Abscisic acid (ABA) is a key phytohormone regulating plant responses to abiotic stress, with ABA-responsive element binding factor/ABA-responsive element binding protein (ABF/AREB) transcription factors serving as central mediators in ABA signaling pathways. Despite their importance, a systematic characterization of this gene family in the economically important forage crop Medicago sativa L. (alfalfa) has been lacking. In this study, we carried out genome-wide identification of ABF/AREB family members in alfalfa using integrated homology-based and domain-based search strategies. Subsequent analyses included phylogenetic classification, examination of the gene structures and conserved motifs, prediction of promoter cis-regulatory elements, and expression profiling using public RNA sequencing (RNA-seq) datasets, complemented by experimental validation under drought, salt, and cold stress conditions. In total, 46 MsABF genes were identified and phylogenetically grouped into seven distinct clades. Family expansion appears to have been driven primarily by segmental duplication events. Promoter analysis uncovered 826 cis-regulatory elements, exhibiting distinct distributions across phylogenetic groups. Expression analysis revealed both tissue-preferential patterns and significant induction of specific members, such as MsABF18, MsABF20, and MsABF25, under multiple abiotic stresses. This study firstly provides the genome-wide overview of the MsABF gene family in alfalfa, elucidating its genomic organization, evolutionary dynamics, and transcriptional responses to environmental challenges. Our findings establish a valuable genomic resource and identify candidate genes for future functional studies aimed at understanding and improving stress adaptation in alfalfa.

Type-B Arabidopsis Response Regulators (ARRs) are essential transcription factors in cytokinin signaling pathway, regulating plant growth, development, and stress responses. However, the type-B ARRs gene family has not been fully characterized in the model legume Medicago truncatula. Here, we systematically identified and functionally analyzed 15 type-B MtARR genes distributed across six chromosomes of the M. truncatula genome. Phylogenetic analysis classified these genes into four distinct clades, with all members containing conserved response regulator and MYB-like DNA-binding domains. Comprehensive promoter analysis identified abundant cis-acting elements associated with abiotic stress responses and developmental processes, suggesting complex regulatory networks. Expression profiling across different tissues demonstrated widespread MtARR activity in various tissues, with distinct induction patterns under drought, salt, and cold stress. Notably, MtARR4/10 exhibited coordinated upregulation across multiple stresses, while MtARR15 displayed an opposite response. Hormone treatment experiments revealed differential responses to ABA, MeJA, SA, IAA, ETH, and 6-BA, indicating multifaceted roles in phytohormone signaling networks. Functional validation through heterologous expression in yeast confirmed that MtARR7/10 enhance stress tolerance, while MtARR15 reduces it. Transcriptional autoactivation assays verified the transcription factor activity of MtARR10, with both N-terminal domains contributing to autoactivation. Collinearity analysis revealed four gene pairs involved in duplication events, with Ka/Ks ratios below 1.0 indicating purifying selection pressure. These findings provide the first comprehensive characterization of the type-B MtARR gene family and establish a foundation for understanding their roles in legume stress adaptation and hormone signaling, with potential applications for improving stress tolerance in forage crops.

Alternaria toxins, as emerging mycotoxins, are common contaminants in apples and apple products. Nevertheless, the responsible Alternaria species, their ability to produce toxins, and the genes responsible for toxin biosynthesis remain ambiguous. In this study, 255 isolates of Alternaria were sampled from apple production areas in 14 provinces across China. Morphological and molecular biological identification indicated that toxin-producing isolates of Alternaria on apples consisted of 251 isolates of Alternaria alternata and 4 isolates belonging to the A. arborescens species complex (AASC). Using an UPLC-MS/MS detection method, the production of five types of Alternaria toxins-alternariol, alternariol monomethyl ether, altenuene, altertoxin I, and tentoxin-was assessed in all isolates. It was found that 254 isolates could produce more than one type of toxin, with altertoxin I having the highest detection rate and altenuene the lowest. PacBio-sequencing was conducted on the EOD115 isolate, and transcriptome sequencing was performed on toxin-producing variants EOD115 and EOD45-2, identifying the biosynthesis genes for these five Alternaria toxins using methods such as homologous alignment. These discoveries provide foundational genetic insights and theoretical data for future research on managing Alternaria toxins in apples.

