Skeletal muscle satellite cells are myogenic stem cells situated between the plasmalemma and basal lamina of muscle fibers. As the key cellular component for muscle regeneration, they play a central role in muscle injury repair. These cells exhibit remarkable heterogeneity, which manifests in diverse gene expression profiles, spatial distributions, and differentiation potentials. Satellite cells are quiescent under physiological conditions. However, upon muscle injury or external stimuli, they rapidly activate, proliferate, and differentiate to initiate muscle repair. A thorough understanding of the functional characteristics and regulatory mechanisms of satellite cells not only elucidates the genetic basis of muscle development but also provides theoretical support for improving meat quality and preventing muscle diseases in farm animals. This review summarizes the biological features and functions of skeletal muscle satellite cells, with a detailed introduction to research strategies including in vitro co-culture and omics technologies applied in satellite cell studies. It focuses on their heterogeneity and the regulatory network of proliferation and differentiation. Furthermore, this review summarizes the relevant research progress and provides an outlook for the field of farm animals, thereby offering a reference for a deeper understanding of muscle growth and meat quality improvement in livestock and poultry.

The alternation of day and night is a primary driver for the evolution of an intrinsic circadian clock in mammals. This clock rhythmically regulates the expression of clock-controlled genes through transcriptional-translational negative feedback loops, thereby synchronizing the organism's physiological functions with environmental cycles. As the largest metabolic and motor organ in the body, skeletal muscle exerts a direct impact on the meat production performance and meat quality of livestock through its growth and development. Recent studies have revealed that core clock genes play a pivotal regulatory role in the proliferation and myogenic differentiation of skeletal muscle satellite cells. Therefore, this review focuses on elucidating the molecular mechanisms by which the circadian clock regulates these processes in satellite cells. It further explores how external factors, such as environmental cues, feeding cycles, the gut microbiota, and exercise, influence skeletal muscle development via the circadian clock. Finally, the article discusses production strategies for translating circadian regulatory mechanisms into practical applications. This review provides new insights for enhancing livestock meat production efficiency and achieving precision healthy farming.

With the deep integration of genomics and quantitative genetics, the practical application of genomic selection techniques in livestock breeding continues to expand. Particularly since the turn of the 21st century, iterative advancements in sequencing technology have reduced the cost of whole-genome genotyping, accelerating the adoption of genomic selection across a broader range of livestock species. This approach demonstrates significant potential in enhancing the production performance of selected populations, strengthening disease resistance within herds, and optimising breeding strategies.This paper provided a systematic review of the research progress and application of genomic selection technology in livestock breeding. The full paper revolves around: (1) the principles and development history of the technology, elucidating the methodological framework for genome-wide molecular marker screening and genetic effect assessment; (2) the methods and models used in the technology, involving traditional genomic estimated breeding value (GEBV) assessment methods such as best linear unbiased prediction (BLUP) and Bayesian method, as well as new methods of machine learning, such as support vector machines and random forests; (3) the methods to improve the accuracy of genome selection, considerations of across breeds, combining new methods and approaches such as genome-wide association analysis to increase the reliability and usability of the technology; (4) analysis of the current status of application, focusing on the great potential of the technology in improving the production performance of livestock, breeding for disease resistance and optimising breeding strategies. This paper constructed a three-dimensional analysis system of technology principle-application practice-trend development to provide theoretical support for the development of livestock breeding technology in the context of smart agriculture.

The gene editing technology based on CRISPR/Cas9 is of great help to the basic biological research, disease treatment, and the improvement of animal and plant production traits. However, the editing efficiency varies greatly among loci. The reason for this is that chromatin conformation is one of the influencing factors. Based on this, researchers have conducted extensive research on the optimization of CRISPR/Cas9 gene editing technology based on chromatin conformational regulation. In order to promote the development of this field, this paper reviews the proteins and small molecules related to chromatin conformation regulation and their application progress in CRISPR/Cas9 gene editing optimization, aiming to provide some theoretical and technical reference for the research on gene editing efficiency improvement.

