Moso bamboo flavone synthase I gene PeFNSI1, encoding protein and application thereof

Abstract

The invention discloses a phyllostachys pubescens flavone synthase I gene PeFNSI1, an encoded protein and an application of the phyllostachys pubescens flavone synthase I gene PeFNSI1, belongs to the technical field of genetic engineering, and particularly relates to the phyllostachys pubescens flavone synthase I gene PeFNSI1, the nucleotide sequence of the phyllostachys pubescens flavone synthase I gene PeFNSI1 is shown as SEQ ID NO.1, and the application of the phyllostachys pubescens flavone synthase I gene PeFNSI1 in enhancing The invention reveals for the first time that flavone synthetase I (PeFNSI) coded by the phyllostachys pubescens PeFNSI1 gene can specifically catalyze flavone precursor naringenin to be converted into apigenin and catalyze eriodictyol to be converted into luteolin, and can activate a'bifunctional 'regulation element of a flavone synthesis and salicylic acid defense pathway at the same time by reprogramming salicylic acid metabolic flux, so that the flavone synthetase I (PeFNSI) can be used for preparing the apigenin and the luteolin. The multi-element synergistic effect of flavone synthesis, salicylic acid metabolic flux regulation and control and disease resistance enhancement is achieved, the limitation of an existing disease resistance strategy is broken through, and a brand new molecular tool is provided for plant disease resistance breeding.

Description

A gene PeFNSI1 of bamboo flavonoid synthase I, its encoded protein and its application Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a bamboo flavonoid synthase I gene PeFNSI1, its encoded protein, and its applications. Background Technology

[0002] Plants face biotic and abiotic stresses such as pathogen infection and drought during their growth cycle, severely restricting crop yield and quality improvement. Flavonoids, as secondary metabolites, are core functional substances for plants to resist external stresses. Moso bamboo (Phyllostachys edulis) is one of the most produced economic bamboo species in my country. Its leaves are rich in characteristic C-glycosyl flavonoids such as scutellarin, isoscutellarin, and vitexin, exhibiting significant antioxidant and stress-resistance activities, and are a key material basis for the high-value utilization of bamboo resources. Flavonoid synthase I (FNSI), as a key enzyme in the flavonoid pathway, catalyzes the conversion of flavanones (such as naringenin) into flavonoid backbones (such as apigenin), and is an essential precursor step in the biosynthesis of C-glycosyl flavonoids. However, functional analysis of bamboo-derived FNSI genes remains scarce, and their regulatory role in plant defense networks is still unclear.

[0003] Salicylic acid (SA), a key molecule in plant defense signaling pathways, plays a crucial role in the precise regulation of its metabolic flux, which is a core mechanism for initiating disease resistance responses in plants. The interaction between flavonoids and the salicylic acid signaling pathway has been confirmed as an important regulatory network for plant stress resistance. Currently, the identification of key genes in flavonoid biosynthesis and the association between core enzyme genes regulating flavonoid synthesis and salicylic acid metabolic flux remain unclear. In model plants such as Arabidopsis and rice, FNSI genes have only been reported to participate in flavonoid accumulation (e.g., AtFNSI), without involvement in salicylic acid metabolic regulation. As a core signaling molecule for acquired resistance in plants, the regulation of salicylic acid metabolic flux mainly focuses on synthetic genes such as ICS1 and PAL or glycosylation genes such as UGT74F2. No flavonoid synthases have been found to actively "reprogram" the direction of SA metabolic flux. Traditional disease resistance breeding relies on single-pathway genes (e.g., NPR1), which makes it difficult to achieve synergistic effects between flavonoids and SA signaling, and cannot meet the high-efficiency requirements of crop stress resistance breeding. Therefore, identifying the key genes in bamboo that regulate flavonoid synthesis and clarifying their functions in enhancing plant disease resistance and regulating salicylic acid metabolic flux is of great significance for molecular breeding of plant stress resistance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a flavonoid synthase I gene, PeFNSI1, encoded protein, and its applications in moso bamboo. It reveals for the first time that the flavonoid synthase I (PeFNSI) encoded by the moso bamboo PeFNSI1 gene can specifically catalyze the conversion of the flavonoid precursor naringenin to apigenin and sennaol to luteolin. Furthermore, through reprogramming of salicylic acid metabolic flux, it is a "dual-function" regulatory element that simultaneously activates flavonoid synthesis and salicylic acid defense pathways. This achieves synergistic regulation of flavonoid synthesis and salicylic acid metabolic flux, enhancing disease resistance through multiple synergistic effects. This breakthrough overcomes the limitations of existing disease resistance strategies and provides a novel molecular tool for plant disease resistance breeding.

