MicroRNA expression profiles reveal wool development and fineness regulation in Gansu alpine fine-wool sheep.

The development of wool has a complex regulatory mechanism both influenced by genetic and environmental factors. MicroRNAs (miRNA) were involved in various biological processes of animals, and may play an important role in the regulation of wool development. In this study, we comprehensively analyzed and identified the histological parameters of hair follicles, as well as the miRNAs, target genes, pathways, and Gene Ontology terms related to wool fineness regulation and wool growth and development using HE staining and RNA-Seqs methods. Both coarse (group C, mean fiber diameter (MFD) = 22.26 ± 0.69 µm, n = 6) and fine (group F, MFD = 16.91 ± 0.29 µm, n = 6) of Gansu alpine fine-wool sheep with different wool fineness were used in this study. The results showed that the primary follicle diameter and secondary wool fiber diameter in group C were significantly higher than those in group F (P < 0.05). And the number of primary and secondary hair follicles in group C was significantly lower than that in group F (P < 0.05). Furthermore, a total of 67 DE miRNAs and 290 potential DE miRNAs target genes were screened in the skin tissues of sheep from groups F and C, and some potential target genes related to wool fineness regulation were screened, such as CDH2, KRT82, FOXN1, LOC101106296, KRT20, MCOLN3, KRT71, and TERT. These genes were closely related to Glutathione metabolism, epidermal cell differentiation, keratinization, and regulation of hair cycle. Moreover, the regulatory network of miRNAs-mRNAs suggested that miRNAs (miR-129-x, novel m0079-3p, miR-2484-z, novel m0025-5P, etc.) may play a key role in the wool development and wool fineness regulation of Gansu alpine fine-wool sheep. In summary, this study expands the existing miRNAs database and provides new information for studying the regulation of wool development in Gansu alpine fine wool sheep.

Comparative proteomic discovery of salt stress response in alfalfa roots and overexpression of MsANN2 confers salt tolerance.

Soil salinity constrains growth, development and yield of alfalfa (Medicago sativa L.). To illustrate the molecular mechanisms responsible for salt tolerance, a comparative proteome analysis was explored to characterize protein profiles of alfalfa seedling roots exposed to 100 and 200 mM NaCl for three weeks. There were 52 differentially expressed proteins identified, among which the mRNA expressions of 12 were verified by Real-Time-PCR analysis. The results showed increase in abundance of ascorbate peroxidase, POD, CBS protein and PR-10 in salt-stressed alfalfa, suggesting an effectively antioxidant and defense systems. Alfalfa enhanced protein quality control system to refold or degrade abnormal proteins induced by salt stress through upregulation of unfolded protein response (UPR) marker PDIs and molecular chaperones (eg. HSP70, TCP-1, and GroES) as well as the ubiquitin-proteasome system (UPS) including ubiquitin ligase enzyme (E3) and proteasome subunits. Upregulation of proteins responsible for calcium signal transduction including calmodulin and annexin helped alfalfa adapt to salt stress. Specifically, annexin (MsANN2), a key Ca2+-binding protein, was selected for further characterization. The heterologous of the MsANN2 in Arabidopsis conferred salt tolerance. These results provide detailed information for salt-responsive root proteins and highlight the importance of MsANN2 in adapting to salt stress in alfalfa.

Genome-wide identification and analysis of abiotic stress responsiveness of the mitogen-activated protein kinase gene family in Medicago sativa L.

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa. RESULTS: In this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs. Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses. CONCLUSION: This study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance.

Multi-omics integrative analysis provided new insights into alkaline stress in alfalfa.

Saline-alkali stress is one of the main abiotic stresses that limits plant growth. Salt stress has been widely studied, but alkaline salt degradation caused by NaHCO3 has rarely been investigated. In the present study, the alfalfa cultivar 'Zhongmu No. 1' was treated with 50 mM NaHCO3 (0, 4, 8, 12 and 24 h) to study the resulting enzyme activity and changes in mRNA, miRNA and metabolites in the roots. The results showed that the enzyme activity changed significantly after alkali stress treatment. The genomic analysis revealed 14,970 differentially expressed mRNAs (DEMs), 53 differentially expressed miRNAs (DEMis), and 463 differentially accumulated metabolites (DAMs). Combined analysis of DEMs and DEMis revealed that 21 DEMis negatively regulated 42 DEMs. In addition, when combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEMs and DAMs, we found that phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism and plant hormone signal transduction played important roles in the alkali stress response. The results of this study further elucidated the regulatory mechanism underlying the plant response to alkali stress and provided valuable information for the breeding of new saline-alkaline tolerance plant varieties.