Mastitis is one of the major threats that affects the production performance of dairy livestock. However, the traditional antibiotic application strategy would lead to drug residues and resistance. This review systematically summarized research progress on mastitis-regulating genes to explore novel prevention and treatment strategies, including inflammatory pathway genes (Toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB)), chemokine genes (interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α)), antimicrobial genes (lysozyme (LYZ), β-defensin-4 (DEFB4), lactoferrin (LF)), and apoptosis-autophagy genes such as B cell lymphoma-2 (Bcl2), Bcl-2-associated X protein (BAX), autophagy-related gene 5 (ATG5), microRNA-223 (miR-223) and other apoptosis-autophagy genes. And this review summarized advances in genetic modification models for mastitis regulation, including mice (Mus musculus), dairy goats (Capra hircus), and dairy cows (Bos taurus). This review provides a technical reference for the novel breeding materials creation of mastitis-resistant dairy livestock and the theoretical guidance for the development of targeted prevention and control strategies for mastitis.

Myostatin (MSTN) is a key factor widely present in higher animals, primarily involved in the regulation of muscle development and lipid metabolism. Loss-of-function mutations or downregulated expression of MSTN can consistently induce a significant "double-muscling" phenotype in various animal models. Recent studies have further revealed that in animals with MSTN gene mutations, the structure and function of their gut microbiota undergo specific remodeling, which serves as a central mediator to significantly regulate the host's energy metabolism and muscle-fat balance. As the "second genome" of the host, gut microbes have been demonstrated to profoundly influence host metabolism and physiological functions. This review focusing on the important metabolic regulator MSTN, systematically reviews research progress on the changes in gut microbiota of MSTN-mutated animals, the correlation between MSTN activity and gut microbiota in non-genetically edited animal models, the mechanisms by which MSTN inhibition mediates gut microbiota remodeling to influence host metabolism, and the potential and challenges of exploring probiotics targeting the MSTN pathway. Through this perspective, the review aimed to elucidate the interaction between host genetic traits and gut microbial communities, providing a comprehensive scientific reference and clear research directions for the efficient development and utilization of genetically edited animal resources with significant economic value (particularly in meat animal breeding and production enhancement), especially their unique and regulable microbiome resources.

The Surfeit locus protein 4 (SURF4) gene, a member of the highly conserved surfeit gene family, functions as a cargo receptor that mediates protein transport and participates in lipid synthesis and metabolism. This study aimed to clone the SURF4 gene sequence from goats (Capra hircus), analyze its tissue expression profile, and investigate its role in intramuscular adipocyte differentiation. Using one-year-old Jianzhou Big-ear male goats as experimental subjects, this study performed RT-PCR to clone the complete CDS of goat SURF4 and conducted bioinformatics analysis of the obtained sequence. qPCR was employed to construct the tissue expression profile. An overexpression vector (pEGFP-N1-SURF4) was constructed and transfected into intramuscular adipocytes to evaluate its effects on adipocyte differentiation through morphological (Oil Red O staining) and molecular analyses.The results showed that the cloned goat SURF4 gene sequence was 1 089 bp, with an 810 bp CDS region encoding 269 amino acids. SURF4 was ubiquitously expressed in heart, liver, spleen, and lung tissues, with significantly higher expression in the liver (P<0.01). Overexpression of SURF4 markedly promoted lipid accumulation in intramuscular adipocytes and activated adipogenic pathways: mRNA levels of CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) were dramatically upregulated (P<0.01), while sterol regulatory element-binding protein 1 (SREBP1) transcription increased significantly (P<0.05). Key lipid synthesis markers—adipocyte fatty acid-binding protein 4 (FABP4) and fatty acid synthase (FASN)—also showed extreme upregulation (P<0.01).In conclusion, SURF4 overexpression enhances intramuscular adipocyte differentiation in goats. This study provides a theoretical foundation for elucidating the biological role and molecular mechanisms of SURF4 in regulating intramuscular fat deposition.

