Lycoris radiata NAC8 gene and application of the coded protein in improving salt stress tolerance and anthocyanin synthesis of lycoris radiata

Abstract

This invention discloses the application of the LrNAC8 gene and its encoded protein in improving the salt tolerance and anthocyanin synthesis of Lycoris radiata, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the LrNAC8 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. Experiments show that overexpression of LrNAC8 in Lycoris radiata increases the activity of antioxidant enzymes in leaves, significantly enhancing its salt tolerance; silencing LrNAC8 leads to a decrease in antioxidant enzyme activity and weakened salt tolerance. Furthermore, overexpression of LrNAC8 in Lycoris radiata cloves increases anthocyanin content. The LrNAC8 gene provided by this invention can significantly improve the salt tolerance and anthocyanin content of plants, which is of great significance for revealing the molecular mechanism of Lycoris radiata's stress resistance and for the breeding of high-quality salt-tolerant varieties of Lycoris radiata.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the Lycoris radiata LrNAC8 gene and its encoded protein in improving the salt stress tolerance and anthocyanin synthesis of Lycoris radiata. Background Technology

[0002] Lycoris is a group of perennial bulbous flowering plants in the Amaryllidaceae family with significant ecological adaptability and economic value. These plants are not only renowned for their unique biological characteristic of flowers and leaves not appearing together, but their bulbs are also rich in alkaloids such as galantamine and lycorine, which are widely used in the treatment of neurological diseases, highlighting their medicinal potential. However, soil salinization limits the cultivation, promotion, and ecological restoration applications of Lycoris plants. Salt stress inhibits plant growth and causes cell damage through mechanisms such as disrupting ion homeostasis, inducing osmotic imbalance, and triggering reactive oxygen species (ROS) bursts. Therefore, elucidating the molecular mechanisms of salt tolerance in Lycoris plants, especially the functions of their core regulatory factors, is of great significance for improving the adaptability of plants in saline-alkali areas.

[0003] Anthocyanins, as key products of flavonoid metabolism, possess dual biological functions in plants. On the one hand, the diversity of their chemical structures (such as hydroxylation and glycosylation modifications) directly determines petal color, making them important targets for flower color breeding in horticultural plants. On the other hand, anthocyanins can enhance plant tolerance to abiotic stresses by scavenging ROS, regulating antioxidant enzyme activity, and chelating excess metal ions. Studies have shown that salt stress can significantly induce anthocyanin accumulation in plants, but the molecular regulatory network remains controversial. Traditional research has focused on the transcriptional regulation of anthocyanin synthesis pathway structural genes (such as CHS, DFR, and ANS) by the MYB-bHLH-WD40 (MBW) complex. However, recent studies have discovered that NAC (NAM-ATAF1 / 2-CUC2) family transcription factors may play a pivotal role in secondary metabolism and stress resistance regulation by integrating developmental and stress signals. The NAC family may participate in plant environmental adaptation through metabolic and stress resistance co-modulation modules, but its functional mechanisms in Lycoris genus have not yet been systematically elucidated.

[0004] Although Lycoris species exhibit natural tolerance to saline-alkali environments, research on the molecular mechanisms of salt tolerance is still in its early stages. Previous transcriptome data indicate that salt stress can specifically induce the expression of some NAC family members in Lycoris, but the downstream regulatory networks of these genes and their association with anthocyanin synthesis remain unclear. Notably, the polychromatic phenomenon of Lycoris petals (such as variegation of blue and red) is closely related to the spatiotemporal accumulation pattern of anthocyanins, and salt stress may further affect plant stress resistance by regulating anthocyanin metabolism. However, whether NAC transcription factors simultaneously mediate anthocyanin synthesis and salt tolerance in Lycoris, and their molecular pathways of action, remain unresolved scientific questions. Summary of the Invention

[0005] To address the issues that the molecular mechanisms of salt tolerance in Lycoris radiata are still unclear in the prior art, and whether NAC transcription factors simultaneously mediate anthocyanin synthesis and salt tolerance remains ambiguous, the technical problem to be solved by this invention is to provide a Lycoris radiata LrNAC8 gene and its encoded protein. Another technical problem to be solved by this invention is to provide the application of the aforementioned gene in regulating salt tolerance and anthocyanin accumulation in Lycoris radiata.

[0006] To address the aforementioned problems in the existing technology, the technical solution adopted by the present invention is as follows:

[0007] A Lycoris LrNAC8 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The protein encoded by the Lycoris LrNAC8 gene has the amino acid sequence shown in SEQ ID NO.2.

