Attention-based generative adversarial networks improve prognostic outcome prediction of cancer from multimodal data.

The prediction of prognostic outcome is critical for the development of efficient cancer therapeutics and potential personalized medicine. However, due to the heterogeneity and diversity of multimodal data of cancer, data integration and feature selection remain a challenge for prognostic outcome prediction. We proposed a deep learning method with generative adversarial network based on sequential channel-spatial attention modules (CSAM-GAN), a multimodal data integration and feature selection approach, for accomplishing prognostic stratification tasks in cancer. Sequential channel-spatial attention modules equipped with an encoder-decoder are applied for the input features of multimodal data to accurately refine selected features. A discriminator network was proposed to make the generator and discriminator learning in an adversarial way to accurately describe the complex heterogeneous information of multiple modal data. We conducted extensive experiments with various feature selection and classification methods and confirmed that the CSAM-GAN via the multilayer deep neural network (DNN) classifier outperformed these baseline methods on two different multimodal data sets with miRNA expression, mRNA expression and histopathological image data: lower-grade glioma and kidney renal clear cell carcinoma. The CSAM-GAN via the multilayer DNN classifier bridges the gap between heterogenous multimodal data and prognostic outcome prediction.

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.

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 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.

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.

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 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.

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.

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.

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.

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.

Functional differentiation of the ovine preadipocytes -insights from gene expression profiling.

The preadipocytes differentiation is a vital process of lipogenesis; exploring the molecular mechanisms of lipogenesis contributes to improve the meat quality and final commercial income. Lipogenesis has been widely reported in other livestock, but little is known about the gene expression profiles at different stages during preadipocytes differentiation in sheep. In this study, ovine preadipocytes were cultured in vitro and then induced to begin differentiation. Then, the gene expression profiles of preadipocytes collected on day 0 (D0), day 2 (D2), and day 8 (D8) of differentiation were analyzed by RNA-seq technology. According to the findings, 2254 differentially expressed genes (DEGs) were found in D2 vs D0; 1817 DEGs and 1902 DEGs were found in D8 vs D0 and D8 vs D2, respectively. The DEGs were found to be enriched in several biological processes, including focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, steroid biosynthesis, and MAPK signaling pathway, according to Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The regulatory network of the DEGs related to ovine preadipocytes differentiation was systematically constructed, which showed that hub genes might modulate ovine preadipocytes differentiation. In summary, preadipocyte differentiation is regulated by several key genes, including ACACB, CXCL6, SREBF1, INSIG1, APOE, GJA1, CDH11, SYNE1, PCSK1, S100A4, FN1, PLIN2, CXCL6, FN1, PTX3, and FABP3. This study provides a deeper knowledge of the roles of genes in sheep lipogenesis by revealing global gene expression profiles during preadipocyte differentiation.

Integrated transcriptome analysis reveals roles of long non-coding RNAs (lncRNAs) in caprine skeletal muscle mass and meat quality.

Long non-coding RNAs (lncRNAs) play important roles in the growth and development of skeletal muscle. However, there is limited information on goats. In this study, expression profiles of lncRNAs in Longissimus dorsi muscle from Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with divergent meat yield and meat quality were compared using RNA-sequencing. Based on our previous microRNA (miRNA) and mRNA profiles obtained from the same tissues, the target genes and binding miRNAs of differentially expressed lncRNAs were obtained. Subsequently, lncRNA-mRNA interaction networks and a ceRNA network of lncRNA-miRNA-mRNA were constructed. A total of 136 differentially expressed lncRNAs were identified between the two breeds. Fifteen cis target genes and 143 trans target genes were found for differentially expressed lncRNAs, and they were enriched in muscle contraction, muscle system process, muscle cell differentiation, and p53 signaling pathway. A total of 69 lncRNA-trans target gene pairs were constructed, with close relationship with muscle development, intramuscular fat deposition, and meat tenderness. A total of 16 lncRNA-miRNA-mRNA ceRNA pairs were identified, of which some reportedly associated with skeletal muscle development and fat deposition were found. The study will provide an improved understanding of the roles of lncRNAs in caprine meat yield and meat quality.

Overexpression of the elongation factor MtEF1A1 promotes salt stress tolerance in Arabidopsis thaliana and Medicago truncatula.

BACKGROUND: Elongation factor 1 A (EF1A), an essential regulator for protein synthesis, has been reported to participate in abiotic stress responses and environmental adaption in plants. However, the role of EF1A in abiotic stress response was barely studied in Medicago truncatula. Here, we identified elongation factor (EF) genes of M. truncatula and studied the salt stress response function of MtEF1A1 (MTR_6g021805). RESULTS: A total of 34 EF genes were identified in the M. truncatula genome. Protein domains and motifs of EFs were highly conserved in plants. MtEF1A1 has the highest expression levels in root nodules and roots, followed by the leaves and stems. Transgenic Arabidopsis thaliana overexpressing MtEF1A1 was more resistant to salt stress treatment, with higher germination rate, longer roots, and more lateral roots than wild type plant. In addition, lower levels of H2O2 and malondialdehyde (MDA) were also detected in transgenic Arabidopsis. Similarly, MtEF1A1 overexpressing M. truncatula was more resistant to salt stress and had lower levels of reactive oxygen species (ROS) in leaves. Furthermore, the expression levels of abiotic stress-responsive genes (MtRD22A and MtCOR15A) and calcium-binding genes (MtCaM and MtCBL4) were upregulated in MtEF1A1 overexpressing lines of M. truncatula. CONCLUSION: These results suggested that MtEF1A1 play a positive role in salt stress regulation. MtEF1A1 may realize its function by binding to calmodulin (CaM) or by participating in Ca2+-dependent signaling pathway. This study revealed that MtEF1A1 is an important regulator for salt stress response in M. truncatula, and provided potential strategy for salt-tolerant plant breeding.