Growth factor receptor-bound protein 10 (GRB10), as a key gene regulating growth and development as well as energy metabolism, plays a significant role in immune regulation. Blood physiological indicators serve as important reference criteria for assessing animal's health status, In practical production, monitoring blood physiological indicators enables timely understanding of animal's health status and immune capacity,while alterations in immune performance also influence blood physiological indicators. In this study, a total of 1 086 healthy Hu sheep (Ovis aries) with clear pedigrees were selected as research subjects. The correlations among their hematological and physiological indices were analyzed, and the effects of GRB10 gene polymorphisms on these indices were investigated. Results revealed that there were strong positive correlations among similar blood physiological indicators, weaker correlations between platelet traits and red blood cell/white blood cell traits, and significant negative correlations among white blood cell-related traits. The coefficient of variation for blood physiological indicators in the experimental group ranged from 6.50% to 66.67%, indicating high variability. PCR amplification, Sanger sequencing, and AQP genotyping of the GRB10 gene identified the mutation site GRB10 g.6478288 G>A located in the sixth intron, exhibiting moderate polymorphism.The association analysis results indicated that individuals with the GG genotype exhibit significantly higher mean corpuscular hemoglobin concentration (MCHC), total white blood cell count (WBC), lymphocyte count (LYMPH1), eosinophil count (EO1), and reticulocyte hemoglobin equivalent (RET_He) were significantly higher than those of AA genotype individuals (P<0.05). Conversely, GG genotype individuals exhibited significantly lower hematocrit (HCT), mean corpuscular volume (MCV), and red blood cell hemoglobin (RBC_He) levels compared to AA genotype individuals (P<0.05). In summary, the GRB10 g.6478288 G>A variant may serve as a molecular marker for sheep-related blood physiological indicators. This study provides theoretical guidance for enhancing sheep immune performance and increasing breeding efficiency, while offering scientific basis for disease prevention and treatment.

The invariant chain (Ii), a key immunoregulatory molecule in antigen processing and presentation, plays a critical role in regulating the assembly and transport of major histocompatibility complex (MHC) class Ⅰ/Ⅱ molecules, endosomal sorting, and antigen peptide loading. Vesicle transport through interaction with t-SNAREs 1B (Vti1b), a crucial regulatory factor in endosomal membrane fusion, participates in antigen processing and presentation pathways by mediating endosome-lysosome fusion and autophagosome maturation; however, the relationship between Ii and Vti1b remains unclear. This study focused on Vti1b and Ii of Mus musculus aiming to clarify their subcellular localization and protein-protein interaction, and to investigate the effects of Vti1b under conditions of low and high intracellular expression on the expression levels of Ii and its related MHC molecules. First, Vti1b molecules from different mouse cell types and growth phases of M. musculus were cloned, followed by amino acid sequence alignment; Subsequently, Vti1b and the P31/P41 isoforms of the Ii gene were separately cloned into eukaryotic expression vectors. Laser confocal microscopy was employed to analyze the subcellular colocalization of Vti1b and Ii in Raw264.7 cells. Meanwhile, co-immunoprecipitation (Co-IP), Pull-down and Western blot assays were used to investigate their protein-protein interactions in eukaryotic cells. Finally, through siRNA-mediated gene silencing and eukaryotic recombinant plasmid transfection for Vti1b overexpression, qPCR was used to detect the transcriptional level changes of Vti1b in Raw264.7 cells, as well as the transcription levels of Ii and MHC-associated genes under low and overexpression states of this gene. Results showed that the amino acid sequences of Vti1b exhibited only 1~2 amino acid variations across different mouse cell types and growth phases. Vti1b and Ii proteins were found to interact and colocalize in late endosomes of Raw264.7 cells. When Vti1b gene transcription was significantly downregulated to 42% of the normal control level (P<0.05), the transcription levels of Ii, MHC Ⅰα, and MHC Ⅱβ genes were significantly reduced to 45% (P<0.01), 48% (P<0.01), and 47% (P<0.05) of the control group, respectively. The Vti1b gene was overexpressed by 40.88-fold, and the transcriptional level of MHCⅡβ gene was significantly upregulated, reaching 154% of that in the control group (P<0.01). In contrast, the transcriptional levels of Ii and MHCⅠα genes showed no significant difference compared with the control group. The transcriptional level of Vti1b was correlated with those of Ii and its MHC-associated molecule genes, particularly showing that reduced Vti1b expression led to downregulation of Ii and MHC-related gene transcription. This results clarified the co-localization and protein interaction between Vti1b and Ii in late endosomes, confirmed that the Vti1b gene exerts a regulatory effect on the expression of Ii and MHC-related genes. This study provides an important basis for elucidating their cell biological functions and immunological mechanisms.

