Application of BpHSF gene family for regulating and controlling selenium metabolism of broussonetia papyrifera

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to application of a BpHSF gene family for regulating and controlling selenium metabolism of broussonetia papyrifera. The invention aims to systematically identify the broussonetia papyrifera HSF gene family and analyze the regulation mechanism of the broussonetia papyrifera HSF gene family under Se stress. Through whole genome analysis, 18 BpHSF genes are identified, and remarkable characteristics of the BpHSF genes in evolution conservative property and subfamily specificity are disclosed. RT-qPCR analysis shows that different BpHSF members have functional differentiation in regulation and control of selenium metabolism of the broussonetia papyrifera. A subcellular localization verification result shows that the three candidate BpHSF are mainly localized in a cell nucleus. A PPI network and AME prove that a typical HSF binding motif is significantly enriched in a promoter of a core hub gene of a Se metabolic network, which indicates that HSFs as a core transcriptional regulation factor preferentially regulates key nodes of Se absorption, transport and detoxification. According to the invention, a model is established, a BpHSF family utilizes coordinated HSFA / HSFB functional divergence to accurately regulate and control Se steady state, and the discoveries provide key candidate genes for molecular breeding of Se biologically-enhanced broussonetia papyrifera.

Description

Application of the BpHSF gene family for regulating selenium metabolism in paper mulberry Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to the application of the BpHSF gene family for regulating selenium metabolism in paper mulberry. Background Technology

[0002] Paper mulberry (Broussonetia papyrifera) is a perennial deciduous plant belonging to the genus Broussonetia in the family Moraceae. Rich in flavonoids, polysaccharides, and other plant active ingredients, it exhibits significant potential in antibacterial, anti-inflammatory, and antioxidant properties. The bark, leaves, and fruit of the paper mulberry can all be used medicinally. Leaf extracts can effectively inhibit various pathogens, while alkaloids in its fruit can significantly inhibit the activity of cancer cells. Paper mulberry grows rapidly, is highly adaptable, and has a high protein content, making it a promising new feed resource that can improve milk quality in dairy goats and enhance the growth and immunity of lambs. Furthermore, as a typical pioneer tree species, paper mulberry possesses significant ecological restoration potential, enabling rapid colonization and efficient adaptation in adverse environments such as heavy metal pollution, drought, and salinity.

[0003] Selenium (Se) is an essential trace element for mammals, participating in the synthesis of antioxidant proteins such as glutathione peroxidase, and playing a role in regulating immunity, anti-cancer activity, and thyroid metabolism. Globally, approximately one billion people face the risk of insufficient selenium intake, leading to health problems such as Keshan disease and Kashin-Beck disease. Plants, as the main source of selenium in the food chain, have a selenium accumulation capacity that directly impacts human nutritional security. Plants primarily absorb selenium in the inorganic form of selenate (SeO4). 2- ) and selenite (SeO3) 2- Selenium is primarily composed of inorganic selenium, with selenate, due to its structural similarity to sulfate, being actively absorbed and transported mainly through high-affinity sulfate transporters (SULTRs). Once inside the plant, this inorganic selenium is converted into organic selenium forms, such as selenomethionine (SeMet), via a pathway similar to sulfur assimilation. This process is a crucial mechanism for selenium detoxification and accumulation in plants. Although selenium is not essential for plants, appropriate concentrations can significantly enhance plant stress resistance and promote growth. Studies have shown that alfalfa (Medicago sativa) treated with nano-selenium exhibits significantly increased nitrogen accumulation, with photosynthetic efficiency and stress resistance being enhanced simultaneously. Appropriate exogenous selenium treatment can significantly promote the growth and nutritional quality of broccoli heads; selenium yeast and sodium selenite can effectively increase selenium content in plants and promote the accumulation of nutrients such as soluble sugars and vitamin C. It is worth noting that in our previous studies, we found that the ability of paper mulberry leaves to accumulate sodium selenate is twice that of sodium selenite. Foliar spraying with 800 μmol / L sodium selenate can increase the selenium content in the leaves to as high as 130.0 mg / kg DW, suggesting that paper mulberry may have a more efficient mechanism for the absorption and transport of selenate.