[0009] A biological material, said biological material being an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the Lycoris LrNAC8 gene.

[0010] In some embodiments, the recombinant vector is an overexpression vector pBIN-GFP4-LrNAC8 or a silencing vector pTRV2-LrNAC8.

[0011] In some embodiments, the recombinant bacteria are Agrobacterium or Saccharomyces cerevisiae.

[0012] Application of the Lycoris LrNAC8 gene or the biological material in regulating Lycoris salt tolerance and / or promoting Lycoris anthocyanin synthesis.

[0013] In some embodiments, the application is to improve the salt stress tolerance of Lycoris radiata and / or promote the synthesis of anthocyanins in Lycoris radiata by overexpressing the LrNAC8 gene.

[0014] In some embodiments, the application includes the following steps: constructing a recombinant plant expression vector of the Lycoris LrNAC8 gene, and then introducing the recombinant plant expression vector into Lycoris plants or Lycoris tissues to cultivate transgenic Lycoris plants or tissues with improved salt stress tolerance and / or anthocyanin synthesis.

[0015] In some embodiments, the application is to reduce salt tolerance of Lycoris radiata by silencing or downregulating LrNAC8 gene expression.

[0016] In some embodiments, the application includes the following steps: constructing a silencing vector for the Lycoris LrNAC8 gene, and then introducing the silencing vector into Lycoris plants or Lycoris tissues to obtain transgenic Lycoris plants or tissues with reduced salt stress tolerance.

[0017] Compared with the prior art, the beneficial effects of this application are as follows:

[0018] This invention is the first to clone the LrNAC8 gene from Lycoris radiata and verify its function through yeast heterologous expression, transient overexpression in Lycoris radiata leaves, and VIGS silencing experiments. The results showed that LrNAC8 is located in the cell nucleus. Overexpression of LrNAC8 under salt stress significantly increased the activity of antioxidant enzymes such as SOD, POD, and CAT in Lycoris radiata leaves, maintained chlorophyll homeostasis, and enhanced the plant's salt tolerance. Conversely, VIGS silencing of LrNAC8 led to decreased antioxidant enzyme activity, reduced chlorophyll content, and significantly weakened salt tolerance. Furthermore, this invention also found that overexpression of LrNAC8 in Lycoris radiata petals significantly promoted anthocyanin accumulation, turning white petals red, and upregulated the expression of structural genes in anthocyanin synthesis pathways such as C4H, LAR, and DFR. These results indicate that LrNAC8 is a positive regulator of salt tolerance and anthocyanin synthesis in Lycoris radiata, possessing a dual function of improving the plant's stress resistance and quality.

[0019] This invention provides new gene resources and clear regulatory targets for salt-tolerant molecular breeding and flower color improvement in Lycoris genus plants. The constructed overexpression vector and VIGS silencing vector can be directly used for breeding new Lycoris varieties. Simultaneously, this invention reveals the pivotal role of NAC transcription factors in the cross-regulatory network of stress response and secondary metabolism, enriching the molecular biology theory of plant stress resistance and possessing significant application value for improving the cultivation adaptability and ornamental quality of Lycoris in saline-alkali areas. Attached Figure Description

[0020] Figure 1 The phylogenetic tree analysis results for LrNAC8 are shown in the figure.

[0021] Figure 2The diagram shows the expression pattern analysis of the LrNAC8 gene. In the diagram, A shows the expression pattern of the LrNAC8 gene in different tissues; B shows the expression changes of the LrNAC8 gene in Lycoris radiata after abiotic stress and plant hormone treatment, with the LrTIP41 gene as an internal reference.

[0022] Figure 3 Figure showing the results of subcellular localization experiments of LrNAC8 protein in Nicotiana benthamiana cells;

[0023] Figure 4 Figure showing the experimental results of heterologous expression of LrNAC8 in Saccharomyces cerevisiae to enhance salt tolerance;

[0024] Figure 5 Agrobacterium tumefaciens, which overexpressed the vector OE-LrNAC8, was used to infect fresh Lycoris radiata leaves by leaf tip injection. Phenotypic images of Lycoris radiata leaves after salt stress treatment in 0 mM, 100 mM, 200 mM, 300 mM and 400 mM NaCl were obtained.

