Identification and expression analysis of dmc1 in Chinese soft-shell turtle (Pelodiscus sinensis) during gametogenesis.

DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3' UTR, a 22 bp 5' UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86-88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles' Dmc1 and close to the reptiles', but far away from the teleost's. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells' meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells' development and differentiation in Chinese soft-shell turtles, even in the reptiles.

RNAseqCovarImpute: a multiple imputation procedure that outperforms complete case and single imputation differential expression analysis.

Missing covariate data is a common problem that has not been addressed in observational studies of gene expression. Here, we present a multiple imputation method that accommodates high dimensional gene expression data by incorporating principal component analysis of the transcriptome into the multiple imputation prediction models to avoid bias. Simulation studies using three datasets show that this method outperforms complete case and single imputation analyses at uncovering true positive differentially expressed genes, limiting false discovery rates, and minimizing bias. This method is easily implemented via an R Bioconductor package, RNAseqCovarImpute that integrates with the limma-voom pipeline for differential expression analysis.

Identification and expression profiling of the bone morphogenetic protein gene family based on pearl culture in mantle and visceral mass of Hyriopsis cumingii.

Bone morphogenetic proteins (BMPs) play an important biological role in pearl biomineralization in pearl mussels. In this study, based on the genome data of the triangular sail mussel (Hyriopsis cumingii), the genome-wide identification and bioinformatic analysis of BMP gene family were performed, and the expression pattern of the BMP genes was investigated by the insertion experiments. The results showed that a total of 12 BMP gene family members (BMP2a/2b, BMP3, BMP5a/5b, BMP7a/7b/7c, BMP9, BMP10a/10b, and BMP11) were identified, which were unevenly distributed on chromosome 3/14/18, encoding 169-583 amino acids, with molecular weights ranging from 19.32 to 65.99 kDa. BMP2a, BMP7b, and BMP10a were distributed, respectively, in the cytoplasm, endoplasmic reticulum and mitochondria, others were distributed in the nucleus. qRT-PCR results showed the significant tissue specificity in BMPs gene expression. The HcBMPs were differentially expressed in the mantle and visceral mass, and the expression level was higher in the visceral mass. The expressing trends of HcBMPs were not consistent between the mantle and visceral mass insertion, suggesting that HcBMPs may perform different functions. We also found that insertion surgery in the mantle and visceral mass significantly alters the expression profiling of the BMP gene family. Insertion of the mantle induced the biomineralization function of BMP2a, BMP7a, and BMP7b, while BMP3 and BMP10b played opposite roles in visceral mass insertion. Visceral mass insertion could suppress BMP9 expression at 5 d and BMP5b expression at 90 d after insertion This work lays the foundation and data support for the preliminary elucidation of regulatory role and mechanism of HcBMPs in the pearl-cultivating process of mantle and visceral mass.

Diurnal.plant.tools in 2024: expanding to Marchantia polymorpha and four angiosperms.

Diurnal gene expression is a pervasive phenomenon occurring across all kingdoms of life, orchestrating adaptive responses to daily environmental fluctuations and thus enhancing organismal fitness. Our understanding of the plant circadian clock is primarily derived from studies in Arabidopsis and direct comparisons are difficult due to differences in gene family sizes. To this end, the identification of functional orthologs based on diurnal and tissue expression is necessary. The diurnal.plant.tools database constitutes a repository of gene expression profiles from 17 members of the Archaeplastida lineage, with built-in tools facilitating cross-species comparisons. In this database update, we expand the dataset with diurnal gene expression from 4 agriculturally significant crop species and Marchantia, a plant of evolutionary significance. Notably, the inclusion of diurnal gene expression data for Marchantia enables researchers to glean insights into the evolutionary trajectories of the circadian clock and other biological processes spanning from algae to angiosperms. Moreover, integrating diurnal gene expression data with datasets from related gene co-expression databases, such as CoNekt-Plants and CoNekt-Stress, which contain gene expression data for tissue and perturbation experiments, provides a comprehensive overview of gene functions across diverse biological contexts. This expanded database serves as a valuable resource for elucidating the intricacies of diurnal gene regulation and its evolutionary underpinnings in plant biology.

Overexpression of two DELLA subfamily genes MiSLR1 and MiSLR2 from mango promotes early flowering and enhances abiotic stress tolerance in Arabidopsis.

