Identification and Characterization of circRNAs in Non-Lactating Dairy Goat Mammary Glands Reveal Their Regulatory Role in Mammary Cell Involution and Remodeling.

This study conducted transcriptome sequencing of goat-mammary-gland tissue at the late lactation (LL), dry period (DP), and late gestation (LG) stages to reveal the expression characteristics and molecular functions of circRNAs during mammary involution. A total of 11,756 circRNAs were identified in this study, of which 2528 circRNAs were expressed in all three stages. The number of exonic circRNAs was the largest, and the least identified circRNAs were antisense circRNAs. circRNA source gene analysis found that 9282 circRNAs were derived from 3889 genes, and 127 circRNAs' source genes were unknown. Gene Ontology (GO) terms, such as histone modification, regulation of GTPase activity, and establishment or maintenance of cell polarity, were significantly enriched (FDR < 0.05), which indicates the functional diversity of circRNAs' source genes. A total of 218 differentially expressed circRNAs were identified during the non-lactation period. The number of specifically expressed circRNAs was the highest in the DP and the lowest in LL stages. These indicated temporal specificity of circRNA expression in mammary gland tissues at different developmental stages. In addition, this study also constructed circRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) regulatory networks related to mammary development, immunity, substance metabolism, and apoptosis. These findings help understand the regulatory role of circRNAs in mammary cell involution and remodeling.

Analysis of the main antioxidant enzymes in the roots of Tamarix ramosissima under NaCl stress by applying exogenous potassium (K+).

Introduction: Salinization affects more than 25% of the world's arable land, and Tamarix ramosissima Ledeb (T. ramosissima), the representative of Tamarix plants, is widely grown in salinized soil. In contrast, less is known about the mechanism of potassium's antioxidative enzyme activity in preventing NaCl stress damage to plants. Method: This study examined changes in root growth for T. ramosissima at 0h, 48h, and 168h, performed antioxidant enzyme activity assays, transcriptome sequencing, and non-targeted metabolite analysis to understand changes in their roots as well as changes in the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Quantitative real-time PCR (qRT-PCR) was used to identify differentially expressed genes (DEGs) and differential metabolites associated with antioxidant enzyme activities. Result: As the time increased, the results showed that compared with the 200 Mm NaCl group, the root growth of the 200 mM NaCl + 10 mM KCl group increased, the activities of SOD, POD and CAT increased the most, but the contents of hydrogen peroxide (H2O2) and Malondialdehyde (MDA) increased less. Meanwhile, 58 DEGs related to SOD, POD and CAT activities were changed during the application of exogenous K+ for 48h and 168h in T. ramosissima. Based on association analysis of transcriptomic and metabolomic data, we found coniferyl alcohol, which can act as a substrate to label catalytic POD. It is worth noting that Unigene0013825 and Unigene0014843, as POD-related genes, have positively regulated the downstream of coniferyl alcohol, and they have a significant correlation with coniferyl alcohol. Discussion: In summary, 48h and 168h of exogenous K+ applied to the roots of T. ramosissima under NaCl stress can resist NaCl stress by scavenging the reactive oxygen species (ROS) generated by high salt stress by enhancing the mechanism of antioxidant enzyme activity, relieving NaCl toxicity and maintaining growth. This study provides genetic resources and a scientific theoretical basis for further breeding of salt-tolerant Tamarix plants and the molecular mechanism of K+ alleviating NaCl toxicity.

Analysis of the Antioxidant Mechanism of Tamarix ramosissima Roots under NaCl Stress Based on Physiology, Transcriptomic and Metabolomic.