Genome-wide identification and characterization of filamentation temperature-sensitive H (FtsH) genes and expression analysis in response to multiple stresses in Medicago truncatula.

BACKGROUND: Filamentation temperature-sensitive H (FtsH) is an AAA+ ATP-dependent protease that plays a vital role in plant environmental adaption and tolerance. However, little is known about the function of the FtsH gene family in the most important legume model plant, Medicago truncatula. METHODS AND RESULTS: To identify and investigate the potential stress adaptation roles of FtsH gene family in M. truncatula, we conducted a series of genome-wide characterization and expression analyses. Totally, twenty MtFtsH genes were identified, which were unevenly distributed across eight chromosomes and classified into six evolution groups based on their phylogenetic relationships, with each group containing similar structures and motifs. Furthermore, MtFtsH genes exhibited a high degree of collinearity and homology with leguminous plants such as alfalfa and soybean. Multiple cis-elements in the upstream region of MtFtsH genes were also identified that responded to light, abiotic stress, and phytohormones. Public RNA-seq data indicated that MtFtsH genes were induced under both salt and drought stresses, and our transcript expression analysis showed that MtFtsH genes of MtFtsH1, MtFtsH2, MtFtsH4, MtFtsH9, and MtFtsH10 were up-regulated after ABA, H2O2, PEG, and NaCl treatments. These results suggest that MtFtsH genes may play a critical role in drought and high salt stress responses and the adaption processes of plants. CONCLUSIONS: This study provides a systematic analysis of FtsH gene family in M. truncatula, serving as a valuable molecular theoretical basis for future functional investigations. Our findings also extend the pool of potential candidate genes for the genetic improvement of abiotic stress tolerance in legume crops.

Genome-Wide Identification and Phylogenetic and Expression Analyses of the PLATZ Gene Family in Medicago sativa L.

The PLATZ family is a novel class of plant-specific zinc finger transcription factors with important roles in plant growth and development and abiotic stress responses. PLATZ members have been identified in many plants, including Oryza sativa, Zea mays, Triticum aestivum, Fagopyrum tataricum, and Arabidopsis thaliana; however, due to the complexity of the alfalfa reference genome, the members of the PLATZ gene family in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed. In this study, 55 Medicago sativa PLATZ genes (MsPLATZs) were identified in the alfalfa "Xinjiangdaye" reference genome. Basic bioinformatic analysis was performed, including the characterization of sequence lengths, protein molecular weights, genomic positions, and conserved motifs. Expression analysis reveals that 7 MsPLATZs are tissue-specifically expressed, and 10 MsPLATZs are expressed in all examined tissues. The transcriptomic expression of these genes is obvious, indicating that these MsPLATZs have different functions in the growth and development of alfalfa. Based on transcriptome data analysis and real-time quantitative PCR (RT-qPCR), we identified 22, 22, and 21 MsPLATZ genes that responded to salt, cold, and drought stress, respectively, with 20 MsPLATZs responding to all three stresses. This study lays a foundation for further exploring the functions of MsPLATZs, and provides ideas for the improvement of alfalfa varieties and germplasm innovation.

Characterization of the Heat Shock Transcription Factor Family in Medicago sativa L. and Its Potential Roles in Response to Abiotic Stresses.

Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The HSF gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 HSF genes (MsHSFs) in the tetraploid alfalfa genome ("Xinjiangdaye" reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 MsHSFs and tissue-wide expression for 28 MsHSFs. Based on transcriptomic data analysis, 21, 11 and 27 MsHSFs responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT-PCR, MsHSF27/33 expression gradually increased with cold, salt and drought stress condition duration; MsHSF6 expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of MsHSFs and for genetic improvement of stress resistance in alfalfa.

Interaction between Rumen Epithelial miRNAs-Microbiota-Metabolites in Response to Cold-Season Nutritional Stress in Tibetan Sheep.