Gibberellic acids (GAs) are a group of endogenous phytohormones that play important roles in plant growth and development. SLENDER RICE (SLR) serves as a vital component of the DELLA gene family, which plays an irreplaceable role in regulating plant flowering and height, as well as stress responses. SLR gene has not been reported in mango, and its function is unknown. In present study, two DELLA subfamily genes MiSLR1 and MiSLR2 were identified from mango. MiSLR1 and MiSLR2 were highly expressed in the stems of the juvenile stage, but were expressed at a low level in flower buds and flowers. Gibberellin treatment could up-regulate the expression of MiSLR1 and MiSLR2 genes, but gibberellin biosynthesis inhibitor prohexadione-calcium (Pro-Ca) and paclobutrazol (PAC) treatments significantly down-regulated the expression of MiSLR1, while MiSLR2 was up-regulated. The expression levels of MiSLR1 and MiSLR2 were up-regulated under both salt and drought treatments. Overexpression of MiSLR1 and MiSLR2 genes significantly resulted early flowering in transgenic Arabidopsis and significantly up-regulated the expression levels of endogenous flower-related genes, such as SUPPRESSOR OF CONSTANS1 (SOC1), APETALA1 (AP1), and FRUITFULL (FUL). Interestingly, MiSLR1 significantly reduced the height of transgenic plants, while MiSLR2 gene increased. Overexpression of MiSLR1 and MiSLR2 increased seed germination rate, root length and survival rate of transgenic plants under salt and drought stress. Physiological and biochemical detection showed that the contents of proline (Pro) and superoxide dismutase (SOD) were significantly increased, while the contents of malondialdehyde (MDA) and H2O2 were significantly decreased. Additionally, protein interaction analysis revealed that MiSLR1 and MiSLR2 interacted with several flowering-related and GA-related proteins. The interaction between MiSLR with MiGF14 and MiSOC1 proteins was found for the first time. Taken together, the data showed that MiSLR1 and MiSLR2 in transgenic Arabidopsis both regulated the flowering time and plant height, while also acting as positive regulators of abiotic stress responses.

T cell receptor clonotype in tumor microenvironment contributes to intratumoral signaling network in patients with colorectal cancer.

Single-cell RNA sequencing (scRNA-seq) has contributed to understanding cellular heterogeneity and immune profiling in cancer. The aim of the study was to investigate gene expression and immune profiling in colorectal cancer (CRC) using scRNA-seq. We analyzed single-cell gene expression and T cell receptor (TCR) sequences in 30 pairs of CRC and matched normal tissue. Intratumoral lymphocytes were measured with digital image analysis. CRC had more T cells, epithelial cells, and myeloid cells than normal colorectal tissue. CRCs with microsatellite instability had more abundant T cells than those without microsatellite instability. Immune cell compositions of CRC and normal colorectal tissue were inversely correlated. CD4 + or CD8 + proliferating T cells, CD4 + effector memory T cells, CD8 + naïve T cells, and regulatory T cells of CRC showed higher TCR clonal expansion. Tumor epithelial cells interacted with immune cells more strongly than normal. T cells, myeloid cells, and fibroblasts from CRCs of expanded T cell clonotypes showed increased expression of genes related to TNF and NFKB signaling and T cell activation. CRCs of expanded T cell clonotypes also showed stronger cellular interactions among immune cells, fibroblasts, and endothelial cells. Pro-inflammatory CXCL and TNF signaling were activated in CRCs of expanded T cell clonotype. In conclusion, scRNA-seq analysis revealed different immune cell compositions, differential gene expression, and diverse TCR clonotype dynamics in CRC. TCR clonality expansion is associated with immune activation through T cell signaling and chemokine signaling. Patients with CRCs of expanded clonotype can be promising candidates for immunotherapy.

RankCompV3: a differential expression analysis algorithm based on relative expression orderings and applications in single-cell RNA transcriptomics.