[0005] To achieve the above objectives, the present invention provides a bamboo flavonoid synthase I gene PeFNSI1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides a protein encoded by the PeFNSI1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides a biomaterial comprising the PeFNSI1 gene, wherein the biomaterial is an expression cassette, an expression vector, or an engineered bacterium.

[0008] Preferably, the biological material is an expression vector with the PeFNSI1 gene inserted into it, and the expression vector is one or more of pC1300-35S or pCAMBIA1301.

[0009] The present invention also provides the application of the PeFNSI1 gene, the protein encoded by the PeFNSI1 gene, or the biomaterial in enhancing plant disease resistance.

[0010] Preferably, the plant is rice, wheat, or tobacco.

[0011] Preferably, by overexpressing the PeFNSI1 gene, the expression level of the protein encoded by the PeFNSI1 gene is increased, which promotes the synthesis of flavonoids, promotes the accumulation of salicylic acid, activates plant defense signaling pathways, enhances plant resistance to pathogens, and enhances plant disease resistance.

[0012] The present invention also provides the application of the PeFNSI1 gene, the protein encoded by the PeFNSI1 gene, or the biological material in promoting flavonoid synthesis, thereby promoting flavonoid synthesis in rice, wheat, tobacco, Saccharomyces cerevisiae, or Escherichia coli, wherein the flavonoids are apigenin and / or luteolin, and the expression level of the protein encoded by the PeFNSI1 gene is increased by overexpressing the PeFNSI1 gene, thereby promoting flavonoid synthesis.

[0013] The present invention also provides the application of the PeFNSI1 gene, the protein encoded by the PeFNSI1 gene, or the biological material in promoting the accumulation of salicylic acid in plants, wherein the plant is rice, wheat, or tobacco. By overexpressing the PeFNSI1 gene, the expression level of the protein encoded by the PeFNSI1 gene is increased, the salicylic acid biosynthesis pathway is activated, salicylic acid glycosylation inactivation is inhibited, salicylic acid metabolic flux is reconstructed, and active free salicylic acid is accumulated.