[0025] Figure 6 To select the activities of SOD (superoxide dismutase), CAT (catalase), and POD (peroxidase) and chlorophyll content in samples (CK, OE-LrNAC8) after 36 h of NaCl treatment;

[0026] Figure 7 Phenotypic diagrams of fresh leaves of Lycoris radiata infected with the virus-induced gene silencing (VIGS) vector pTRV2-LrNAC8 of the LrNAC8 gene by leaf tip injection, after being cut into equal lengths and subjected to salt stress treatment in 0 mM, 100 mM, 200 mM, 300 mM and 400 mM NaCl respectively.

[0027] Figure 8 The figure shows the results of chlorophyll content and antioxidant enzyme (superoxide dismutase SOD, catalase CAT and peroxidase POD) detection in Lycoris radiata leaf samples (TRV1 / TRV2, TRV1 / TRV2-LrNAC8) after treatment with 0 mM NaCl and 200 mM NaCl for 72 h.

[0028] Figure 9 The results of Agrobacterium truncatum overexpressing vector OE-LrNAC8 infecting white petals of Lycoris radiata using the petal tip injection method are shown in the image. (A) is a comparison of infected Lycoris radiata petals with the control; (B) is a comparison of anthocyanin content in infected Lycoris radiata petals with the control; and (C) is a comparison of the expression levels of anthocyanin synthesis-related genes in infected Lycoris radiata petals with the control. Detailed Implementation

[0029] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sam Brook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or as recommended in the manufacturer's instructions.

[0030] Example 1

[0031] Obtaining LrNAC8 candidate gene

[0032] The Pearson correlation coefficients between differentially expressed genes and differentially expressed metabolites were calculated using the Corr package. Correlation analysis was performed on differentially expressed genes and differentially expressed anthocyanin metabolites in petals at three flowering stages of Lycoris longituba. First, the structural genes for differentially expressed anthocyanin synthesis and all differentially expressed transcription factors were analyzed for correlation. Genes with correlation Pearson > 0.8 and significance p < 0.05 were selected to construct a correlation network diagram. The results showed that 144 differentially expressed transcription factors were significantly correlated with anthocyanin synthesis.

[0033] To further identify the NAC transcription factors involved in anthocyanin synthesis in Lycoris radiata, correlation analysis was performed on differentially expressed anthocyanins, structural genes involved in differential anthocyanin synthesis, and differentially expressed NAC transcription factors. Genes with correlation Pearson scores > 0.85 and significance values ​​p < 0.05 were selected to construct a correlation network diagram. The results showed that 33 differentially expressed anthocyanins, 32 structural genes involved in anthocyanin synthesis, and 17 NAC transcription factors were significantly correlated. Among them, Cluster-75739.10 (LrNAC8) was identified as a pivot gene that significantly regulates anthocyanin synthesis and was used as a candidate gene for functional validation.

[0034] Example 2

[0035] Cloning and sequence characterization of LrNAC8

[0036] Using cDNA from Lycoris longituba leaves as a template, the LrNAC8 gene was cloned. Sequence cloning analysis showed that the LrNAC8 gene CDS sequence was 837 bp long (sequence shown in SEQ ID NO.1), encoding 279 amino acids (sequence shown in SEQ ID NO.2). RNA was extracted from Lycoris longituba leaves using an RNA extraction kit (Biotake), and then used as a template, the RNA was used for reverse transcription using the PrimeScript reverse transcription kit. TMcDNA was obtained by reverse transcription using the RT Reagent Kit (TaKaRa). Using the cDNA as a template, PCR was performed with primer pairs LrNAC8-F (5'-ATGTCTGGTGGTGATTTGCA-3' (SEQ ID NO.3)) and LrNAC8-R (5'-AAACGGCTTCTGCAGGTACA-3' (SEQ ID NO.4)) to clone the coding region sequence of the LrNAC8 gene. The PCR reaction system was 50 μL, containing 25 μL of high-fidelity Taq enzyme 2×Phanta MaxMaster Mix (Dye Plus), 2 μL each of upstream and downstream primers LrNAC8-F and LrNAC8-R, 1 μL of cDNA template, and 20 μL of water. The PCR program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 50℃ annealing for 15 s, 72℃ extension for 2 min, 35 cycles, and a final extension at 72℃ for 5 min.

