Full-length cDNA Research Articles

A c-type lectin HcLec1 with dual function of immunology and mineralization from the freshwater oyster (Hyriopsis cumingii Lea)

BackgroundShell and pearl formation in bivalves is a sophisticated biomineralization process that encompasses immunological and mineralization aspects, particularly during shell repair and the initial stages of pearl cultivation when a nucleus is inserted. Here, we describe a novel C-type lectin, HcLec1, isolated and characterized from the freshwater pearl mussel Hyriopsis cumingii Lea. MethodsImmune challenge, RNA interference (RNAi) experiments, ELISA, and antibacterial assays were employed to investigate the role of HcLec1 in innate immunity. We also established shell damage repair and pearl nucleus insertion models to examine the impact of HcLec1 on the biomineralization process in Hyriopsis cumingii Lea. In vitro calcium carbonate crystallization assays were conducted to explore the direct role of HcLec1 in calcium carbonate crystal formation.ResultsThe HcLec1 gene sequence is a full-length cDNA of 1552 bp, encoding 240 amino acids. HcLec1 comprises an N-terminal signal peptide and a carbohydrate-recognition domain (CRD), with QPD (Gln-Pro-Asp) and MND (Met-Asn-Asp) motifs for polysaccharide binding. Tissue expression analysis showed that HcLec1 is predominantly expressed in the gill tissue of Hyriopsis cumingii Lea under normal conditions, and its expression is significantly elevated in both gill and pearl sac tissues following nucleus insertion for pearl cultivation (P < 0.05). After immune stimulation with Aeromonas hydrophila and lipopolysaccharides (LPS), HcLec1 expression levels significantly increased in both cases (P < 0.01), indicating a role in bivalve innate immunity. RNA interference (RNAi)-mediated knockdown of HcLec1 led to a significant decrease in the expression levels of immune-related genes (WAP, α2m, and Lyso) and mineralization-related genes (CA, CHS, Nacrein, and Pif) (P < 0.05). In animal models for shell damage and nucleus insertion in pearl cultivation, HcLec1 showed a consistent expression pattern, with an initial significant decrease followed by a marked increase, peaking at day 14 (P < 0.05). This suggests a role for HcLec1 in pearl formation and shell repair. The recombinant HcLec1 protein demonstrated binding affinity to LPS and PGN, a robust ability to agglutinate Escherichia coli, Staphylococcus aureus, Aeromonas veronii, and Aeromonas hydrophila, and significantly inhibited bacterial growth (P < 0.05). Moreover, rHcLec1 promoted calcite crystal formation in saturated calcium carbonate solutions and altered crystal morphology.DiscussionThe HcLec1 gene plays a pivotal role in both innate immunity and biomineralization in the triangle sail mussel. This study enhances our understanding of the functional diversity of C-type lectins and provides a foundation for future studies on shell repair and pearl growth.

Intraspecific variation and functional study of VERL polymorphism in Pacific abalone (Haliotis discus hannai Ino) and giant abalone (H. gigantea Gmelin).

Sperm and eggs have interacting proteins on their surfaces that affect their compatibility during fertilization. These gamete recognition proteins (GRPs) are typically polymorphic within species, resulting in variations in gamete affinity. This study presents the full-length sequence of VERL, an egg vitelline envelope receptor for sperm lysin, in Pacific abalone (Haliotis discus hannai Ino) and giant abalone (Haliotis gigantea Gmelin). The full-length VERL cDNA sequence in Pacific abalone (11,373 bp) encodes 3790 amino acids, most of which are organized into 21 tandem repeats. In giant abalone, the VERL spans 9405 bp and encodes 3134 amino acids, including 17 tandem repeats. By resolving the nucleotide polymorphisms from resequencing data into haplotypes, high allelic diversity was found in VERL domain repeats 1-2 in both species. Further analysis of intraspecific variation in this region revealed that the alleles of VERL repeats 1-2 in Pacific abalone clustered into two clades, yet no significant divergence was observed among the three Pacific abalone geographic populations. In contrast, alleles in giant abalone were subdivided into two distinct branches, separated by a maximum of 12 amino acid substitutions. Notably, this study provides the first evidence of assortative mating between male and female gametes based on VERL genotypes in giant abalone. Overall, this study revealed the amino acid polymorphism of VERL in two abalone species and its functional effects on fertilization potential. These findings provide a foundation for future research on gamete recognition mechanisms and support selective breeding strategies to enhance abalone reproductive efficiency.

