Application of tea tree CsNAC25 gene in negative regulation of plant cold resistance

Abstract

The application discloses application of a tea tree CsNAC25 gene in negative regulation of plant cold resistance, the gene belongs to a tea tree NAC transcription factor family, and a nucleotide sequence of the gene is shown as SEQ ID NO. 1. The tea tree CsNAC25 gene negatively regulates cold resistance of the tea tree, and gene expression is down-regulated under low-temperature induction. Overexpression of the gene in tobacco weakens cold resistance of the transgenic plant, and silencing of CsNAC25 expression in the tea tree by using a VIGS technology makes the cold resistance of the tea tree be significantly improved. Silencing of CsNAC25 effectively reduces wilting degree and ion leakage rate of tea seedling leaves under low-temperature stress, simultaneously activates a key CBF signal path, and promotes accumulation of an osmotic regulator. The application is expected to effectively improve the cold resistance of the tea tree when low-temperature disasters such as cold spring occur, and has important theoretical significance and wide application prospect for guaranteeing stable yield and quality of the tea industry.

Description

TECHNICAL FIELD

[0001] The application relates to the field of genetic engineering, in particular to application of a tea tree CsNAC25 gene in plant cold resistance BACKGROUND

[0002] Camellia sinensis (L.) O. Kuntze is regarded as an important woody economic crop in many countries, mainly growing in tropical and subtropical regions. However, tea tree has weak resistance to low temperature, which makes it vulnerable to frost, and thus affects its growth, development, yield and quality. Extreme low temperature in winter and sudden "early spring cold" in late spring often cause significant economic losses to the tea industry. How to protect the yield and quality of tea under low temperature stress has become an important bottleneck problem restricting the stable development of the tea industry. Although some cold-resistant genes have been reported in the field of tea tree, the application of these cold-resistant genes is mostly in the form of heterologous overexpression of target genes, such as obtaining tobacco or Arabidopsis plants with improved cold resistance by using transgenic technology. It is not uncommon for transgenic plants to inhibit plant growth and development while improving cold resistance, which limits the application of these cold-resistant genes as a whole. At present, there are few applications of cold-resistant genes in tea tree species by using genetic engineering methods to improve the cold resistance of tea tree. Therefore, further in-depth study of the cold resistance regulation network of tea tree and screening and mining of effective and applicable tea tree cold-resistant genes not only provide new strategies and methods for tea tree to cope with low temperature stress, but also have important practical significance for improving tea quality and ensuring the safe production of the entire tea industry.

[0003] When tea tree responds to low temperature stress, it produces complex responses at multiple levels, including cell, physiology, biochemistry and molecular levels. These responses involve complex interactions between CBF-related or independent regulation networks, transcription factors, kinases and hormone signaling pathways. Among them, CBF is a key positive regulator of low temperature regulation pathway, and activation of CBF pathway often activates the downstream osmotic regulation system, thereby improving the cold resistance of plants. As an important plant hormone, ethylene has been reported to reduce the cold tolerance of plants in tomato and Arabidopsis under low temperature stress. The NAC superfamily is a highly conserved group of transcription factors in plants, which plays a variety of regulatory roles in plant growth, development and stress response. Previous studies have found that tea tree CsNAC25 transcription factor is involved in regulating secondary cell wall biosynthesis and trichome formation in tea tree. However, there is no evidence that CsNAC25 is related to the cold resistance of tea tree, and the specific role of CsNAC25 in the cold signal pathway of tea tree and its application in improving the cold resistance of tea tree are not clear. SUMMARY

[0004] The application provides application of a tea tree CsNAC25 gene in cold resistance of plants, and provides a new strategy for improving cold resistance of tea trees.

[0005] The application provides application of a tea tree CsNAC25 gene in cold resistance of plants, and provides a new strategy for improving cold resistance of tea trees.

[0006] The CsNAC25 gene is involved in regulation of an ethylene biosynthesis pathway through a CBF-dependent pathway under cold stress, thereby negatively regulating cold tolerance of the tea tree.

[0007] The application is silencing the CsNAC25 gene, thereby positively improving cold resistance of the tea tree.

[0008] The CsNAC25 gene is silenced, and cold-induced CsCBF1 and CsCBF3 expression is significantly up-regulated, and ethylene biosynthesis-related genes CsACS1 and CsACS2 and ethylene signal transduction-related genes CsERF1 and CsERF2 are significantly down-regulated.

[0009] The silencing method is VIGS technology, antisense oligonucleotide gene silencing technology, nanomaterial delivery or microRNA interference, and the CsNAC25 gene is silenced by using an agrobacterium liquid vacuum infiltration method, an in-vitro spraying agrobacterium liquid method or an agrobacterium liquid direct injection method.

[0010] The silencing method is VIGS technology: a CsNAC25 interference fragment is constructed on a pTRV2 vector by using a homologous recombination method, a recombinant plasmid pTRV2-CsNAC25 is obtained, and then agrobacterium liquid containing the pTRV2-CsNAC25 is introduced into plants, so that the CsNAC25 gene of the plants is silenced.

[0011] The CsNAC25 interference fragment is 357 bp of 28-384 in the sequence listing SEQ ID NO. 1.

[0012] The plant is a tea tree.

[0013] According to the embodiment of the application, the tea tree CsNAC25 gene is a tea tree NAC transcription factor family gene, and the nucleotide sequence of the tea tree CsNAC25 gene is shown in the sequence listing SEQ ID NO. 1.

[0014] According to the embodiment of the application, the CsNAC25 gene is overexpressed in a plant body to reduce low-temperature tolerance of transgenic tobacco, and the CsNAC25 gene is inhibited to express to improve cold resistance of the tea tree.