[0014] The present invention also provides the application of the PeFNSI1 gene, the protein encoded by the PeFNSI1 gene, or the biological material in enhancing plant resistance to pathogens, wherein the plant is rice, wheat, or tobacco, and the pathogen is *Pseudomonas syringae*. By overexpressing the PeFNSI1 gene, the expression level of the protein encoded by the PeFNSI1 gene is increased, thereby enhancing the plant's resistance to *Pseudomonas syringae*.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention provides a bamboo flavonoid synthase I gene PeFNSI1, its encoded protein, and its application. This invention is the first to isolate and identify the bamboo flavonoid synthase I gene PeFNSI1 from bamboo, clarifying that it is a key gene for the synthesis of flavonoids apigenin and luteolin in bamboo, filling the gap in the discovery of key genes for bamboo flavonoid synthesis. The PeFNSI1 gene has a dual function: it can regulate the synthesis of bamboo flavonoids and can also be reprogrammed into salicylic acid metabolic flux, significantly increasing flavonoid and salicylic acid synthesis through a positive feedback loop. Compared to existing single-function stress-resistance genes, PeFNSI1 gene enhances plant disease resistance, exhibiting higher stress resistance efficiency and stronger targeting. Overexpression of the PeFNSI1 gene significantly improves the resistance to pathogens. The application of the PeFNSI1 gene can overcome the limitations of traditional breeding cycles and poor targeting, enabling the rapid cultivation of new crop varieties with strong disease resistance and wide environmental adaptability. Furthermore, the flavonoids accumulated in the cultivated transgenic crops possess potential biological activities such as antioxidant and anti-inflammatory properties, enhancing the nutritional and economic value of the crops. This provides a solid theoretical foundation and experimental evidence for the practical application of the gene, with broad application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 shows the expression patterns of genes in *Phyllostachys edulis* under different stress conditions. In the figure, a represents the expression level of PePAL7 under abiotic stress, b represents the expression level of PePAL7 under simulated disease resistance signals, c represents the expression level of PePAL7 under drought stress, d represents the expression level of PePAL7 under salt stress, e represents the expression level of Pe4CL1 under abiotic stress, f represents the expression level of Pe4CL1 under simulated disease resistance signals, g represents the expression level of Pe4CL1 under drought stress, and h represents the expression level of Pe4CL1 under drought stress. 1. Expression levels under salt stress, i represents PeCHS7 expression under abiotic stress, j represents PeCHS7 expression under simulated disease resistance signals, k represents PeCHS7 expression under drought stress, l represents PeCHS7 expression under salt stress, m represents PeFNSI1 expression under abiotic stress, n represents PeFNSI1 expression under simulated disease resistance signals, o represents PeFNSI1 expression under drought stress, and p represents PeFNSI1 expression under salt stress. q represents the expression level of PeFNSI2 under abiotic stress conditions, r represents the expression level of PeFNSI2 under simulated disease resistance signals, s represents the expression level of PeFNSI2 under drought stress conditions, t represents the expression level of PeFNSI2 under salt stress conditions, u represents the expression level of PeF3H2 under abiotic stress conditions, v represents the expression level of PeF3H2 under simulated disease resistance signals, w represents the expression level of PeF3H2 under drought stress conditions, x represents the expression level of PeF3H2 under salt stress conditions, and y represents the expression level of PeF3H2 under salt stress conditions. 5. Expression levels under abiotic stress conditions, z represents the expression level of PeF3'5'H5 under simulated disease resistance signals, a' represents the expression level of PeF3'5'H5 under drought stress conditions, b' represents the expression level of PeF3'5'H5 under salt stress conditions, c' represents the expression level of PeLDOX4 under abiotic stress conditions, d' represents the expression level of PeLDOX4 under simulated disease resistance signals conditions, e' represents the expression level of PeLDOX4 under drought stress conditions, and f' represents the expression level of PeLDOX4 under salt stress conditions. The figures show the expression levels under various conditions. " "and" "Represents significant difference analysis; Figure 2 shows the subcellular localization analysis of PeFNSI1 and PeFNSI2, where a is a representative confocal image of cells transiently expressing 35S-PeFNSI1::eGFP, and b is a representative confocal image of cells transiently expressing 35S-PeFNSI2::eGFP. From left to right, they are green fluorescent protein, cytoplasmic marker, chlorophyll autofluorescence, bright field, and merged coverage, with a scale bar of 20 μm; Figure 3 shows the in vivo enzyme activity assay of PeFNSI1 and PeFNSI2 in yeast, where a is the product composition of naringenin as a substrate analyzed by HPLC. In the figure, PeFNSI1+Nar is the PeFNSI1 substrate group with naringenin, PeFNSI2+Nar is the PeFNSI2 substrate group with naringenin, EV+Nar is the empty vector substrate group with naringenin, Nar standard is the naringenin standard group, and Api is the product composition of naringenin. The standard represents the apigenin standard group; b represents the product composition of luteolin as a substrate analyzed by HPLC; PeFNSI1+Erio represents PeFNSI1 with luteolin as a substrate; PeFNSI2+Erio represents PeFNSI2 with luteolin as a substrate; EV+Erio represents the empty vector with luteolin as a substrate; Erio standard represents the luteolin standard group; Lute standard represents the luteolin standard group; c represents the product composition of pinocembrin as a substrate analyzed by HPLC; PeFNSI1+Pino represents PeFNSI1 with pinocembrin as a substrate; PeFNSI2+Pino represents PeFNSI2 with pinocembrin as a substrate; EV+Pino represents the empty vector with pinocembrin as a substrate; Pino standard represents the pinocembrin standard group; Chry... The standard represents the hesperidin standard group; d represents the product composition of hesperidin as a substrate analyzed by HPLC; PeFNSI1+Hesp represents PeFNSI1 with hesperidin as a substrate; PeFNSI2+Hesp represents PeFNSI2 with hesperidin as a substrate; EV+Hesp represents the empty vector with hesperidin as a substrate; Hesp standard represents the hesperidin standard group; Dio standard represents the geraniol standard group; e represents the molecular docking score heatmap; f represents the secondary