Viperin and Its Effect on SVCV Replication in Common Carp, Cyprinus carpio.

Viperin is an interferon-stimulated gene (ISG) that plays an important role in the congenital antiviral immunity of vertebrates. In this study, the common carp viperin (cc-viperin) gene is characterized, and we determine whether it has the ability to inhibit spring viremia of carp virus (SVCV) replication in EPC cells. The results showed that the full-length cDNA of the cc-viperin gene was 1044 bp and it encoded 348 amino acids. The cc-viperin sequence contained a leucine zipper in the N-terminal, a CxxxCxxC motif in the SAM domain, and a conservative C-terminus. The cc-viperin gene's nucleotide and amino acid sequence alignment revealed that cc-viperin displayed relatively high sequence identity compared with other species. Phylogenetic analysis displayed the close relation of cc-viperin with Carassius auratus and Mylopharyngodon piceus. Subcellular localization analysis indicated that the cc-viperin protein was located in the cytoplasm. The gene expression results showed that cc-viperin was expressed in all of the tissues tested. Its expression level significantly increased in EPC cells after 24 h to 72 h compared to the control during SVCV infection. Moreover, cc-viperin significantly inhibited SVCV replication when it was overexpressed, whereas it increased SVCV replication when it had reduced expression in EPC cells, respectively. To summarize, the results obtained in this work show that cc-viperin shares similar sequence characteristics with other vertebrates, and it could inhibit SVCV replication in EPC cells, displaying an antiviral effect in common carp.

Molecular and biological characterization of infectious full-length cDNA clones of two viruses in Paris yunnanensis, including a novel potyvirus

Paris yunnanensis, also named as Rhizoma Paridis in the Chinese Pharmacopeia, is a perennial Chinese medicinal herb commonly grown in Southwest China. However, several viruses have been found infecting this plant in recent years. Using high-throughput sequencing (HTS) and Sanger sequencing, this study obtained the complete genome sequences of three capillovirus isolates and one potyvirus isolate. Genomic and phylogenetic analyses revealed that these three capillovirus isolates are the same virus as the newly reported capillovirus, Paris polyphylla chlorotic mottle virus. The newly found potyvirus isolate shares 52.4–68.9% nucleotide sequence identity with other known potyviruses and thus, is grouped into the bean common mosaic virus subgroup. Based on the nucleotide sequence identity, we consider this virus a novel potyvirus species and propose ‘Paris potyvirus 5’ (ParPV-5) as its common name, and ‘Potyvirus shilinense’ as its species name. To characterize their biological features, two infectious clones, representing the two viruses, have been constructed through homologous recombination or yeast homologous recombination, and inoculated to several species plants, respectively. The results showed both of the viruses can infect P. yunnanensis and Nicotiana benthamiana. In addition, Paris polyphylla chlorotic mottle virus (PpCMV) can infect N. tabacum var. Xanthi nc, Cucurbita moschata, and Capsicum annuum, and ParPV-5 can infect Cucumis sativus L. and Bidens pilosa L. However, except mild leaf deformation exhibited on the PpCMV-inoculated C. moschata plants, no obvious symptom were observed in these plants including P. yunnanensis. A total of 179 field P. yunnanensis leaf samples from four counties in 2020–2021, and all 640 P. yunnanensis plants from a whole study plot of Lijiang in 2024, were tested using RT-PCR and specific primers, the results showed that PpCMV is a potential preponderant species in some regions, and ParPV-5 has the possible transmission from the original site to other regions.