Since its introduction to China for more than two decades, the Landes goose (Anser anser domesticus) has formed adapted populations in several regions of the country. This study conducted whole-genome sequencing for 30 individual Jining Landes goose and downloaded the genomic data for 46 samples representing Jiangxi and French Landes goose, along with swan goose (Anser cygnoides) and grey goose (Anser anser), from the NCBI database, and then proceeded to analyze the genetic structure, runs of homozygosity (ROH), and inbreeding coefficients based on the mitochondrial genome and the whole genome information. The objective of this study was to ascertain the genetic structure and diversity of various populations of Landes geese, with the aim of providing theoretical reference for their conservation, development, and utilization in China. The results showed that the Landes goose populations in Jining and Jiangxi exhibited a conspicuous genetic affinity with the French Landes goose. However, these populations evolved to possess a more distinct population structure over the course of their long-term utilization in China. Furthermore, a degree of differentiation emerged between the 2 Landes goose populations in Jining and Jiangxi. The 3 Landes goose populations were observed to exhibit high genetic diversity. However, a comparison of the genetic diversity of the Jining Landes goose population with those of the Jiangxi and French Landes goose populations revealed that the former was lower. These findings indicated that the Jining Landes goose population experienced a greater degree of genetic diversity compared to the Jiangxi and French populations. In regard to the genetic structure, the Jining Landes goose sustained its distinct lineage, initially developing 2 relatively autonomous subgroups. A subset of this group undergone a gradual differentiation from the Jiangxi and French Landes goose groups. The Jining Landes goose group exhibited higher numbers and lengths of ROH compared to the Jiangxi and French groups. This finding suggested that artificial selection and inbreeding played a more significant role in the evolution of the Jining Landes goose group. The degree of inbreeding in the Jining Landes goose group was higher than that of the Jiangxi and French Landes goose groups which indicated that artificial selection and inbreeding played a more significant role in the Jining Landes goose group. The inbreeding coefficient of the Jining Landes goose group was found to be higher than that of the other 2 groups, however, the overall inbreeding level was found to be similar to that of other local goose breeds in China. The results of this study provide a theoretical basis for the conservation and utilization of the Landes goose in China.

Ningdu yellow chicken (Gallus gallus domesticus), as an important local high-quality meat chicken breed in China, its core breeding and industrial value are mainly reflected in the outstanding meat quality traits. To investigate the effects of SNP sites and their haplotypes in partial sequences of the cluster of differentiation 36 (CD36) gene on the meat quality traits of Ningdu yellow chicken, this study selected 250 107-day-old Ningdu Yellow roosters as the experimental flock. Polymorphisms in the CD36 gene were detected, and the associations of SNPs and haplotype groups with meat quality traits were analyzed using SAS 8.1 software. Further validation was conducted by qRT-PCR, and the transcription factor binding patterns at the associated sites were predicted using the JASPAR online software (https://jaspar.elixir.no/). The results showed that a total of 9 SNP sites were identified in partial exon 11 and intron 10 of the CD36 gene in Ningdu yellow chicken. Genetic diversity analysis revealed that, except for the C11336291T and A11336367T sites, all other sites were in Hardy-Weinberg equilibrium (HWE). Association analysis indicated that the C11336291T, G11336340C, A11336343C, T11336353C, G11336358A, A11336367T, and C11336479T sites in the CD36 gene were significantly or extremely significantly associated with breast muscle pH value. Among these, the A11336343C, T11336353C, and G11336358A sites exhibited high linkage disequilibrium. qRT-PCR validation results showed that at the 5 sites of C11336291T, A11336343C, T11336353C, G11336358A, and A11336367T, CD36 gene expression levels differed significantly among individuals with different genotypes. Transcription factor prediction results suggested that polymorphisms in the CD36 gene led to changes in certain transcription factor binding sites, and the A11336343C, T11336353C, and G11336358A sites and their resulting haplotypes in the CD36 gene might serve as potential genetic markers for selecting breast muscle pH value in Ningdu yellow chicken. This study provides a theoretical basis for the molecular marker-assisted breeding of the meat quality traits of Ningdu yellow chicken.