BACKGROUND: Effective identification of differentially expressed genes (DEGs) has been challenging for single-cell RNA sequencing (scRNA-seq) profiles. Many existing algorithms have high false positive rates (FPRs) and often fail to identify weak biological signals. RESULTS: We present a novel method for identifying DEGs in scRNA-seq data called RankCompV3. It is based on the comparison of relative expression orderings (REOs) of gene pairs which are determined by comparing the expression levels of a pair of genes in a set of single-cell profiles. The numbers of genes with consistently higher or lower expression levels than the gene of interest are counted in two groups in comparison, respectively, and the result is tabulated in a 3 × 3 contingency table which is tested by McCullagh's method to determine if the gene is dysregulated. In both simulated and real scRNA-seq data, RankCompV3 tightly controlled the FPR and demonstrated high accuracy, outperforming 11 other common single-cell DEG detection algorithms. Analysis with either regular single-cell or synthetic pseudo-bulk profiles produced highly concordant DEGs with the ground-truth. In addition, RankCompV3 demonstrates higher sensitivity to weak biological signals than other methods. The algorithm was implemented using Julia and can be called in R. The source code is available at https://github.com/pathint/RankCompV3.jl . CONCLUSIONS: The REOs-based algorithm is a valuable tool for analyzing single-cell RNA profiles and identifying DEGs with high accuracy and sensitivity.

Role of Rubus chingii BBX gene family in anthocyanin accumulation during fruit ripening.

The B-box (BBX) family, which is a class of zinc finger transcription factors, exhibits special roles in plant growth and development as well as in plants' ability to cope with various stresses. Even though Rubus chingii is an important traditional medicinally edible plant in east Asia, there are no comprehensive studies of BBX members in R. chingii. In this study, 32 RcBBX members were identified, and these were divided into five groups. A collinearity analysis showed that gene duplication events were common, and when combined with a motif analysis of the RcBBX genes, it was concluded that group V genes might have undergone deletion of gene fragments or mutations. Analysis of cis-acting elements revealed that each RcBBX gene contained hormone-, light-, and stress-related elements. Expression patterns of the 32 RcBBX genes during fruit ripening revealed that highest expression occurred at the small green fruit stage. Of note, the expression of several RcBBX genes increased rapidly as fruit developed. These findings, combined with the expression profiles of anthocyanin biosynthetic genes during fruit ripening, allowed us to identify the nuclear-targeted RcBBX26, which positively promoted anthocyanin production in R. chingii. The collective findings of this study shed light on the function of RcBBX genes in different tissues, developmental stages, and in response to two abiotic stresses.

Monitoring of parasite Orobanche cumana using Vis-NIR hyperspectral imaging combining with physio-biochemical parameters on host crop Helianthus annuus.

KEY MESSAGE: This study provided a non-destructive detection method with Vis-NIR hyperspectral imaging combining with physio-biochemical parameters in Helianthus annuus in response to Orobanche cumana infection that took insights into the monitoring of sunflower weed. Sunflower broomrape (Orobanche cumana Wallr.) is an obligate weed that attaches to the host roots of sunflower (Helianthus annuus L.) leading to a significant reduction in yield worldwide. The emergence of O. cumana shoots after its underground life-cycle causes irreversible damage to the crop. In this study, a fast visual, non-invasive and precise method for monitoring changes in spectral characteristics using visible and near-infrared (Vis-NIR) hyperspectral imaging (HSI) was developed. By combining the bands sensitive to antioxidant enzymes (SOD, GR), non-antioxidant enzymes (GSH, GSH + GSSG), MDA, ROS (O2-, OH-), PAL, and PPO activities obtained from the host leaves, we sought to establish an accurate means of assessing these changes and conducted imaging acquisition using hyperspectral cameras from both infested and non-infested sunflower cultivars, followed by physio-biochemical parameters measurement as well as analyzed the expression of defense related genes. Extreme learning machine (ELM) and convolutional neural network (CNN) models using 3-band images were built to classify infected or non-infected plants in three sunflower cultivars, achieving accuracies of 95.83% and 95.83% for the discrimination of infestation as well as 97.92% and 95.83% of varieties, respectively, indicating the potential of multi-spectral imaging systems for early detection of O. cumana in weed management.

Anti-inflammatory and in silico docking studies of Litsea wightiana (Nees) Hook.f. (Lauraceae) leaf constituents.