Recovery of Recombinant Marburg Virus by Reverse Genetics.

Reverse genetics systems can be used to study parts or all of the viral life cycle. Recovery systems are reverse genetics systems used for the generation of infectious recombinant viruses that can carry targeted mutations or express reporter genes. The former can help address basic research questions to better understand certain steps of viral replication, while recombinant viruses expressing reporter genes, such as luciferases or fluorescent proteins, can be used for drug screening assays. Accordingly, recombinant viruses can be of great use for a variety of research purposes, including antiviral compound screenings and development of other countermeasures if no virus isolates are available. In this protocol, we describe a T7-driven reverse genetics system to generate recombinant Marburg virus from full-length cDNA.

FMRFamide G protein-coupled receptors (GPCR) in the cuttlefish Sepiella japonica: Identification, characterization and expression profile

FMRFamide is a ubiquitous neuromodulator in the animal kingdom. Once FMRFamide or similar neuropeptides bind to their G protein-coupled receptors (GPCR), a series of signal transduction events are triggered, thereby mediating various physiological effects. FMRFamide had been reported to be involved in the regulation of sexual maturation in Sepiella japonica. In this research, the full-length cDNA of FMRFamide G protein-coupled receptor of S. japonica (SjFaGPCR) was cloned. The sequence is 1396 bp long and encodes a protein consisting of 418 amino acid residues, lacking a signal peptide at the N-terminal region. The 3D structure of SjFaGPCR was predicted using Todarodes pacificus rhodopsin as a template, and the result indicated the presence of seven transmembrane regions. Multiple sequence alignments and phylogenetic trees indicated that SjFaGPCR is conserved among invertebrates, and shares highly similar sequence characteristics with other cephalopods. In situ hybridization (ISH) results revealed that significant signals of SjFaGPCR were detected in the central medulla and the granular layer cells of the optic lobe, and were also observed in the supraesophageal and subesophageal masses of the brain. Meanwhile, quantitative real-time PCR (qRT-PCR) results showed that a higher expression level of SjFaGPCR mRNA was detected in the brain and optic lobe of female cuttlefish at stage III and stage VI, and also in the brain (stage V) and optic lobe (stages IV and V) of male cuttlefish than that in other tissues. The co-localization results demonstrated that fluorescence signals of SjFMRFamide and SjFaGPCR were overlapped in HEK293 cells, suggesting a possible interaction between the SjFMRFamide and SjFaGPCR. These findings provide molecular support for further exploring the roles of FMRFamide and FaGPCR in the reproductive regulation of S. japonica.

Decoding Vitellogenin Subtype Responses: A Molecular Approach to Biomarkers of Endocrine Disruption in Scatophagus argus

Vitellogenins (Vtgs) are key yolk precursor proteins in fish, serving as critical indicators of gonadal maturation in females and reliable biomarkers for detecting xeno-oestrogenic pollution, particularly through their expression in juveniles or males. The vtg gene family comprises multiple subtypes that are species-specific, necessitating precise characterisation and quantification for effective use as biomarkers in studies on estrogenic endocrine-disrupting chemicals (EEDCs). In this study, we successfully cloned and characterised the full-length cDNAs of three vtg subtypes (vtgAa, vtgAb, and vtgC) from Scatophagus argus. Differential expression analysis revealed that vtgAb exhibited the highest responsiveness to 17α-ethynylestradiol (EE2) exposure, with a 3-fold increase in vivo at 10.0 μg/g EE2 and a 30-fold increase in vitro at 10−7 mol/L EE2. The expression patterns were dose- and time-dependent, with peak expression observed 72 h post-exposure. While in vivo assays indicated moderate upregulation, in vitro experiments demonstrated significantly higher expression, attributed to direct hepatocyte interaction with EE2. These findings confirm vtgAb as the most responsive subtype to oestrogen exposure in S. argus and highlight the species’ tolerance to EE2, as compared to more sensitive species like Danio rerio. This study shows the evolutionary conservation of vtg transcripts across teleost species and reinforces the importance of subtype-specific characterisation to advance their application as biomarkers for EEDCs, with significant implications for environmental monitoring and pollution regulation.

