Full-length Coding Sequence Research Articles

WSSV induces Rubicon expression to regulate innate immune response in Penaeus vannamei

Rubicon, the RUN domain Beclin-1-interacting and cysteine-rich domain-containing protein plays an important role in facilitating viral replication. In this study, an involvement of P. vannamei Rubicon or PvRUBCN during white spot syndrome virus (WSSV) infection and its roles in regulation of apoptosis and innate immune response were investigated. The full-length coding sequence of PvRUBCN was 3708 bp encoding the protein of 1235 amino acids. PvRUBCN contained three conserved domains including the RUN, the PI3K-binding, and the Rubicon homology domains. PvRUBCN was grouped with the closely related RUBCN from the Penaeid species with more than 95 % identity. It was highly expressed in nerve followed by intestine, and gill. Its expression was induced upon WSSV challenge at 12 and 48 hpi. Suppression of PvRUBCN by dsRNA upon WSSV challenge resulted in more than 80 % reduction in PvRUBCN mRNA expression. Knockdown of PvRUBCN mRNA significantly decreased the WSSV copy number and prolonged the survival rate of WSSV-infected shrimp. In addition, the caspase3, a key regulator of the apoptosis pathway was significantly down-regulated. The interferon like genes including Vago4 was dramatically decreased at 72 hpi whereas Vago5 demonstrated slight reduction at 24 hpi and increase at 72 and 120 hpi, respectively. On the other hand, the expressions of the genes involved in the prophenoloxidase pathway including PPO1 and PPO2 were not changed. Taken together, PvRUBCN played important roles in the antiviral immunity in response to WSSV infection.

Exploring Functional Gene XsPDAT1’s Involvement in Xanthoceras sorbifolium Oil Synthesis and Its Acclimation to Cold Stress

Phospholipid: diacylglycerol acyltransferase (PDAT) is crucial in triacylglycerol (TAG) synthesis as it represents the final rate-limiting step of the acyl-CoA-independent acylation reaction. PDAT not only regulates lipid synthesis in plants, but also plays an important function in improving stress tolerance. In this study, the full-length coding sequence (CDS) of XsPDAT1, totaling 2022 base pairs and encoding 673 amino acids, was cloned from Xanthoceras sorbifolium. The relative expression of XsPDAT1 was significantly and positively correlated with oil accumulation during seed kernel development; there were some differences in the expression patterns under different abiotic stresses. Transgenic Arabidopsis thaliana plants overexpressing XsPDAT1 were obtained using the Agrobacterium-mediated method. Under low-temperature stress, the transgenic plants exhibited a smaller decrease in chlorophyll content, a smaller increase in relative conductivity, and a larger increase in POD enzyme activity and proline content in the leaves compared with the wild type. Additionally, lipid composition analysis revealed a significant increase in unsaturated fatty acids, such as oleic (C18:1) and linoleic (C18:2), in the seeds of transgenic plants compared to the wild type. These results suggest that XsPDAT1 plays a dual role in regulating the ratio of fatty acid composition and low-temperature stress in plants.

UBE2J1 suppresses interferon signaling by facilitating the ubiquitination and degradation of IRF7

Ubiquitin-binding enzyme E2J1 (UBE2J1) is a predominant member of the UBE2 family. In mammals, it is involved in antiviral immunity, ER-misfolded protein degradation, and ubiquitination. However, the function of UBE2J1 in teleost has not been elucidated. In the present study, we cloned the homolog of UBE2J1 in triploid hybrid fish and investigated its regulatory role in the antiviral interferon (IFN) signaling. We determined that the full-length coding sequence of the triploid fish UBE2J1 (3nUBE2J1) consists of 939 base pairs and encodes 313 amino acids. Upon SVCV infection, the mRNA levels of 3nUBE2J1 were increased in the spleen, liver, kidney, and the caudal fin cell line of triploid fish. Knockdown of 3nUBE2J1 in host cells and fish enhanced their antiviral response and curtailed SVCV replication. Conversely, overexpression of 3nUBE2J1 inhibited the activation of IFN promoter induced by SVCV, poly (I:C), or RLR pathway factors. Further studies revealed that 3nUBE2J1 interacted with 3nIRF7 and suppressed both the protein level and antiviral activity of 3nIRF7. Finally, we demonstrated that 3nUBE2J1 enhanced the K27- and K29-linked ubiquitination of 3nIRF7, leading to its degradation. Collectively, our findings indicate that triploid fish UBE2J1 serves as a novel negative regulator of IFN signaling by inhibiting 3nIRF7.