[0004] HSF (heat shock factor) transcription factors were initially defined as transcriptional regulators of heat shock proteins (HSPs). However, mounting evidence suggests that HSFs play a crucial role in plant growth and development, as well as in responses to various abiotic stresses (such as low temperature, drought, hypoxia, and soil salinization) and biotic stresses (pathogen infection). HSF proteins contain a highly conserved DNA-binding domain (DBD) at their N-terminus, a helical-turn-helical motif that is essential for interaction with heat shock elements (HSES, 5'-AGAAnnTTCT-3'). Following the DBD is an oligomerization domain (ODorHR-A / B region), linked by a 15-80 amino acid linker in a heptapeptide repeat pattern containing hydrophobic amino acid residues. This repeater is responsible for protein-protein interactions during transcriptional activation and also participates in nuclear import and export. Based on the insertion fragment between the OD sequence HR-A and HRB of the oligomerization domain, plant HSF proteins are classified into three classes: A, B, and C. In angiosperms, HSF proteins are further divided into 16 subfamilies, including A1-A9, B1-B5, and C1-C2. The C-terminal activation domain of class A HSFs possesses a short peptide motif (AHA activation domain) and is specifically present in the HSFA subfamily, mediating its transcriptional activation function. Except for the B5 subfamily, the C-terminus of class B HSFs contains a highly conserved tetrapeptide repression domain (LFGV). Most HSF proteins also contain nuclear localization signals (NLS) and nuclear export signals (NES) in their C-terminal regions, which play a role in the assembly of the nuclear import complex and the receptor-mediated export complex. Plant HSF proteins were first identified in tomato (Solanum lycopersicum). To date, the HSF family has been identified in various plants; for example, Arabidopsis thaliana contains 22 HSF members, tomato contains 24 members, rice and wheat contain 25 and 56 members respectively, and carrots and peppers contain 35 and 25 HSF members respectively. It is noteworthy that selenium stress, as a specific abiotic stress, also involves the regulation of HSF transcription factors. Previous studies have shown that HSF transcription factors also play a crucial role in the sensing of selenium stress signals and metabolic responses. In barley, HSF may enhance the plant's tolerance to selenite stress by regulating organic acid metabolism; while in wheat, nano-selenium treatment can induce the expression of HSFA4A, thereby triggering specific signaling pathways and regulating selenium metabolism-related processes. Our previous research found that paper mulberry has a very strong selenium accumulation capacity. Further transcription factor studies have shown that BpNAC59 and BpNAC62 specifically respond to sodium selenite, while BpNAC55 specifically responds to selenate, suggesting that all three are involved in the regulation of selenium metabolism; in addition, BpMYB135 has also been identified as a key candidate gene.At the transport level, recent studies have identified the key sulfate transporter gene BpSULTR3;1, which is significantly upregulated under selenium treatment, and its overexpression has been shown to effectively enhance the selenium accumulation capacity of plants. Notably, at the metabolic level, our genome-wide identification of the HMT gene family revealed the crucial role of BpHMT2 in converting inorganic selenium into organic forms (such as selenomethionine) to enhance selenium accumulation and tolerance. Although these genes have been successively discovered in the absorption, transport, and metabolism of selenium in mulberry trees, the gene regulatory network and molecular mechanisms of the BpHSF transcription factor family, as the core regulators of stress responses, under selenium stress have not yet been systematically elucidated. Summary of the Invention

[0005] The purpose of this invention is to address existing problems by providing an application of the BpHSF gene family for regulating selenium metabolism in paper mulberry.

[0006] The present invention is achieved through the following technical solution: an application of the BpHSF gene family for regulating selenium metabolism in paper mulberry, wherein the BpHSF gene family includes 18 members from BpHSF01 to BpHSF18, which regulate selenium metabolism in paper mulberry by controlling at least one of the processes of selenium absorption, transport, assimilation or tolerance in paper mulberry.

[0007] Furthermore, the BpHSF gene family is divided into three subgroups: HSFA, HSFB, and HSFC. The HSFA subgroup includes BpHSF02, BpHSF03, BpHSF06, BpHSF08, BpHSF11, BpHSF12, BpHSF15, BpHSF16, BpHSF17, and BpHSF18; the HSFB subgroup includes BpHSF04, BpHSF05, BpHSF07, BpHSF09, BpHSF10, BpHSF13, and BpHSF14; and the HSFC subgroup is BpHSF01.

[0008] Furthermore, the expression of at least one member of the BpHSF gene family is significantly positively or negatively correlated with the total selenium content of Broussonetia papyrifera.

[0009] Furthermore, the gene positively correlated with total selenium content is at least one of BpHSF13, BpHSF12, and BpHSF09; the gene negatively correlated with total selenium content is at least one of BpHSF06, BpHSF03, BpHSF01, BpHSF07, BpHSF04, and BpHSF18.

[0010] Furthermore, the BpHSF09 and BpHSF12 proteins are located in the cell nucleus, and the BpHSF13 protein is located in both the cell nucleus and the cell membrane.

[0011] Furthermore, the BpHSF gene family regulates the expression of the structural genes by binding to the heat shock element in the promoter of the selenium metabolism structural gene through its encoded protein.

[0012] Furthermore, the selenium metabolism structural gene is at least one of the ABC transporter, SAT, SULTR, HMT, or GST family genes.

[0013] A method for regulating selenium metabolism in paper mulberry, comprising regulating the expression of at least one BpHSF gene in paper mulberry, wherein the BpHSF gene is any of the genes described above.

[0014] Furthermore, the regulatory mechanisms include at least one of overexpression, silencing, editing, or exogenously induced expression.