BACKGROUND: MYB transcription factors play a crucial regulatory role in plant growth and stress response. The gene EpMYB, obtained from Endocarpon pusillum, a dominant lichen in the Tengger Desert, was transferred to creeping bentgrass to explore its effects on plant growth and response to abiotic stress. RESULTS: Compared to wild-type (WT), transgenic (TG) plants exhibited a faster growth rate, a significantly higher number of leaves per tiller, increased internode length, and longer maximum leaf length. However, some of the leaves were severely twisted. Additionally, the antioxidant enzyme content, lignin content and drought tolerance of the TG plants was significantly enhanced. RNA-seq analysis revealed that differentially expressed genes (DEGs) in the TG-vs-WT were primarily associated with pathways such as photosynthesis, wax biosynthesis, lipid metabolism, and flavonoid biosynthesis. By comparing the TG (Drought treatment)-vs-TG and WT (Drought treatment)-vs-WT groups, numerous DEGs related to growth, development, and stress tolerance were identified, including aldehyde decarbonylase gene(CER1), lignin synthesis gene (HCT), adenylate dimethylallyltransferase gene (IPT), peroxin-10 gene (PEX10), among others. These results suggest that the EpMYB gene enhances the drought tolerance of transgenic creeping bentgrass by regulating photosynthesis, antioxidant enzyme activity, lignin synthesis, wax synthesis, and lipid metabolism. CONCLUSION: These findings suggest that the EpMYB gene functions as a positive regulator of plant growth and development, while also playing a crucial role in the plant’s response to drought stress. Furthermore, this study demonstrates the feasibility of selecting specific functional genes from stress-tolerant microorganisms and applying them to plants to enhance stress resistance.

Zoysiagrass stands out as a crucial native turfgrass due to its exceptional abiotic stress tolerance, extensive adaptability, and high ornamental value. In this study, we generated a high-quality chromosome-level genome assembly of Compadre (COM) zoysiagrass, leveraging PacBio SMRT sequencing and Hi-C scaffolding technologies. The resulting genome assembly (312.42 Mb) is anchored on 20 chromosomes, with a Scaffold N50 of 18.72 Mb. In total, 49,074 genes and 306,768 repeat sequences were annotated in the assembled genome. The first chromosome-scale genome of Zoysia japonica ‘Compadre’ provides a critical genetic resource for cold-tolerant turfgrass breeding through identifying stress-responsive candidate genes. Additionally, we have successfully established a cell nucleus extraction and library construction protocol tailored for zoysiagrass ATAC-seq technology, and a total of 80 low temperature tolerance candidate genes were preliminarily identified via ATAC-seq and RNA-seq profiling, thereby initiating the exploration of turfgrass epigenomics.

Abscisic acid (ABA) is a pivotal phytohormone involved in regulating various aspects of plant growth, development, and responses to environmental stress. The Cytochrome P450 family member ABA 8'-hydroxylase (8'OH-ABA) is proposed to play a central role in the catabolic degradation of ABA. In the present study, the 8'OH-ABA gene from Medicago truncatula was isolated and functionally characterized using transgenic overexpression approaches. Under non-stress conditions, plants overexpressing 8'-OH-ABA displayed notable phenotypic variations compared to wild-type plants, including altered leaf morphology, an extended lifespan, and delayed flowering. Scanning electron microscopy (SEM) analysis revealed a reduction in cell spacing and curvature at the leaf margins, which was attributed to the smaller size of epidermal cells in the stem, ultimately contributing to a slower growth rate. Furthermore, these overexpressing plants exhibited heightened sensitivity to drought stress, an effect closely associated with 8'OH-ABA expression. Transcriptome analysis revealed 3,814 differentially expressed genes (DEGs), with 13 genes enriched in the "abscisic acid-activated signaling pathway" and 29 in the "carotenoid biosynthesis" pathway. Notably, we identified genes directly linked to ABA responses, including the ABA 8'-hydroxylase CYP707A2, the transcription factor gene MYC2, and the cytochrome P450 enzyme CYP78A5, which regulates organ size and leaf development. Collectively, these findings indicated the regulatory role of 8’OH-ABA in plant development and drought stress response, thereby highlighting the importance of ABA signaling in these biological processes.