Identification, Molecular Characterization and Preliminary Functional Analysis of Three SLC27A Full-CDS cDNAs from Water Buffalo (Bubalus bubalis)

Background: Solute carrier family 27 family (SLC27As) have been identified to be involved in long chain fatty acid uptake in various tissues and cells, but their function in buffalo is unclear. Buffalo is an important dairy animal in some parts of the world. Buffalo milk contains more fat, protein, lactose and minerals than cow’s milk. The objective of this study was to identify the buffalo SLC27A1, 2 and 6 genes and to describe their characteristics, structure and basic function. Methods: The full-length coding sequences (CDSs) of SLC27A1, 2 and 6 were isolated from Binglangjiang buffalo and bioinformatics analyses were used to provide insights into the physicochemical characteristics, structure and function of their encoded proteins. Result: The CDSs of buffalo SLC27A1, 2 and 6 were 1941 bp, 1863 bp and 1905 bp in length, respectively, encoding 646, 620 and 634 amino acid residues accordingly. Their proteins FATP1, 2 and 6 have no signal peptide but a transmembrane helix with the N-terminus located outside the membrane and the C-terminus located in cytoplasmic side. All three proteins contain AMP-binding domains that belonged to AFD_class_I superfamily and may play a crucial role in the transport of fatty acids. The amino acid sequences of buffalo FATP1, 2 and 6 showed more than 88.6% identity with other species of Bovidae and more than 76.4% identity with other species of mammals. The phylogenetic tree showed that buffalo FATP1, 2 and 6 clustered in their respective clades and buffalo was more closely genetically related to other species of the Bovidae family. These findings suggest that buffalo FATPs are more similar in function to other species. The present study will provide a theoretical basis for further investigation of the functions of FATP1, 2 and 6 in buffalo lipid metabolism, especially milk fat metabolism.

Molecular cloning and characterization of duck Annexin A2 and its effect on DTMUV replication

Annexin A2 (ANXA2) is a multifaceted protein implicated in various stages of viral infections, particularly in envelope virus replication through mechanisms such as endocytosis and exocytosis. This study delves into the characterization and functional dynamics of duck ANXA2 (duANXA2). We successfully cloned the full-length coding sequence of duANXA2 and conducted a detailed structural analysis. The open reading frame (ORF) of duANXA2 is 1020 bp, encoding 339 amino acids and featuring 4 conserved domains. Phylogenetic tree analysis indicates that duANXA2 is most closely related to Gallus gallus, with significantly lesser homology to fish species. We evaluated the tissue-specific expression of duANXA2 in healthy ducks, noting its ubiquitous presence but varying expression levels across different organs, with notably high expression in the esophagus and immune organs. Upon infecting duck embryo fibroblast (DEF) cells with the duck Tembusu virus (DTMUV), a flavivirus causing ducks substantial mortality and a dramatic decline in egg production, we observed a pronounced upregulation of duANXA2. Functional assays demonstrated that overexpression of duANXA2 in DEF cells augments DTMUV replication, while its interference markedly reduces DTMUV replication. These findings underscore the role of duANXA2 as a facilitator of DTMUV replication, presenting it as a potential target for therapeutic intervention in managing DTMUV infections.

