African lungfish genome sheds light on the vertebrate water-to-land transition.

Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.

Integrative omics analysis elucidates the genetic basis underlying seed weight and oil content in soybean.

Synergistic optimization of key agronomic traits by traditional breeding has dramatically enhanced crop productivity in the past decades. However, the genetic basis underlying coordinated regulation of yield- and quality-related traits remains poorly understood. Here, we dissected the genetic architectures of seed weight and oil content by combining genome-wide association studies (GWAS) and transcriptome-wide association studies (TWAS) using 421 soybean (Glycine max) accessions. We identified 26 and 33 genetic loci significantly associated with seed weight and oil content by GWAS, respectively, and detected 5,276 expression quantitative trait loci (eQTLs) regulating expression of 3,347 genes based on population transcriptomes. Interestingly, a gene module (IC79), regulated by two eQTL hotspots, exhibited significant correlation with both seed weigh and oil content. Twenty-two candidate causal genes for seed traits were further prioritized by TWAS, including Regulator of Weight and Oil of Seed 1 (GmRWOS1), which encodes a sodium pump protein. GmRWOS1 was verified to pleiotropically regulate seed weight and oil content by gene knockout and overexpression. Notably, allelic variations of GmRWOS1 were strongly selected during domestication of soybean. This study uncovers the genetic basis and network underlying regulation of seed weight and oil content in soybean and provides a valuable resource for improving soybean yield and quality by molecular breeding.

Identification of BMAL1-Regulated circadian genes in mouse liver and their potential association with hepatocellular carcinoma: Gys2 and Upp2 as promising candidates.

Identification and functional analysis of key genes regulated by the circadian clock system will provide a comprehensive understanding of the underlying mechanisms through which circadian clock disruption impairs the health of living organisms. The initial phase involved bioinformatics analysis, drawing insights from three RNA-seq datasets (GSE184303, GSE114400, and GSE199061) derived from wild-type mouse liver tissues, which encompassed six distinct time points across a day. As expected, 536 overlapping genes exhibiting rhythmic expression patterns were identified. By intersecting these genes with differentially expressed genes (DEGs) originating from liver RNA-seq data at two representative time points (circadian time, CT: CT2 and CT14) in global Bmal1 knockout mice (Bmal1-/-), hepatocyte-specific Bmal1 knockout mice (L-Bmal1-/-), and their corresponding control groups, 80 genes potentially regulated by BMAL1 (referred to as BMAL1-regulated genes, BRGs) were identified. These genes were significantly enriched in glycolipid metabolism, immune response, and tumorigenesis pathways. Eight BRGs (Nr1d1, Cry1, Gys2, Homer2, Serpina6, Slc2a2, Nmrk1, and Upp2) were selected to validate their expression patterns in both control and L-Bmal1-/- mice livers over 24 h. Real-time quantitative polymerase chain reaction results demonstrated a comprehensive loss of rhythmic expression patterns in the eight selected BRGs in L-Bmal1-/- mice, in contrast to the discernible rhythmic patterns observed in the livers of control mice. Additionally, significant reductions in the expression levels of these selected BRGs, excluding Cry1, were also observed in L-Bmal1-/- mice livers. Chromatin immunoprecipitation (ChIP)-seq (GSE13505 and GSE39860) and JASPAR analyses validated the rhythmic binding of BMAL1 to the promoter and intron regions of these genes. Moreover, the progression of conditions, from basic steatosis to non-alcoholic fatty liver disease, and eventual malignancy, demonstrated a continuous gradual decline in Bmal1 transcripts in the human liver. Combining the aforementioned BRGs with DEGs derived from human liver cancer datasets identified Gys2 and Upp2 as potential node genes bridging the circadian clock system and hepatocellular carcinoma (HCC). In addition, CCK8 and wound healing assays demonstrated that the overexpression of human GYS2 and UPP2 proteins inhibited the proliferation and migration of HepG2 cells, accompanied by elevated expression of p53, a tumor suppressor protein. In summary, this study systematically identified rhythmic genes in the mouse liver, and a subset of circadian genes potentially regulated by BMAL1. Two circadian genes, Gys2 and Upp2, have been proposed and validated as potential candidates for advancing the prevention and treatment of HCC.