Cloning and overexpression of Zea mays cystatin 2 (ccii) gene in Bacillus subtilis to reduce root-knot nematode infection in cucumber plants in Iraq

Root-knot nematodes significantly reduce cucumber yields, prompting the development of a genetic engineering strategy as an alternative to chemical nematicides. In this study, the maize CCII gene was cloned into the pHT1469 shuttle vector and transformed into the Bacillus subtilis expression system to produce a cystatin that interferes with nematode digestive proteins. The full-length CCII cDNA sequence was optimized for overexpression in B. subtilis, driven by the Pgrac promoter, resulting in a protein of 134 amino acids with a deduced molecular mass of 15.01 kDa. After optimization, the relative adaptiveness in the heterologous B. subtilis system improved by 6.72% compared to non-optimized codons. Purification results using immobilized metal affinity chromatography (IMAC) indicated that the third fraction exhibited the highest inhibition activity, and the purified cystatin was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be approximately 15 kDa. Treatments with nematode juveniles showed the highest mortality percentages at 30% and 40% concentrations of purified cystatin isolated from both competent and wild B. subtilis cells, yielding mortality rates of 65%, 73.7%, 75.3%, and 89% respectively after 72 h. Furthermore, the recombinant cystatin isolated from the first B. subtilis (wild) strain had a greater impact on juvenile mortality than the second strain. This study aimed to clone and express corn cystatin in heterologous B. subtilis and evaluate its effectiveness in reducing nematode populations, exploring its potential as a biocontrol agent.

Systematic identification of sugarcane vacuolar H+-translocating pyrophosphatase (VPP) gene family and the role of ScVPP1 in salt resistance.

A total of 24 genes of vacuolar H+-translocating pyrophosphatases H+-PPases (VPP) genes were identified in Saccharum spontaneum AP85-441 and the ScVPP1-overexpressed Arabidopsis plants conferred salt tolerance. The vital role of vacuolar H+-translocating pyrophosphatases H+-PPases (VPP) genes involved in plants in response to abiotic stresses. However, the understanding of VPP functions in sugarcane remained unclear. In this study, a total of 24 VPP genes (SsaVPP1-SsaVPP24) were identified in the Saccharum spontaneum genome of haploid clone AP85-441. These genes were distributed in two phylogenetic groups. The SsaVPPs displayed diverse physio-chemical and gene structure attributes. The SsaVPP family genes have expanded through segmental duplication (20 gene pairs) rather than tandem duplication. A full-length cDNA of ScVPP1 was cloned from the sugarcane cultivar ROC22 and shared 99.48% sequence identity (amino acid) with homologous gene SsaVPP21 from AP85-441. In ROC22, the ScVPP1 gene was considerably upregulated by NaCl and ABA treatments among leaf, root, and stem tissues, while this gene was exclusively upregulated in the root with PEG treatment. Under NaCl and ABA stresses, yeast cells transfected by the ScVPP1 plasmid showed distinct growth rates compared to control yeast cells transfected by the empty vector. In transgenic Arabidopsis lines overexpressing ScVPP1, the seed gemination and survival rate were enhanced under NaCl treatment but not underABA stress as compared to wild-type plants. These results suggested that the ScVPP1 gene conferred tolerance to slat and may be used as a salt resistance gene source for sugarcane breeding.