Background Zoysiagrass is renowned for its drought resistance and serves as an exceptional domestic turfgrass in China. However, the changes in chromatin accessibility during drought in zoysiagrass are not well understood. Methods We assessed the drought tolerance of six ecotypes zoysiagrass varieties based on their growth characteristics and physiological traits under drought conditions. Additionally, we utilized an integrated multi-omics strategy, encompassing whole-genome sequencing (WGS), RNA sequencing (RNA-seq), Assay for Transposase Accessible Chromatin using high-throughput sequencing (ATAC-seq), and RT-qPCR verification experiments, to gain a deeper understanding of the chromatin accessibility patterns linked to gene expression in response to drought stress in zoysiagrass. Results The correlation analysis between proline levels and drought tolerance in zoysiagrass revealed that the variety 'X4' exhibited notably high drought resistance compared to the other six zoysiagrass varieties. The KEGG pathway enrichment analysis revealed that zoysiagrass responded to environmental stress by regulating stress response and antioxidant defense pathways. Notably, the expression levels of genes Zja03G031540 and Zja11G000860 were notably increased in the 'X4' zoysiagrass genotype with improved drought tolerance compared to the 'X1' zoysiagrass genotype with reduced drought tolerance. This study suggested that 63 high-confidence genes related to drought stress and 6 motifs regulating drought responses were unearthed. The study discovered a positive correlation between ATAC-seq peak intensity and gene expression levels. The expression of high-confidence genes was linked to zoysiagrass resistance evaluation and phenotypic traits, implying that these genes are involved in responding to external drought stress. Conclusions This study combined ATAC-seq and RNA-seq technologies for the first time to identify drought-related genes expression in zoysiagrass, elucidating the grass adaptation to environmental stress and the regulatory mechanisms underlying stress responses, and laying the groundwork for zoysiagrass improvement and breeding.

Light is not only an important environmental cue for photosynthetic energy production but also for plant growth and development. ELONGATED HYPOCOTYL 5 (HY5) is a key component of light signaling. Yeast two-hybrid screening identified a novel interacting partner ZjSTO of ZjHY5 (GenBank accession number: OP748368) in Zoysia japonica. The BiFC assay demonstrated their interaction within the chloroplast. Similar to low-intensity white light treatment, ectopic expression of ZjHY5 caused a reduction in grana diameter and a reduction in the number of membrane layers per stack. The light intensity dependence of grana stack modifications is consistent with the needs of plants throughout the period of transition from darkness to light. The results demonstrated that the ectopic expression of ZjHY5 exhibited a more light-adapted growth pattern than the control. The leaves of UBI::ZjHY5 transgenic plants were substantially thinner and narrower than the control. Additionally, the morphology of granum and thylakoids was altered. Unknown are the mechanisms through which light cues influence endogenous developmental processes in plants. Electron microscopy and transcriptome analysis revealed that ectopic expression of ZjHY5 dramatically altered the shape of thylakoids and photosynthetic-related proteins.

[Objective]bHLH transcription factors are numerous and widely participate in plant growth,development,and stress responses.The experiment used Medicago truncatula R108 as the material to clone the MtbHLH25 gene and preliminarily explored its function,which will help to further study the function of M.truncatula bHLH transcription factors.[Methods]MtbHLH25 gene and its promoter were cloned from M.truncatula using PCR technology.Yeast expression vector was constructed and transferred to Y2H Gold yeast strain using LiAc transformation method.Vectors for subcellular localiza-tion assays were constructed and transferred into Agrobacterium EHA105 through freeze-thaw method.The Agrobacterium was injected into tobacco epidermal cells and gene expression was observed using SP8 laser confocal microscopy.The spatiotemporal expression of MtbHLH25 in M.truncatula was observed using fluorescence quantitative PCR technology.[Results]The MtbHLH25 gene and its promoter were cloned from M.truncatula with 882 bp,encoding 293 amino acids.Promoter analysis revealed that it con-tains ABA response elements,MeJA response elements,GA response elements,and SA response ele-ments.(2)The evolutionary tree showed that the MtbHLH25 protein was highly homologous with the bHLH proteins in Vicia faba and V.villosa Roth.(3)The MtbHLH25 protein was localized in the nucle-us.(4)The yeast self-activation test showed that the MtbHLH25 protein has self-activation activity.(5)MtbHLH25 was expressed in roots,stems,leaves,flowers,and fruits of M.truncatula,with the high-est expression in roots.Exogenous SA,MeJA,ABA,GA,and salt stress decreased the expression of Mt-bHLH25,indicating that SA,MeJA,ABA,GA,and salt stress had a negative regulatory effect on the expression of MtbHLH25.Drought stress increased the expression of MtbHLH25,indicating that this transcription factor might play a positive role in drought stress.[Conclusion]The MtbHLH25 gene may be sensitive to salt stress and play a positive regulatory role in drought stress.The MtbHLH25 protein has self-activating activity and may have an activating effect on the downstream reporter genes.