[0037] The coding sequence fragment of the cloned LrNAC8 gene was recovered and ligated into the pTOPO001 vector (Beijing Jinsha Biotechnology Co., Ltd.) at room temperature for 5 min. This ligation was then introduced into competent DH5α cells of *E. coli* (Sangon Biotech) and cultured on solid LB medium (containing 50 μg / mL ampicillin) for selection. After overnight culture at 37°C, single clones were picked, and positive clones were verified by PCR. Confirmed positive strains were cultured in liquid LB medium (containing 50 μg / mL ampicillin) for 12 h, and plasmids were extracted. PCR verification of positive clones using primers LrNAC8-F and LrNAC8-R yielded the plasmid pTOPO001-T-LrNAC8. Sequencing of this plasmid yielded the full-length coding sequence of the LrNAC8 gene. The full-length coding sequence of the LrNAC8 gene is 837 bp, encoding a 279-amino acid protein (i.e., LrNAC8). The full-length nucleotide sequence of the coding region of the LrNAC8 gene is shown in SEQ ID NO.1, and the amino acid sequence of the LrNAC8 gene is shown in SEQ ID NO.2.

[0038] Phylogenetic analysis of LrNAC8 with species such as asparagus (Asparagus officinalis), coconut (Cocos nucifera), yam (Dioscorea alata), iris (Iris pallida), Siberian cabbage (Brassica napus), tomato (Solanumlycopersicum), rice (Oryza sativa), and upland cotton (Gossypium hirsutum) showed that LrNAC8 is most closely related to the NAC gene in Iris pallida (Iris aceae), followed by asparagus and coconut. Figure 1 Homologous protein sequence alignment revealed that the LrNAC8 protein sequence possesses a conserved domain of the ATAF subfamily.

[0039] Example 3

[0040] Analysis of the expression pattern of Lycoris radiata NAC transcription factor LrNAC8

[0041] Real-time PCR expression analysis of the LrNAC8 gene was performed using cDNA from different Lycoris radiata tissues as templates. Real-time PCR was performed according to the SYBR Premix Ex Taq™ II (TAKARA) kit. The reaction mixture consisted of 12.5 μL SYBR Micelles, 1 μL each of forward and reverse primers, 1 μL template, and sterile distilled water to a final volume of 25 μL. The reaction program was: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 40 cycles; 72℃ for 5 min. The forward primer for LrNAC8 RtPCR was 5'-GCGTTGTACGGGGAGAAAGA-3' (SEQ ID NO. 5), and the reverse primer was 5'-GGGGCCTTCCCCGAATAAAA-3' (SEQ ID NO. 6). TIP41 was used as an internal reference gene. The forward primer was 5'-GCAACCATCCAAAGTTTAACTGCT-3' (SEQ ID NO.7), and the reverse primer was 5'-AATGTGCAAGCAGGGCTAGTAA-3' (SEQ ID NO.8). PCR reactions were performed on a Bio-Rad iCycler iQ5 Real-time PCR instrument. Using 2... -△△Ct The method was used to normalize the relative expression levels of the LrNAC8 gene in different samples.

[0042] Expression analysis in different tissues showed that LrNAC8 had the highest expression level in roots, followed by petals, with lower expression levels in other tissues. Figure 2In a study involving Lycoris radiata seedlings, various abiotic stresses and plant hormone treatments were applied, including drought (20% PEG 6000), salt stress (200 mM NaCl), low temperature (4℃) and high temperature (40℃) stress, abscisic acid (ABA, 0.1 mM), and methyl jasmonate (MeJA, 0.1 mM) solutions. The control group received dH₂O. Roots of all treated seedlings were sampled at 6, 12, and 24 hours post-treatment. The coordinated expression of the LrNAC8 gene under various stress conditions and its role in ABA signaling were observed, indicating its involvement in the salt stress response of Lycoris radiata. LrNAC8 was ultimately identified as a key regulator of Lycoris radiata's response to abiotic stress, particularly salt stress. Figure 2 (B)