Isolation and Characterization of Some Genes Involved in Essential Oil Synthesis in Endemic Sütçüler Thyme (Origanum minutiflorum)

Origanum minutiflorum, is an endemic species found in the Sütçüler district of Isparta province, Turkey. This study aimed to isolate and characterize the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (OmDXR) and terpene synthase 1 (OmTPS-1) genes, which play roles in essential oil biosynthesis in O. minutiflorum. For this purpose, total RNA was isolated from thyme leaves, and the sequence information of the target genes was obtained using gene-specific primers. The sequences were compared with those from other species in the NCBI database using BLAST programs. The nucleotide sequences were translated into protein sequences using ExPASy Tools, the three-dimensional structures of the encoded proteins were constructed with 3D SWISS-MODEL, and phylogenetic relationships of nucleotide and protein sequences were analyzed using the MEGA X version 11 software package. The results showed that the full-length cDNA nucleotide sequence of the OmDXR gene is 1,700 base pairs (bp), and its nucleotide sequence displayed 74.22%-81.07% similarity to nucleotide sequences from other species in previous studies, while the protein sequence showed 70.92%-72.95% similarity. The full-length cDNA nucleotide sequence of the OmTPS-1 gene was determined to be 1.836 bp, and its nucleotide sequence exhibited 75.99%-89.58% similarity to nucleotide sequences from other species, while the protein sequence showed 51.66%-69.88% similarity. This study represents the first molecular-based research on the O. minutiflorum species. Therefore, it will provide a valuable resource for future studies.

Development of single nucleotide polymorphisms in key genes of taurine and betaine metabolism in Crassostrea hongkongensis and their association with content-related traits.

Taurine and betaine are important nutrients in Crassostrea hongkongensis and have many important biological properties. To investigate the characteristics of taurine and betaine contents and identify SNPs associated with traits in the C.hongkongensis, we cloned the full-length cDNA of key genes in taurine and betaine (unpublished data) metabolism, determined taurine and betaine content and gene expression in different tissues and months of specimen collection, and developed SNPs in the gene coding region. We cloned the full-length cDNA of cysteine dioxygenase (ChCDO) and cysteine sulfite decarboxylase (ChCSAD), which are key genes involved in taurine metabolism in C. hongkongensis, and found that betaine and taurine contents and the expression of key genes were regulated by seawater salinity. A total of 47 SNP markers were developed in the coding regions of ChCSAD, ChCDO, ChCDH, ChBADH, and ChBHMT using gene fragment resequencing and FLDAS-PCR. Through association analysis in a population of C. hongkongensis in the Maowei Sea, Guangxi, nine SNPs were found to be associated with taurine content, and one SNP was associated with betaine content. Haploid and linkage disequilibrium analyses showed that SNPs in ChCDO formed one linkage group with three haplotypes: ACACA, GTTTG, and GTACA. The average taurine content of the corresponding individuals was 873.88, 838.99, and 930.72 ng/g, respectively, indicating the GTACA haplotype has a significant advantage in terms of taurine content. We identified SNPs associated with taurine and betaine contents in C.hongkongensis for the first time, and found the GTACA haplotype in the ChCDO coding region has a significant advantage in taurine content. These loci and haplotypes can serve as potential molecular markers for the molecular breeding of C. hongkongensis.

GLUT2 gene from Penaeus monodon: Molecular characterization, expression and association with tolerance to low salinity stress

The full-length cDNA sequence of Penaeus monodon glucose transporter-2 (PmGLUT2) was cloned in this study using the RACE method. Quantitative real-time PCR was used to analyze the differential expression of PmGLUT2 during the development of P. monodon larvae in different tissues and under low salinity stress. The PmGLUT2 cDNA exhibited a total length of 2018 base pairs, with 94 base pairs located in the 5’ untranslated region (UTR) and 352 base pairs in the 3’ UTR. Additionally, the sequence contained 29 base poly (A) tails and 1572 base pairs within the open reading frame (ORF), capable of encoding 523 amino acids. Through a comparative analysis of the amino acid sequences of PmGLUT2 and LvGLUT2, it was determined that PmGLUT2 had 94.77% homology with Tret1 gene of Litopenaeus vannamei, and 60.54% homology with GLUT2 gene of L. vannamei. The results indicated that there was a fluctuation in PmGLUT2 expression levels from zygote to postlarva development, with initial reduction followed by an increase, although the difference was not statistically significant. According to the molting analysis, the highest expression level of PmGLUT2 was observed in the hepatopancreas during the premolt period, while the gill and gut exhibited peak expression levels during the intermolt period. PmGLUT2 was found to be most abundant in lymph tissue, followed by gill tissue, and least abundant in ootheca, according to the tissue expression analyses. Following 96 hours of acute salt stress, there was a notable inhibition in the expression of PmGLUT2 in the hepatopancreas and gills. Additionally, the expression level in the gills at 96 hours was significantly lower compared to the baseline level at 0 hours.