Cadmium (Cd), present in agricultural soil, poses a substantial threat to public health through the food chain, adversely affecting food crops and yield. This study examined the role of silicon (Si) in alleviating Cd stress in maize (Zea mays L.). Germinated uniform seedlings were transplanted into 20 L pots filled with a lowconcentration nutrient solution. At the three fully expanded leaves stage (21 days after transplanting), plants were treated with Si (1000 mu\M as K2SO3) and Cd (10 mu M as CdCl2). Plants were assessed 0, 1, and 28 days after treatments. The results demonstrate that Si application leads to a reduction in Cd concentration and content in maize shoot tissue. Additionally, Si treatment positively influences the activity of antioxidant enzymes (peroxidase, superoxidase dismutase, catalase) and plant hormones (abscisic acid, gibberellic acid, salicylic acid) in maize leaves. Furthermore, Si application enhances plant growth, increases levels of antioxidant substances, improves gas exchange parameters, boosts chlorophyll content, and enhances fluorescence. Weighted gene coexpression network analysis (WGCNA) and identification of transcription factors (TFs) and structural genes among differentially expressed genes (DEGs) reveal physiological correlations and enriched signaling pathways, particularly those related to metabolism and biosynthesis. Si actively modulates the formation of metabolites such as arginine and proline, starch and sucrose, as well as biosynthetic pathways for secondary metabolites like benzoxazinoid and carotenoid. Additionally, Si exerts regulatory influence over pathways such as the MAPK signaling pathway and plant hormone signal transduction in plants subjected to Cd stress. These findings unveil the molecular mechanisms underpinning the alleviation of Cd-induced stress in maize leaves due to Si application. Consequently, Si application not only mitigates Cd stress in maize but also reduces the risk of Cd entering the food chain.

BACKGROUND:Isopentenyltransferases (IPT) serve as crucial rate-limiting enzyme in cytokinin synthesis, playing a vital role in plant growth, development, and resistance to abiotic stress. RESULTS:Compared to the wild type, transgenic creeping bentgrass exhibited a slower growth rate, heightened drought tolerance, and improved shade tolerance attributed to delayed leaf senescence. Additionally, transgenic plants showed significant increases in antioxidant enzyme levels, chlorophyll content, and soluble sugars. Importantly, this study uncovered that overexpression of the MtIPT gene not only significantly enhanced cytokinin and auxin content but also influenced brassinosteroid level. RNA-seq analysis revealed that differentially expressed genes (DEGs) between transgenic and wild type plants were closely associated with plant hormone signal transduction, steroid biosynthesis, photosynthesis, flavonoid biosynthesis, carotenoid biosynthesis, anthocyanin biosynthesis, oxidation-reduction process, cytokinin metabolism, and wax biosynthesis. And numerous DEGs related to growth, development, and stress tolerance were identified, including cytokinin signal transduction genes (CRE1, B-ARR), antioxidase-related genes (APX2, PEX11, PER1), Photosynthesis-related genes (ATPF1A, PSBQ, PETF), flavonoid synthesis genes (F3H, C12RT1, DFR), wax synthesis gene (MAH1), senescence-associated gene (SAG20), among others. CONCLUSION:These findings suggest that the MtIPT gene acts as a negative regulator of plant growth and development, while also playing a crucial role in the plant's response to abiotic stress.