Transcriptional Regulation Analysis Provides Insight into the Function of GSK3β Gene in Diannan Small-Ear Pig Spermatogenesis.

Glycogen synthase kinase-3β (GSK3β) not only plays a crucial role in regulating sperm maturation but also is pivotal in orchestrating the acrosome reaction. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine GSK3β expression patterns in adult Diannan small-ear pig (DSE) testes. We identified the most important transcript ENSSSCT00000039364 of GSK3β, obtaining its full-length coding sequence (CDS) spanning 1263 bp. Gene structure analysis located GSK3β on pig chromosome 13 with 12 exons. Protein structure analysis reflected that GSK3β consisted of 420 amino acids containing PKc-like conserved domains. Phylogenetic analysis underscored the evolutionary conservation and homology of GSK3β across different mammalian species. The evaluation of the protein interaction network, KEGG, and GO pathways implied that GSK3β interacted with 50 proteins, predominantly involved in the Wnt signaling pathway, papillomavirus infection, hippo signaling pathway, hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, endometrial cancer, basal cell carcinoma, and Alzheimer's disease. Functional annotation identified that GSK3β was involved in thirteen GOs, including six molecular functions and seven biological processes. ceRNA network analysis suggested that DSE GSK3β was regulated by 11 miRNA targets. Furthermore, qPCR expression analysis across 15 tissues highlighted that GSK3β was highly expressed in the testis. Subcellular localization analysis indicated that the majority of the GSK3β protein was located in the cytoplasm of ST (swine testis) cells, with a small amount detected in the nucleus. Overall, our findings shed new light on GSK3β's role in DSE reproduction, providing a foundation for further functional studies of GSK3β function.

Identification of Phytophthora cinnamomi CRN effectors and their roles in manipulating cell death during Persea americana infection

The oomycete Phytophthora cinnamomi is a devastating plant pathogen with a notably broad host range. It is the causal agent of Phytophthora root rot (PRR), arguably the most economically important yield-limiting disease in Persea americana (avocado). Despite this, our understanding of the mechanisms P. cinnamomi employs to infect and successfully colonize avocado remains limited, particularly regarding the pathogen’s ability to maintain its biotrophic and necrotrophic lifestyles during infection. The pathogen utilises a large repertoire of effector proteins which function in facilitating and establishing disease in susceptible host plants. Crinkling and necrosis effectors (CRN/Crinklers) are suspected to manipulate cell death to aid in maintenance of the pathogens biotrophic and necrotrophic lifestyles during different stages of infection. The current study identified 25 P. cinnamomi CRN effectors from the GKB4 genome using an HMM profile and assigned putative function to them as either cell death inducers or suppressors. Function was assigned to 10 PcinCRNs by analysing their RNA-seq expression profiles, relatedness to other functionally characterised Phytophthora CRNs and tertiary protein predictions. The full-length coding sequences for these PcinCRNs were confirmed by Sanger sequencing, six of which were found to have two divergent alleles. The presence of alleles indicates that the proteins encoded may perform contradicting functions in cell death manipulation, or function in different host plant species. Overall, this study provides a foundation for future research on P. cinnamomi infection and cell death manipulation mechanisms.

Characterization of three lamp genes from largemouth bass (Micropterus salmoides): molecular cloning, expression patterns, and their transcriptional levels in response to fast and refeeding strategy.