Accurate quantification of active components in complex yeast culture (CYC) is essential for evaluating its quality and functionality. This study established quantitative analytical methods for 3 key small molecule metabolites—N-acetylglutamic acid (NAG), arachidonic acid (ARA), and taurine (Tau)—which were previously identified through non-targeted metabolomics by this research group. To address the complex matrix of CYC, specific methods were developed: Solid-phase extraction combined with high-performance liquid chromatography was used for the highly polar NAG; dansyl chloride pre-column derivatization followed by high-performance liquid chromatography was applied for the zwitterionic Tau; and methylation derivatization coupled with gas chromatography-flame ionization detection was employed for the lipid-soluble ARA. The results demonstrated good linearity (R²>0.999) for all 3 methods, with precision (relative standard deviation<2%), repeatability (relative standard deviation<5%), and spike recovery rates (81.87%~106.95%) all meeting analytical requirements. The developed methods were applied to analyze a laboratory-prepared CYC sample, revealing contents of NAG, ARA, and Tau as (253.80±8.41), (19.72±0.26), and (230.51±4.69) mg/kg (relative standard deviation≤3.31%), respectively. Significant variations were observed in commercially available yeast culture products, with concentration ranges of 38.28~502.31 mg/kg for NAG, 2.60~73.36 mg/kg for ARA, and 28.11~146.44 mg/kg for Tau. The established methods were accurate, reproducible, and suitable for routine quality monitoring of NAG, ARA, and Tau in CYC, providing a reliable analytical foundation for standardizing applications and optimizing production processes.

Foot-and-mouth disease (FMD), caused by the Foot-and-mouth disease virus (FMDV), is a highly contagious disease among cloven-hoofed animals, posing a serious threat to the development of the livestock industry. In order to develop a novel epitope vaccine for FMD, this study, based on the gene sequence of the prevalent swine (Sus scrofa) O-type FMDV strain (O/MYA98/BY/2010), constructed a recombinant expression plasmid pColdⅡ-7B2T containing 7 B-cell epitopes and 2 T-cell epitopes. The recombinant plasmid was co-expressed with the molecular chaperone TF16 in Escherichia coli, followed by a series of analytical procedures including affinity purification, SDS-PAGE, and Western blot analysis. The recombinant protein was then used to immunise BALB/c mice (Mus musculus), after which its humoral and cellular immune responses were evaluated. The results demonstrated that the 7B2T recombinant protein, prepared using the molecular chaperone TF16, was expressed in a soluble form with a molecular weight of approximately 26 kD and could induce a specific immune response with FMDV-positive serum. Furthermore, the 7B2T protein was found to induce significant specific IgG production in the host, with an average neutralising antibody titer of 1∶96. In addition, the 7B2T protein had been observed to induce robust cellular immune responses in murine models, promoting the differentiation of CD4+ and CD8+ T lymphocyte subsets within the spleen. Lymphocyte proliferation assays and enzyme-linked immunospot assays (ELISPOTS) revealed that the splenic lymphocyte proliferation levels and interferon-γ (IFN-γ) spot counts in the 7B2T protein-treated group were extremely significantly higher than those in the inactivated vaccine (IV) group and PBS group (P<0.01), while the IL-4 spot counts were comparable to those of the IV group. In summary, the present study demonstrated that the soluble multi-epitope 7B2T recombinant protein, prepared using the molecular chaperone TF16, was capable of inducing both humoral and cellular immune responses in mice. This study provides experimental ideas and data references for the preparation of FMD multi-epitope recombinant proteins and the development of multi-epitope vaccines.

The prohibitins (PHB) are proteins characterized by SPFH domain, which play an important role in regulating plant development and senescence, and responding to biotic and abiotic stresses. High-temperature stress is one of the main abiotic stresses limiting wheat (Triticum aestivum) growth and yield increase. To identify key genes contributing to high-temperature stress in wheat, in this study, a comprehensive genome-wide analysis of the PHB gene family was performed. Using bioinformatics methods, the physiochemical properties, phylogenetic evolution, chromosome localization, collinearity and cis-elements in promoter sequences were all systematically analyzed in this study. Combined with transcriptome sequencing data and qRT-PCR, the expression patterns of PHB gene family members under high-temperature stress were also studied. In total, 61 TaPHB genes were identified in wheat genome, and all of them contained conserved PHB motifs. Subcellular localization prediction results suggested that most of the PHB gene members were localized in cytoplasm, others localized in mitochondria, chloroplasts and cell membranes, respectively. The phylogenetic tree analysis suggested that the PHB gene family could be classified into 5 clades, which were unevenly distributed on 21 chromosomes of wheat. Collinear analysis revealed that there were collinear relationships among a total of 44 gene pairs in wheat; In promoter regions of the PHB gene family, cis-acting elements involved in hormone regulation, light and abiotic stress response were identified. Combined transcriptome data analysis and qRT-PCR, 5 PHB gene members responding to high-temperature stress in leaves and roots of wheat were identified, which might play an important role in regulating wheat resistant to high-temperature stress. This study provides a theoretical basis for the in-depth analysis of the functions of PHB gene family members in wheat's response to high-temperature stress.

