRNA transcripts of PcRab5 and PcRab6 were widely detected in various tissues of P. clarkii, with the highest expression levels in hemocytes and gill. The gene expression levels of PcRab5 and PcRab6 were significantly induced in hepatopancreas, gill, and intestines of P. clarkii after WSSV and Aeromonas hydrophila infection, respectively. RNA interference experiment was designed to further investigate the roles of PcRab5 and PcRab6 during pathogen infection. The results showed that the mortality rate of crayfish was influenced by silenced PcRab5 and PcRab6 after challenged by WSSV or A. hydrophila. These results suggested that PcRab5 and PcRab6 were involved in bacterial and viral infection in the immune response of P. clarkii

Genome-wide identification of NF-YA gene family in cotton and the positive role of GhNF-YA10 and GhNF-YA23 in salt tolerance

Nuclear factor YA (NF-YA) genes play important roles in many biological processes, such as leaf growth, nitrogen nutrition, drought resistance, and salt stress. The functions of NF-YA genes in cotton have not been elucidated. The current study identified a total of 16, 16, 31, and 29 genes from Gossypium raimondii, G. arboretum, G. barbadense, and G. hirsutum, respectively. The NF-YA genes in cotton were phylogenetically classified into 4 groups. Analysis of gene structure, conserved motifs and multiple sequence alignments supported the evolutionary conservation of NF-YA family genes in cotton. Analysis of the expression patterns of GhNF-YAs in cotton suggested that GhNF-YAs play important roles in plant growth, development, and stress responses. The quantitative real-time PCR (qRT-PCR) validation of selected genes suggested that GhNF-YA genes are induced in response to salt, drought, ABA, and MeJA treatments. GhNF-YA genes may regulate salt and drought stress via the ABA or MeJA pathway. Silencing of GhNF-YA10 and GhNF-YA23 significantly reduced the salt tolerance of cotton seedlings, indicating that these genes participate in the regulation of the response of cotton to salt stress. These results establish a foundation for subsequent functional studies of the NF-YA gene family in cotton.