Lysosomes-associated membrane proteins (LAMPs), a family of glycosylated proteins and major constituents of the lysosomal membranes, play a dominant role in various cellular processes, including phagocytosis, autophagy and immunity in mammals. However, their roles in aquatic species remain poorly known. In the present study, three lamp genes were cloned and characterized from Micropterus salmoides. Subsequently, their transcriptional levels in response to different nutritional status were investigated. The full-length coding sequences of lamp1, lamp2 and lamp3 were 1251bp, 1224bp and 771bp, encoding 416, 407 and 256 amino acids, respectively. Multiple sequence alignment showed that LAMP1-3 were highly conserved among the different fish species, respectively. 3-D structure prediction, genomic survey, and phylogenetic analysis were further confirmed that these genes are widely existed in vertebrates. The mRNA expression of the three genes was ubiquitously expressed in all selected tissues, including liver, brain, gill, heart, muscle, spleen, kidney, stomach, adipose and intestine, lamp1 shows highly transcript levels in brain and muscle, lamp2 displays highly expression level in heart, muscle and spleen, but lamp3 shows highly transcript level in spleen, liver and kidney. To analyze the function of the three genes under starvation stress in largemouth bass, three experimental treatment groups (fasted group and refeeding group, control group) were established in the current study. The results indicated that the expression of lamp1 was significant induced after starvation, and then returned to normal levels after refeeding in the liver. The expression of lamp2 and lamp3 exhibited the same trend in the liver. In addition, in the spleen and the kidney, the transcript level of lamp1 and lamp2 was remarkably increased in the fasted treatment group and slightly decreased in the refed treatment group, respectively. Collectively, our findings suggest that three lamp genes may have differential function in the immune and energetic organism in largemouth bass, which is helpful in understanding roles of lamps in aquatic species.

Multi-omics analysis reveals promiscuous O-glycosyltransferases involved in the diversity of flavonoid glycosides in Periploca forrestii (Apocynaceae)

Flavonoid glycosides are widespread in plants, and are of great interest owing to their diverse biological activities and effectiveness in preventing chronic diseases. Periploca forrestii, a renowned medicinal plant of the Apocynaceae family, contains diverse flavonoid glycosides and is clinically used to treat rheumatoid arthritis and traumatic injuries. However, the mechanisms underlying the biosynthesis of these flavonoid glycosides have not yet been elucidated. In this study, we used widely targeted metabolomics and full-length transcriptome sequencing to identify flavonoid diversity and biosynthetic genes in P. forrestii. A total of 120 flavonoid glycosides, including 21 C-, 96 O-, and 3 C/O-glycosides, were identified and annotated. Based on 24,123 full-length coding sequences, 99 uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) were identified and classified into 14 groups. Biochemical assays revealed that four UGTs exhibited O-glycosyltransferase activity toward apigenin and luteolin. Among them, PfUGT74B4 and PfUGT92A8 were highly promiscuous and exhibited multisite O-glycosylation or consecutive glycosylation activities toward various flavonoid aglycones. These four glycosyltransferases may significantly contribute to the diversity of flavonoid glycosides in P. forrestii. Our findings provide a valuable genetic resource for further studies on P. forrestii and insights into the metabolic engineering of bioactive flavonoid glycosides.

Variation of gene expression of fatty acid acyl CoA reductase associated with wax secretion of a scale insect, Ericerus pela, and identification of its regulation factors through the accessible chromatin analyses and yeast one-hybrid.

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 107 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI.

Vegetative cell wall protein OsGP1 regulates cell wall mediated soda saline-alkali stress in rice.

Plant growth and development are inhibited by the high levels of ions and pH due to soda saline-alkali soil, and the cell wall serves as a crucial barrier against external stresses in plant cells. Proteins in the cell wall play important roles in plant cell growth, morphogenesis, pathogen infection and environmental response. In the current study, the full-length coding sequence of the vegetative cell wall protein gene OsGP1 was characterized from Lj11 (Oryza sativa longjing11), it contained 660 bp nucleotides encoding 219 amino acids. Protein-protein interaction network analysis revealed possible interaction between CESA1, TUBB8, and OsJ_01535 proteins, which are related to plant growth and cell wall synthesis. OsGP1 was found to be localized in the cell membrane and cell wall. Furthermore, overexpression of OsGP1 leads to increase in plant height and fresh weight, showing enhanced resistance to saline-alkali stress. The ROS (reactive oxygen species) scavengers were regulated by OsGP1 protein, peroxidase and superoxide dismutase activities were significantly higher, while malondialdehyde was lower in the overexpression line under stress. These results suggest that OsGP1 improves saline-alkali stress tolerance of rice possibly through cell wall-mediated intracellular environmental homeostasis.