[0043] Example 4

[0044] LrNAC8 subcellular localization analysis

[0045] The localization of a protein at the cellular level is crucial for elucidating its function in an organism. Bioinformatics prediction analysis using the WoLF PSORT subcellular localization prediction website (https: / / wolfpsort.hgc.jp / ) indicates that LrNAC8 is a protein located in the cell nucleus. The LrNAC8 sequence (pBIN-GFP4-LrNAC8-F: 5'-ATTTTACGAACGATAGGGTACCATGTCTGGTGGTGATTTGCA-3' (SEQ ID NO. 9); (pBIN-GFP4-LrNAC8-R: 5'-GCCCTTGCTCACCATGGATCCAAACGGCTTCTGCAGGTACA-3' (SEQ ID NO. 10)) was amplified from cDNA of Lycoris radiata leaves using primers containing restriction enzyme sites Pst1 and Kpn1. The purified fragment was ligated into the plant binary expression vector pBIN-GFP4. The constructed pBIN-GFP4-LrNAC8 was then chemically transformed into Agrobacterium GV3101 competent cells. GFP (green fluorescent protein) was used as a reporter protein to investigate the subcellular localization of LrNAC8 in plant cells. Figure 3 DAPI staining was used as a nuclear marker, with cells expressing GFP alone serving as a control. The localization of LrNAC8 in plant organelles was determined by transiently expressing the LrNAC8-GFP fusion protein in tobacco leaf cells and combining it with DAPI staining. Results showed that GFP was diffusely distributed throughout the entire cell in the control cells, while the green fluorescence carried by LrNAC8-GFP completely overlapped with the blue nuclear fluorescence of DAPI in the superposition field. Figure 3This indicates that LrNAC8 is located in the cell nucleus.

[0046] Example 5

[0047] LrNAC8 positively regulates the tolerance of Saccharomyces cerevisiae to salt stress.

[0048] Primers containing EcoRI and XbaI restriction sites (pYES2-LrNAC8F:5'-CGGAATTCATGTCTGGTGGTGATTTGCA-3' (SEQ ID NO.11); pYES2-LrNAC8R:5'-GCTCTAGAAAACGGCTTCTGCAGGTACA-3' (SEQ ID NO.12)) were designed based on the open reading frame of LrNAC8. The target fragment was then amplified from Lycoris radiata leaf cDNA using PCR. The LrNAC8 fragment with restriction sites was amplified using TA cloning, and the sticky-end fragment containing EcoRI and XbaI cleavage sites was recovered and purified by double enzyme digestion (EcoRI and XbaI). The purified fragment was constructed into the pYES2 yeast expression vector, and the pYES2-LrNAC8 plasmid was introduced into competent yeast G19 cells using chemical transformation. Recombinant yeast G19::pYES2- After LrNAC8 and control yeast pYES2 were induced to express in galactose-containing SC / -Ura medium, they were inoculated at a ratio of 1% (v / v) into media containing 0, 500, 700, and 900 mmol·L⁻¹, respectively. -1 The recombinant yeast G19::pYES2-LrNAC8 was cultured under stress in NaCl-containing SC / -Ura (containing 2% glucose) medium. The OD values ​​of the induced recombinant yeast G19::pYES2-LrNAC8 and the control yeast pYES2 were compared. 600 Adjust to a uniform consistency, then dilute to 10⁻⁶ with SC / -Ura culture medium. -0 10 -1 10 -2 10 -3 and 10 -4 Concentration. Take 5 μL of the diluted bacterial suspension and drop it onto a container containing 500 mmol·L⁻¹. -1 700 mmol·L -1 and 900 mmol·L -1 The samples were incubated on a NaCl-stressed medium at 28°C for 5 days, and then observed and photographed. The results showed that in a medium containing 500 mmol·L⁻¹ NaCl... -1 700 mmol·L -1 and 900 mmol·L - 1 The number of recombinant yeast in NaCl solid culture medium was significantly higher than that of control yeast (0 mmol·L⁻¹). -1These results indicate that heterologous expression of LrNAC8 protein has a positive regulatory effect on improving the salt tolerance of yeast. Figure 4 Therefore, LrNAC8 may be a positive regulator of salt stress response.

[0049] Example 6

[0050] Construction of the overexpression vector pBIN-GFP4-LrNAC8

[0051] To analyze the function of LrNAC8, an overexpression vector p35S-LrNAC8-GFP was constructed, and overexpressing Arabidopsis plants were obtained. The specific process is briefly described below.

[0052] First, primers with restriction endonuclease sites Pst1 and Kpn1 were designed, with the following sequences:

[0053] pBIN-GFP4-LrNAC8-F:5'-TTTACGAACGATAGGGTACCATGTCTGGTGGTGATTTGCA-3' (SEQ ID NO.9);

[0054] pBIN-GFP4-LrNAC8-R:5'-GCCCTTGCTCACCATGGATCCAAACGGCTTCTGCAGGTACA -3' (SEQ ID NO. 10);

[0055] Second, using the cDNA sample prepared in Example 1 as a template, PCR amplification was performed, and the amplification product was purified and recovered.