MicroRNA-148a Targets DNMT1 and PPARGC1A to Regulate the Viability, Proliferation, and Milk Fat Synthesis of Ovine Mammary Epithelial Cells.

In this study, the expression profiles of miR-148a were constructed in eight different ovine tissues, including mammary gland tissue, during six different developmental periods. The effect of miR-148a on the viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells (OMECs) was investigated, and the target relationship of miR-148a with two predicted target genes was verified. The expression of miR-148a exhibited obvious tissue-specific and temporal-specific patterns. miR-148a was expressed in all eight ovine tissues investigated, with the highest expression level in mammary gland tissue (p < 0.05). Additionally, miR-148a was expressed in ovine mammary gland tissue during each of the six developmental periods studied, with its highest level at peak lactation (p < 0.05). The overexpression of miR-148a increased the viability of OMECs, the number and percentage of Edu-labeled positive OMECs, and the expression levels of two cell-proliferation marker genes. miR-148a also increased the percentage of OMECs in the S phase. In contrast, transfection with an miR-148a inhibitor produced the opposite effect compared to the miR-148a mimic. These results indicate that miR-148a promotes the viability and proliferation of OMECs in Small-tailed Han sheep. The miR-148a mimic increased the triglyceride content by 37.78% (p < 0.01) and the expression levels of three milk fat synthesis marker genes in OMECs. However, the miR-148a inhibitor reduced the triglyceride level by 87.11% (p < 0.01). These results suggest that miR-148a promotes milk fat synthesis in OMECs. The dual-luciferase reporter assay showed that miR-148a reduced the luciferase activities of DNA methyltransferase 1 (DNMT1) and peroxisome proliferator-activated receptor gamma coactivator 1-A (PPARGC1A) in wild-type vectors, suggesting that they are target genes of miR-148a. The expression of miR-148a was highly negatively correlated with PPARGC1A (r = -0.789, p < 0.001) in ovine mammary gland tissue, while it had a moderate negative correlation with DNMT1 (r = -0.515, p = 0.029). This is the first study to reveal the molecular mechanisms of miR-148a underlying the viability, proliferation, and milk fat synthesis of OMECs in sheep.

Circular RNA_015343 sponges microRNA-25 to regulate viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells via INSIG1.

In our previous study, circ_015343 was found to inhibit the viability and proliferation of ovine mammary epithelial cells (OMECs) and the expression levels of milk fat synthesis marker genes, but the regulatory mechanism underlying the processes is still unclear. Accordingly in this study, the target relationships between circ_015343 with miR-25 and between miR-25 with insulin induced gene 1 (INSIG1) were verified, and the functions of miR-25 and INSIG1 were investigated in OMECs. The dual-luciferase reporter assay revealed that miR-25 mimic remarkably decreased the luciferase activity of circ_015343 in HEK293T cells cotransfected with a wild-type vector, while it did not change the activity of circ_015343 in HEK293T cells cotransfected with a mutant vector. These suggest that cic_015343 can adsorb and bind miR-25. The miR-25 increased the viability and proliferation of OMECs, and the content of triglycerides in OMECs. In addition, INSIG1 was found to be a target gene of miR-25 using a dual-luciferase reporter assay. Overexpression of INSIG1 decreased the viability, proliferation, and level of triglycerides of OMECs. In contrast, the inhibition of INSIG1 in expression had the opposite effect on activities and triglycerides of OMECs with overexpressed INSIG1. A rescue experiment revealed that circ_015343 alleviated the inhibitory effect of miR-25 on the mRNA and protein abundance of INSIG1. These results indicate that circ_015343 sponges miR-25 to inhibit the activities and content of triglycerides of OMECs by upregulating the expression of INSIG1 in OMECs. This study provided new insights for understanding the genetic molecular mechanism of lactation traits in sheep.