Genome-Wide Association Study and Identification of Candidate Genes Associated with Seed Number per Pod in Soybean.

Soybean (Glycine max [L.] Merr.) is one of the primary sources of plant protein and oil for human foods, animal feed, and industrial processing. The seed number per pod generally varies from one to four and is an important component of seed number per unit area and seed yield. We used natural variation in 264 landraces and improved cultivars or lines to identify candidate genes involved in the regulation of seed number per pod in soybean. Genome-wide association tests revealed 65 loci that are associated with seed number per pod trait. Among them, 11 could be detected in multiple environments. Candidate genes were identified for seed number per pod phenotype from the most significantly associated loci, including a gene encoding protein argonaute 4, a gene encoding histone acetyltransferase of the MYST family 1, a gene encoding chromosome segregation protein SMC-1 and a gene encoding exocyst complex component EXO84A. In addition, plant hormones were found to be involved in ovule and seed development and the regulation of seed number per pod in soybean. This study facilitates the dissection of genetic networks underlying seed number per pod in soybean, which will be useful for the genetic improvement of seed yield in soybean.

Hypoxia activation attenuates progesterone synthesis in goat trophoblast cells via NR1D1 inhibition of StAR expression†.

Trophoblast plays a crucial role in gestation maintenance and embryo implantation, partly due to the synthesis of progesterone. It has been demonstrated that hypoxia regulates invasion, proliferation, and differentiation of trophoblast cells. Additionally, human trophoblasts display rhythmic expression of circadian clock genes. However, it remains unclear if the circadian clock system is present in goat trophoblast cells (GTCs), and its involvement in hypoxia regulation of steroid hormone synthesis remains elusive. In this study, immunofluorescence staining revealed that both BMAL1 and NR1D1 (two circadian clock components) were highly expressed in GTCs. Quantitative real-time PCR analysis showed that several circadian clock genes were rhythmically expressed in forskolin-synchronized GTCs. To mimic hypoxia, GTCs were treated with hypoxia-inducing reagents (CoCl2 or DMOG). Quantitative real-time PCR results demonstrated that hypoxia perturbed the mRNA expression of circadian clock genes and StAR. Notably, the increased expression of NR1D1 and the reduction of StAR expression in hypoxic GTCs were also detected by western blotting. In addition, progesterone secretion exhibited a notable decline in hypoxic GTCs. SR9009, an NR1D1 agonist, significantly decreased StAR expression at both the mRNA and protein levels and markedly inhibited progesterone secretion in GTCs. Moreover, SR8278, an NR1D1 antagonist, partially reversed the inhibitory effect of CoCl2 on mRNA and protein expression levels of StAR and progesterone synthesis in GTCs. Our results demonstrate that hypoxia reduces StAR expression via the activation of NR1D1 signaling in GTCs, thus inhibiting progesterone synthesis. These findings provide new insights into the NR1D1 regulation of progesterone synthesis in GTCs under hypoxic conditions.

MSX1 Regulates Goat Endometrial Function by Altering the Plasma Membrane Transformation of Endometrial Epithelium Cells during Early Pregnancy.