Overexpression of the NbZFP1 encoding a C3HC4-type zinc finger protein enhances antiviral activity of Nicotiana benthamiana

Viral diseases are crucial determinants affecting tobacco cultivation, leading to a substantial annual decrease in production. Previous studies have demonstrated the regulatory function of the C3HC4 family of plant zinc finger proteins in combating bacterial diseases. However, it remains to be clarified whether this protein family also plays a role in regulating resistance against plant viruses. In this study, the successful cloning of the zinc finger protein coding gene NbZFP1 from Nicotiana benthamiana has been achieved. The full-length coding sequence of NbZFP1 is 576 bp. Further examination and analysis of this gene revealed its functional properties. The induction of NbZFP1 transcription in N. benthamiana has been observed in response to TMV, CMV, and PVY. Transgenic N. benthamiana plants over-expressing NbZFP1 demonstrated a notable augmentation in the production of chlorophyll a (P < 0.05). Moreover, NbZFP1-overexpressing tobacco exhibited significant resistance to TMV, CMV, and PVY, as evidenced by a decrease in virus copies (P < 0.05). In addition, the defense enzymes activities of PAL, POD, and CAT experienced a significant increase (P < 0.05). The up-regulated expression of genes of NbPAL, NbNPR1 and NbPR-1a, which play a crucial role in SA mediated defense, indicated that the NbZFP1 holds promise in enhancing the virus resistance of tobacco plant. Importantly, the results demonstrate that NbZFP1 can be considered as a viable candidate gene for the cultivation of crops with enhanced virus resistance.

Molecular characterization and expression pattern of Rubisco activase gene GhRCAβ2 in upland cotton (Gossypium hirsutum L.).

Rubisco activase (RCA) is a pivotal enzyme that can catalyse the activation of Rubisco in carbon assimilation pathway. Many studies have shown that RCA may be a potential target for genetic manipulation aimed at enhancing photosynthetic efficiency and crop yield. To understand the biological function of the GhRCAβ2 gene in upland cotton, we cloned the coding sequence (CDS) of the GhRCAβ2 gene and investigated its sequence features, evolutionary relationship, subcellular localization, promoter sequence and expression pattern. The bioinformatics tools were used to analyze the sequence features of GhRCAβ2 protein. Transient transformation of Arabidopsis mesophyll protoplasts was performed to determine the subcellular localization of the GhRCAβ2 protein. The expression pattern of the GhRCAβ2 gene was examined by analyzing transcriptome data and using the quantitative real-time PCR (qRT-PCR). The full-length CDS of GhRCAβ2 was 1317bp, and it encoded a protein with a chloroplast transit peptide. The GhRCAβ2 had two conserved ATP-binding domains, and did not have the C-terminal extension (CTE) domain that was unique to the RCA α-isoform in plants. Evolutionarily, GhRCAβ2 was clustered in Group A, and had a close evolutionary relationship with the soybean RCA. Western blot analysis demonstrated that GhRCAβ2 was immunoreactive to the RCA antibody displaying a molecular weight similar to that of the RCA β-isoform. The GhRCAβ2 protein was found in chloroplast, aligning with its role as a vital enzyme in the process of photosynthesis. The GhRCAβ2 gene had a leaf tissue-specific expression pattern, and the yellow-green leaf mutant exhibited a decreased expression of GhRCAβ2 in comparison to the wild-type cotton plants. The GhRCAβ2 promoter contained several cis-acting elements that respond to light, phytohormones and stress, suggesting that the expression of GhRCAβ2 may be regulated by these factors. An additional examination of stress response indicated that GhRCAβ2 expression was influenced by cold, heat, salt, and drought stress. Notably, diverse expression pattern was observed across different stress conditions. Additionally, low phosphorus and low potassium stress may result in a notable reduction in the expression of GhRCAβ2 gene. Our findings will establish a basis for further understanding the function of the GhRCAβ2 gene, as well as providing valuable genetic knowledge to improve cotton photosynthetic efficiency and yield under challenging environmental circumstances.