Current study aimed to disclose the anti-inflammatory potential of the methanolic leaf extracts of L. wightiana (LWME). The in vitro studies focused on enzyme inhibition assays targeting the key enzymes such as cyclooxygenase, lipoxygenase and nitric oxide synthase and revealed that LWME effectively inhibited the activity of these enzymes. Gene expression studies confirmed the anti-inflammatory effect, demonstrating down regulation of genes associated with inflammation and key proinflammatory factors such as COX-2, TNF-α, IL-6 and NFkB. In vivo anti-inflammatory experiments by carrageenan-induced paw edoema method in model animals and inflammation was found to be reduced by 10% concentration of extract and significant at P˂0.001 level. GCMS and LCMS analysis were conducted and the resulted compounds were docked against target proteins indicated that most of the bioactive compounds showed better binding affinity with enzymes in which the dicentrinone showed higher affinity and it may be useful in the treatment of several ailments.

Genome-wide identification and expression analysis of the U-box E3 ubiquitin ligase gene family related to bacterial wilt resistance in tobacco (Nicotiana tabacum L.) and eggplant (Solanum melongena L.).

The E3 enzyme in the UPS pathway is a crucial factor for inhibiting substrate specificity. In Solanaceae, the U-box E3 ubiquitin ligase has a complex relationship with plant growth and development, and plays a pivotal role in responding to various biotic and abiotic stresses. The analysis of the U-box gene family in Solanaceae and its expression profile under different stresses holds significant implications. A total of 116 tobacco NtU-boxs and 56 eggplant SmU-boxs were identified based on their respective genome sequences. Phylogenetic analysis of U-box genes in tobacco, eggplant, tomato, Arabidopsis, pepper, and potato revealed five distinct subgroups (I-V). Gene structure and protein motifs analysis found a high degree of conservation in both exon/intron organization and protein motifs among tobacco and eggplant U-box genes especially the members within the same subfamily. A total of 15 pairs of segmental duplication and 1 gene pair of tandem duplication were identified in tobacco based on the analysis of gene duplication events, while 10 pairs of segmental duplication in eggplant. It is speculated that segmental duplication events are the primary driver for the expansion of the U-box gene family in both tobacco and eggplant. The promoters of NtU-box and SmU-box genes contained cis-regulatory elements associated with cellular development, phytohormones, environment stress, and photoresponsive elements. Transcriptomic data analysis shows that the expression levels of the tobacco and eggplant U-box genes in different tissues and various abiotic stress conditions. Using cultivar Hongda of tobacco and cultivar Yanzhi of eggplant as materials, qRT-PCR analysis has revealed that 15 selected NtU-box genes and 8 SmU-box may play important roles in response to pathogen Ras invasion both in tobacco and eggplant.

Cell-type-specific expression analysis of liver transcriptomics with clinical parameters to decipher the cause of intrahepatic inflammation in chronic hepatitis B.

Functional cure for chronic hepatitis B (CHB) remains challenging due to the lack of direct intervention methods for hepatic inflammation. Multi-omics research offers a promising approach to understand hepatic inflammation mechanisms in CHB. A Bayesian linear model linked gene expression with clinical parameters, and population-specific expression analysis (PSEA) refined bulk gene expression into specific cell types across different clinical phases. These models were integrated into our analysis of key factors like inflammatory cells, immune activation, T cell exhaustion, chemokines, receptors, and interferon-stimulated genes (ISGs). Validation through multi-immune staining in liver specimens from CHB patients bolstered our findings. In CHB patients, increased gene expression related to immune cell activation and migration was noted. Marker genes of macrophages, T cells, immune-negative regulators, chemokines, and ISGs showed a positive correlation with serum alanine aminotransferase (ALT) levels but not hepatitis B virus DNA levels. The PSEA model confirmed T cells as the source of exhausted regulators, while macrophages primarily contributed to chemokine expression. Upregulated ISGs (ISG20, IFI16, TAP2, GBP1, PSMB9) in the hepatitis phase were associated with T cell and macrophage infiltration and positively correlated with ALT levels. Conversely, another set of ISGs (IFI44, ISG15, IFI44L, IFI6, MX1) mainly expressed by hepatocytes and B cells showed no correlation with ALT levels. Our study presents a multi-omics analysis integrating bulk transcriptomic, single-cell sequencing data, and clinical data from CHB patients to decipher the cause of intrahepatic inflammation in CHB. The findings confirm that macrophages secrete chemokines like CCL20, recruiting exhausted T cells into liver tissue; concurrently, hepatocyte innate immunity is suppressed, hindering the antiviral effects of ISGs.