[0056] Third, the pBIN-GFP4-GFP vector was digested with pst1 and kpn1 double enzymes, and the digestion products were purified.

[0057] Fourth, the PCR amplification product and the enzyme-digested vector were ligated by homologous recombination to construct the pBIN-GFP4-LrNAC8 overexpression vector (OE-LrNAC8);

[0058] Fifth, the heat shock transformation method was used to transform the ligation product into Escherichia coli DH5α, and Kan+ (kanamycin, 50 μg / mL) resistance screening was performed. Positive colonies were selected for PCR detection. Colonies that were correctly identified by PCR were amplified and sent for sequencing. Plasmids were extracted from the bacterial solutions that were correctly sequenced for later use.

[0059] Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101 and stored at -80℃ for later use.

[0060] Example 7

[0061] Construction of the silent VIGS vector pTRV2-LrNAC8

[0062] To analyze the function of LrNAC8, the silent VIGS vector pTRV2-LrNAC8 of LrNAC08 was constructed. The specific process is briefly described below.

[0063] First, primers with restriction endonuclease sites Xbal I and Kpn I were designed, with the following sequences:

[0064] LrNAC8-pTRV2 -Xbal IF:5'-GTAAGGTTACCGAATTCTCTAGAATGTCTGGTGGTGATTTGCA-3' (SEQ ID NO. 13);

[0065] LrNAC8-pTRV2 - Kpn IR: 5'-CTCGAGACGCGTGAGCTCGGTACCAAACGGCTTCTGCAGGTACA -3' (SEQ ID NO. 14);

[0066] Second, using the cDNA sample prepared in Example 1 as a template, PCR amplification was performed, and the amplification product was purified and recovered.

[0067] Third, the pTRV2 vector was double-digested with Xbal I and kpn1, and the digestion products were purified.

[0068] Fourth, the PCR amplification product and the enzyme-digested vector were ligated by homologous recombination to construct the pTRV2-LrNAC8 overexpression vector;

[0069] Fifth, the heat shock transformation method was used to transform the ligation product into Escherichia coli DH5α, and Kan+ (kanamycin, 50 μg / mL) resistance screening was performed. Positive colonies were selected for PCR detection. Colonies that were correctly identified by PCR were amplified and sent for sequencing. Plasmids were extracted from the bacterial solutions that were correctly sequenced for later use.

[0070] Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101 and stored at -80℃ for later use.

[0071] Example 8

[0072] Agrobacterium infection of Lycoris leaves and petals

[0073] (1) Agrobacterium GV3101 containing empty vector plasmid, overexpression plasmid, and silencing vector were cultured separately on YEB (25 mg / L Rif, 50 mg / L Kana) solid medium. After culturing at 28℃ for 48 h, a single colony of freshly cultured Agrobacterium EHA105 transformant containing the vector plasmid was picked and added to 3 mL of YEB liquid medium (25 mg / L Rif, 50 mg / L Kana), and cultured overnight at 28℃ and 200 rpm / min in a shaker.

[0074] (2) Add 1 mL of the cultured bacterial suspension to 100 mL of YEB liquid medium (25 mg / L Rif, 50 mg / L Kana), and incubate at 28℃ and 200 rpm / min for 1 day. Detect the OD of the bacterial suspension. 600 When the concentration is around 1.0, take 30 mL, centrifuge at 6000 rpm for 10 min to collect bacterial cells, and add an equal volume of infection resuspension to bring the final OD600 concentration to 1.0.

[0075] (3) Preparation of infection resuspension: 100 mL of resuspension contains 1 mL of MgCl2 solution (1 mol / L), 1 mL of MES solution (1 mol / L), and 200 µL of acetylsalicylic acid solution (1 mol / L). The solution is diluted with sterile distilled water and should be prepared and used immediately.

[0076] (4) Preparation of infection solution: Incubate the infection suspension in the dark for 2-3 hours and use it to infect the leaves or petals of Lycoris radiata.

[0077] (5) Agrobacterium-mediated infection: The method for infecting Lycoris radiata leaves or petals is the needle injection method. First, draw up the infection solution cultured in step (4), and carefully insert the needle of the syringe into the leaf vein vascular bundle at the tip of the leaf or the tip of the Lycoris radiata petal. Do not insert it too deeply, and pinch the needle firmly with your fingers to slowly push the bacterial solution in until the entire leaf is soaked. It should be noted that gloves and syringes need to be changed when injecting different bacterial solutions.