ToxCodAn-Genome: an automated pipeline for toxin-gene annotation in genome assembly of venomous lineages.

The rapid development of sequencing technologies resulted in a wide expansion of genomics studies using venomous lineages. This facilitated research focusing on understanding the evolution of adaptive traits and the search for novel compounds that can be applied in agriculture and medicine. However, the toxin annotation of genomes is a laborious and time-consuming task, and no consensus pipeline is currently available. No computational tool currently exists to address the challenges specific to toxin annotation and to ensure the reproducibility of the process. Here, we present ToxCodAn-Genome, the first software designed to perform automated toxin annotation in genomes of venomous lineages. This pipeline was designed to retrieve the full-length coding sequences of toxins and to allow the detection of novel truncated paralogs and pseudogenes. We tested ToxCodAn-Genome using 12 genomes of venomous lineages and achieved high performance on recovering their current toxin annotations. This tool can be easily customized to allow improvements in the final toxin annotation set and can be expanded to virtually any venomous lineage. ToxCodAn-Genome is fast, allowing it to run on any personal computer, but it can also be executed in multicore mode, taking advantage of large high-performance servers. In addition, we provide a guide to direct future research in the venomics field to ensure a confident toxin annotation in the genome being studied. As a case study, we sequenced and annotated the toxin repertoire of Bothrops alternatus, which may facilitate future evolutionary and biomedical studies using vipers as models. ToxCodAn-Genome is suitable to perform toxin annotation in the genome of venomous species and may help to improve the reproducibility of further studies. ToxCodAn-Genome and the guide are freely available at https://github.com/pedronachtigall/ToxCodAn-Genome.

Long-read and short-read RNA-seq reveal the transcriptional regulation characteristics of PICK1 in Baoshan pig testis.

PICK1 plays a crucial role in mammalian spermatogenesis. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine PICK1 expression patterns in adult Baoshan pig (BS) testes. We identified the most important transcript ENSSSCT00000000120 of PICK1, obtaining its full-length coding sequence (CDS) spanning 1254 bp. Gene structure analysis located PICK1 on pig chromosome 5 with 14 exons. Protein structure analysis reflected that PICK1 consisted of 417 amino acids containing two conserved domains, PDZ and BAR_PICK1. Phylogenetic analysis underscored the evolutionary conservation and homology of PICK1 across different mammalian species. Evaluation of protein interaction network, KEGG, and GO pathways implied that interacted with 50 proteins, predominantly involved in glutamatergic synapses, amphetamine addiction, neuroactive ligand-receptor interactions, dopaminergic synapses, and synaptic vesicle recycling, and PICK1 exhibited significant correlation with DLG4 and TBC1D20. Functional annotation identified that PICK1 was involved in 9 GOs, including seven cellular components and two molecular functions. ceRNA network analysis suggested BS PICK1 was regulated by seven miRNA targets. Moreover, qPCR expression analysis across 15 tissues highlighted that PICK1 was highly expressed in the bulbourethral gland and testis. Subcellular localization analysis in ST (Swine Tesits) cells demonstrated that PICK1 significantly localized within the cytoplasm. Overall, our findings shed new light on PICK1's role in BS reproduction, providing a foundation for further functional studies of PICK1.

Biochemical features of the Cry4B toxin of Bacillus thuringiensis subsp. israelensis and its interaction with BT-R3, a bitopic cadherin G-protein coupled receptor in Anopheles gambiae.