protein structure analysis of the PeFNS family; g represents the docking result of PeFNSI1 with naringenin; h represents the docking result of PeFNSI2 with naringenin; i represents the docking result of PeFNSI1 with sennaol; j represents the docking result of PeFNSI2 with sennaol.Figure 4 shows the statistical results of phenotypic and physiological responses of tobacco leaves to Pst DC3000 inoculation. In Figure a, leaf phenotypic representation is shown, where WT represents wild-type tobacco leaves, EV represents empty vector control leaves, OE represents tobacco leaves overexpressing PeFNSI1, Pst represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment. The scale bar is 1 cm. Figure b shows DAB-stained leaf representation, where WT represents wild-type tobacco leaves, EV represents empty vector control leaves, OE represents tobacco leaves overexpressing PeFNSI1, Pst represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment. The scale bar is 1 cm. Figure c shows disease symptom representation, where WT represents wild-type tobacco leaves, EV represents empty vector control leaves, OE represents tobacco leaves overexpressing PeFNSI1, and Pst represents Pst DC3000 inoculation. DC3000 inoculation, MgCl2 represents MgCl2 treatment, the scale bar is 1 cm, d is the relative electrolyte leakage at 24 h, WT in the figure is wild-type tobacco leaf group, EV is empty vector control leaf group, PeFNSI1-OE is tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, MgCl2 represents MgCl2 treatment, "; "and" "This represents the analysis of significant differences. e represents the relative electrolyte leakage at 48h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "and" "This represents the analysis of significant differences. f is the maximum quantum yield of PSII at 24h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "This represents the analysis of significant differences. g represents the maximum quantum yield of PSII at 48h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "This represents the analysis of significant differences. h is the bacterial population quantified by colony formation units at 72h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "Represents significant difference analysis, i is the photoinduced kinetic curve of effective quantum yield at 24h, where WT represents wild-type tobacco leaf group, EV represents empty vector control leaf group, OE represents tobacco leaf group overexpressing PeFNSI1, Pst represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment; j is the photoinduced kinetic curve of non-photochemical quenching at 24h, where WT represents wild-type tobacco leaf group, EV represents empty vector control leaf group, OE represents tobacco leaf group overexpressing PeFNSI1, Pst represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment; k is the photoinduced kinetic curve of effective quantum yield at 48h, where WT represents wild-type tobacco leaf group, EV represents empty vector control leaf group, OE represents tobacco leaf group overexpressing PeFNSI1, and Pst represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." DC3000 inoculation, MgCl2 represents MgCl2 treatment, l is the photo-induced kinetic curve of non-photochemical quenching at 48h, WT in the figure represents wild-type tobacco leaf group, EV represents empty vector control leaf group, OE represents tobacco leaf group overexpressing PeFNSI1, Pst represents Pst DC3000 inoculation, MgCl2 represents MgCl2 treatment; Figure 5 shows the effect of PeFNSI1 overexpression on salicylic acid metabolic flux, where a is the loudness expression level of PeFNSI1 at 0h, WT in the figure represents wild-type tobacco leaf group, EV represents empty vector control leaf group, PeFNSI1-OE represents tobacco leaf group overexpressing PeFNSI1, " "This represents the analysis of significant differences. b represents the loudness expression level of PeFNSI1 at 24h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, PstDC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "This represents the significance analysis, where c is the relative expression level of salicylic acid-related genes at 0h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, and PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1." " "and" "This represents the analysis of significant differences. d represents the transcriptional level of NbBBO1 at 24h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "and" "This represents the analysis of significant differences. e represents the transcriptional level of NbBSH1 at 24h, f represents the transcriptional level of NbSAT1 at 24h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "and" "This represents the analysis of significant differences. g represents the transcriptional level of NbICS1 at 24h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." " "and" "This represents the analysis of significant differences. h represents the transcriptional level of NbPR1 at 24h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "and" "This represents the analysis of significant differences. i represents the transcriptional level of NbNPR1 at 24h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "and" "This represents the analysis of significant differences. j represents the content of free SA, SAG, apigenin, and luteolin at 0h. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, k represents the MS1 and MS2 spectra of SA, and l represents the MS1 and MS2 spectra of SAG. Figure 6 shows the quantitative analysis and MS / MS spectra of salicylic acid and flavonoids at 24h. In the figure, a represents free salicylic acid, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "and" "This represents the analysis of significant differences. b represents salicylic acid glyceride. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "and" "This represents the analysis of significant differences. c represents apigenin. WT in the figure represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, Pst DC3000 represents PstDC3000 inoculation, and MgCl2 represents MgCl2 treatment." "This represents the analysis of significant differences. d represents luteolin. In the figure, WT represents the wild-type tobacco leaf group, EV represents the empty vector control leaf group, PeFNSI1-OE represents the tobacco leaf group overexpressing PeFNSI1, PstDC3000 represents Pst DC3000 inoculation, and MgCl2 represents MgCl2 treatment." "Represents the significance analysis of differences, e is the LC-MS / MS spectrum of apigenin, f is the LC-MS / MS spectrum of luteolin; Figure 7 is a schematic diagram of the action mode of the flavonoid-salicylic acid "feedforward amplification loop" proposed in this invention. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The bamboo used in this invention is sourced from commercially available seeds, which are then sown and cultivated in a laboratory artificial climate chamber.