Heterologous Expression of Chrysanthemum TCP Transcription Factor CmTCP13 Enhances Salinity Tolerance in Arabidopsis.

Plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) proteins play critical roles in plant development and stress responses; however, their functions in chrysanthemum (Chrysanthemum morifolium) have not been well-studied. In this study, we isolated and characterized the chrysanthemum TCP transcription factor family gene CmTCP13, a homolog of AtTCP13. This gene encoded a protein harboring a conserved basic helix-loop-helix motif, and its expression was induced by salinity stress in chrysanthemum plants. Subcellular localization experiments showed that CmTCP13 localized in the nucleus. Sequence analysis revealed the presence of multiple stress- and hormone-responsive cis-elements in the promoter region of CmTCP13. The heterologous expression of CmTCP13 in Arabidopsis plants enhanced their tolerance to salinity stress. Under salinity stress, CmTCP13 transgenic plants exhibited enhanced germination, root length, seedling growth, and chlorophyll content and reduced relative electrical conductivity compared with those exhibited by wild-type (WT) plants. Moreover, the expression levels of stress-related genes, including AtSOS3, AtP5CS2, AtRD22, AtRD29A, and AtDREB2A, were upregulated in CmTCP13 transgenic plants than in WT plants under salt stress. Taken together, our results demonstrate that CmTCP13 is a critical regulator of salt stress tolerance in plants.

Identification of Pathogenic Pathways for Recurrence of Focal Segmental Glomerulosclerosis after Kidney Transplantation.

Primary focal segmental glomerulosclerosis (FSGS) is a disease of the podocytes and glomerulus, leading to nephrotic syndrome and progressive loss of renal function. One of the most serious aspects is its recurrence of disease in over 30% of patients following allogeneic kidney transplantation, leading to early graft loss. This research investigates the individual genetic predispositions and differences in the immune responses leading to recurrence of FSGS after transplantation. We performed exome sequencing on six patients with recurrent FSGS to identify variants in fifty-one genes and found significant variations in the alpha-2-macroglobulin (A2M). Immunoblotting was used to investigate effects of specific gene variants at the protein level. Further expression analysis identified A2M, exophilin 5 (EXPH5) and plectin (PLEC) as specific proteins linked to podocytes, endothelial cells, and the glomerulus. Subsequent protein array screening revealed the presence of non-HLA-specific antibodies, including TRIM21, after transplantation. Using Metascape for pathway and process enrichment analysis, we focused on the IL-17 signaling and chemotaxis pathways. ELISA measurements showed significantly elevated IL-17 levels in patients with recurrent FSGS (32.30 ± 9.12 pg/mL) compared to individuals with other glomerular diseases (23.16 ± 2.49 pg/mL; p < 0.01) and healthy subjects (22.28 ± 0.94 pg/mL; p < 0.01), with no significant difference in plasma CCL2/MCP-1 levels between groups. This study explores the molecular dynamics underlying recurrence of FSGS after transplantation, offering insights into potential biomarkers and therapeutic targets for the future development of individualized treatments for transplant patients.

Divergent Roles of the Auxin Response Factors in Lemongrass (Cymbopogon flexuosus (Nees ex Steud.) W. Watson) during Plant Growth.

Auxin Response Factors (ARFs) make up a plant-specific transcription factor family that mainly couples perception of the phytohormone, auxin, and gene expression programs and plays an important and multi-faceted role during plant growth and development. Lemongrass (Cymbopogon flexuosus) is a representative Cymbopogon species widely used in gardening, beverages, fragrances, traditional medicine, and heavy metal phytoremediation. Biomass yield is an important trait for several agro-economic purposes of lemongrass, such as landscaping, essential oil production, and phytoremediation. Therefore, we performed gene mining of CfARFs and identified 26 and 27 CfARF-encoding genes in each of the haplotype genomes of lemongrass, respectively. Phylogenetic and domain architecture analyses showed that CfARFs can be divided into four groups, among which groups 1, 2, and 3 correspond to activator, repressor, and ETTN-like ARFs, respectively. To identify the CfARFs that may play major roles during the growth of lemongrass plants, RNA-seq was performed on three tissues (leaf, stem, and root) and four developmental stages (3-leaf, 4-leaf, 5-leaf. and mature stages). The expression profiling of CfARFs identified several highly expressed activator and repressor CfARFs and three CfARFs (CfARF3, 18, and 35) with gradually increased levels during leaf growth. Haplotype-resolved transcriptome analysis revealed that biallelic expression dominance is frequent among CfARFs and contributes to their gene expression patterns. In addition, co-expression network analysis identified the modules enriched with CfARFs. By establishing orthologous relationships among CfARFs, sorghum ARFs, and maize ARFs, we showed that CfARFs were mainly expanded by whole-genome duplications, and that the duplicated CfARFs might have been divergent due to differential expression and variations in domains and motifs. Our work provides a detailed catalog of CfARFs in lemongrass, representing a first step toward characterizing CfARF functions, and may be useful in molecular breeding to enhance lemongrass plant growth.