The cadherin G-protein coupled receptor BT-R3 in the mosquito Anopheles gambiae is a single membrane-spanning α-helical (bitopic) protein that represents the most abundant and functionally diverse group of membrane proteins. Binding of the Cry4B toxin of Bacillus thuringiensis subsp. israelensis (Bti) to BT-R3 triggers a Mg2+-dependent signalling pathway in the mosquito that involves stimulation of G protein α-subunit, which subsequently launches a coordinated signalling cascade involving Na+/K+-ATPase. Described in this study is the behaviour of the Cry4B purified active protein toxin in solution relative to its protoxin predecessor produced by Bti as well as identification of the region within BT-R3 of An. gambiae to which the toxin binds. The relationship and behaviour of protoxin and toxin were ascertained in vitro by solubility studies in an alkaline environment like that of the mosquito larval midgut. To identify the specific toxin-binding site within BT-R3, the full-length coding sequence of the bt-r3 gene was amplified and cloned in pENTR/D-TOTO and subcloned in pXINSECT-DEST38 resulting in recombinant pXINSECT-DEST38-bt-r3. Cytotoxicity was analysed using Trichoplusia ni High Five™ insect cells transfected with the pXINSECT-DEST38-bt-r3 plasmid rendering them susceptible to the Cry4B toxin. Truncation mutational analyses, receptor-toxin binding studies and live cell experiments were used to elucidate the toxin-binding site in BT-R3. The N-terminal half of the Cry4B protoxin was cleaved releasing active Cry4B toxin. The nontoxic C-terminal portion was degraded into small peptide fragments. The receptor BT-R3 contained a single toxin-binding site--a 106-amino acid polypeptide bounded by Ile1359 and Ser1464 (1359IS1464) localized in the 11th cadherin repeat of the receptor. The structural features of the toxin-binding site are critical to the specificity, selectivity and affinity of the active toxin and for the design and development of novel Bti-based biopesticides.

Molecular cloning, expression and Insilco analysis of drought stress inducible MYB transcription factor encoding gene from C4 plant Eleusine coracana

Drought is one of the key abiotic stresses that critically influences the crops by restraining their growth and yield potential. Being sessile, plant tackle the detrimental effects of drought stress by modulating the cellular state by changing the gene expression. The transcriptional syndicate essentially drives such alteration of gene expression. Transcription factors (TF) are the key regulatory protein that controls the expression of their target gene by binding to the cis-regulatory elements present in the promoter region. Myb-TF, ubiquitously present in all eukaryotes belong to one of the largest TF family, and play a wide array of biological functions in plants, including anthocyanin biosynthesis, vasculature system, cell signalling, seed maturation and abiotc stress responses. The present performed isolation and molecular cloning of full length Myb TF from Eleusine corocana. The isolated full-length coding sequence has 1053 bp and 350 aa was submitted to NCBI (Accession number MT312253). The transcript level of EcMYB increases under different abiotic stress treatments including dehydration, salinity, and high-temperature stress. The promoter region of EcMyb1 was found to be enriched in stress-responsive cis-regulatory elements such as DRE, HSE, ABRE etc. In phylogenetic analysis, EcMyb1 appeared to have high homology with its monocot orthologs particularly Sateria italica, Hordeum vulgare, Saccharum barberi and Oryza sativa. The three-dimension protein structure was generated based on homology modeling and structural aspects were discussed. Further, Insilco analysis was conducted to explore the physiological properties, subcellular localization, potential posttranslational modification sites (phosphorylation and glycosylation sites), and molecular and biological function of the full-length protein. Overall, the expression profiling and Insilco analysis of EcMyb1 strongly indicated its potential role in abiotic stress response in Eleusine corocana.