A genome-wide association study reveals novel loci and candidate genes associated with plant height variation in Medicago sativa.

BACKGROUND: Plant height (PH) is an important agronomic trait influenced by a complex genetic network. However, the genetic basis for the variation in PH in Medicago sativa remains largely unknown. In this study, a comprehensive genome-wide association analysis was performed to identify genomic regions associated with PH using a diverse panel of 220 accessions of M. sativa worldwide. RESULTS: Our study identified eight novel single nucleotide polymorphisms (SNPs) significantly associated with PH evaluated in five environments, explaining 8.59-12.27% of the phenotypic variance. Among these SNPs, the favorable genotype of chr6__31716285 had a low frequency of 16.4%. Msa0882400, located proximal to this SNP, was annotated as phosphate transporter 3;1, and its role in regulating alfalfa PH was supported by transcriptome and candidate gene association analysis. In addition, 21 candidate genes were annotated within the associated regions that are involved in various biological processes related to plant growth and development. CONCLUSIONS: Our findings provide new molecular markers for marker-assisted selection in M. sativa breeding programs. Furthermore, this study enhances our understanding of the underlying genetic and molecular mechanisms governing PH variations in M. sativa.

Genome-Wide Identification, Phylogenetic and Expression Analysis of Expansin Gene Family in Medicago sativa L.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

Overexpression of MsDREB1C Modulates Growth and Improves Forage Quality in Tetraploid Alfalfa (Medicago sativa L.).

DREB has been reported to be involved in plant growth and response to environmental factors. However, the function of DREB in growth and development has not been elucidated in alfalfa (Medicago sativa L.), a perennial tetraploid forage cultivated worldwide. In this study, an ortholog of MtDREB1C was characterized from alfalfa and named MsDREB1C accordingly. MsDREB1C was significantly induced by abiotic stress. The transcription factor MsDREB1C resided in the nucleus and had self-transactivation activity. The MsDREB1C overexpression (OE) alfalfa displayed growth retardation under both long-day and short-day conditions, which was supported by decreased MsGA20ox and upregulated MsGA2ox in the OE lines. Consistently, a decrease in active gibberellin (GA) was detected, suggesting a negative effect of MsDREB1C on GA accumulation in alfalfa. Interestingly, the forage quality of the OE lines was better than that of WT lines, with higher crude protein and lower lignin content, which was supported by an increase in the leaf-stem ratio (LSR) and repression of several lignin-synthesis genes (MsNST, MsPAL1, MsC4H, and Ms4CL). Therefore, this study revealed the effects of MsDREB1C overexpression on growth and forage quality via modifying GA accumulation and lignin synthesis, respectively. Our findings provide a valuable candidate for improving the critical agronomic traits of alfalfa, such as overwintering and feeding value of the forage.

Can energy saving and emission reduction policies promote green transformation of industrial enterprises--The Case of China.

In the context of the continued advancement of the green economy transition, the proactive pursuit of carbon emissions reduction and the early attainment of carbon neutrality goals have emerged as essential components in promoting high-quality economic development. Not only does it contribute to the creation of a community of human destiny, but it is also vital to the realization of sustainable development for human civilization. A dynamic evolutionary game model, which encompasses the interactions among government, enterprises, and the public, was constructed to examine the inherent impact mechanisms of the behavior of three players on the development of a green economy under the context of energy saving and emission reduction subsidies. The results showed that the incentive and punishment mechanisms served as effective tools for harmonizing the interests of system members. Within the mechanisms, the public demonstrated a higher sensitivity to rewards, while enterprises exhibited greater responsiveness to fines. Consequently, the government could influence the behavior of enterprises by incentivizing the public to serve as a third-party inquiry and oversight body. Simultaneously, the government could encourage enterprises to expedite green technology innovation by employing a combination of incentive and punishment mechanisms.

Evolutionary genomics of climatic adaptation and resilience to climate change in alfalfa.