[0078] (6) Post-injection culture: After infection, it needs to be cultured in the dark at 18℃ for 1-2 days, and then grown in an environment of 23-28℃.

[0079] (7) Real-time PCR analysis: RNA was extracted from the leaves or petals of Lycoris radiata that were overexpressed or silenced, and LrNAC8 expression was detected using exogenous gene-specific primers.

[0080] Example 9

[0081] Yeast Function Validation of LrNAC8

[0082] To verify whether LrNAC8 plays a role in salt stress resistance in plants, this application constructed a pYES2-LrNAC8 yeast recombinant expression vector. This vector was transformed into the salt-sensitive yeast strain G19. Yeast cells containing the recombinant plasmid were serially diluted 10, 100, 1000, and 10,000 times, and then cultured for 36 hours (30℃) on SD-Ura medium containing 0 mM, 500 mM, 700 mM, and 900 mM NaCl, respectively, and their phenotypes were observed. The results showed that, compared with the control, the yeast strain overexpressing LrNAC8 exhibited significant salt tolerance with increasing salt concentration. Figure 4 The above results indicate that LrNAC8 has a positive regulatory effect on salt tolerance.

[0083] Example 10

[0084] Overexpression of LrNAC8 increases tolerance to Lycoris salt stress.

[0085] Agrobacterium containing the overexpression vector OE-LrNAC8 was used to infect fresh Lycoris radiata leaves via leaf tip injection. The injected leaves were cut into equal lengths and subjected to salt stress treatment in 0 mM, 100 mM, 200 mM, 300 mM, and 400 mM NaCl. The results showed that with increasing salt concentration, the salt tolerance of leaves overexpressing the LrNAC8 gene significantly improved, manifested as less yellowing, with the most pronounced phenotype observed after 36 h of treatment with 200 mM NaCl. Figure 5 Similarly, this example also measured the activities of SOD (superoxide dismutase), CAT (catalase), and POD (peroxidase), as well as chlorophyll content, in samples (CK, OE-LrNAC8) after treatment with 0 mM NaCl and 200 mM NaCl for 36 h. The results showed that overexpression of LrNAC8 could further activate the salt tolerance and maintain chlorophyll homeostasis in leaves by enhancing SOD, CAT, and POD activities. Figure 6 ).

[0086] Example 11

[0087] Statistical analysis of salt stress phenotypes in VIGS-silenced LrNAC8 leaves

[0088] Furthermore, to further verify whether LrNAC8 plays a role in the salt stress response of Lycoris radiata, this application constructed a virus-induced gene silencing (VIGS) vector pTRV2-LrNAC8 for the LrNAC8 gene. Agrobacterium containing the silencing vector was injected into fresh Lycoris radiata leaves via leaf tip injection. The injected leaves were cut into equal lengths and subjected to salt stress treatment with 0 mM, 100 mM, 200 mM, 300 mM, and 400 mM NaCl, respectively. The results showed that, compared with the control, the salt tolerance of Lycoris radiata leaves with the LrNAC8 gene silenced decreased significantly with increasing NaCl concentration, manifested as more severe leaf yellowing. Figure 7 The phenotype was most pronounced after treatment with 200 mM NaCl for 72 h.

[0089] Therefore, in this embodiment, samples (TRV1 / TRV2, TRV1 / TRV2-LrNAC8) treated with 0 mM NaCl and 200 mM NaCl for 72 h were selected for chlorophyll content and antioxidant enzyme (superoxide dismutase SOD, catalase CAT and peroxidase POD) activity detection.

[0090] The results showed that salt stress significantly induced an increase in SOD activity in Lycoris radiata leaves. However, in both the control and salt stress treatment groups, the SOD levels in the LrNAC8 gene VIGS samples were significantly lower than those in the TRV1 / TRV2 (CK) samples. Figure 8 Similarly, salt stress significantly induced an increase in POD activity in Lycoris radiata leaves, consistent with the trend in SOD activity. However, salt stress treatment did not increase CAT activity in Lycoris radiata. POD activity was significantly higher in the TRV1 / TRV2 treatment under salt stress than in the control group, while the POD activity in the TRV1 / TRV2-LrNAC8 treatment showed little difference between the control group and under salt stress. Chlorophyll content decreased in all three treatments under salt stress, with the TRV1 / TRV2-LrNAC8 treatment showing the most significant decrease. This result suggests that silencing LrNAC8 with VIGS results in lower salt tolerance in the leaves.