There is a serious problem with soil salinization that affects the growth and development of plants. Tamarix ramosissima Ledeb (T. ramosissima), as a halophyte, is widely used for afforestation in salinized soils. At present, there are few reports on the antioxidant mechanism of T. ramosissima under NaCl stress. In this study, we learned about the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and hydrogen peroxide (H2O2) and malondialdehyde (MDA) content changes in T. ramosissima. We also mined the relevant metabolic pathways in the antioxidant mechanism, candidate key genes, and their related differential metabolites and verified them using quantitative real-time PCR (qRT-PCR). The results show that the SOD, POD, and CAT activities, and the H2O2 and MDA content reached the highest values in the roots of T. ramosissima. Simultaneously, 92 differentially expressed genes (DEGs) related to antioxidant enzyme activities changed during 48 and 168 h of NaCl stress, and these DEGs were mainly upregulated in 168 h. Based on the association analysis of transcriptomic and metabolomic data, we found Unigene0089358 and Unigene0007782 as genes related to key enzymes in the flavonoid biosynthesis pathway. They were located in the upstream positive regulation at 48 and 168 h under NaCl stress, and their respective related metabolites (phloretin and pinocembrin) were involved in resistance to NaCl stress, and they were significantly correlated with their respective metabolites. In conclusion, at 48 and 168 h under NaCl stress, the roots of T. ramosissima resist NaCl stress by enhancing enzymatic and nonenzymatic antioxidant mechanisms, scavenging ROS generated by high-salt stress, alleviating NaCl toxicity, and maintaining the growth of T. ramosissima. This study provides genetic resources and a scientific theoretical basis for further breeding of salt-tolerant Tamarix plants and the molecular mechanism of antioxidants to alleviate NaCl toxicity.

Transcriptome Analysis of Goat Mammary Gland Tissue Reveals the Adaptive Strategies and Molecular Mechanisms of Lactation and Involution.

To understand how genes precisely regulate lactation physiological activity and the molecular genetic mechanisms underlying mammary gland involution, this study investigated the transcriptome characteristics of goat mammary gland tissues at the late gestation (LG), early lactation (EL), peak lactation (PL), late lactation (LL), dry period (DP), and involution (IN) stages. A total of 13,083 differentially expressed transcripts were identified by mutual comparison of mammary gland tissues at six developmental stages. Genes related to cell growth, apoptosis, immunity, nutrient transport, synthesis, and metabolism make adaptive transcriptional changes to meet the needs of mammary lactation. Notably, platelet derived growth factor receptor beta (PDGFRB) was screened as a hub gene of the mammary gland developmental network, which is highly expressed during the DP and IN. Overexpression of PDGFRB in vitro could slow down the G1/S phase arrest of goat mammary epithelial cell cycle and promote cell proliferation by regulating the PI3K/Akt signaling pathway. In addition, PDGFRB overexpression can also affect the expression of genes related to apoptosis, matrix metalloproteinase family, and vascular development, which is beneficial to the remodeling of mammary gland tissue during involution. These findings provide new insights into the molecular mechanisms involved in lactation and mammary gland involution.

Characterization of long noncoding RNA in nonlactating goat mammary glands reveals their regulatory role in mammary cell involution and remodeling.

Long noncoding RNA (lncRNA) can regulate mammary gland development and lactation physiological activities. However, the molecular genetic mechanisms of lncRNA in mammary gland involution and cell remodeling remain unclear. This work analyzed the expression characteristics and molecular functions of lncRNA in goat mammary gland tissue at the late lactation (LL), dry period (DP), and late gestation (LG) stages. Sequencing results showed that 3074 lncRNAs were identified in non-lactating goat mammary gland tissue. Statistical analysis of lncRNA length characteristics and exon number found that goat lncRNAs were shorter in length, had fewer exons, and significantly lower expression levels than those of protein-coding genes. 331 differentially expressed lncRNAs were identified in the three comparison groups (LLvsDP, DPvsLG, and LLvsLG), which indicated that the lncRNAs expression at the transcriptional level were changed during mammary involution. Interestingly, lncRNAs were more actively expressed during the dry period compared to lactation, suggesting that lncRNAs in mammary glands are developmentally specific. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that lncRNAs could regulate immune function, cell proliferation, apoptosis, hormones, substance metabolism, transport, and intercellular communication in the mammary gland through various action modes. Among them, cis-acting lncRNAs enhanced the protection of mammary gland health during the dry period and late gestation. The above reflects the particular mechanisms of lncRNA to adapt to the developmental needs of mammary involution and remodeling. Furthermore, in the lncRNA-miRNA-mRNA network associated with mammary gland development, the expression of LOC102168552 was higher in late gestation than in the dry period and late lactation. Its expression was positively correlated with PRLR and negatively correlated with chi-miR-324-3p. Overexpression of LOC102168552 in goat mammary epithelial cells cultured in vitro could up-regulate PRLR to activate the prolactin signaling pathway by competitively binding to chi-miR-324-3p, promoting cell proliferation, reducing cell cycle arrest in the G1 / S phase, and inhibiting apoptosis. However, overexpression of LOC102168552 alone did not affect mammary cell growth status and the prolactin signaling pathway. This indicates that LOC102168552 must rely on chi-miR-324-3p to inhibit mammary cell apoptosis. In conclusion, the above analysis revealed that lncRNAs in goat mammary tissue are differentially expressed at different stages of involution. As expected, lncRNAs adaptively regulate various physiological activities during mammary gland involution through multiple modes of action, in preparation for a new round of lactation. These findings provide a reference and help further understand the regulatory role of lncRNAs in mammary cell involution and remodeling.