Given the escalating impact of climate change on agriculture and food security, gaining insights into the evolutionary dynamics of climatic adaptation and uncovering climate-adapted variation can empower the breeding of climate-resilient crops to face future climate change. Alfalfa (Medicago sativa subsp. sativa), the queen of forages, shows remarkable adaptability across diverse global environments, making it an excellent model for investigating species responses to climate change. In this study, we performed population genomic analyses using genome resequencing data from 702 accessions of 24 Medicago species to unravel alfalfa's climatic adaptation and genetic susceptibility to future climate change. We found that interspecific genetic exchange has contributed to the gene pool of alfalfa, particularly enriching defense and stress-response genes. Intersubspecific introgression between M. sativa subsp. falcata (subsp. falcata) and alfalfa not only aids alfalfa's climatic adaptation but also introduces genetic burden. A total of 1671 genes were associated with climatic adaptation, and 5.7% of them were introgressions from subsp. falcata. By integrating climate-associated variants and climate data, we identified populations that are vulnerable to future climate change, particularly in higher latitudes of the Northern Hemisphere. These findings serve as a clarion call for targeted conservation initiatives and breeding efforts. We also identified pre-adaptive populations that demonstrate heightened resilience to climate fluctuations, illuminating a pathway for future breeding strategies. Collectively, this study enhances our understanding about the local adaptation mechanisms of alfalfa and facilitates the breeding of climate-resilient alfalfa cultivars, contributing to effective agricultural strategies for facing future climate change.

Molecular Characteristics of JAK2 and Its Effect on the Milk Fat and Casein Synthesis of Ovine Mammary Epithelial Cells.

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2, 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1-JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 (CSN1S2), casein beta (CSN2), and casein kappa (CSN3) but down-regulated casein alpha s1 (CSN1S1) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

Expression Localization of the KRT32 Gene and Its Association of Genetic Variation with Wool Traits.

Changes in keratin gene expression and spatiotemporal regulation determine the compositional content and cellular localization of wool keratin, thereby affecting wool traits. Therefore, keratin gene family member 32 (KRT32) was selected for a study using RT-qPCR, immunofluorescence, and penta-primer amplification refractory mutation system (PARMS) techniques. The results showed that KRT32 mRNA was highly expressed in the skin and localized to the inner root sheath (IRS), outer root sheath (ORS) and dermal papilla (DP). Sequencing results identified eight SNPs in KRT32, and association analyses revealed that the variations were significantly associated with multiple traits in wool (p < 0.05), including MFD, CF and MFC. The constructed haplotype combination H2H3 has higher CF and smaller MFD than other haplotype combination (p < 0.05). In conclusion, KRT32 can be used as a candidate gene for molecular genetic improvement of wool in Gansu Alpine Fine-wool sheep.

Noninvasive Models to Assess Liver Inflammation and Fibrosis in Chronic HBV Infected Patients with Normal or Mildly Elevated Alanine Transaminase Levels: Which One Is Most Suitable?

The prevalence of substantial inflammation or fibrosis in treatment-naïve patients with chronic hepatitis B (CHB) and normal alanine transaminase (ALT) levels is high. A retrospective analysis was conducted on 559 consecutive patients with hepatitis B virus infection, who underwent liver biopsy, to investigate the value of noninvasive models based on routine serum markers for evaluating liver histology in CHB patients with normal or mildly elevated ALT levels and to provide treatment guidance. After comparing 55 models, we identified the top three models that exhibited excellent performance. The APGA model, based on the area under the receiver operating characteristic curve (AUROC), demonstrated a superior ability to evaluate significant (AUROC = 0.750) and advanced fibrosis (AUROC = 0.832) and demonstrated a good performance in assessing liver inflammation (AUROCs = 0.779 and 0.874 for stages G ≥ 2 and G ≥ 3, respectively). APGA also exhibited significant correlations with liver inflammation and fibrosis stage (correlation coefficients, 0.452 and 0.405, respectively (p < 0.001)). When the patients were stratified into groups based on HBeAg status and ALT level, APGA consistently outperformed the other 54 models. The other top two models, GAPI and XIE, also outperformed models based on other chronic hepatitis diseases. APGA may be the most suitable option for detecting liver fibrosis and inflammation in Chinese patients with CHB.

Screening and identification of lncRNAs in preadipocyte differentiation in sheep.