Identification and Expression Analysis of Cytochrome P450 Genes Probably Involved in Triterpenoid Saponins Biosynthesis in Astragalus mongholicus.

Cytochromes P450 (P450s) are one of the largest enzymatic protein families and play critical roles in the synthesis and metabolism of plant secondary metabolites. Astragaloside IV (AS-IV) is one of the primary active components in Astragalus herbs, exhibiting diverse biological activities and pharmacological effects. However, P450s involved in the astragaloside biosynthesis have not been systematically analyzed in Astragalus mongholicus (A. mongholicus). In this study, we identified 209 P450 genes from the genome of A. mongholicus (AmP450s), which were classified into nine clans and 47 families and performed a systematic overview of their physical and chemical properties, phylogeny, gene structures and conserved motifs. Weighted gene co-expression network analysis (WGCNA) revealed that AmP450s are critical in the astragaloside biosynthesis pathway. The expression levels of these AmP450s were verified by quantitative real-time PCR (qRT-PCR) analysis in the root, stem and leaf, showing that most AmP450s are abundant in the root. Additionally, the correlation analysis between gene expressions and AS-IV content showed that twelve AmP450s, especially CYP71A28, CYP71D16 and CYP72A69, may have significant potential in the biosynthesis of astragaloside. This study systematically investigates the P450s of A. mongholicus and offers valuable insights into further exploring the functions of CYP450s in the astragaloside biosynthesis pathway.

Identification, Expression, Characteristic Analysis, and Immune Function of Two Akirin Genes in Grass Carp (Ctenopharyngodon idella).

Intensive aquaculture of grass carp often leads to decreased immunity and increased disease prevalence, resulting in economic losses. Improving grass carp immunity is therefore a critical strategy for addressing these challenges. Akirin reportedly participates in myogenesis, growth, and immune responses. However, its role in grass carp remains unclear. Herein, we isolated akirins from the spleen of grass carp and analyzed their tissue-specific expression. Akirin expression was detected following treatment with poly (I:C), LPS, and Aeromonas hydrophila (A. hydrophila). The immunological function of the akirin protein was evaluated in head kidney leukocytes (HKLs). The results revealed that the coding sequence (CDS) of akirin1 is 570 bp, encoding 189 amino acids. There was one predicted nuclear localization signal (NLS) and two predicted α- helix domains. The CDS of akirin2 is 558 bp, encoding 185 amino acids. There were two predicted NLSs and two predicted α-helix domains. Tissue-specific expression analysis showed that akirins are widely detected in grass carp tissues. akirin1 was highly detected in the brain, kidneys, heart, spleen, and gonads, while akirin2 was highly detected in the brain, liver, gonads, kidneys, spleen, and heart. The mRNA levels of akirins were promoted after treatment with poly (I:C), LPS, and A. hydrophila. Recombinant akirin proteins were produced in Escherichia coli (E. coli). il-1ß, ifnγ, il-6, tnfα, il-4, iκbα, and nfκb were markedly increased in grass carp HKLs by treatment with the akirin protein. These results suggest that akirins play a role in the immunological regulation of grass carp.

Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer Chilo sacchariphagus (Lepidoptera: Pyralidae).