Sweet potato (Ipomoea batatas) is an important food, industrial, and feed crop. The contents and composition of starch and soluble sugars are key determinants of sweet potato quality. Vacuolar invertase (VIN) genes are closely associated with starch-sugar metabolism; however, their functions and regulatory mechanisms in the starch-sugar metabolism of sweet potato storage roots remain to be systematically elucidated. This study conducted a systematic investigation of the vacuolar invertase gene family in sweet potato to elucidate its role in starch-sugar metabolism and carbon partitioning of storage roots. Three IbVIN members were cloned from 7 sweet potato genotypes, their sequences were analyzed bioinformatically, phylogenetic tree was built, and protein secondary/tertiary structures and conserved domains were predicted. Expression patterns were profiled across cultivars, developmental stages, and tissues using qPCR, starch and soluble sugars were quantified by high-performance liquid chromatography (HPLC), and correlation analyses were performed. This study also cloned the 1.5~2.5 kb upstream promoter regions of IbVINs, predicted cis-acting elements, and carried out subcellular localization assays. In Arabidopsis thaliana, Agrobacterium-mediated transgenic lines overexpressing IbVIN1 were generated, and starch and soluble sugar contents were measured in leaves and seeds. The results showed that IbVIN1, IbVIN2, and IbVIN3 all contained the conserved GH32 domain and the catalytic motifs NDPNG, RDP, and WEC, with structural differences among the proteins. The 3 genes were highly expressed in storage roots but weakly expressed in stems, peaking during early-mid root swelling (65~80 d after transplanting), indicating tissue- and stage-specific patterns. At 95 d after transplanting, IbVIN1 and IbVIN2 correlated negatively with starch content and positively with hexoses content, while IbVIN3 correlated positively with hexoses content. At 125 d, all 3 genes showed significant negative correlations with hexoses content, suggesting dynamic regulation of carbon allocation. All 3 promoter types harbored light-, hormone-, and stress-responsive elements, with typeⅡuniquely containing the low-temperature responsive element (LTR) and typeⅢ containing an auxin-responsive element (AuxRE). Subcellular localization confirmed that all 3 proteins resided in the vacuole. Overexpression of IbVIN1 in A. thaliana significantly decreased leaf starch and increased leaf soluble sugars, while promoting the accumulation of both starch and soluble sugars in seeds. Collectively, these findings reveal that the IbVIN family exhibits spatiotemporal expression and stage-dependent roles in carbon partitioning during storage-root development, clarifies its regulatory function in starch-sugar metabolism, and provides a theoretical basis and candidate targets for sweet potato quality improvement and molecular breeding.

Isosteroidal alkaloids are the main active phytochemicals in Fritillaria hupehensis. Sterol C-methyltransferase 1 (SMT1) is an important rate limiting enzyme, playing a significant role in regulating phytosterol biosynthesis. In this study, FhSMT1 gene (GenBank No. PV826664) was successfully cloned from the bulbs of F. hupehensis, based on the gene sequence identified from the prior transcriptomic analysis. Bioinformatics analysis, protein expression profiling, tissue-specific expression analysis, and isosteroidal alkaloid content detection were conducted to study the function of FhSMT1 protein and its expression characteristics. The results showed that the CDS sequence of FhSMT1 gene was 483 bp, which encoded a protein characterized by instability, consisting of 160 amino acids with molecular weight of 17.9 kD and pI of 8.85. There were no signal peptide and transmembrane domains in this protein. The protein had a classical SMT domain, which was a typical SMT family member. The subcellular localization results indicated that FhSMT1 protein was located on the nucleus and the cytoplasmic compartment. On this basis, an expression vector was constructed for prokaryotic expression, and the recombinant expression vector transformed into Escherichia coli. The recombinant protein was obtained through inducing expression with 0.5 mmol/L isopropyl-β-D-thiogalactoside (IPTG) at 16 ℃. His-FhSMT1 and pET28a-SUMO-FhSMT1-His proteins with higher expression were purified using imidazole concentrations with 100 and 250 mmol/L, respectively. FhSMT1 exhibited a tissue-specific expression gradientwithleaf>stem>bulb, consistenting the distribution patterns of peiminine and peimisine, which indicated that FhSMT1 may be involved in regulating the peiminine and peimisine biosynthesis in F. hupehensis. This study provides a theoretical basis for further analysis of the FhSMT1 function in the isosteroidal alkaloid biosynthesis.