[0024] Example 1 (I) Cloning and Sequence Analysis of the PeFNSI1 Gene: Sample Preparation: Moso bamboo seedlings were cultured in 1 / 4 Hoglund nutrient solution at 25±2°C, 70±10% relative humidity, and under natural light. To ensure strict comparability with the publicly available transcriptome sequencing dataset (search number GSE169067), all stress treatments were strictly performed according to the parameters in the dataset: Moso bamboo seedlings were treated with 1 μM abscisic acid (ABA), 1 mM salicylic acid (SA), 25% polyethylene glycol 6000 (PEG 6000), and 200 mM sodium chloride (NaCl) for 0, 3, 8, and 24 h, respectively. Whole plants were collected, rapidly frozen in liquid nitrogen, and stored at -80°C for analysis.

[0025] Reverse transcription-quantitative PCR (qRT-PCR): Specific primers were designed using Primer Premier 5.0 (as shown in Table 1).

[0026] Table 1 Primer sequences for qRT-PCR and gene cloning

[0027] Total RNA was extracted using a total RNA extraction kit (Tanon, TR205-D-200). First-strand cDNA was synthesized using a PrimeScript™ RT reverse transcription kit (Takara, RR037A). qRT-PCR was performed using Roche premixed PCR solution. The reaction mixture consisted of 1 μL cDNA, 0.3 μL forward and reverse primers, 5 μL SYBR Master Mix, and 3.4 μL ddH2O. PeTIP41 was used as an internal control gene for *Phyllostachys edulis*. Three biological replicates were set up, using 2... −ΔΔCt The relative expression levels of target genes were calculated using a relative quantification method. To determine statistical significance, the differences in relative gene expression levels between the experimental group and the corresponding control group were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc test, and GraphPad Prism (8.0.2) was used for analysis. The significance level was indicated in the correlation plot by using "". "This indicates that a p-value < 0.05 is considered statistically significant compared to the control group." "P<0.01 indicates a highly significant difference." "P<0.001 indicates that the difference is extremely significant."

[0028] The expression patterns of flavonoid biosynthesis genes in *Phyllostachys edulis* under stress conditions were investigated using qRT-PCR. The expression of eight key genes (PePAL7, Pe4CL1, PeCHS7, PeFNSI1, PeFNSI2, PeF3H2, PeF3'5'H5, and PeLDOX4) under four stress modes (ABA, SA, PEG 6000, and NaCl) was detected (Figure 1). The results are as follows: The PeFNSI1 gene exhibits broad stress responsiveness. PeFNSI1 was the gene among the eight tested genes that showed an overall upregulation trend under all four stress treatments. ABA (abiotic stress: low temperature / drought simulation): generally upregulated; SA (disease resistance signal simulation): initially upregulated then rapidly downregulated (early response); PEG (drought stress): continuously upregulated, reaching a peak at 24 h; NaCl (salt stress): strongly induced upregulation. This indicates that P... eFNSI1 is a broad-spectrum stress response gene associated with multiple stress signaling pathways. Its functional differentiation from its homologs reveals completely opposite expression patterns between eFNSI1 and eFNSI2 under all four treatments. Upregulation of eFNSI1 indicates an active response to stress, while significant downregulation of eFNSI2 may indicate suppression. Within the FNSI family, eFNSI1 plays a major role in stress response. This functional differentiation suggests that eFNSI1 is a regulatory node in the flavonoid synthesis pathway in response to adversity and a key signaling pathway against disease. The pathway showed a positive response. SA is a core signaling molecule in plant systemic acquired resistance. After SA treatment, PeFNSI1 in bamboo seedlings was significantly upregulated at 3h and 8h, indicating that PeFNSI1 responded significantly to SA signaling. This suggests that PeFNSI1 may be involved in SA-mediated plant immune responses. This result is consistent with the conclusion that PeFNSI1 enhances disease resistance by remodeling SA metabolic flux, demonstrating from different perspectives that it can act as a molecular switch regulating disease resistance. Regarding its dominant role in abiotic stresses (drought and salt stress), P... eFNSI1 was continuously upregulated to a 24-hour peak, while PeCHS7 and PeF3H2 in the same pathway were downregulated. Under NaCl simulated salt stress, PeFNSI1, along with upstream genes such as PePAL7 and Pe4CL1, was strongly induced. Under drought and salt stress, metabolic flux may be redirected, preferentially flowing to the flavonoid synthesis branch catalyzed by PeFNSI1 to produce more antioxidant flavonoids (such as apigenin) to scavenge free radicals. This indicates that PeFNSI1 is a key target for metabolic reprogramming under abiotic stress.

