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The cloning and characterization of the complete coding sequence of the Clarias magur SRD5A1 (CmSRD5A1) gene, which encodes an enzyme responsible for regulating steroid levels by converting testosterone into 5α-dihydrotestosterone (DHT), have been successfully achieved. DHT plays a vital role in enabling the complete expression of testosterone's actions in neuroendocrine tissues. The ORF of the full-length cDNA sequence of SRD5A1 was 795 bp, translating into 265 amino acids, with a total length of 836 bp including UTRs. Like other vertebrates, the signal peptide analysis revealed that SRD5A1 is a non-secretory protein, and hydropathy profiles indicated that it is hydrophobic in nature. The 3D structure of CmSRD5A1 sequence generated above was predicted using highly accurate AlphaFold 2 in Google Colab online platform. CmSRD5A1 contains seven transmembrane helices connected by six loops, with the N-termini located on the periplasmic side and C-termini on the cytosolic side. Structural superimposition with known bacterial and human SRD5As showed very high structural similarity. The electrostatic potential calculation and surface analysis of CmSRD5A1 revealed the presence of a large cavity with two openings one highly electropositive towards the cytosolic side and another relatively neutral towards the transmembrane region. The structural comparison revealed that the electropositive side of the cavity should bind to NADPH and the steroid hormone in the hydrophobic environment. Polar residues binding to NADPH are highly conserved and the same as known strictures. The conserved residues involved in hydrogen bonding with the ketone group at C-3 in the steroids hence fevering Δ4 double-bond reduction are identified as E66 and Y101. Our findings showed that SRD5A1 expression was lower during the spawning phase than the preparatory phase in female fish, while the administration of Ovatide (a GnRH analogue) resulted in up-regulation of expression after 6 h of injection in the ovary. In males, the lowest expression was observed during the preparatory phase and peaked at 16 h post- Ovatide injection in the testis. The expression of SRD5A1 in the brain of female fish was slightly higher during the Ovatide stimulation phase than the spawning phase. This study represents the first report on the cloning and characterization of the full-length cDNA of SRD5A1 in Indian catfish. [ABSTRACT FROM AUTHOR]

The rice Hybrid Proline Rich Protein (HyPRP) encoding gene, OsHyPRP16 expression exhibit early upregulation in response to Magnaporthe oryzae inoculation. Here, we functionally characterized the OsHyPRP16 promoter through deletion analysis in transgenic Arabidopsis using GUS (β-glucuronidase) reporter assay. The promoter fragments, sequentially deleted from the 5′ end could induce differential GUS activity in response to stresses induced by different hormones and abiotic stress conditions. In addition, a strong GUS induction was observed in M. oryzae inoculated transgenic Arabidopsis. Based on the insilico and stress-inducibility of D1 promoter fragment against various phytohormones and rice blast fungus, and with no basal activity under control conditions, we rationally selected D1 promoter fragment to drive the expression of a major rice blast resistance gene; Pi54 in the genetic background of blast susceptible TP309 rice line. The D1 promoter fragment was able to induce the expression of Pi54 at immediate-early stages of M. oryzae infection in transgenic rice. The transgenic plants with Pi54 under the control of D1 promoter fragment displayed complete resistance against M. oryzae infection as compared to control plants. The present study suggests that the D1 fragment of OsHyPRP16 promoter is a valuable tool for breeding and development of rice lines with early-inducible and pathogen-responsive enhanced disease resistance. • Functional characterization of rice HyPRP16 promoter was performed. • Deletion fragment (D1) exhibits distinct stress-response with no basal activity. • D1 driven R gene (Pi54) induce early upon M. oryzae infection in transgenic rice. • Early induction of Pi54 impart resistance against rice blast disease. [ABSTRACT FROM AUTHOR]

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association between chilling and methylation changes was observed, which suggested that chilling acquisition during dormancy in apple is likely to affect the epigenetic regulation through DNA methylation. [ABSTRACT FROM AUTHOR]

Apple is one of the most important fruit crops and the availability of recently sequenced genome facilitates research on gene–trait relationship. Quantitative real-time PCR (qRT-PCR) is a sensitive technique to study gene expression, but its accuracy largely depends on stability of reference gene used for data normalization. Therefore, present study was aimed to identify and validate suitable reference gene in apple. Expression stabilities of 10 housekeeping genes, which are commonly used as reference genes in qRT-PCR studies were evaluated in samples of different developmental stages, various tissue types and under diverse biotic stress conditions in apple. The PCR efficiency of all the genes was found to be ranging between 94–107.6%. Analysis using geNorm, NormFinder and Bestkeeper programs demonstrated that their expression stabilities vary among sample sets. However, protein phosphatase 2A ( PP2A ), ribosomal protein L2 ( RPL2 ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) were found to be suitable reference genes across all the tissue types and under different biotic stress conditions, while 18S ribosomal RNA ( 18S ), β-tubulin ( TUB ) and ubiquitin ( UBQ ) were found to be the least stable genes. Moreover, a combination of different reference genes was suggested for different sample sets. These, results suggest that selection of suitable reference gene depends on the tissue type and development stage or disease condition. Further, use of more than one reference genes in respective tissue types of apple is suggested to accurately normalize qRT-PCR data, in future. [ABSTRACT FROM AUTHOR]

Nucleotide binding site leucine-rich repeats (NBS-LRR) disease resistance proteins play an important role in plant defense against pathogen attack. A number of recent studies have been carried out to identify and characterize NBS-LRR gene families in many important plant species. In this study, we identified NBS-LRR gene family comprising of 1015 NBS-LRRs using highly stringent computational methods. These NBS-LRRs were characterized on the basis of conserved protein motifs, gene duplication events, chromosomal locations, phylogenetic relationships and digital gene expression analysis. Surprisingly, equal distribution of Toll/interleukin-1 receptor (TIR) and coiled coil (CC) (1∶1) was detected in apple while the unequal distribution was reported in majority of all other known plant genome studies. Prediction of gene duplication events intriguingly revealed that not only tandem duplication but also segmental duplication may equally be responsible for the expansion of the apple NBS-LRR gene family. Gene expression profiling using expressed sequence tags database of apple and quantitative real-time PCR (qRT-PCR) revealed the expression of these genes in wide range of tissues and disease conditions, respectively. Taken together, this study will provide a blueprint for future efforts towards improvement of disease resistance in apple. [ABSTRACT FROM AUTHOR]

The ß-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixedlinkage glucans [MLG - ß-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Grampositive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16MLG cleaved various types of ß-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94MLG and ß-glucosidases (BpGH3-AR8MLG and BpGH3-X62MLG). Using targeted deletion, we demonstrated BpSBPMLG is essential for B. producta growth on barley ß-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581T, Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16MLG. Disentangling the ß-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism. [ABSTRACT FROM AUTHOR]