Human cell-expressed tag-free rhMFG-E8 as an effective radiation mitigator

Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and the possibility of antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 will have suitable structural and functional properties to be developed as a safe and effective novel biologic to treat inflammatory diseases including radiation injury. We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing an experimental rodent model of radiation injury, i.e., partial body irradiation (PBI). HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE with the standard human MFG-E8 loaded as control and, mass spectrometry followed by analysis using MASCOT for peptide mass fingerprint. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with a terminal elimination half-life in circulation of at least 1.45 h. In the 15 Gy PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 calculated using probit analysis was 1.058. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI suggesting it as a potential therapeutic candidate for a medical countermeasure for radiation injury. Our new human cell-expressed tag-free rhMFG-E8 has proper structural and functional properties to be further developed as a safe and effective therapy to treat victims of severe acute radiation injury.

Molecular characterization of gonadotropin-releasing hormone receptor and its expression profiling in response to HCG stimulation in striped murrel, Channa striata (Bloch, 1793)

The gonadotropin releasing hormone receptors (GnRHR) play an important role in the activation of GnRH and elicit the release of gonadotropins that plays an important role in maturation and spawning. In the present work, molecular characterization of the GnRHR gene of snakehead murrel, Channa striata was carried out. The full-length cDNA of GnRH receptors of the 1581 bp sequence, which encodes 440 amino acids was identified by RACE-PCR. Signal peptide analysis confirmed that GnRHR is a non-secretory protein, and hydropathy profiles indicated its hydrophobic nature. The deduced amino acid sequences of the GnRHR showed the expected conserved domains, which include seven membrane-spanning domains, two potential N-linked glycosylation sites (631 Asp, 561 Asp), and 32 sites phosphorylation sites of serine and threonine residues. A homology search of GnRHR cDNA sequence revealed 88 % identity with Channa argus, followed by 85 % and 83 % sequence identities with Lates calcarifer, and Anabas testudineus at the protein level. Phylogenetic analysis depicted a close association with Channa argus and other fish species. Our findings showed differential GnRHR expression during different reproductive stages, with higher expression after HCG administration. Expression profiling revealed that GnRHR is highly expressed in the brain and testis at 16 h post-HCG injection, while it was neutrally regulated in female ovarian tissues. This study provides full-length coding sequence information of the GnRHR gene and will help to understand the reproductive biology of murrel using expression studies and devise strategies for induced breeding.

Cloning and Functional Analysis of NtHDR in ‘Jinzhanyintai’ of Narcissus tazetta var. chinensis M.Roem

The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-pyrophosphate reductase, a key enzyme at the end of the methylerythritol 4-phosphate pathway (MEP), was cloned from the petals of Narcissus tazetta var. Chinensis ‘Jinzhanyintai’ and named NtHDR (accession number OQ739816). NtHDR exhibits a full-length coding sequence spanning 1380 base pairs, encoding a total of 460 amino acids. Through phylogenetic-tree analysis, it has been determined that NtHDR shared the closest evolutionary relationship with monocotyledons, specifically Asparagus officinalis and Zingiber officinale. Utilizing quantitative real-time PCR analysis, it was observed that NtHDR exhibited significant expression in both petals and corona, with expression levels varying throughout the flowering process of Narcissus. Specifically, in petals, NtHDR expression demonstrated a pattern of initial increase followed by subsequent decrease, while in corona, it consistently increased. Subsequent subcellular-localization experiments indicated that NtHDR was localized within the chloroplasts. To investigate the functional impact of NtHDR, a stable transformation was performed, where NtHDR was introduced into Nicotiana benthamiana. The resulting transgenic N. benthamiana flowers were analyzed using solid-phase microextraction coupled with gas chromatography–mass spectrometry (SPME-GC-MS) to characterize their volatile components. The analysis revealed the presence of the monoterpene compound linalool, as well as the phenylpropanoids benzyl alcohol and phenylethanol, within the floral fragrance components of the transgenic N. benthamiana plants. However, these compounds were absent in the floral fragrance components of wild-type tobacco plants, thus highlighting the impact of NtHDR transformation on the floral scent profile.