[0029] Total RNA was extracted from bamboo leaves, and cDNA was synthesized. The complete coding sequence (CDS) of the PeFNSI1 gene was amplified by PCR using specific primers. Sequencing analysis confirmed that the nucleotide sequence of this gene, as shown in SEQ ID NO.1, encodes a protein containing 348 amino acids (SEQ ID NO.2). The encoded protein is bamboo flavonoid synthase I, which has a typical 2-oxoglutarate-dependent dioxygenase domain. The PeFNSI1 gene specifically catalyzes key steps in the synthesis of flavonoids in bamboo, converting the flavonoid precursor naringenin into apigenin and sennaol into luteolin. Both of these flavonoids are flavonoid aglycones in bamboo and are core functional substances for plant stress resistance.

[0030] Bioinformatics analysis predicts that the PeFNSI1 protein does not contain a transmembrane domain or an N-terminal signal peptide and is mainly located in the cytoplasm.

[0031]

[0032] The amino acid sequence of bamboo flavonoid synthase I, encoded by the PeFNSI1 gene, is shown in SEQ ID NO.2. SEQ ID NO.2: MAPAIAKPLLSDLVAQSGQVPPGHIRPVGDRPDLANVDHVSGAGIPLIDLKQLDGPERHKVVEAIGSACETDGFFMVTNHGIPEAVIEGMLRVAREFFHLPESERLKCYSDDPKKAIRLSTSFNVRTEKVNNWRDFLRLHCYPLESFIDQWPSNPPAFRQVVGTYSTEARALALRLLEAISESLGLERGHMVTAMGRHAQHMAVNYYPPCPQPELTYGLPGHKDPNAITLLLQDGVSGLQVQRDGRWVAVNPVPNALVINIGDQVQALSNDRYKSVLHRVIVNSESERISVPTFYCPSPDAMIVPADALVDDSHPLAYRPFTYQEYFDEFWNMGLNSASCLDRFRLIE.

[0033] (II) In vitro enzyme activity verification of PeFNSI1 gene: Construction of recombinant expression vector: The correctly sequenced PeFNSI1 gene coding region sequence (SEQ ID NO.1) was inserted into the HindIII / XbaI restriction site of yeast expression vector pYES2 (purchased from Shanghai Zeye Biotechnology Co., Ltd.) to construct recombinant vector pYES2-PeFNSI1, with empty pYES2 vector as negative control.

[0034] Yeast transformation and induced expression: The recombinant vector pYES2-PeFNSI1 and the empty vector pYES2 were introduced into the Saccharomyces cerevisiae INVSC1 strain (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by electroporation. Positive transformants were cultured in SD-Ura medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.) and then transferred to SD-Ura medium containing galactose for induced expression at 28°C for 48 h.

[0035] Enzyme activity reaction and product detection: The substrate naringenin and sennaol (final concentration 20 μmol / L) were added to the induced yeast culture medium, and the reaction was carried out at 28℃ for 12 h. Then, an equal volume of ethyl acetate was added to extract the product.

[0036] HPLC analysis revealed the following results, as shown in Figures 3a to 3f: characteristic peaks of apigenin and luteolin were detected in the pYES2-PeFNSI1 recombinant vector-transformed group, while no corresponding products were found in the pYES2 empty vector negative control group, confirming the enzyme catalytic function of PeFNSI1.

[0037] (III) Subcellular localization verification of PeFNSI1 gene: Using cDNA from moso bamboo (Phyllostachys edulis) as a template, PCR amplification was performed using PeFNSI1 gene-specific primers designed with Primer Premier 5.0. The amplification system used 2×Prime Star (Takara, R040A), and the 25μL amplification system contained 12.5μL 2×Prime Star Mix, 1μL 10μM forward primer, 1μL 10μM reverse primer, 1μL 50ng / μL cDNA template, and 8.5μL ddH2O. The amplification program was set as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and a final extension at 72℃ for 5 min. After the amplification products were detected by 1% agarose gel electrophoresis, the target band was excised and purified by gel recovery using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 according to the kit instructions to obtain the purified PeFNSI1 gene.