Molecular cloning, expression and functional analysis of foxl2 from Chinese sturgeon (Acipenser sinensis) in relation to sex differentiation

IntroductionOur understanding of the regulatory mechanisms that govern the development of sexual characteristics in sturgeon, particularly the Chinese sturgeon (Acipenser sinensis), is limited. Foxl2, a member of the forkhead/HNF-3-related transcription factor family, is known for its role in precise gene regulation within specific tissues and its control over ovarian aromatase, a key enzyme in ovarian development and maturation. This study aimed to investigate the sexually dimorphic expression of foxl2 during the molecular and morphological sex differentiation stages in Chinese sturgeon.MethodsWe successfully isolated the full-length cDNAs of foxl2 in Chinese sturgeon. We then examined the expression profiles of this gene across various somatic tissues in both female and male specimens.ResultsFoxl2 was predominantly expressed in the ovulatory organs of two-year-old Chinese sturgeon, with significantly lower expression in other somatic tissues. We observed sexual dimorphism in foxl2 expression within gonads that had achieved morphological differentiation.DiscussionThe results suggest that foxl2 expression is intricately linked to the process of ovarian differentiation in Chinese sturgeon. Furthermore, we propose that the mRNA quantities of foxl2 could serve as an accurate biomarker for sex determination in sturgeon during their early growth stages.

Polymorphisms in the transit peptide of phytoene synthase ZmPSY1 link to the white color of grain endosperm in maize inbred lines

The yellow and orange color of Zea mays L. grain is determined by the presence of carotenoids, the first enzyme of the biosynthesis pathway of which is phytoene synthase PSY. In this study, we analyzed allelic variants of the ZmPSY1 gene in accessions of yellow-grain and white-grain maize inbred lines of domestic selection. In four lines with different grain colors, full-length ZmPSY1 cDNAs were amplified and sequenced, and their variability was characterized. In the cDNA sequence of ZmPSY1 from white-grain lines, nonsynonymous SNPs were found that lead to substitutions of four amino acid residues (L47I, W52S, E53D and A54V) in the N-terminal transit peptide responsible for the plastid localization of the enzyme. A primer system has been developed for PCR identification of the ZmPSY1 allele type in maize accessions. Testing of primers on 44 maize lines showed the presence of the wild-type ZmPSY1 allele and the absence of the mutant allele in the genome of all 22 yellow-grain lines analyzed. The mutant ZmPSY1 allele was detected in the genome of 41% of the 22 tested white-grain lines. The use of the developed primer system may be promising in the selection of corn with altered carotenoid content in the grain endosperm.

Functional Study of PTSMAD4 in the Spermatogenesis of the Swimming Crab Portunus trituberculatus.

Portunus trituberculatus holds significant economic value. The spermatogenesis is regulated by numerous signaling pathways. Among them, the TGF-β signaling pathway plays an important role in the development of testes and spermatogenesis. Smad4 is a Co-Smad protein that forms a complex with R-Smad to regulate the expression of target genes. The sperm structure in crustaceans differs greatly from that in mammals, with mature sperm lacking tails. Our previous studies have reported the function of R-Smad in the spermatogenesis of P. trituberculatus. In this study, we cloned the full-length cDNA sequence of PTSMAD4; immunofluorescence technology revealed that PTSMAD4 is expressed throughout all stages of spermatogenesis. We knocked down the expression of PTSMAD4 in P. trituberculatus using RNAi technology, and the immunofluorescence results show abnormal co-localization and weakened signals of PTSMAD4 and PTSMAD2. Additionally, transcriptome sequencing results enriched functional genes and pathways related to spermatogenesis. This study indicates that PTSMAD4 may participate in the spermatogenesis process through its involvement in signal transduction. This research not only lays the foundation for further study of the function of the TGF-β signaling pathway in spermatogenesis but also provides a theoretical basis for further investigation of the spermatogenesis mechanism in crustaceans.