Evidence suggests that the 14-3-3 protein plays a role in a wide variety of physiological activities in plants. However, its specific role in Medicago truncatula remains unclear. For this study, a Mt14-3-3 gene was isolated from M. truncatula for further research. Subcellular localization data indicates that this gene is localized in both the cytoplasm and nucleus. Furthermore, the gene's response to multiple hormonal and abiotic stress treatments suggests its potential involvement in hormonal pathways and abiotic stress response mechanisms. When overexpressed, Mt14-3-3 caused Arabidopsis to flower approximately 6 days earlier than the wild type on average. Additionally, the overexpression resulted in enhanced growth resilience under both drought and salt stress conditions when compared to the wild type. The expression levels of relevant genes showed significant variances, further indicating the gene's potential role. Moreover, a MtNAC2 protein known to interact with the Mt14-3-3 protein was identified and validated using yeast two-hybrid experiments. Further evidence was provided by bimolecular fluorescence complementation (bifc) experiment. Analysis of transcriptome data revealed that Mt14-3-3 may play a key role in multiple biological pathways, including valine, leucine, and isoleucine degradation, protein processing in the endoplasmic reticulum, and plant hormone signal transduction. In summary, our study provides new insights into the role of 14-3-3 genes in plant physiology.

Poa pratensis is a widely cultivated turf grass with strong cold and drought resistance. While the chloroplast genome of P. pratensis has been sequenced, its mitochondrial genome remains unexplored. This study assembled the mitochondrial genome of P. pratensis, revealing a total length of 447,463 bp and a guanine-cytosine content of 44.41%. Its main structure comprises a single circular molecule. Annotation results revealed the presence of 56 genes, including 34 unique protein-coding genes, 19 tRNA genes, and three rRNA genes. Additionally, we investigated codon usage bias, repeats, sequence migration, and RNA editing events in the genome. Furthermore, we constructed a phylogenetic tree based on the mitochondrial genomes of P. pratensis and 20 related plants. Notably, synteny analysis results demonstrated that the mitochondrial genome of P. pratensis exhibited greater variation and more significant rearrangement than those of its close relatives. This study reports the complete mitochondrial genome of P. pratensis for the first time, providing a solid foundation for further research into the genetics of bluegrass.

酵母双杂交和单杂交技术是生物大分子互作和调控研究中的高效分子生物学技术,是蛋白质互作或蛋白质与DNA互作筛选的手段.本研究旨在构建高容量的日本结缕草(Zoysia japonica)酵母文库,以便后续深入挖掘日本结缕草相关基因,进行基因功能及基因网络研究.本研究应用不同组织来源以及不同生长时期的日本结缕草,通过SMART技术构建了高容量的酵母杂交cDNA文库.质量检测结果显示:次级文库库容为4.0×106 cfu·mL-1,总克隆数为1.6×107,重组率>95％.最终获得的Y187酵母文库工作液细胞密度为3×107 cells·mL-1,插入片段具有良好的多态性.以日本结缕草类胡萝卜素裂解双加氧酶ZjCCD7作为诱饵,从构建的酵母文库中筛选到一个与Zj CCD7互作的蛋白Zj atpG,证明该文库质量较高,符合酵母杂交筛选试验标准,可适用于调控和互作蛋白筛选.本研究为筛选日本结缕草的互作蛋白以及基因功能研究提供了基础.

Medicago is a genus of legumes (Fabaceae) that resemble common clovers with pinnately trifoliate leaves and spirally coiled seed pods, and Medicago sativa is a famous forage crop throughout the world. In this study, we systematically assembled the complete plastid genomes of 18 Medicago species, representing 35 Medicago accessions, whose genome size ranged from similar to 119 to 125 kb, and identified one novel inverted repeat (IR) in two accessions of Medicago soleirolii (PI537242 and PI537243), albeit of no IRs in the most accessions. We built a phylogenetic tree based on common protein-coding sequences of 55 Medicago accessions in 38 species, which were placed into five clades with a divergence since 9.37 million years ago. Global alignment revealed independent genome evolution events, including eight inversions in nine species and four intron losses (ILs) in 10 species, among which four inversions and two ILs have not been reported previously. Within 109-111 unique genes, ndhA, rpl2, and ycf3 were under positive selection in 54 Medicago accessions. Finally, by aligning chloroplast genes against the nuclear genome assembly of M. sativa cultivar "Zhongmu No.1", we found that a large number of chloroplast gene fragments were horizontally transferred to nuclear chromosomes in alfalfa, especially on the chr3:47518422-48722257 coordinates of chromosome 3. Our comprehensive exploration of Medicago chloroplast genomes provided insights for the understanding of Medicago diversity and their genomic evolution events.