[0015] Compared with existing technologies, this invention has the following advantages: 1. This application systematically identifies HSF transcription factors in the whole genome of *Broussonetia papyrifera* and analyzes their physicochemical properties, phylogenetic relationships, and interspecific collinearity characteristics in depth. By detecting the expression profile of the BpHSF gene under selenium stress, core response members are further screened and their cellular functional properties are verified through subcellular localization. To further explore the potential regulatory role of BpHSF in selenium metabolism, this study identifies key BpHSFs and their potential target genes that are significantly associated with total selenium content based on functional annotation and expression correlation analysis of structural genes in selenium metabolism in *Broussonetia papyrifera*, and constructs the corresponding co-expression and protein interaction network. On this basis, AME motif enrichment analysis is carried out in combination with the network topology of key genes in selenium metabolism. The results reveal that multiple typical HSF binding elements are significantly enriched in selenium metabolism hub genes, suggesting that BpHSF may participate in the transcriptional regulation of key links such as selenium absorption, transport, and detoxification in a sequence-specific manner. In summary, this application reveals the potential function of BpHSF in the selenium response of Broussonetia papyrifera from multiple levels, including genome structure, expression regulation, network association, and cis-elements, providing important key gene resources and theoretical basis for molecular breeding of selenium-enriched Broussonetia papyrifera.

[0016] 2. This application presents a genome-wide analysis of the HSF gene family in Broussonetia papyrifera and establishes a complex regulatory model controlling Se homeostasis. Eighteen BpHSF members were identified, demonstrating significant functional differences between the HSFA and HSFB subgroups. HSFB members may promote Se accumulation, while HSFA members mediate negative feedback and detoxification. The discovery of membrane-nuclear dual localization of BpHSF13 suggests a novel mechanism by which HSFs act as early signaling sensors in response to Se-induced stress. Furthermore, network analysis confirmed that HSF-binding elements are significantly enriched in the promoters of core Se metabolic center genes (such as GST and ABC), indicating that HSFs have preferential regulatory roles at the most critical nodes in the Se network. In summary, the findings of this application provide a detailed molecular framework for the HSF-mediated Se regulatory network, offer valuable insights into the adaptive evolution of Se accumulation in Broussonetia papyrifera, and identify promising targets for future bio-enhancement strategies. Attached Figure Description

[0017] Figure 1 shows the phylogenetic analysis of HSFs from Broussonetia papyrifera, Arabidopsis thaliana, rice, and poplar; Figure 2 shows the phylogenetic tree, gene structure, and conserved motif analysis of BpHSFs; Figure 3 shows the chromosome distribution and collinearity analysis of BpHSFs; Figure 4 shows the cis-regulatory element analysis of the promoter region of BpHSFs; Figure 5 shows the correlation between BpHSFs and selenium content and their co-expression modules with selenium metabolism genes; Figure 6 shows the effect of different concentrations of exogenous selenium treatment on the expression levels of nine BpHSF transcription factors; Figure 7 shows the subcellular localization of BpHSF09, BpHSF12, and BpHSF13; Figure 8 shows the network topology and motif analysis of BpHSFs. Detailed Implementation

[0018] To further explain the present invention, the following specific embodiments are described.

[0019] The plant material and treatments used young leaves of *Broussonetia papyrifera* 'Kegou101' cultivated in a greenhouse in Jingzhou, Hubei Province (30.35°N, 112.15°E) as experimental material. Uniformly growing plants were selected and randomly assigned to 7 experimental groups, with 3 replicates per group and 10 plants per replicate. Sodium selenate and sodium selenite were applied to the leaves at concentrations of 200 μM, 400 μM, and 800 μM, respectively. The control group was treated with deionized water. 100 ml of the solution was sprayed onto both the upper and lower surfaces of the leaves weekly for a total of 3 treatments. Samples were taken on day 28, and the leaves were rapidly frozen in liquid nitrogen and stored at -80°C.

[0020] All experimental data are presented as mean ± standard error of three biological replicates. Statistical analysis was performed using Excel 2019 and Origin (v2024, https: / / www.originlab.com / ). One-way ANOVA and Tukey's test were used to analyze for significant differences between groups; p < 0.05 was considered significant. Graphs and charts were generated using Origin 2024 (Origin Lab) or Tbtools.

[0021] Example 1: Identification, Physicochemical Properties Analysis, and Subcellular Localization Prediction of BHPSF. The Bulbus genome was obtained from publicly available data from NCBI (Bioproject: PRJNA437223) and assembled internally. The Hidden Markov Model (PF00447) of the HSF domain was obtained from the Pfam database (http: / / pfam.xfam.org). Protein sequences containing the HSF domain were retrieved using the HMMER 3.0 online database, and TBtools was used to screen for sequences containing the HSF domain (E value e ≤ 10). -5 Candidate transcription factors were selected, and the intersection of the two was taken. Sequence integrity was verified using NCBI_CDD (https: / / www.ncbi.nlm.nih.gov / cdd / ) and SMART (https: / / smart.embl.de / ), and subsequent sequences with incomplete domains were removed. The basic physicochemical properties of BpHSF transcription factors were analyzed using the online website ExPASy (https: / / web.expasy.org / protparam / ). The subcellular localization of BpHSF proteins was predicted using the online tool WoLF PSORT web server (https: / / wolfpsort.hgc.jp / ).