Construction and Characterization of a Full-Length Infectious cDNA Clone of a Strain of Watermelon Mosaic Virus Isolated from Melon.

Watermelon mosaic virus (WMV), a member of the genus Potyvirus, causes serious economic losses in cucurbit crops. For molecular biological studies of viruses, it is necessary to construct an infectious clone that can facilitate gene functional analysis. In this study, we constructed an infectious cDNA clone of WMV genomic RNA by Gibson assembly and evaluated its virulence and symptoms on a variety of host plants. A WMV isolate (WMV-IS-me2) collected from melon in Iksan, Jeonbuk Province, Korea in 2015 caused mosaic symptoms on leaves. Four overlapping PCR fragments of the full-length genome of this isolate and the pJL89 binary vector with overhanging ends of 40 bp were amplified by reverse transcription PCR and joined in a single isothermal reaction. The complete nucleotide sequence of the infectious cDNA clone of WMV was determined and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101. Following agro-infiltration, an infectious clone (pWMV-M, KACC95145P) systemically induced mosaic symptoms on watermelon (Citrullus lanatus), zucchini (Cucurbita pepo), melon (Cucumis melo), and Nicotiana benthamiana plants. This infectious clone may be useful for screening cucurbit varieties resistant to WMV, as well as for studying viral gene function.

Splice site and de novo variants can cause PLCG2-associated immune dysregulation with cold urticaria (PLAID-CU).

Phospholipase Cγ2 (PLCγ2) is an important signaling molecule that receives and transmits signals from various cell surface receptors in most hematopoietic lineages. Variants of PLCG2 cause PLCγ2-associated immune dysregulation (PLAID), a family of conditions that are classified by mutational effect. PLAID with cold urticaria (PLAID-CU) is caused by in-frame deletions of PLCG2 that are dominant negative at physiologic temperatures but become spontaneously active at sub-physiologic temperatures. To identify genetic lesions that cause PLAID by combining RNA sequencing of full-length PLCG2 with whole genome sequencing. We studied nine probands with antibody deficiency and a positive evaporative cooling test, together with two known PLAID-CU patients and three healthy subjects. Illumina sequencing was performed on full-length PLCG2 cDNA synthesized from peripheral blood mononuclear cell RNA and whole genome sequencing was used to identify genetic lesions. Novel alternate transcripts were overexpressed in the Plcg2-deficient DT40 cell overexpression system. ERK phosphorylation was quantified by flow cytometry with and without BCR crosslinking. Two probands expressed novel alternative transcripts of PLCG2 with in-frame deletions. Proband 1, expressing PLCG2 without exons 18-19, carried a splice site mutation in intron 19. Proband 2, expressing PLCG2 without exons 19-22, carried a 14kb de novo deletion of PLCG2. DT40 cells overexpressing the exon 18-19 or exon 19-22 deletions failed to phosphorylate ERK in response to BCR crosslinking. In addition to autosomal dominant genomic deletions, de novo deletions and splice site mutations of PLCG2 can also cause PLAID-CU. All of these can be identified by cDNA-based sequencing.

Antimicrobial activity and immunomodulation of four novel cathelicidin genes isolated from the tiger frog Hoplobatrachus rugulosus.