MSX1 is an important member of the muscle segment homeobox gene (Msh) family and acts as a transcription factor to regulate tissue plasticity, yet its role in goat endometrium remodeling remains elusive. In this study, an immunohistochemical analysis showed that MSX1 was mainly expressed in the luminal and glandular epithelium of goat uterus, and the MSX1 expression was upregulated in pregnancy at days 15 and 18 compared with pregnancy at day 5. In order to explore its function, goat endometrial epithelial cells (gEECs) were treated with 17 ß-estrogen (E2), progesterone (P4), and/or interferon-tau (IFNτ), which were used to mimic the physiological environment of early pregnancy. The results showed that MSX1 was significantly upregulated with E2- and P4-alone treatment, or their combined treatment, and IFNτ further enhanced its expression. The spheroid attachment and PGE2/PGF2α ratio were downregulated by the suppression of MSX1. The combination of E2, P4, and IFNτ treatment induced the plasma membrane transformation (PMT) of gEECs, which mainly showed the upregulation of N-cadherin (CDH2) and concomitant downregulation of the polarity-related genes (ZO-1, α-PKC, Par3, Lgl2, and SCRIB). The knockdown of MSX1 partly hindered the PMT induced by E2, P4, and IFNτ treatment, while the upregulation of CDH2 and the downregulation of the partly polarity-related genes were significantly enhanced when MSX1 was overexpressed. Moreover, MSX1 regulated the CDH2 expression by activating the endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) pathway. Collectively, these results suggest that MSX1 was involved in the PMT of the gEECs through the ER stress-mediated UPR pathway, which affects endometrial adhesion and secretion function.

Transcription Factor Nrf2 Modulates Lipopolysaccharide-Induced Injury in Bovine Endometrial Epithelial Cells.

Endometritis in high-yield dairy cows adversely affects lactation length, milk quality, and the economics of dairy products. Endoplasmic reticulum stress (ERS) in bovine endometrial epithelial cells (BEECs) occurs as a consequence of diverse post-natal stressors, and plays a key role in a variety of inflammatory diseases. Nuclear-factor-erythroid-2-related factor 2 (Nrf2) is an important protective regulatory factor in numerous inflammatory responses. However, the mechanism by which Nrf2 modulates inflammation by participating in ERS remains unclear. The objective of the present study was to explore the role of Nrf2 in lipopolysaccharide (LPS)-induced injury to BEECs and to decipher the underlying molecular mechanisms of this injury. The expression of Nrf2- and ERS-related genes increased significantly in bovine uteri with endometritis. Isolated BEECs were treated with LPS to stimulate the inflammatory response. The expression of Nrf2 was significantly higher in cells exposed to LPS, which also induced ERS in BEECs. Activation of Nrf2 led to enhanced expression of the genes for the inflammation markers TNF-α, p65, IL-6, and IL-8 in BEECs. Moreover, stimulation of Nrf2 was accompanied by activation of ERS. In contrast, Nrf2 knockdown reduced the expression of TNF-α, p65, IL-6, and IL-8. Additionally, Nrf2 knockdown decreased expression of ERS-related genes for the GRP78, PERK, eIF2α, ATF4, and CHOP proteins. Collectively, our findings demonstrate that Nrf2 and ERS are activated during inflammation in BEECs. Furthermore, Nrf2 promotes the inflammatory response by activating the PERK pathway in ERS and inducing apoptosis in BEECs.

A Comparative Proteomic Analysis to Explore the Influencing Factors on Endometritis Using LC-MS/MS.

The inflammatory system activated by uterine infection is associated with decreased fertility. Diseases can be detected in advance by identifying biomarkers of several uterine diseases. Escherichia coli is one of the most frequent bacteria that is involved in pathogenic processes in dairy goats. The purpose of this study was to investigate the effect of endotoxin on protein expression in goat endometrial epithelial cells. In this study, the LC-MS/MS approach was employed to investigate the proteome profile of goat endometrial epithelial cells. A total of 1180 proteins were identified in the goat Endometrial Epithelial Cells and LPS-treated goat Endometrial Epithelial Cell groups, of which, 313 differentially expressed proteins were accurately screened. The proteomic results were independently verified by WB, TEM and IF techniques, and the same conclusion was obtained. To conclude, this model is suitable for the further study of infertility caused by endometrial damage caused by endotoxin. These findings may provide useful information for the prevention and treatment of endometritis.