[0022] Eighteen BpHSF genes were identified in the Broussonetia papyrifera genome and renamed BpHSF01–BpHSF18 based on their chromosomal locations. Physicochemical analysis of the BpHSF proteins showed that all BpHSF proteins are hydrophilic, with amino acid counts ranging from 218 to 844, molecular weights ranging from 24.67 to 90.41 kDa, and theoretical isoelectric points ranging from 4.57 to 9.02. Instability coefficient analysis revealed that 89% of the BpHSF proteins were unstable (instability coefficient greater than 40). Subcellular localization prediction showed that all members were located in the cell nucleus (Table 1).

[0023] Table 1 Physicochemical properties of BpHSF protein

[0024] Example 2: Phylogenetic Tree Analysis of the HSF Family To investigate the evolutionary relationship between the BpHSFs family and other plant HSFs, we downloaded the protein sequences of Arabidopsis thaliana and rice HSFs from HEATSTER (https: / / applbio.biologie.uni-frankfurt.de / hsf / heatster / hsf_visualization_tool.php). The poplar HSF protein was obtained from the study of Zhao et al. (Zhao K, Dang H, Zhou L, Hu J, Jin X, Han Y, Wang S, 2023. Genome-Wide Identification and Expression Analysis of the HSFGene Family in Poplar. Forests 14, 510. https: / / doi.org / 10 / gtwzdq). Multiple sequence alignment was performed using ClustalW2, and a phylogenetic tree was constructed using the One Step Build a ML Tree software in TBtools. The phylogenetic tree was then formatted on the evolview online website (http: / / www.evolgenius.info / evolview / # / treeview).

[0025] This study constructed a phylogenetic tree using 92 HSF protein sequences, including 18, 21, 25, and 28 HSFs from Broussonetia papyrifera, Arabidopsis thaliana, rice, and poplar, respectively. Based on the classification of HSFs in other species, BpHSFs from Broussonetia papyrifera were divided into three subgroups: HSFA, HSFB, and HSFC (Figure 1). The distribution of BpHSFs differed significantly among the different subfamilies. HSFA contained the most BpHSFs, accounting for 55.6%, followed by HSFB (38.9%), while HSFC had the least, accounting for only 5.6%. Subsequently, BpHSFs were subdivided into 9 subfamilies (A1-A9) according to evolutionary relationships, HSFB was subdivided into 4 subfamilies (B1-B4), and HSFC did not require further subdivision because this subfamilie contains only one BpHSF member (BpHSF01) (Table 1).

[0026] In Figure 1: different background colors represent different subfamily classifications, and red stars represent BpHSFs of paper mulberry.

[0027] Table 2 Classification of BpHSFs of Paper Mulberry

[0028] Example 3: Analysis of the gene structure and conserved motifs of the encoded protein of BpHSF. Based on the genome annotation file, the exon-intron structure of the BpHSF gene was visualized using TBtools software. The conserved motifs of the BpHSF protein were analyzed using the MEME online tool (https: / / meme-suite.org / meme / tools / meme).

[0029] The phylogenetic tree of the BpHSF family is shown in Figure 2A. The branching structure of this tree is highly consistent with the phylogenetic tree constructed based on HSF protein sequences from multiple species (Figure 1), further demonstrating the correctness and reliability of the evolutionary tree constructed in this study. Structural analysis of the BpHSF genes showed that all exons of the BpHSF genes were disrupted by introns, with approximately 77.8% of the BpHSF genes containing only one intron. BpHSF18 had the most introns (9), while the number of introns in the remaining genes ranged from 2 to 9 (Figure 2B). Furthermore, domain prediction of BpHSFs using NCBI_CDD revealed that all BpHSFs contain a typical HSF domain (Figure 2C), further demonstrating the reliability of the gene identification results. Conserved motifs are highly conserved short sequences or structural units in biological macromolecules, directly determining their key biological functions and maintaining structural stability. Analysis of conserved motifs in BpHSFs revealed that each BpHSF protein contains between 4 and 8 conserved motifs (Figure 2D). Motif 2 is present in all BpHSF proteins, while all members except BpHSF16 contain motif 1. Furthermore, motifs 4 and 9 are also present in most BpHSF proteins. In addition, some conserved motifs are only present in specific subfamilies, exhibiting family specificity. For example, motif 5 is only present in the HSFB subfamily of BpHSF04, BpHSF05, BpHSF07, BpHSF09, BpHSF10, BpHSF13, and BpHSF14, suggesting that it may perform specific functions within particular subfamilies.