[0091] Example 12

[0092] Overexpression of LrNAC8 increases anthocyanin synthesis in Lycoris petals.

[0093] To investigate whether LrNAC8 regulates anthocyanin synthesis in Lycoris radiata petals, white-petaled Lycoris longituba was selected as the material. An overexpression vector containing LrNAC8 was constructed and successfully transformed into Agrobacterium tumefaciens EHA105. Agrobacterium tumefaciens containing the overexpression vector OE-LrNAC8 was injected into the white petals of Lycoris longituba via petal tip injection. The results showed that, compared to uninjected petals, the petals injected with the overexpression vector OE-LrNAC8 were slightly redder, and the anthocyanin content measurements were consistent with the phenotypic results. Figure 9 Therefore, it is hypothesized that LrNAC8 positively regulates anthocyanin synthesis in Lycoris petals.

[0094] Then, to further clarify the role of LrNAC8 in anthocyanin synthesis in Lycoris radiata, qRT-PCR was used to detect changes in the expression levels of LrNAC8 and structural genes in the anthocyanin synthesis pathway in petals overexpressing LrNAC8. The results are as follows: Figure 9 As shown, compared with uninjected petals, the expression level of LrNAC8 in overexpression petals was significantly increased (approximately 700-fold), indicating that the OE-LrNAC8 vector was successfully expressed in petals. Furthermore, the expression levels of structural genes in most anthocyanin synthesis pathways were upregulated, including DFR1, DFR2, CHI, OMT, F3'H1, F3'H2, C4H, LAR, and CHS1, with C4H and LAR showing the highest increases. This result suggests that LrNAC8 may indirectly participate in anthocyanin synthesis by influencing structural genes in the anthocyanin synthesis pathway.

[0095] Biological functions of LrNAC8, a NAC transcription factor in Lycoris radiata. Experiments showed that LrNAC8 is located in the cell nucleus and enhances plant adaptability through a dual pathway: 1) In salt-sensitive yeast and Lycoris radiata leaves, overexpression of LrNAC8 significantly improved salt tolerance, while VIGS silencing led to decreased stress resistance; in salt stress response, it reduced ROS accumulation and maintained chlorophyll homeostasis by activating the expression of antioxidant enzyme genes such as SOD and POD; 2) In secondary metabolism, LrNAC8 specifically regulates key genes in the anthocyanin synthesis pathway (such as C4H and LAR), promoting petal color development and synergistically enhancing antioxidant capacity. Simultaneously, overexpression of LrNAC8 resulted in color development of white petals in Lycoris radiata, with an anthocyanin content doubling, providing direct evidence for flower color improvement.

[0096] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A Lycoris LrNAC8 gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the Lycoris LrNAC8 gene according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, The biological material is an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the Lycoris LrNAC8 gene as described in claim 1.

4. The biomaterial according to claim 3, characterized in that, The recombinant vector is either the overexpression vector pBIN-GFP4-LrNAC8 or the silencing vector pTRV2-LrNAC8.

5. The biomaterial according to claim 3, characterized in that, The recombinant bacteria are Agrobacterium or Saccharomyces cerevisiae.

6. The application of the Lycoris LrNAC8 gene of claim 1 or the biomaterial of claim 3 in regulating Lycoris salt tolerance and / or promoting Lycoris anthocyanin synthesis.

7. The application according to claim 6, characterized in that, The application involves improving the salt tolerance of Lycoris radiata and / or promoting anthocyanin synthesis by overexpressing the LrNAC8 gene.

8. The application according to claim 7, characterized in that, The application includes the following steps: constructing a recombinant plant expression vector of the Lycoris LrNAC8 gene as described in claim 1, and then introducing the recombinant plant expression vector into Lycoris plants or Lycoris tissues to cultivate transgenic Lycoris plants or tissues with improved salt stress tolerance and / or enhanced anthocyanin synthesis.

9. The application according to claim 6, characterized in that, The application involves reducing salt tolerance of Lycoris radiata by silencing or downregulating LrNAC8 gene expression.

10. The application according to claim 9, characterized in that, The application includes the following steps: constructing a silencing vector for the Lycoris LrNAC8 gene as described in claim 1, and then introducing the silencing vector into Lycoris plants or Lycoris tissues to cultivate transgenic Lycoris plants or tissues with reduced salt stress tolerance.

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