Background Plants possess mitochondrial genomes that are large and complex compared to animals. Despite their size, plant mitochondrial genomes do not contain significantly more genes than their animal counterparts. Studies into the sequence and structure of plant mitochondrial genomes heavily imply that the main mechanism driving replication of plant mtDNA, and offer valuable insights into plant evolution, energy production, and environmental adaptation. Results This study presents the first comprehensive analysis of Agrostis stolonifera ’s mitochondrial genome, characterized by a branched structure comprising three contiguous chromosomes, totaling 560,800 bp with a GC content of 44.07%. Annotations reveal 33 unique protein-coding genes (PCGs), 19 tRNA genes, and 3 rRNA genes. The predominant codons for alanine and glutamine are GCU and CAA, respectively, while cysteine and phenylalanine exhibit weaker codon usage biases. The mitogenome contains 73, 34, and 23 simple sequence repeats (SSRs) on chromosomes 1, 2, and 3, respectively. Chromosome 1 exhibits the most frequent A-repeat monomeric SSR, whereas chromosome 2 displays the most common U-repeat monomeric SSR. DNA transformation analysis identifies 48 homologous fragments between the mitogenome and chloroplast genome, representing 3.41% of the mitogenome’s total length. The PREP suite detects 460 C-U RNA editing events across 33 mitochondrial PCGs, with the highest count in the ccmFn gene and the lowest in the rps7 gene. Phylogenetic analysis confirms A. stolonifera ’s placement within the Pooideae subfamily, showing a close relationship to Lolium perenne , consistent with the APG IV classification system. Numerous homologous co-linear blocks are observed in A. stolonifera ’s mitogenomes and those of related species, while certain regions lack homology. Conclusions The unique features and complexities of the A. stolonifera mitochondrial genome, along with its similarities and differences to related species, provide valuable insights into plant evolution, energy production, and environmental adaptation. The findings from this study significantly contribute to the growing body of knowledge on plant mitochondrial genomes and their role in plant biology.

BackgroundAUX/IAA is an essential signaling molecule and has great physiological importance in various plants, but its function in Zoysia japonica remains unknown.Methods and resultsGenome-wide identification and analysis of AUX/IAA genes used bioinformatics methods to investigate the ZjIAA genes' expression of exogenous IAA hydroponics treatment for 2 h by qRT-PCR, control and exogenous IAA treated zoysia were subjected to transcriptome sequencing. ZjIAAs were distributed across the 13 subfamilies by phylogenetic analysis with Oryza sativa and Arabidopsis thaliana. Multiple sequence alignment revealed that the majority of genes were non-canonical ZjIAAs with incomplete domain. The optimal growth concentration of the IAA hormone was 0.05 mM, and the qRT-PCR analysis revealed that eight ZjIAAs were differentially expressed, with seven genes considerably upregulating and one gene significantly downregulating. The result of transcriptome sequencing revealed that 515 differentially expressed genes (DEGs) were identified, with 344 upregulated genes and 171 downregulated genes. A total of 18 genes were annotated as involved in the plant hormone signal transduction pathway. And 8 ZjIAAs exhibited distinct expressions, 7 upregulated, and only one downregulated, according to the qRT-PCR study.ConclusionsGenome-wide identification and analysis increased the understanding of the evolution and function of the IAA family in zoysia. DEGs of control and treatment with 0.05 mM exogenous IAA hormone were investigated by transcriptome sequencing. ZjIAAs had substantial variations in the expression of associated genes, with the majority of genes upregulated and 18 genes implicated in plant hormone signal transduction.