Studies of preadipocyte differentiation and fat deposition in sheep have mainly focused on functional genes, and with no emphasis placed on the role that long non-coding RNAs (lncRNAs) may have on the activity of those genes. Here, the expression profile of lncRNAs in ovine preadipocyte differentiation was investigated and the differentially expressed lncRNAs were screened on day 0 (D0), day 2(D2) and day 8(D8) of ovine preadipocyte differentiation, with their target genes being predicted. The competing endogenous RNA (ceRNA) regulatory network was constructed by GO and KEGG enrichment analysis for functional annotation, and some differentially expressed lncRNAs were randomly selected to verify the RNA-Seq results by RT-qPCR. In the study, a total of 2517 novel lncRNAs and 3943 known lncRNAs were identified from ovine preadipocytes at the three stages of differentiation, with the highest proportion being intergenic lncRNAs. A total of 3455 lncRNAs were expressed at all three stages of preadipocyte differentiation, while 214, 226 and 228 lncRNAs were uniquely expressed at day 0, day 2 and day 8, respectively. By comparing the expression of the lncRNAs between the three stages of differentiation stages, a total of 405, 272 and 359 differentially expressed lncRNAs were found in D0-vs-D2, D0-vs-D8, and D2-vs-D8, respectively. Functional analysis revealed that the differentially expressed lncRNAs were enriched in signaling pathways related to ovine preadipocyte differentiation, such as mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase protein kinase B (PI3K-Akt) pathway, and the transforming growth factor beta (TGF-ß) pathway. In summary, lncRNAs from preadipocytes at different stages of differentiation in sheep were identified and screened using RNA-Seq technology, and the regulatory mechanisms of lncRNAs in preadipocyte differentiation and lipid deposition were explored. This study provides a theoretical reference for revealing the roles of lncRNAs in ovine preadipocyte differentiation and also offers a theoretical basis for further understanding the regulatory mechanisms of ovine preadipocyte differentiation.

The GARP family transcription factor MtHHO3 negatively regulates salt tolerance in Medicago truncatula.

High salinity is one of the detrimental environmental factors restricting plant growth and crop production throughout the world. This study demonstrated that the GARP family transcription factor MtHHO3 is involved in response to salt stress and abscisic acid (ABA) signaling in Medicago truncatula. The transcription of MtHHO3 was repressed by salt, osmotic stress, and ABA treatment. The seed germination assay showed that, overexpression of MtHHO3 in Arabidopsis thaliana caused hypersensitivity to salt and osmotic stress, but increased resistance to ABA inhibition. Overexpression of MtHHO3 in M. truncatula resulted in decreased tolerance of salinity, while loss-of-function mutants mthho3-1 and mthho3-2 were more resistant to salt stress compared with wild-type plants. qRT-PCR analyses showed that MtHHO3 downregulated the expression of genes in stress and ABA responsive pathways. We further demonstrated that MtHHO3 repressed the transcription of the pathogenesis-related gene MtPR2 by binding to its promoter. Overall, these results indicate that MtHHO3 negatively regulates salt stress response in plants and deepen our understanding of the role of the GARP subfamily transcription factors in modulating salt stress and ABA signaling.

A genome-wide study of the lipoxygenase gene families in Medicago truncatula and Medicago sativa reveals that MtLOX24 participates in the methyl jasmonate response.

BACKGROUND: Lipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid. RESULTS: In this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress. CONCLUSIONS: We identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago.

Molecular Genetic Characteristics of the Hoxc13 Gene and Association Analysis of Wool Traits.

Homobox C13 (Hoxc13) is an important transcription factor in hair follicle cycle development, and its deletion had been found in a variety of animals leading to abnormal hair growth and disruption of the hair follicle system. In this study, we used immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PCR (RT-qPCR), and Kompetitive Allele-Specific PCR (KASP) genotyping to investigate molecular genetic characteristics of the Hoxc13 gene in Gansu alpine fine-wool sheep. The results revealed that Hoxc13 was significantly expressed during both the anagen and catagen phases (p < 0.05). It was found to be highly expressed predominantly in the dermal papillae and the inner and outer root sheaths, showing a distinct spatiotemporal expression pattern. Two single nucleotide polymorphisms (SNPs) in the exon 1 of Hoxc13, both the individual locus genotypes and the combined haplotypes were found to be correlated with wool length (p < 0.05). It was determined the mutations led to changes in mRNA expression, in which higher expression of this gene was related with longer wool length. In summary, this unique spatiotemporal expression pattern of the Hoxc13 gene may regulate the wool length of Gansu alpine fine-wool sheep, which can be used as a molecular genetic marker for wool traits and thus improve the breed.