Alr Gene in Brucella suis S2: Its Role in Lipopolysaccharide Biosynthesis and Bacterial Virulence in RAW264.7.

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella's survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

Circadian clock regulates granulosa cell autophagy through NR1D1-mediated inhibition of ATG5.

Autophagy of granulosa cells (GCs) is involved in follicular atresia, which occurs repeatedly during the ovarian development cycle. Several circadian clock genes are rhythmically expressed in both rodent ovarian tissues and GCs. Nuclear receptor subfamily 1 group D member 1 (NR1D1), an important component of the circadian clock system, is involved in the autophagy process through the regulation of autophagy-related genes. However, there are no reports illustrating the role of the circadian clock system in mouse GC autophagy. In the present study, we found that core circadian clock genes (Bmal1, Per2, Nr1d1, and Dbp) and an autophagy-related gene (Atg5) exhibited rhythmic expression patterns across 24 h in mouse ovaries and primary GCs. Treatment with SR9009, an agonist of NR1D1, significantly reduced the expression of Bmal1, Per2, and Dbp in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, Nr1d1 knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of Bmal1-/- mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited Atg5 transcription by binding to two putative retinoic acid-related orphan receptor response elements within the promoter. In addition, rapamycin-induced autophagy and ATG5 expression were partially reversed by SR9009 treatment in mouse GCs. Taken together, our current data demonstrated that the circadian clock regulates GC autophagy through NR1D1-mediated inhibition of ATG5 expression, and thus, plays a role in maintaining autophagy homeostasis in GCs.

HSP70-3 Interacts with Phospholipase Dδ and Participates in Heat Stress Defense.

Heat shock proteins (HSPs) function as molecular chaperones and are key components responsible for protein folding, assembly, translocation, and degradation under stress conditions. However, little is known about how HSPs stabilize proteins and membranes in response to different hormonal or environmental cues in plants. Here, we combined molecular, biochemical, and genetic approaches to elucidate the involvement of cytosolic HSP70-3 in plant stress responses and the interplay between HSP70-3 and plasma membrane (PM)-localized phospholipase Dδ (PLDδ) in Arabidopsis (Arabidopsis thaliana). Analysis using pull-down, coimmunoprecipitation, and bimolecular fluorescence complementation revealed that HSP70-3 specifically interacted with PLDδ. HSP70-3 bound to microtubules, such that it stabilized cortical microtubules upon heat stress. We also showed that heat shock induced recruitment of HSP70-3 to the PM, where HSP70-3 inhibited PLDδ activity to mediate microtubule reorganization, phospholipid metabolism, and plant thermotolerance, and this process depended on the HSP70-3-PLDδ interaction. Our results suggest a model whereby the interplay between HSP70-3 and PLDδ facilitates the re-establishment of cellular homeostasis during plant responses to external stresses and reveal a regulatory mechanism in regulating membrane lipid metabolism.

BtpB inhibits innate inflammatory responses in goat alveolar macrophages through the TLR/NF-κB pathway and NLRP3 inflammasome during Brucella infection.

Brucella species are infectious facultative intracellular pathogens. They have evolved multiple strategies to thwart immune responses and replicate in macrophages for chronic persistence in the host. As a Brucella effector, BtpB is transferred into target cells through the type IV secretion system. BtpB, a Toll/interleukin-1 receptor domain-containing protein, blocks host innate immune responses by interfering with Toll-like receptor signaling. However, the intracellular targets and their activated downstream pathways remain unclear. In this study, we constructed a strain of Brucella suis S2 with a deletion in the gene for BtpB, ΔbtpB, and the complemented strain, C-ΔbtpB with a restored copy of the btpB gene. The bacterial growth curves and stress resistance results showed that BtpB did not affect B. suis S2 growth. Infection of alveolar macrophages with WT and ΔbtpB strains showed that BtpB inhibited TLR2 and TLR4 expression and attenuated NLRP3 inflammasome activation. BtpB also attenuated secretion of the Brucella-induced proinflammatory cytokines, IL-1ß, IL-6, and TNF-α, in alveolar macrophages while up-regulating IL-10 expression. In general, the results confirmed that BtpB specifically inhibits TLR2/TLR4 and disrupts NLRP3 signaling pathways to inhibit host immune responses in early Brucella infections.