Effects of Exogenous (K+) Potassium Application on Plant Hormones in the Roots of Tamarix ramosissima under NaCl Stress.

Abiotic stresses such as salt stress seriously affect the growth and yield of plants. Tamarix ramosissima Lcdcb (T. ramosissima) is a widely cultivated halophyte in saline-alkali areas of the world. As an essential element for plant growth and development, K+ plays an irreplaceable role in improving the tolerance of plants to salt stress. However, there are few reports on the mechanism of K+ in promoting plant hormones to reduce the damage of NaCl stress to T. ramosissima. In this study, we sequenced the transcriptome of the roots of T. ramosissima which were treated with exogenous potassium (K+) for 0 h, 48 h and 168 h under NaCl stress, according to the changes in the expression levels of differentially expressed genes (DEGs) in T. ramosissima roots. Key candidate genes and metabolic pathways related to plant hormones were mined for analysis and further verified by quantitative real-time PCR (qRT-PCR). The results showed that under NaCl stress for 48 h and 168 h, there were a large number of DEGs in the roots of T. ramosissima, and the expression levels changed over time. In particular, we found that 56 plant hormone-related genes were annotated to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and with the increase of time, their expression levels were mainly up-regulated and involved in the related metabolic pathways to resist NaCl stress. It is worth noting that 7 DEGs related to abscisic acid (ABA), 28 DEGs related to auxin, 1 DEG related to ethylene (ET), and 1 DEG related to cytokinin (CK) were added within 168 h of exogenous potassium, and they were involved in alleviating the root damage of T. ramosissima under NaCl stress and played an important role. In addition, we found the plant hormone signal transduction pathway, which plays an important role in resistance to NaCl stress. As a result of this study, the molecular mechanism of plant hormones involved in applying exogenous potassium under NaCl stress is further understood, resulting in a better understanding of how exogenous potassium can alleviate the damage caused by NaCl under stress in T. ramosissima.

Effects of Exogenous Potassium (K+) Application on the Antioxidant Enzymes Activities in Leaves of Tamarix ramosissima under NaCl Stress.