[0038] The purified PeFNSI1 PCR product was ligated into the pGEM-T Easy vector via TA. A 10 μL ligation system contained 0.5 μL of 50 ng / μL pGEM-T Easy vector, 4 μL of purified PeFNSI1 gene, 5 μL of 2×T4 DNA Ligase Buffer, and 0.5 μL of 350 U / μL T4 DNA ligase. After mixing, the system was incubated overnight at 4°C. 5 μL of the ligation product was added to 100 μL of thawed *E. coli* DH5α competent cells. After incubating on ice for 30 min, the cells were heat-shocked at 42°C for 90 s, rapidly cooled on ice for 3 min, and then 900 μL of antibiotic-free LB broth was added. The cells were incubated at 37°C and 200 rpm for 1 h using a shaker. 200 μL of the revived bacterial culture was then evenly spread onto LB solid medium containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12 h. Positive clones were selected and inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin. The medium was cultured at 37°C and 200 rpm for 12 h. The recombinant plasmid was extracted and verified using a plasmid miniprep kit to obtain the pGEM-T-PeFNSI1 recombinant plasmid.

[0039] Subcellular localization vector construction: Primers containing homologous recombination sites were designed to amplify the PeFNSI1 gene using pGEM-T-PeFNSI1 as a template. The amplified gene was then inserted into the linearized pC1300-35S-eGFP vector (purchased from Shanghai Maokang Biotechnology Co., Ltd.) to construct the recombinant vector 35S-PeFNSI1::eGFP.

[0040] Agrobacterium transformation and transient expression in tobacco: The recombinant vector was transformed into Agrobacterium GV3101 (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and positive single colonies were picked and cultured to OD200. 600 The concentration was 0.5, which infected leaves of 4-5 week old Nicotiana benthamiana.

[0041] Fluorescence observation: 48 hours after infection, the lower epidermis of tobacco leaves was taken and the fluorescence signal was observed using a laser confocal microscope.

[0042] The results are shown in Figure 2a and Figure 2b. The eGFP fluorescence is mainly distributed in the cytoplasm and overlaps with the cytoplasmic marker protein signal, confirming that PeFNSI1 is located in the cytoplasm.

[0043] (iv) Verification of disease resistance and salicylic acid metabolic flux regulation function of PeFNSI1 gene: Preparation of transiently transformed tobacco: Agrobacterium tumefaciens GV3101 was transformed into 35S-PeFNSI1::eGFP recombinant vector and empty vector respectively, and then infected tobacco leaves to obtain tobacco leaves overexpressing PeFNSI1 (OE group) and empty vector control leaves (EV group), with wild-type tobacco leaves as WT group.

[0044] Pathogen inoculation: 48 hours after infection, *Pseudomonas syringae* Pst DC3000 (purchased from Zhili Zhongte Biotechnology Co., Ltd.) was inoculated onto tobacco leaves. 600 =0.005), with MgCl2 treatment as a blank control.

[0045] Disease resistance identification: 72 hours after inoculation, leaf lesion phenotypes were observed, and bacterial titers, relative electrolyte leakage rate and chlorophyll fluorescence parameters (Fv / Fm, Y(II), NPQ) were measured.

[0046] The results are shown in Figures 4a to 4l. The leaf lesion area in the OE group was significantly smaller than that in the EV and WT groups, the bacterial titer was significantly reduced, the electrolyte leakage rate was low, the photosynthetic efficiency was stable, and the disease resistance was significantly enhanced. The accumulation of reactive oxygen species (ROS) in the leaves was inhibited, the cell membrane integrity was maintained, and the photosynthetic efficiency was stable, indicating that overexpression of the PeFNSI1 gene can significantly enhance plant disease resistance.

[0047] Salicylic acid metabolic flux analysis: Metabolite profiles of PeFNSI1 overexpressing plants were analyzed by HPLC and LC-MS / MS.