[0030] Figure 2 shows: (A) the phylogenetic relationship of the BpHSF family, with different colors representing different subfamilies; (B) the gene structure of BpHSF, with gray lines representing introns and green squares representing exons; (C) the conserved domains of BpHSF, with gray representing protein length and yellow representing conserved domains; and (D) the analysis of conserved motifs in the BpHSF protein, with black lines representing protein length and different colored blocks representing different motifs.

[0031] Example 4: Chromosome Localization and Collinearity Analysis. The location information of BpHSF genes was mapped to the corresponding chromosomes using the genomic annotation file of *Mulberry*, and visualized using TBtools software. To further explore the evolutionary relationships of BpHSF genes in *Mulberry*, we downloaded *Arabidopsis thaliana* genome data from the Ensembl database (https: / / plants.ensembl.org / index.html) and obtained *Morus alba* genome data from the study by Ma Bi et al. (Ma B, Wang H, Liu J, Chen L, Xia X, Wei W, Yang Z, Yuan J, Luo Y, He N, 2023. The gap-free genome of mulberry elucidates the architecture and evolution of polycentric chromosomes. Horticulture Research 10, uhad111. https: / / doi.org / 10 / gt54n4). We used MCScanX to analyze and visualize collinearity within *Mulberry* and with other species.

[0032] Chromosome localization analysis revealed that BpHSF genes were present on all 11 chromosomes except chr5 and chr12, exhibiting a non-uniform distribution (Figure 3A). Chr1 chromosome contained the most BpHSF genes (4); chr2, chr4, chr6, and chr13 each contained 2 BpHSF genes; and chr3, chr7, chr8, chr9, chr10, and chr11 each contained only 1 BpHSF gene. The number of genes in plants is not constant, and fragment duplication plays a crucial role in gene expansion and plant adaptation to the environment. However, no fragment duplication genes were detected in the HSFs of *Broussonetia papyrifera*. To further explore the evolutionary relationships of *Broussonetia papyrifera* BpHSFs, we examined the collinearity between the *Broussonetia papyrifera* BpHSF family and *Morus alba* and *Arabidopsis thaliana*. The results showed that in the BpHSF family of paper mulberry, there were 3 pairs of collinear genes with mulberry, while there was only 1 pair of collinear genes with the model plant Arabidopsis thaliana (Figure 3B).

[0033] Figure 3 shows: (A) the distribution of BpHSF genes on chromosomes in *Broussonetia papyrifera*, with the scale bar on the left indicating the length (Mb) of constitutive tree chromosomes. (B) the collinearity between the BpHSF family of *Broussonetia papyrifera* and *Morus alba* and *Arabidopsis thaliana*. The gray lines in the background represent collinear gene pairs between species, while each pink line represents a gene pair that is collinearly related to the BpHSF gene. Different colored blocks represent different chromosomes.

[0034] Example 5: Cis-regulatory element analysis of the BpHSF promoter. The DNA sequence of 2000 bp upstream of the start codon ATG of the BpHSF gene was extracted from the genome of Broussonetia papyrifera and then uploaded to PlantCARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) to predict the possible cis-regulatory elements in the promoter of the BpHSF gene.

[0035] To predict the potential regulatory features of the BpHSF gene, we systematically analyzed the cis-acting elements in the promoter region 2000 bp upstream of its start codon ATG (Figure 4). Overall, these elements can be categorized into four main types: light-responsive, stress-responsive, hormone-responsive, and development-responsive, with light-responsive and hormone-responsive elements being the most abundant. Among light-responsive elements, G-box is the most prevalent type, distributed in most BpHSF promoters, followed by Box4 and GATA-motif (Figure 4B), suggesting that the BpHSF family as a whole has a high potential for sensing light signals. Hormone-responsive elements mainly involve pathways such as gibberellin (GA), abscisic acid (ABA), methyl jasmonic acid (MeJA), auxin (IAA), and salicylic acid (SA). MeJA-responsive elements were the most numerous (44), followed by GA-responsive elements (17), indicating that BpHSF may be located at a key intersection of the jasmonic acid and gibberellin signaling networks and is synergistically regulated by multiple hormones. Among the stress response categories, low-temperature-related LTRs were the most numerous elements. Typical cis-elements associated with drought-induced and wound-related stresses were also detected, suggesting that BpHSFs are closely related to temperature stress and tolerance to abiotic / mechanical stress. A total of 19 developmental-related elements were identified. Although relatively few BpHSF members carry these elements, it suggests that some family members may be involved in transcriptional regulation specific to developmental stages or tissues.

[0036] Figure 4: (A) Distribution of cis-elements on the BpHSF promoter. Some elements may overlap; different colored blocks represent different element types. (B) Statistical analysis of cis-acting elements in the BpHSF gene family promoter. The horizontal axis represents the type of cis-acting element, and the vertical axis represents the number of BpHSF family members containing that specific element.