Cathelicidin is a family of antimicrobial peptides in vertebrates that plays an important role in resistance and immunization against pathogenic microorganisms. In the present study, the full-length cDNA sequences of four novel cathelicidins (cathelicidin-1 to cathelicidin-4) in the tiger frog Hoplobatrachus rugulosus, encoding 153, 188, 132, and 160 amino acids, respectively, were firstly cloned by rapid amplification of the cDNA ends (RACE) technique. Sequence comparison and phylogenetic tree analysis indicated that the structures of the four cathelicidins are highly diverse. Afterwards, the tissue distribution profiles and antimicrobial patterns of cathelicidins in H. rugulosus were determined by real-time PCR. The four cathelicidins showed tissue-specific distribution patterns in the healthy frogs, and the transcriptional levels of cathelicidins exhibited a tissue- and time-dependency profile in the frogs challenged with pathogenic bacteria Aeromonas hydrophila for 72 h. The synthetic peptides of cathelicidin-1 and cathelicidin-2 exhibited broad-spectrum in vitro antimicrobial activity, and cathelicidins exerted antimicrobial activities through excessive induction of reactive oxygen species and direct disruption of the microbial membrane structure. In addition, the intraperitoneal injection of cathelicidin proteins significantly increased the marine medaka Oryzias melastigma resistance to bacterial challenges. The existence of multiple cathelicidins, their distinct tissue distribution patterns, and the inducible expression profiles suggest a sophisticated, highly redundant, and multilevel network of antimicrobial defense mechanisms in tiger frogs. This study provides evidence that cathelicidins have antimicrobial and immunomodulatory activities, and cathelicidins derived from H. rugulosus have potential therapeutic applications against pathogenic infections in aquaculture.

Function of PxGrp78 in the Reproduction of Plutella xylostella.

Glucose-regulated protein 78 (Grp78), a crucial molecular chaperone in the endoplasmic reticulum, has been extensively investigated in vertebrates. However, its functional exploration in insects remains limited. This study cloned the full-length cDNA sequence of Grp78 in Plutella xylostella (L.), which is 2583 bp long. The open reading frame (ORF) is 2004 bp in length and encodes a total of 667 amino acids, including three conserved characteristic sequences of the HSP70 family. PxGrp78 is expressed in various developmental stages of P. xylostella, with the highest expression observed in third instar larvae and higher expression in female adults compared to male adults. The interference with PxGrp78 in female adults was found to significantly reduce the quantity of egg laying and the hatching rate, as well as shorten the oviposition period and down-regulate the expression of the PxVg gene. These results suggested an important role for PxGrp78 in the reproduction of P. xylostella.

Cloning and characterization of the thioredoxin reductase 1 gene in Hyriopsis cumingii and its regulatory mechanism by Nrf2

The thioredoxin system, consisting of thioredoxin reductases and thioredoxin, plays a crucial role in defending against oxidative stress. Despite its importance, limited research has been conducted on this system in bivalves. In this study, we aimed to clone and characterize the thioredoxin reductase 1 gene from Hyriopsis cumingii (HcTrxR1) and to elucidate its interaction with the nuclear factor erythroid 2-related factor 2 (Nrf2) of H. cumingii (HcNrf2) using a combination of gene cloning, bioinformatics, RNA interference (RNAi), activator/inhibitor treatments, and dual-luciferase reporter assays. We successfully cloned the full-length cDNA of HcTrxR1, which consisted of a 1788 bp open reading frame encoding a 595-amino acid protein. Sequence analysis revealed high conservation of HcTrxR1 compared to homologs in other bivalve species. The expression of HcTrxR1 mRNA was detected across various tissues, with the highest levels observed in the gonads and hemolymph. RNAi and activator/inhibitor experiments demonstrated that HcNrf2 positively regulated the expression of HcTrxR1. Dual-luciferase reporter assays identified two antioxidant response elements in the promoter region of HcTrxR1, which were critical for HcNrf2 binding and transcriptional activation. Additionally, a polyclonal antibody against the HcTrxR1 protein was generated and confirmed for specificity. These findings underscore the regulatory role of Nrf2 in the thioredoxin system of bivalves, offering novel insights into the antioxidant mechanisms in H. cumingii. The study provides a molecular framework that may inform environmental monitoring and conservation efforts in aquatic ecosystems.