MYB （V-myb Avian myeloblastosis viral Oncogene Homolog） transcription factors， with numerous members and diverse functions， play important roles in the developmental processes of plants. To investigate the subcellular localization and self-activation properties of the MsMYB33 gene in Medicago sativa， the MsMYB33 gene was cloned from M. sativa， which had an open reading frame of 1，641 bp and encoded a polypeptide with 546 amino acids. Phylogenetic analysis showed that MsMYB33 protein was most closely related to the CaMYB33 protein in Cicer arietinum. MsMYB33 was fused with yellow fluorescent protein （YFP） for expression， and the results showed that the MsMYB33 protein was localized in the nucleus. A pGBKT7-MYB33 yeast expression vector was successfully constructed by DNA recombination technology and transformed into the Y2HGold yeast strain. Yeast self-activation assays showed that MsMYB33 had self-activation activity， and further analysis showed that the activation domain was located at the C-terminus. This study provides a scientific basis for further research on the MsMYB33 gene and the MYB transcription factor family in M. sativa.

纳米抗体是一种新型的蛋白质工程抗体,其体积小、稳定性强、亲和力高等特性为科学研究提供了新的可能性.FLAG标签是一种广泛应用于生物学研究中的短肽标签,在生物学研究中具有重要的作用.为了制备FLAG标签的纳米抗体,利用酵母双杂交技术筛选出具有高亲和力的纳米抗体,并对制备的FLAG纳米抗体进行性能检验.通过DNA重组技术,构建包含FLAG的诱饵载体,利用酵母双杂交技术,在驼源纳米抗体酵母文库中,筛选针对FLAG标签纳米抗体.在酵母文库中筛选出5 个单一的候选抗体DNA序列,为了排除载体本身表达蛋白序列对抗体筛选带来的干扰,通过"点对点"验证方法排除非特异性杂交的可能,通过该操作确认所筛选的 5 株纳米抗体均能与FLAG标签发生特异性亲和反应.为了制备纳米抗体,构建了 5 个纳米抗体原核表达载体,并利用大肠杆菌体系进行表达.通过SDS-PAGE和Western杂交(WB)分析,结果显示,成功获得 2 株可溶性表达的抗FLAG标签蛋白纳米抗体.对这 2 株纳米抗体与商品化的常规FLAG标签抗体进行效果比对,结果显示制备的 2 株纳米抗体与商品化抗体均能够识别FLAG多肽及含有FLAG标签的融合蛋白,且在特异性上没有明显区别,表明制备的FLAG纳米抗体具有较好的应用前景.基于酵母双杂交技术,成功筛选并制备了FLAG标签纳米抗体,这一成果不仅丰富了纳米抗体的类型,也为抗体开发及应用提供了新的途径.该研究为进一步研究FLAG标签在生物学研究中的应用,以及纳米抗体在生物工程中的应用提供有力支持.

MADS-box protein SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) were certified as key regulators of flowering and other developmental traits that are important for agricultural production in various plant species. Legumes are one of the most important industrial crops, commonly used for food, feed, and fuel production. Therefore, to fill the gap of the functions for SOC1 homologs in legume (Medicago truncatula) were of great values in increasing productions. Here, a SOC1-like gene in M. truncatula, MtSOC1c, was functionally characterized. In MtSOC1c-overexpressing plants, it had a considerably earlier flowering time and reduced biomass. Additionally, the ectopic expression of MtSOC1c had an obvious effect on seed development, resulting in smaller pods, lesser thorns, smaller embryo cells and seeds, darker color, and delayed germination. Further, RNA-seq analysis identified 416 differentially expressed genes (DEGs), including 230 upregulated and 186 downregulated DEGs. Enrichment analysis showed that 23 DEGs were enriched in the photosynthesis process. Among the 11 DEGs enriched in “plant hormone signaling transduction”, eight DEGs were involved in abscisic acid signaling pathway, including HVA22 and ATHB. Moreover, MtSOC1c could physically interact with other three MADS-box factors, MtAGL15 and MtAGL16a/b, whose Arabidopsis homologs also participate in seed development regulation. Concludingly, our findings certificated MtSOC1 also had crucial roles mainly in flowering and seed development by affecting ABA transduction, probably mediated by interacting with other functional MADS factors in M. truncatula. These findings provide insights into SOC1 function and its underlying mechanism in M. truncatula.