Chilo sacchariphagus (Lepidoptera: Pyralidae) is an economically important sugarcane pest. Although numerous studies were conducted on the physiological responses in C. sacchariphagus, little is known regarding the genes regulating these physiological processes. Gene expression analysis by qRT-PCR can offer a significant indication for functional gene studies. To our knowledge, the reference genes of C. sacchariphagus have not been screened or evaluated, which hinders the functional gene study. In the present study, the stability of seven reference genes (ß-ACT, GAPDH, BTF3, 28S, RPL7, EF1α, and SDHA) was evaluated in C. sacchariphagus under different experimental conditions, including tissues (antenna, head, thorax, abdomen, leg, and wing), temperatures (4 °C, 25 °C, and 37 °C) and sexes (male and female), through RefFinder, which integrates four algorithms (Normfinder, BestKeeper, ΔCt method, and geNorm). The findings suggested that the combination of ß-ACT and RPL7 is ideal to analyze gene expressions in different tissues and at distinct temperatures, and EF1α and SDHA were suitable reference genes for comparing gene expressions between sexes. Finally, the expression profiles of CsacPBP1 gene were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. This study represents the first kind in screening out suitable reference genes for gene expression analysis in C. sacchariphagus. Information from this study is poised to galvanize future inquiry into the gene expression of C. sacchariphagus, an economically important pest of sugarcane.

Functional analysis of two caffeoyl-coenzyme 3 a-o-methyltransferase involved in pear lignin metabolism.

Caffeoyl-coenzyme 3 A-O-methyltransferase (CCoAOMT) plays a crucial role in the lignin synthesis in many higher plants. In this study, nine PbCCoAOMT genes in total were identified from pear, and classified into six categories. We treated pear fruits with hormones abscisic acid (ABA) and methyl jasmonate (MeJA) and salicylic acid (SA) and observed differential expression levels of these genes. Through qRT-PCR, we also preliminarily identified candidate PbCCoAOMT gene, potentially involved in lignin synthesis in pear fruits. Additionally, the overexpression of PbCCoAOMT1/2 in Arabidopsis and pear fruits increased in lignin content. Enzymatic assays showed that recombinant PbCCoAOMT1/2 proteins have similar enzymatic activity in vitro. The Y1H (Yeast one-hybrid) and dual luciferase (dual-LUC) experiments demonstrated that PbMYB25 can bind to the AC elements in the promoter region of the PbCCoAOMT1 gene. Our findings suggested that the PbCCoAOMT1 and PbCCoAOMT2 genes may contribute to the synthesis of lignin and provide insights into the mechanism of lignin biosynthesis and stone cell development in pear fruits.

Fine Mapping and Candidate Gene Analysis of Two Major Quantitative Trait Loci, qFW2.1 and qFW3.1, Controlling Fruit Weight in Pepper (Capsicum annuum).

Fruit weight is an important agronomic trait in pepper production and is closely related to yield. At present, many quantitative trait loci (QTL) related to fruit weight have been found in pepper; however, the genes affecting fruit weight remain unknown. We analyzed the fruit weight-related quantitative traits in an intraspecific Capsicum annuum cross between the cultivated species blocky-type pepper, cv. Qiemen, and the bird pepper accession, "129-1" (Capsicum annuum var. glatriusculum), which was the wild progenitor of C. annuum. Using the QTL-seq combined with the linkage-based QTL mapping approach, QTL detection was performed; and two major effects of QTL related to fruit weight, qFW2.1 and qFW3.1, were identified on chromosomes 2 and 3. The qFW2.1 maximum explained 12.28% of the phenotypic variance observed in two F2 generations, with the maximum LOD value of 11.02, respectively; meanwhile, the qFW3.1 maximum explained 15.50% of the observed phenotypic variance in the two F2 generations, with the maximum LOD value of 11.36, respectively. qFW2.1 was narrowed down to the 1.22 Mb region using homozygous recombinant screening from BC2S2 and BC2S3 populations, while qFW3.1 was narrowed down to the 4.61Mb region. According to the transcriptome results, a total of 47 and 86 differentially expressed genes (DEGs) in the candidate regions of qFW2.1 and qFW3.1 were identified. Further, 19 genes were selected for a qRT-PCR analysis based on sequence difference combined with the gene annotation. Finally, Capana02g002938 and Capana02g003021 are the most likely candidate genes for qFW2.1, and Capana03g000903 may be a candidate gene for qFW3.1. Taken together, our results identified and fine-mapped two major QTL for fruit weight in pepper that will facilitate marker-assistant breeding for the manipulation of yield in pepper.