GmAOC4 modulates seed germination by regulating JA biosynthesis in soybean.

KEY MESSAGE: An allene oxide cyclase 4, GmAOC4, was determined by GWAS and RT-PCR to be significantly associated with seed germination in soybean, and regulates seed germination by promoting more JA accumulation. The seed germination phase is a critical component of the plant lifecycle, and a better understanding of the mechanism behind seed germination in soybeans is needed. We used a genome-wide association study (GWAS) to detect a GWAS signal on chromosome 18. In this GWAS signal, SNP S18_56189166 was located within the 3'untranslated region of Glyma.18G280900, which encodes allene oxide cyclase 4 (named GmAOC4). Analysis of real-time PCR demonstrated that expression levels of GmAOC4 in the low-germination variety (KF, carrying SNP S18_56189166-T) were higher than in the high-germination variety (NN, carrying SNP S18_56189166-C). In these two varieties, KF showed a higher JA concentration than NN at 0 and 24 h after imbibition. Moreover, the overexpression of GmAOC4 led to an increase in the concentration of jasmonic acid (JA) in soybean hairy roots and Arabidopsis thaliana. Furthermore, it was found that GmAOC4-OE lines showed less seed germination than the wild type (WT) under normal conditions in Arabidopsis. After 7 days of ABA treatment, transgenic lines exhibited lower seed germination and higher expression levels of AtABI5 compared with WT, indicating that the overexpression of GmAOC4 resulted in hypersensitivity to ABA. Our findings demonstrate that GmAOC4, which promotes more JA accumulation, helps to regulate seed germination in soybeans.

Autophagy Mediates Escherichia Coli-Induced Cellular Inflammatory Injury by Regulating Calcium Mobilization, Mitochondrial Dysfunction, and Endoplasmic Reticulum Stress.

Bovine endometritis is a reproductive disorder that is induced by mucus or purulent inflammation of the uterine mucosa. However, the intracellular control chain during inflammatory injury remains unclear. In the present study, we found that E. coli activated the inflammatory response through the assembly of the NLRP3 inflammasome and activation of the NF-κB p65 subunit in primary bovine endometrial epithelial cells (bEECs). Infection with E. coli also led to an abnormal increase in cytoplasmic calcium and mitochondrial dysfunction. Additionally, live-cell imaging of calcium reporters indicated that the increase in cytosolic calcium mainly was caused by the release of Ca2+ ions stored in the ER and mitochondria, which was independent of extracellular calcium. Cytoplasmic calcium regulates mitochondrial respiratory chain transmission, DNA replication, and biogenesis. Pretreatment with NAC, BAPTA-AM, or 2-APB reduced the expression of IL-1ß and IL-18. Moreover, ERS was involved in the regulation of bovine endometritis and cytosolic calcium was an important factor for regulating ERS in E. coli-induced inflammation. Finally, activation of autophagy inhibited the release of IL-1ß and IL-18, cytochrome c, ATP, ERS-related proteins, and cytoplasmic calcium. Collectively, our findings demonstrate that autophagy mediated E. coli-induced cellular inflammatory injury by regulating cytoplasmic calcium, mitochondrial dysfunction, and ERS.

[Research progress on the regulation of mammalian energy metabolism by the circadian clock system and gut microbiota].