[0037] Example 6: Expression Patterns of BpHSFs and Regulation of Selenium Metabolism-Related Genes 6.1 Expression Profile Characteristics of BpHSFs and Co-expression Analysis with Selenium Metabolism Genes To investigate the response pattern of BpHSF genes to selenium stress, this study used published transcriptome data from Broussonetia papyrifera leaves (GSA: CRA005102) (Chen Q, Zhu C, Guo L, Bu X, Yang W, Cheng S, Cong X, Xu F, 2024. Genome-wide identification of HMT gene family explores BpHMT2 enhancing selenium accumulation and tolerance in Broussonetia papyrifera. TreePhysiology 44, tpae030. https: / / doi.org / 10 / g5w499) to analyze its expression levels under different concentrations (200, 400, and 800 μM) of sodium selenate (Na2SeO4) and sodium selenite (Na2SeO3). Gene expression abundance was measured by FPKM values.

[0038] To further elucidate the potential function of BpHSF in the selenium metabolism regulatory network, we first systematically screened 126 structural genes involved in key metabolic pathways such as selenium uptake, assimilation, and amino acid synthesis based on mulberry tree genome functional annotation. Subsequently, we calculated the Pearson correlation coefficients between the expression levels of all BpHSF genes and the aforementioned selenium metabolism structural genes and the total selenium content under different treatments, with a significance level set at P < 0.05. Based on the correlation analysis results, we selected five key BpHSF genes that showed a strong correlation with total selenium content (|r| ≥ 0.6), along with selenium metabolism genes exhibiting the same correlation characteristics. Finally, we constructed a co-expression matrix using these screened genes and generated a heatmap to visualize the potential regulatory relationship between BpHSF and selenium metabolism structural genes.

[0039] The BpHSF family exhibited significant differentiation and expression patterns in response to different concentrations and forms of selenium. Correlation analysis showed that the FPKM of BpHSF04, BpHSF09, and BpHSF13 were significantly positively correlated with the total selenium content of plants (R>0.6) and were strongly induced by high concentrations of selenium, suggesting that they act as positive regulators driving selenium enrichment (Figure 5A). Conversely, the FPKM of BpHSF12 and BpHSF18 were significantly negatively correlated with the total selenium content (R<-0.6), suggesting that they may maintain intracellular selenium homeostasis through a negative feedback mechanism. To elucidate the molecular network behind this regulation, we screened 126 selenium metabolism-related structural genes from the transcriptome and identified 39 core response genes that were significantly correlated with selenium physiological indicators (|r|>0.6, P<0.05). Co-expression analysis revealed a highly synergistic and differentiated regulatory module between BpHSF and these core genes (Figure 5B). Twenty genes formed a tight co-expression cluster with the "positive regulatory group" (BpHSF04 / 09 / 13), while another 19 genes were specifically highly positively correlated with the "negative regulatory group" (BpHSF12 / 18). This result suggests that BpHSF may finely regulate the balance between selenium uptake and tolerance in Broussonetia papyrifera by modulating different downstream metabolic gene groups.

[0040] Figure 5 shows: (A) A heatmap of the correlation between the BpHSF gene and total selenium content. Red text represents total selenium content, and the color of each cell, from blue to red, indicates an increasing correlation coefficient from negative to positive. The numbers in the cells are Pearson correlation coefficients. (B) A heatmap of co-expression of the BpHSF gene and screened selenium metabolism-related genes, with colors from blue to red indicating increasing correlation. * indicates significance, *P ≤ 0.05, **.

[0041] 6.2 RNA Extraction and RT-qPCR Analysis Total Broussonetia papyrifera RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa, China). RNA purity was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), and reverse transcription was performed using the HiScript cDNA Synthesis Kit (Vazyme, China) to obtain cDNA templates. Quantitative primers were designed using Primer Premier 6 software (Table 3). Ubiquitin was used as an internal control gene, and 2... -ΔΔCt The relative expression level of the BpHSF gene was calculated using this method. Primer synthesis and sequencing of the sequences used in this experiment were performed at Sangon Biotech (Shanghai) Co., Ltd.

[0042] Table 3 Primer sequences used in RT-qPCR

[0043] To verify the reliability of the transcriptome correlation analysis results, nine BpHSF genes with the highest correlation to total selenium content in plants were selected for real-time quantitative RT-qPCR validation. The results showed that the expression trends of most genes were consistent with the transcriptome FPKM data, and the correlation analysis further confirmed their significant association with selenium accumulation (Figure 6). Specifically, BpHSF13, BpHSF12, and BpHSF09 were significantly positively correlated with selenium content (R = 0.487–0.688, P<0.05), indicating that their high expression may promote selenium uptake or conversion. Conversely, BpHSF06, BpHSF03, BpHSF01, BpHSF07, BpHSF04, and BpHSF18 were significantly negatively correlated with selenium content (|R| = 0.426–0.705, P<0.05), suggesting that they may participate in inhibiting selenium enrichment or mediating defense responses under selenium stress. Among them, BpHSF07 showed the strongest negative correlation with selenium (R = -0.705), and may play a key negative regulatory role in the selenium response. Overall, the BpHSF family exhibits a bidirectional differentiation pattern in the selenium response of Broussonetia papyrifera, with different members possibly participating in the promotion of selenium metabolism and the regulation of stress defense, respectively, to jointly maintain selenium homeostasis.