[0048] The results are shown in Figures 5a-5l and 6a-6f. The PeFNSI1 gene significantly induced the expression of key genes (NbBBO1, NbBSH1) in the phenylalanine ammonia-lyase (PAL)-mediated salicylic acid biosynthesis pathway, while inhibiting the expression of the salicylic acid glycosylation inactivation gene (NbSAT1), promoting the accumulation of biologically active free salicylic acid. Overexpressing plants specifically accumulated apigenin (14.19±1.71 μg / g FW) and luteolin (8.44±1.26 μg / g FW). Simultaneously, salicylic acid metabolite analysis showed that overexpressing plants had accumulated basal levels of free salicylic acid (SA) (0.86±0.17 μg / g FW) before infection, reaching 9.44±0.78 μg / g FW 24 h after infection, significantly higher than the wild-type control (5.18±0.66 μg / g FW). Notably, the inactive form of salicylic acid (SAG) was almost undetectable in the overexpressing plants, indicating that metabolic flux was effectively reprogrammed. Molecular mechanism studies: qRT-PCR analysis showed that PeFNSI1 gene overexpression significantly upregulated the expression of salicylic acid biosynthesis genes NbBBO1 (2.2-fold) and NbBSH1 (1.6-fold), while simultaneously inhibiting the SA glycosyltransferase gene NbSAT1 and the heterobranched acid synthase pathway gene NbICS1. Further RNA-seq and WGCNA analyses revealed 37 RNA molecules highly associated with the PeFNSI1 gene (R... 2 The presence of co-expressed genes (>0.90) including three transcription factors and four protein kinase genes indicates a complex regulatory network involved in PeFNSI1-mediated defense responses. Furthermore, the expression levels of salicylic acid downstream defense genes (NbNPR1, NbPR1) were significantly upregulated, with NbPR1 transcription reaching 2.2-fold that of the wild type, suggesting that PeFNSI1 can further enhance plant defense responses by regulating salicylic acid metabolic flux.

[0049] Figure 7 shows the working model of the flavonoid-salicylic acid "feedforward amplification loop" proposed in this invention, which is a working model of stress adaptation mediated by PeFNSI1. PeFNSI1 from moso bamboo catalyzes the conversion of flavanones to flavonoids. In host cells, the increased flavonoids act as a metabolic switch, reprogramming the SA flow by promoting the three-step PAL-derived pathway (via BB: benzyl benzoate and BS: benzyl salicylate) and simultaneously inhibiting NbSAT1-mediated SA to SAG inactivation. The accumulation of the resulting active free SA further triggers the feedforward loop to enhance PeFNSI1 expression, resulting in strong resistance to Pst DC3000.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A gene PeFNSI1 for flavonoid synthase I in bamboo, characterized in that, The nucleotide sequence of the PeFNSI1 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the PeFNSI1 gene as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the PeFNSI1 gene is shown in SEQ ID NO.

2.

3. A biomaterial comprising the PeFNSI1 gene of claim 1, characterized in that, The biological material is an expression cassette, expression vector, or engineered bacteria.

4. The biomaterial according to claim 3, characterized in that, The biological material is a PeFNSI1 gene inserted into an expression vector as a backbone. The expression vector is one or more of pC1300-35S or pCAMBIA1301.

5. The application of the PeFNSI1 gene as described in claim 1, the protein encoded by the PeFNSI1 gene as described in claim 2, or the biomaterial as described in claim 3 in enhancing plant disease resistance.

6. The application according to claim 5, characterized in that, The plant in question is rice, wheat, or tobacco.

7. The application according to claim 5, characterized in that, Overexpression of the PeFNSI1 gene increases the expression level of the protein encoded by the PeFNSI1 gene, promotes flavonoid synthesis, promotes salicylic acid accumulation, activates plant defense signaling pathways, enhances plant resistance to pathogens, and strengthens plant disease resistance.

8. The application of the PeFNSI1 gene as described in claim 1, the protein encoded by the PeFNSI1 gene as described in claim 2, or the biomaterial as described in claim 3 in promoting flavonoid synthesis, characterized in that, Promotes flavonoid synthesis in rice, wheat, tobacco, brewer's yeast or Escherichia coli, wherein the flavonoids are apigenin and / or luteolin, by overexpressing the PeFNSI1 gene to increase the expression level of the protein encoded by the PeFNSI1 gene and promote flavonoid synthesis.

9. The application of the PeFNSI1 gene as described in claim 1, the protein encoded by the PeFNSI1 gene as described in claim 2, or the biomaterial as described in claim 3 in promoting salicylic acid accumulation in plants, characterized in that... The plant is rice, wheat, or tobacco. By overexpressing the PeFNSI1 gene, the expression level of the protein encoded by the PeFNSI1 gene is increased, the salicylic acid biosynthesis pathway is activated, salicylic acid glycosylation inactivation is inhibited, salicylic acid metabolic flux is reconstructed, and active free salicylic acid is accumulated.

10. The application of the PeFNSI1 gene as described in claim 1, the protein encoded by the PeFNSI1 gene as described in claim 2, or the biomaterial as described in claim 3 in enhancing plant resistance to pathogens, characterized in that... The plant is rice, wheat, or tobacco, and the pathogen is *Pseudomonas syringae*. By overexpressing the PeFNSI1 gene, the expression level of the protein encoded by the PeFNSI1 gene is increased, thereby enhancing the plant's resistance to *Pseudomonas syringae*.

Images