The mammalian internal circadian clock system has been evolved to adapt to the diurnal changes in the internal and external environment of the organism to regulate diverse physiological functions, such as the sleep-wake cycle and feeding rhythm, thereby coordinating the rhythmic changes of energy demand and nutrition supply in each diurnal cycle. The circadian clock regulates glucose metabolism, lipid metabolism, and hormones secretion in diverse tissues and organs, including the liver, skeletal muscle, pancreas, heart, and vessels. As a special "organ" of the host, the gut microbiota, together with the intestinal microenvironment (tissues, cells, and metabolites) in a co-evolutionary process, constitutes a micro-ecosystem and plays an important role in the process of nutrient digestion and absorption in the intestine of the host. In recent years, accumulating evidence indicates that the compositions, quantities, colonization, and functional activities of the gut microbiota exhibit significant circadian variations, which are closely related to the changes of various physiological functions under the regulation of host circadian clock system. In addition, several studies have shown that the gut microbiota can produce many important metabolites such as the short-chain fatty acids through the degradation of indigestive dietary fibers. A portion of gut microbiota-derived metabolites can regulate the circadian clock system and metabolism of the host. This article mainly discusses the interaction between the host circadian clock system and the gut microbiota, and highlights its influence on energy metabolism of the host, providing a novel clues and thought for the prevention and treatment of metabolic diseases.

The Determination Method of Satisfactory Consistency of the Interval Number Pairwise Comparisons Matrix Based on Submatrix.

The decision-maker obtains the pairwise comparisons matrix by comparing two entities. In the process of comparing the two entities, the relationship between the two entities and other entities is not considered. In this way, the judgment may be illogical. This paper mainly studies the satisfactory consistency of the interval number pairwise comparisons matrix based on cyclic matrix. Firstly, the illogical judgment entity in the process of the decision-maker's judgment is expressed by the cyclic matrix. There are three entities and four entities to form the cyclic matrix. The relationship and various forms of the cyclic cycle formed by the four entities and the three entities are discussed; then, the satisfactory consistency of the interval number pairwise comparisons matrix is determined by judging whether there is a cyclic matrix in the submatrix of the interval number pairwise comparisons matrix. Finally, two examples are given to verify the rationality and effectiveness of the method.

Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells.

Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0-5 IU/ml) and 22R-OH-cholesterol (0-30 µM) addition stimulated testosterone synthesis in primary goat LCs in a dose-dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real-time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis-related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1-Luc circadian oscillations were clearly observed in dexamethasone-treated goat LCs transfected with the pLV6-Bmal1-Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis-related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability.

Mapping and validation of a major QTL for primary root length of soybean seedlings grown in hydroponic conditions.

BACKGROUND: The root system provides nutrient absorption and is closely related to abiotic stress tolerance, but it is difficult to study the roots under field conditions. This study was conducted to identify quantitative trait loci (QTL) associated with primary root length (PRL) during soybean seedling growth in hydroponic conditions. A total of 103 F7 recombinant inbred lines (RILs) derived from a cross between K099 (short primary root) and Fendou 16 (long primary root) were used to identify QTL for PRL in soybean. The RIL population was genotyped with 223 simple sequence repeats markers covering 20 chromosomes. Phenotyping for primary root length was performed for 3-weeks plants grown in hydoponic conditions. The identified QTL was validated in near isogenic lines and in a separate RIL population. RESULTS: QTL analysis using inclusive composite interval mapping method identified a major QTL on Gm16 between SSR markers Sat_165 and Satt621, explaining 30.25 % of the total phenotypic variation. The identified QTL, qRL16.1, was further confirmed in a segregating population derived from a residual heterozygous line (RHLs-98). To validate qRL16.1 in a different genetic background, QTL analysis was performed in another F6 RIL population derived from a cross between Union (medium primary root) and Fendou 16, in which a major QTL was detected again in the same genomic region as qRL16.1, explaining 14 % of the total phenotypic variation for PRL. In addition, the effect of qRL16.1 was confirmed using two pair of near-isogenic lines (NILs). PRL was significantly higher in NILs possessing the qRL16.1 allele from Fendou 16 compared to allele from K099. CONCLUSIONS: The qRL16.1 is a novel QTL for primary root length in soybean which provides important information on the genetic control of root development. Identification of this major QTL will facilitate positional cloning and DNA marker-assisted selection for root traits in soybean.