[0044] In Figure 6: the left Y-axis of the bar chart represents gene expression level, and the right Y-axis of the line chart represents total selenium content (mg / kg DW). Different lowercase letters indicate significant differences (P≤ 0.05), and the R value indicates the correlation between HSF gene expression level and total selenium content.

[0045] Example 7: Subcellular localization Specific primers were used to amplify BpHSF09, BpHSF12, and BpHSF13 (without stop codons) by PCR. These were then fused to the C-terminus of the pNC-Cam1304-GFP gene using a one-step cloning method to construct the fusion plasmid pNC-1304-BpHSF09 / 12 / 13-GFP (Table 4). Subsequently, the empty eGFP vector and the nuclear marker p2300-35S-H2B-mCherry-OCS (purchased from Bio LABs) were transformed into Agrobacterium tumefaciens GV3101, respectively. The Agrobacterium strain was incubated and resuspended to prepare the working solution. Equal volumes of the fusion construct, the empty eGFP vector, and the mCherry-labeled vector were then injected into Nicotiana benthamiana. After incubation in the dark for 16 h, the plants were exposed to normal light conditions for 2 days, and then observed using a laser confocal microscope (Leica TCS SP8, Germany).

[0046] Table 4 Primer sequences used in PCR

[0047] Given the significant positive correlation between the expression levels of BpHSF09, BpHSF12, and BpHSF13 and total selenium content, it is speculated that these three genes may be positively involved in the absorption and metabolism of selenium. To further verify their biological functions, subcellular localization analysis was performed. The results showed that the nuclear localization markers exhibited red fluorescence signals in all cell nuclei, while the empty vector eGFP was distributed in both the nucleus and cytoplasm. In contrast, in tobacco leaves infected with the BpHSF09-GFP and BpHSF12-GFP fusion vectors, the green fluorescence and mCherry red fluorescence signals completely overlapped in the cell nucleus, indicating that both were localized in the nucleus. Furthermore, the green fluorescence of BpHSF13-GFP was detected not only in the nucleus but also showed a clear signal on the cell membrane (Figure 7).

[0048] Figure 7: Observation results of fluorescence signals of the empty eGFP vector, nuclear marker, and BpHSF09-eGFP, BpHSF12-eGFP, and BpHSF13-eGFP fusion proteins in tobacco leaf cells. Bar = 20 μm.

[0049] Example 8: Protein-protein interaction network and AME enrichment analysis of the BpHSF family. To further identify key Se metabolism genes in the protein-protein interaction (PPI) network, the interaction regulatory network of BpHSF proteins in Broussonetia papyrifera was predicted using the STRING database (https: / / cn.string-db.org / ). Simultaneously, using the Plant TF Binding Motif Shift plugin in Tbtools software, with Arabidopsis thaliana as a model plant, BpHSF binding motifs were screened, and Fimo: Binding Motif Scan was used to preliminarily identify potential target genes of BpHSF in Broussonetia papyrifera. Finally, Cytoscape 3.10.1 was used to visualize the protein-protein interaction network and target gene regulatory network.

[0050] To assess the regulatory tendencies of BpHSF on genes at different topological levels in the Se metabolism network, the PartnerOfMultiEdgedNodePairs index in Cytoscape was used to quantify the connectivity characteristics of network nodes. This index, based on multiple edge pairs, emphasizes robust interactions supported by multiple pieces of evidence, rather than simply the number of edges. All Se metabolism-related genes were ranked according to the PartnerOfMultiEdgedNodePairs values. The top 25 genes were selected as the hub genes (primary sequences), and the bottom 25 genes were selected as the background genes (control sequences), representing high-connectivity and low-connectivity nodes in the network, respectively. Promoter sequences 2,000 bp upstream of the transcription start sites of these 50 genes were extracted and input into the AME (Analysis of Motif Enrichment) module of MEME Suite for motif enrichment analysis. The regulatory characteristics were assessed by comparing the enrichment differences of BpHSF binding elements between the high-connectivity and low-connectivity groups. In the “Select a motif database” option, choose the ARABIDOPSIS (Arabidopsis thaliana) DNA database to improve the accuracy of plant transcription factor identification.

[0051] To systematically elucidate the regulatory network of BpHSF in selenium tolerance in mulberry trees, we constructed protein-protein interaction (PPI) and transcriptional regulatory networks. PPI network topology analysis (Figure 8A) revealed that the BpHSF family exhibits a distinct "core-periphery" hierarchical structure: BpHSF15, BpHSF17, and BpHSF18, as high-connectivity hubs (Degree>15), are located in the innermost layer of the network, suggesting that they may act as integrators of functions within the family, working synergistically with other radially distributed peripheral members.