Saline soil is a worldwide distributed resource that seriously harms plants' growth and development. NaCl is the most widely distributed salt in saline soil. As a typical representative of halophytes, Tamarix ramosissima Lcdcb (T. ramosissima) is commonly grown in salinized soil, and halophytes have different abilities to retain more K+ under salt stress conditions. Halophytes can adapt to different salt environments by improving the scavenging activity of reactive oxygen species (ROS) by absorbing and transporting potassium (K+). In this study, electron microscope observation, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents determination, primary antioxidant enzyme activity determination and transcriptome sequencing analysis were carried out on the leaves of T. ramosissima under NaCl stress at 0 h, 48 h and 168 h. The results showed that H2O2 and MDA contents increased in the 200 mM NaCl + 10 mM KCl and 200 mM NaCl groups, but the content increased the most in the 200 mM NaCl group at 168 h. In addition, the leaves of T. ramosissima in the 200 mM NaCl + 10 mM KCl group had the most salt secretion, and its superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were all higher than those of the 200 mM NaCl group and significantly higher than those of the control group. According to the results of transcriptome sequencing, it was found that the expression of 39 genes related to antioxidant enzyme activity changed significantly at the transcriptional level. Among them, 15 genes related to antioxidant enzyme activities were upregulated, and 24 genes related to antioxidant enzyme activities were downregulated in the leaves of T. ramosissima when exogenous potassium (K+) was applied under NaCl stress for 48 h; when exogenous potassium (K+) was used for 168 h under NaCl stress, 21 antioxidant enzyme activity-related genes were upregulated, and 18 antioxidant enzyme activity-related genes were downregulated in T. ramosissima leaves. Based on the changes of expression levels at different treatment times, 10 key candidates differentially expressed genes (DEGs) (Unigene0050462, Unigene0014843, Unigene0046159, Unigene0046160, Unigene0008032, Unigene0048033, Unigene0004890, Unigene0015109, Unigene0020552 and Unigene0048538) for antioxidant enzyme activities were further screened. They played an important role in applying exogenous potassium (K+) for 48 h and 168 h to the leaves of T. ramosissima in response to NaCl stress. Their expression levels were dominated by upregulation, which enhanced the activity of antioxidant enzymes, and helped T. ramosissima mitigate NaCl poison and resist NaCl stress. Particularly, Unigene0048538 in glutathione S-transferase (GST) activity had the largest log2 fold-change in the comparison groups of 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h and 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl-168 h. Its expression level was upregulated and played an important role in NaCl toxicity. At the same time, the results of the phylogenetic tree analysis showed that Unigene0048538 had the closest genetic distance to Prunus persica in the evolutionary relationship. In summary, with the increase of exogenous potassium (K+) application time under NaCl stress, T. ramosissima can resist high NaCl stress by enhancing antioxidant enzymes' activity and maintaining the growth of T. ramosissima. Still, it is not enough to completely eliminate NaCl poison. This study provides a theoretical basis for the molecular mechanism of salt tolerance and K+ mitigation of NaCl poison by the representative halophyte T. ramosissima in response to NaCl stress.

Analysis of Amino Acids in the Roots of Tamarix ramosissima by Application of Exogenous Potassium (K+) under NaCl Stress.

Soil salinization is one of the main environmental factors affecting plant growth worldwide. Tamarix ramosissima Ledeb. (T. ramosissima) is a halophyte representative that is widely grown in salinized soils. As an important nutrient element for plant growth, K+ plays an important role in improving the tolerance to salt stress, but the mechanism of reducing the damage caused by NaCl stress to T. ramosissima is less reported. Our results show that the proline content and the Log2 fold-change of proline's relative quantification in the roots of T. ramosissima increased over time with the application of exogenous potassium (K+) for 48 h and 168 h under NaCl stress. Moreover, 13 amino-acid-related metabolic pathways were involved in the resistance of T. ramosissima to salt stress. Mainly, the aldehyde dehydrogenase family genes and tryptophan-synthase-related genes were found at 48 h and 168 h with exogenous potassium applied to the roots of T. ramosissima under NaCl stress, and they regulated their related metabolic accumulation in the arginine and proline metabolism pathways, increasing the effectiveness of inducing NaCl tolerance of T. ramosissima. It is noteworthy that alpha-ketobutyric was produced in the roots of T. ramosissima under NaCl stress for 48 h with the application of exogenous potassium, which is one of the most effective mechanisms for inducing salt tolerance in plants. Meanwhile, we found three DEGs regulating alpha-ketobutyric acid. This study provides a scientific theoretical basis for further understanding the molecular mechanism of K+ alleviating the salinity damage to T. ramosissima caused by NaCl.

Transcriptome and Metabonomic Analysis of Tamarix ramosissima Potassium (K+) Channels and Transporters in Response to NaCl Stress.