Bmal1 promotes prostaglandin E2 synthesis by upregulating Ptgs2 transcription in response to increasing estradiol levels in day 4 pregnant mice.

Prostaglandin G/H synthase 2 (PTGS2) is a rate-limiting enzyme in prostaglandin synthesis. The present study assessed the role of the uterine circadian clock on Ptgs2 transcription in response to steroid hormones during early pregnancy. We demonstrated that the core clock genes (Bmal1, Per2, Nr1d1, and Dbp), Vegf, and Ptgs2, and their encoded proteins, have rhythmic expression in the mouse uterus from days 3.5 to 4.5 (D3.5-4.5) of pregnancy. Progesterone (P4) treatment of cultured uterus endometrial stromal cells (UESCs) isolated from mPer2Luciferase reporter gene knock-in mice on D4 induced a phase shift in PER2::LUCIFERASE oscillations. This P4-induced phase shift of PER2::LUCIFERASE oscillations was significantly attenuated by the P4 antagonist RU486. Additionally, the amplitude of PER2::LUCIFERASE oscillations was increased by estradiol (E2) treatment in the presence of P4. Consistently, the mRNA levels of clock genes (Bmal1 and Per2), Vegf, and Ptgs2 were markedly increased by E2 treatment of UESCs in the presence of P4. Treatment with E2 also promoted prostaglandin E2 (PGE2) synthesis by UESCs. Depletion of Bmal1 in UESCs by small-interfering RNA (siRNA) decreased the transcript levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2 compared with nonsilencing siRNA treatment. Bmal1 knockdown also inhibited PGE2 synthesis. Moreover, the mRNA expression levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2, and their respective proteins were significantly decreased in the uterus of Bmal1-/- mice. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.NEW & NOTEWORTHY Rhythmic expression of Bmal1 and Ptgs2 was observed in the uterus isolated from D3.5-4.5 of pregnant mice. E2 increased the expression of Bmal1 and Ptg2 in UESCs isolated from mice on D4. The expression of Ptgs2 was significantly decreased in Bmal1-siRNA treated UESCs. Bmal1 knockdown also inhibited PGE2 synthesis. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.

Identification of soybean phosphorous efficiency QTLs and genes using chlorophyll fluorescence parameters through GWAS and RNA-seq.

MAIN CONCLUSION: Soybean phosphorous efficiency QTLs were identified and candidate genes were predicted using chlorophyll fluorescence parameters through GWAS and RNA-seq. Phosphorus (P) is an essential nutrient element for crop growth and development, lack of P uptake seriously affects yield in various crops. Photosynthesis is the basis of crop production, while it is very sensitive to P deficiency. It is of great importance to study the genetic relationship between photosynthesis and P efficiency to provide genetic insight for soybean improvement. In this study, a genome-wide association study (GWAS) was performed using 292,035 SNPs and the ratios of four main chlorophyll fluorescence parameters of 219 diverse soybean accessions under P deficiency and normal P across three experiments. In total, 52 SNPs in 12 genomic regions were detected in association with the four main chlorophyll fluorescence parameters under sufficient or deficient P levels. Combined it with RNA-seq analysis, we predicted three candidate genes for the significant genomic regions. For example, the expression level of the candidate gene (Glyma.18g092900) in P deficiency tolerant accession was three times higher than that of P deficiency sensitive one under phosphorous deficiency condition. This study provides insight into genetic links between photosynthetic and phosphorous efficiency and further functional analysis will provide valuable information for understanding the underlying genetic mechanism to facilitate marker-assisted breeding in soybean.