[0052] Based on this, and by further combining the cis-acting element prediction results, potential target genes related to Se metabolism that are directly regulated by BpHSF were screened. The results showed that a total of 9 BpHSF transcription factors may target a group of functional genes involved in selenium absorption, transport and detoxification (Figure 8 B). These downstream genes mainly belong to key families such as ABC, SAT, SULTR, HMT and GST.

[0053] To determine whether core selenium metabolism genes are subject to common upstream regulation, we performed AME enrichment analysis on the promoter sequences (TPs) of the 25 selenium metabolism genes with the highest connectivity, and used the 25 genes with the lowest connectivity as the background set (FP). The results showed that AME analysis identified 7 significantly enriched motifs, suggesting that selenium metabolism hub genes possess characteristic cis-regulatory structures (Figure 8C). Among them, the three classic HSF motifs, HSF21, HSFA1E, and HSF6, were significantly enriched (p = 3.18 × 10⁻⁶). -3 Up to 8.93 × 10 -3 The core of these motifs all contain typical TTC–GAA type HSE (heatshock element). These motifs have a total tract response (TP) of 36–40% and were not detected in the background set, indicating significant selective enrichment of selenium metabolism hub genes. In addition to HSFs, two C2H2 zinc finger protein motifs (AT3G46070 and AT5G22890) were also significantly enriched, with AT3G46070 having a TP as high as 68%. However, they were also detected in the background set (FP = 16%), suggesting that zinc finger proteins may jointly participate in the transcriptional regulation of selenium responses. Furthermore, the B3-domain (ABI3 / VP1) motif AT5G25475 appeared in almost all hub genes (TP = 92%), but it also had a high frequency in the background set (FP = 44%), indicating that this motif may reflect a more general promoter structural feature rather than a selenium metabolism hub gene-specific regulatory element. Similarly, the binding motif of MADS-box transcription factor AGL95 was significantly enriched (TP = 52%), but it also appeared in the background set to a certain extent.

[0054] Figure 8: (A) Protein-protein interaction (PPI) network of the BpHSF family analyzed using the STRING database. Node size and color depth correspond to connectivity, highlighting the central hub (inner circle). (B) Regulatory network of BpHSFs and target genes involved in selenium metabolism, with prediction results visualized using Cytoscape 3.10.1. Transcriptional regulatory network between BpHSFs (green nodes) and Se metabolism-related structural genes (yellow nodes). (C) Significantly enriched motifs identified in the promoters of core target genes. Bar charts showing motif Alt IDs, sequence logos, and the proportion of each motif in the target gene (TP) and background gene (FP) with the highest connectivity in the AME.

[0055] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An application of the BpHSF gene family for regulating selenium metabolism in paper mulberry, characterized in that, The BpHSF gene family comprises 18 members, from BpHSF01 to BpHSF18, which regulate selenium metabolism in paper mulberry by controlling at least one of the processes of selenium absorption, transport, assimilation, or tolerance in paper mulberry.

2. The application according to claim 1, characterized in that, The BpHSF gene family is divided into three subgroups: HSFA, HSFB, and HSFC. The HSFA subgroup includes BpHSF02, BpHSF03, BpHSF06, BpHSF08, BpHSF11, BpHSF12, BpHSF15, BpHSF16, BpHSF17, and BpHSF18; the HSFB subgroup includes BpHSF04, BpHSF05, BpHSF07, BpHSF09, BpHSF10, BpHSF13, and BpHSF14; and the HSFC subgroup is BpHSF01.

3. The application according to claim 1 or 2, characterized in that, The expression of at least one member of the BpHSF gene family is significantly positively or negatively correlated with the total selenium content of Broussonetia papyrifera.

4. The application according to claim 3, characterized in that, The gene positively correlated with total selenium content is at least one of BpHSF13, BpHSF12, and BpHSF09; the gene negatively correlated with total selenium content is at least one of BpHSF06, BpHSF03, BpHSF01, BpHSF07, BpHSF04, and BpHSF18.

5. The application according to claim 4, characterized in that, The BpHSF09 and BpHSF12 proteins are located in the cell nucleus, while the BpHSF13 protein is located in both the cell nucleus and the cell membrane.

6. The application according to claim 1, characterized in that, The BpHSF gene family regulates the expression of the structural genes by binding their encoded proteins to heat-shock elements in the promoters of selenium metabolism structural genes.

7. The application according to claim 6, characterized in that, The selenium metabolism structural gene is at least one of the ABC transporter, SAT, SULTR, HMT, or GST family genes.

8. A method for regulating selenium metabolism in paper mulberry, characterized in that, This includes regulating the expression of at least one BpHSF gene in mulberry trees, wherein the BpHSF gene is any one of claims 1-7.

9. The method according to claim 8, characterized in that, The regulatory mechanisms include at least one of overexpression, silencing, editing, or exogenous induction of expression.

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