Potassium ion (K+) channels and transporters are key components of plant K+ absorption and transportation and play an important role in plant growth and development. This study revealed that K+ channels and transporters are involved in the salt tolerance molecular mechanism and metabolites of the halophyte representative plant Tamarix ramosissima (T. ramosissima) in response to NaCl stress, providing a theoretical basis for the mitigation of salt stress using halophytes. Through transcriptome sequencing and metabolite detection analysis of 0 h, 48 h and 168 h by applying exogenous K+ to the roots of T. ramosissima under NaCl stress, 15 high-quality Clean Data bases were obtained, Q20 reached more than 97%, Q30 reached more than 92%, and GC content reached 44.5%, which is in line with further bioinformatics analysis. Based on the Liquid chromatography−mass spectrometry (LC-MS) analysis, the roots of T. ramosissima were exposed to exogenous potassium for 48 h and 168 h under NaCl stress, and 1510 and 1124 metabolites were identified in positive and negative ion mode, respectively. Through orthogonal projections to latent structures discriminant analysis (OPLS-DA) model analysis, its metabolomic data have excellent predictability and stability. The results of this study showed that there were 37 differentially expressed genes (DEGs) annotated as Class 2 K+ channels (Shaker-like K+ channel and TPK channel) and Class 3 K+ transporters (HAK/KUP/KT, HKT and CPAs transporter families). Among them, 29 DEGs were annotated to the gene ontology (GO) database, and the most genes were involved in the GO Biological Process. In addition, the expression levels of Unigene0014342 in the HAK/KUP/KT transporter and Unigene0088276 and Unigene0103067 in the CPAs transporter both first decreased and then increased when treated with 200 mM NaCl for 48 h and 168 h. However, when treated with 200 mM NaCl + 10 mM KCl for 48 h and 168 h, a continuous upward trend was shown. Notably, the expression level of Unigene0016813 in CPAS transporter continued to increase when treated with 200 mM NaCl and 200 mM NaCl + 10 mM KCl for 48 h and 168 h. 3 DEGs, Unigene0088276, Unigene0016813 and Unigene0103067, were dominated by the positive regulation of their related metabolites, and this correlation was significant. The results showed that these DEGs increased the absorption of K+ and the ratio of K+/Na+ under NaCl stress at 48 h and 168 h after adding exogenous potassium and enhanced the salt tolerance of T. ramosissima. Notably, the expression level of Unigene0103067 in the CPAs transporter was consistently upregulated when 200 mM NaCl + 10 mM KCl was treated for 48 h and 168 h. The positive regulatory metabolites were always dominant, which better helped T. ramosissima resist salt stress. Unigene0103067 plays an important role in enhancing the salt tolerance of T. ramosissima and reducing the toxicity of NaCl in roots. Additionally, phylogenetic tree analysis showed that Unigene0103067 and Reaumuria trigyna had the closest genetic distance in the evolutionary relationship. Finally, 9 DEGs were randomly selected for quantitative real-time PCR (qRT-PCR) verification. Their expression trends were completely consistent with the transcriptome sequencing analysis results, proving that this study's data are accurate and reliable. This study provides resources for revealing the molecular mechanism of NaCl stress tolerance in T. ramosissima and lays a theoretical foundation for cultivating new salt-tolerant varieties.

High-Throughput Sequencing Reveals Transcriptome Signature of Early Liver Development in Goat Kids.

As a vital metabolic and immune organ in animals, the liver plays an important role in protein synthesis, detoxification, metabolism, and immune defense. The primary research purpose of this study was to reveal the effect of breast-feeding, weaning transition, and weaning on the gene expression profile in the goat kid liver and to elucidate the transcriptome-level signatures associated with liver metabolic adaptation. Therefore, transcriptome sequencing was performed on liver tissues, which was collected at 1 day (D1), 2 weeks (W2), 4 weeks (W4), 8 weeks (W8), and 12 weeks (W12) after birth in Laiwu black goats at five different time-points, with five goats at each time point. From 25 libraries, a total of 37497 mRNAs were found to be expressed in goat kid livers, and 1271 genes were differentially expressed between at least two of the five time points. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that these genes were annotated as an extracellular region fraction, exhibiting monooxygenase activity, positive regulation of T cell activation, mitotic spindle mid-region assembly, cytokinesis, cytoskeleton-dependent cytokinesis, regulation of cytokinesis, regulation of lymphocyte proliferation, and so on. In addition, it mainly deals with metabolism, endocrine, cell proliferation and apoptosis, and immune processes. Finally, a gene regulatory network was constructed, and a total of 14 key genes were screened, which were mainly enriched for cell growth and development, endocrine, immune, and signal transduction-related pathways. Our results provide new information on the molecular mechanisms and pathways involved in liver development, metabolism, and immunity of goats.