Identification and screening of circular RNAs during adipogenic differentiation of ovine preadipocyte by RNA-seq.

Circular RNAs (circRNAs) are a class of non-coding RNAs that play important roles in preadipocyte differentiation and adipogenesis. However, little is known about genome-wide identification, expression profile, and function of circRNAs in sheep. To investigate the role of circRNAs during ovine adipogenic differentiation, the subcutaneous adipose tissue of Tibetan rams was collected in June 2022. Subsequently, the preadipocytes were immediately isolated from collected adipose tissue and then induced to begin differentiation. The adipocytes samples cultured on days 0, 2, and 8 of preadipocytes differentiation were used to perform RNA sequencing (RNA-seq) analysis to construct the expression profiles of circRNAs. Subsequently, the function of differentially expressed circRNAs was investigated by performing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of their parent genes. Finally, a circRNAs-miRNAs-mRNAs network involved in adipogenic differentiation was been analyzed. As a result, a total of 6,449 candidate circRNAs were identified in ovine preadipocytes. Of these circRNAs identified, 63 candidate circRNAs were differentially expressed among the three differentiation stages and their parent genes were mainly enriched in acetyl-CoA metabolic process, positive regulation of lipid biosynthetic process, positive regulation of steroid biosynthetic process, and focal adhesion pathway (P < 0.05). Based on a circRNAs-miRNAs-mRNAs regulatory network constructed, circ_004977, circ_006132 and circ_003788 were found to function as competing endogenous RNAs (ceRNAs) to regulate ovine preadipocyte differentiation and lipid metabolism. The results provide an improved understanding of functions and molecular mechanisms of circRNAs underlying ovine adipogenesis in sheep.

The moderate fat deposition contributes to improve mutton quality, which is associated with the differentiation of preadipocytes. To investigate roles of circular RNAs (circRNAs) in preadipocyte differentiation, we identified circRNAs on days 0, 2, and 8 of preadipocytes differentiation and compared the expression profile of circRNAs at different adipogenic differentiation stages. A total of 6,449 candidate circRNAs were identified, among which 63 candidate circRNAs were differentially expressed among the three differentiation stages. The parent genes of differentially expressed circRNAs were enriched in several biological process and pathways related to lipid metabolism and synthesis. In addition, several circRNAs may regulate ovine preadipocyte differentiation by interacting with microRNAs (miRNAs). The results reveal the potential roles of circRNAs in adipogenic differentiation of sheep.

Spatiotemporal Expression and Haplotypes Identification of KRT84 Gene and Their Association with Wool Traits in Gansu Alpine Fine-Wool Sheep.

Keratin (K) is a major protein component of hair and is involved in hair growth and development. In this study, we analysed the expression, localization, and polymorphism of the K84 gene (KRT84) in Gansu Alpine Fine-wool sheep using immunofluorescence, RT-qPCR, and PARMS (penta-primer amplification refractory mutation system). Haplotypes of KRT84 were also constructed and their relationship with wool traits analysed. It was revealed that KRT84 was highly expressed in hair follicles, including the inner root sheath, outer root sheath, and hair medulla and at all six lamb ages investigated from 1 to 270 days of age. Three SNPs were detected in KRT84 exon 1, and they formed three haplotypes (named H1, H2, and H3) and six genotypes. Analyses revealed an association between haplotype combinations (diplotypes) and the mean fibre curvature, mean staple length, mean staple strength, mean fibre diameter, the coefficient of variation of fibre diameter, and comfort factor for these sheep. These results suggest that KRT84 is of importance in determining several key traits in Gansu Alpine Fine-wool sheep and that the gene could possibly be used as a genetic marker for wool trait selection in these sheep.