Tibetan sheep are already well adapted to cold season nutrient stress on the Tibetan Plateau. Rumen, an important nutrient for metabolism and as an absorption organ in ruminants, plays a vital role in the cold stress adaptations of Tibetan sheep. Ruminal microbiota also plays an indispensable role in rumen function. In this study, combined multiomics data were utilized to comprehensively analyze the interaction mechanism between rumen epithelial miRNAs and microbiota and their metabolites in Tibetan sheep under nutrient stress in the cold season. A total of 949 miRNAs were identified in the rumen epithelium of both cold and warm seasons. A total of 62 differentially expressed (DE) miRNAs were screened using FC > 1.5 and p value < 0.01, and a total of 20,206 targeted genes were predicted by DE miRNAs. KEGG enrichment analysis revealed that DE miRNA-targeted genes were mainly enriched in axon guidance(ko04360), tight junction(ko04530), inflammatory mediator regulation of TRP channels(ko04750) and metabolism-related pathways. Correlation analysis revealed that rumen microbiota, rumen VFAs and DE miRNAs were all correlated. Further study revealed that the targeted genes of cold and warm season rumen epithelial DE miRNAs were coenriched with differential metabolites of microbiota in glycerophospholipid metabolism (ko00564), apoptosis (ko04210), inflammatory mediator regulation of TRP channels (ko04750), small cell lung cancer (ko05222), and choline metabolism in cancer (ko05231) pathways. There are several interactions between Tibetan sheep rumen epithelial miRNAs, rumen microbiota, and microbial metabolites, mainly through maintaining rumen epithelial barrier function and host homeostasis of choline and cholesterol, improving host immunity, and promoting energy metabolism pathways, thus enabling Tibetan sheep to effectively respond to cold season nutrient stress. The results also suggest that rumen microbiota have coevolved with their hosts to improve the adaptive capacity of Tibetan sheep to cold season nutrient stress, providing a new perspective for the study of cold season nutritional stress adaptation in Tibetan sheep.

LncRNA and Protein Expression Profiles Reveal Heart Adaptation to High-Altitude Hypoxia in Tibetan Sheep.

The Tibetan sheep has an intricate mechanism of adaptation to low oxygen levels, which is influenced by both genetic and environmental factors. The heart plays a crucial role in the adaptation of Tibetan sheep to hypoxia. In the present study, we utilized transcriptomic and proteomic technologies to comprehensively analyze and identify the long non-coding RNAs (lncRNAs), genes, proteins, pathways, and gene ontology (GO) terms associated with hypoxic adaptation in Tibetan sheep at three different altitudes (2500 m, 3500 m, and 4500 m). By integrating the differentially expressed (DE) lncRNA target genes, differentially expressed proteins (DEPs), and differentially expressed genes (DEGs), we were able to identify and characterize the mechanisms underlying hypoxic adaptation in Tibetan sheep. Through this integration, we identified 41 shared genes/proteins, and functional enrichment analyses revealed their close association with lipid metabolism, glycolysis/gluconeogenesis, and angiogenesis. Additionally, significant enrichment was observed in important pathways such as the PPAR signaling pathway, glycolysis/gluconeogenesis, the oxoacid metabolic process, and angiogenesis. Furthermore, the co-expression network of lncRNAs and mRNAs demonstrated that lncRNAs (MSTRG.4748.1, ENSOART00020025894, and ENSOART00020036371) may play a pivotal role in the adaptation of Tibetan sheep to the hypoxic conditions of the plateau. In conclusion, this study expands the existing database of lncRNAs and proteins in Tibetan sheep, and these findings may serve as a reference for the prevention of altitude sickness in humans.

Genome-wide identification, phylogeny and expression analysis of the SPL gene family and its important role in salt stress in Medicago sativa L.

BACKGROUND: SQUAMOSA promoter-binding protein-like (SPL) transcription factors are widely present in plants and are involved in signal transduction, the stress response and development. The SPL gene family has been characterized in several model species, such as A. thaliana and G. max. However, there is no in-depth analysis of the SPL gene family in forage, especially alfalfa (Medicago sativa L.), one of the most important forage crops worldwide. RESULT: In total, 76 putative MsSPL genes were identified in the alfalfa genome with an uneven distribution. Based on their identity and gene structure, these MsSPLs were divided into eight phylogenetic groups. Seventy-three MsSPL gene pairs arose from segmental duplication events, and the MsSPLs on the four subgenomes of individual chromosomes displayed high collinearity with the corresponding M. truncatula genome. The prediction of the cis-elements in the promoter regions of the MsSPLs detected two copies of ABA (abscisic acid)-responsive elements (ABREs) on average, implying their potential involvement in alfalfa adaptation to adverse environments. The transcriptome sequencing of MsSPLs in roots and leaves revealed that 54 MsSPLs were expressed in both tissues. Upon salt treatment, three MsSPLs (MsSPL17, MsSPL23 and MsSPL36) were significantly regulated, and the transcription level of MsSPL36 in leaves was repressed to 46.6% of the control level. CONCLUSION: In this study, based on sequence homology, we identified 76 SPL genes in the alfalfa. The SPLs with high identity shared similar gene structures and motifs. In total, 71.1% (54 of 76) of the MsSPLs were expressed in both roots and leaves, and the majority (74.1%) preferred underground tissues to aerial tissues. MsSPL36 in leaves was significantly repressed under salt stress. These findings provide comprehensive information regarding the SPB-box gene family for improve alfalfa tolerance to high salinity.