Cold shock protein (CSP) is an evolutionarily conserved DNA/RNA binding protein that plays a key role in the establishment of cold tolerance and growth, as well as in the development of plants. In order to understand the family characteristics of CSP gene (PmCSP) in Pinus massoniana, this study used full-length transcriptome data to identify the PmCSP gene family and to analyse its phylogeny, structure and functions, as well as the expression patterns of seedlings in response to cold stress. The results showed that 2 PmCSP gene family members were identified and cloned, both were intronless. The open reading frame of PmCSP1 was 642 bp in length, encoding 213 amino acids residues. The open reading frame of PmCSP2 was 603 bp in length, encoding 200 amino acids residues. The encoded polypeptide chains contained both cold shock domains and CCHC zinc finger domains, but differed in the number of zinc finger domains. PmCSP1 contained 3 domains, while PmCSP2 only contained 2 domains. Phylogenetic analysis showed that PmCSPs were more closely related to P. tabuliformis. Protoplast transient expression analysis showed that PmCSPs proteins were mainly located in the nucleus and cytoplasm. The expression of PmCSPs in the leaves and terminal buds of P. massoniana seedlings were higher than those in other tissues. Under cold stress, PmCSP1 and PmCSP2 were upregulated in the primary needle and secondary needle seedlings, respectively, with PmCSP expression peaking at 8 h, which indicated that PmCSPs were early response genes for P. massoniana seedlings in response to cold stress and may be involved in specific molecular regulation in heteroblastic foliage seedlings. This study provides a theoretical basis for further revealing the functions of the PmCSP gene family and the regulatory mechanisms established for the cold resistance of conifer heteroblastic foliage seedlings.

Respiratory metabolism is closely related to plant tolerance to saline and alkali stress, and ethanol dehydrogenase is a key enzyme for anaerobic respiration in plants, which is up-regulated under saline and alkali stress and plays an important role in plant tolerance to saline and alkali stress. In order to clarify the molecular mechanism of CbADH gene involved in the salinity stress tolerance of Cinnamomum bodinieri, in this study, the promoter sequence of CbADH gene was cloned using the alkali-tolerant Cinnamomum bodinieri line as the material, and the upstream regulatory factors of CbADH gene was screened out by yeast one-hybridization, and the function of the upstream regulatory proteins on the promoter of CbADH gene was verified by the dual luciferase reporter gene technique. The results showed that the length of the obtained CbADH gene promoter fragment was 1 996 bp; the CbADH gene promoter fragment contained light-responsive regulatory elements, ABA-responsive elements, phloem-expression-responsive elements, MeJA-responsive elements, GA-responsive elements, stress-responsive elements, SA response element, healing tissue response element, transcription factor MYB binding site, transcription factor bHLH binding site, and transcription factor WRKY binding site. A total of 17 successfully annotated reciprocal proteins were obtained by yeast one-hybrid technique, mainly including protein kinase superfamily, amino acid transporters, mediator of ABA-regulated dormancy 1 (MARDⅠ) zinc finger structural proteins, Arabidopsis thaliana homeobox 6 (ATHB-6) proteins, trisaccharide phosphoribosylceramide isomerase proteins, late embryogenesis abundant protein (LEA) proteins, tyrosine/dopamine decarboxylases, mannitol dehydrogenase proteins, ATP synthases, and cytochrome B5 family proteins. According to the results of the dual luciferase reporter gene experiment technology, it was shown that the CbATHB6 protein activated the promoter activity of CbADH gene in plants. Based on these findings, the transcription factor CbATHB6 could bind to the promoter sequence of the CbADH gene and respond to salt-alk stress by regulating the expression of the CbADH gene. This study provides a basis for analyzing the molecular mechanism of respiratory metabolism of Cinnamomum bodinieri in response to saline and alkaline stress.