[0015] The core technical scheme of the present application is: by using gene silencing technology, the expression level of CsNAC25 gene in tea tree is reduced, and the cold resistance of tea tree can be significantly improved. In the specific implementation process, the forms including but not limited to VIGS technology (such as using TRV virus vector), antisense oligonucleotide gene silencing technology, nano material delivery (such as CsNAC25-siRNA nanoparticles), microRNA interference (such as designing amiRNA targeting CsNAC25), and engineering bacteria carrying CsNAC25-RNAi carrier can be used, and various gene silencing methods such as agrobacterium liquid vacuum infiltration method, in-vitro spraying agrobacterium liquid method and agrobacterium liquid direct injection method can be used.

[0016] Compared with the prior art, the beneficial effects of the present application are: for the first time, it is found that CsNAC25 is a negative regulator of cold resistance of tea tree, compared with the positive regulator of cold resistance pathway, the negative regulator can be directly applied in the woody plant such as tea tree which is difficult to obtain stable plants by transformation by using gene silencing technology. Silencing CsNAC25 gene in tea tree significantly improves the cold resistance of tea tree. Phenotypic observation finds that after low temperature treatment, the leaves of tea tree with silenced CsNAC25 are delayed compared with the control. Statistical analysis of data finds that the relative electrical conductivity of tea tree leaves with silenced CsNAC25 is significantly lower than that of the control under low temperature stress, and the content of osmotic adjustment substance soluble sugar is increased. The hormone determination result shows that the tea tree with silenced CsNAC25 reduces the content of ethylene in the body. Gene expression analysis finds that silencing CsNAC25 activates the transcription level of CsCBF1 and CsCBF3 genes under low temperature stress, and inhibits the expression of ethylene biosynthesis and signal related genes. The present application first proves that CsNAC25 gene plays an important regulatory role in the process of tea tree responding to low temperature stress, and analyzes the mechanism that CsNAC25 may participate in regulating the ethylene biosynthesis pathway through CBF-dependent pathway under cold stress, thereby negatively regulating the cold tolerance of tea tree. Silencing CsNAC25 positively improves the cold resistance of tea tree. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The expression of CsNAC25 gene under 4℃ low temperature stress was analyzed by fluorescence quantitative PCR technology. The numerical value is represented by mean ± standard deviation (n=3); ** indicates that there is a significant difference between normal growth conditions (**, P<0.01, Student's t-test)

[0018] Figure 2This invention, in Example 3, examines the effect of CsNAC25 overexpression on the cold resistance of transgenic tobacco plants, specifically the model plant tobacco. The results include: (A) Cold-sensitive phenotypes of overexpressing (OE) and control (CK) tobacco plants under normal and low-temperature stress; (B) Relative electrical conductivity of OE and CK tobacco plants under normal and low-temperature stress; (C) Superoxide dismutase activity of OE and CK tobacco plants under normal and low-temperature stress; and (D) Soluble sugar content in OE and CK tobacco plants under normal and low-temperature stress.

[0019] Figure 3 To analyze the relative expression levels of the CsNAC25 gene in the control and two CsNAC25 silencing lines using quantitative real-time PCR. CsGAPDH from tea plantations was used as an internal control. Values ​​are expressed as mean ± standard deviation (n = 3); ** indicates a highly significant difference compared to the control TRV-EV (**, P < 0.01, Student's t-test).

[0020] Figure 4 This invention, Example 4, describes the effect of silencing CsNAC25 on the cold resistance of tea plants. The results include: (A) changes in the wilting phenotype of young leaves before and after low-temperature treatment in control and silenced plants; (B) determination of relative ion permeability in control and silenced plants under normal and low-temperature treatments; (C) determination of soluble sugar content in control and silenced plants under normal and low-temperature treatments; and (D) determination of ethylene content in control and silenced plants under normal and low-temperature treatments. Data represent the mean ± standard deviation of three replicates. ** indicates a highly significant difference in TRV2-EV between the silenced line and the control plant (**, P < 0.01, Student's t-test).

[0021] Figure 5 This study used quantitative real-time PCR to analyze the transcriptional levels of cold response genes and ethylene synthesis-related genes in the control (TRV-EV) and two CsNAC25 silencing lines (TRV-CsNAC25-1 and TRV-CsNAC25-2). Tea plant CsGAPDH was used as an internal control. Values ​​are expressed as mean ± standard deviation (n=3); ** indicates a highly significant difference between the silencing lines and TRV-EV (**, P<0.01, Student's t-test). Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0023] Example 1: Cloning of the CsNAC25 gene from tea plants:

[0024] The CsNAC25 gene is a member of the NAC transcription factor family. Its sequence analysis is as follows: Using the local Guizhou tea variety Qiancha No. 1 as material, tea seedlings were planted at the germplasm resource nursery base of Guizhou University South Campus, Huaxi District, Guiyang City (26°34′N, 106°52′E). RNA was extracted from young leaves using the plant RNA extraction kit from Omega Technology Co., Ltd. The integrity and concentration of RNA were confirmed by agarose gel electrophoresis and NanoDrop nucleic acid quantification. cDNA was then synthesized using the Genestar reverse transcription kit from Xibao Biotechnology Co., Ltd. This cDNA was used as a PCR template to amplify the CsNAC25 gene. The upstream primer (protective base + restriction site + gene sequence) was (5'-TGCTCTAGAATGGGCGATAAGAATGTG-3'), and the downstream primer (protective base + restriction site + gene sequence) was (5'-GCGTCGACATTTGGTAGACTAATTTCGT-3'). PCR amplification was performed using these primers. The PCR reaction system consisted of 50 μL of KOD high-fidelity enzyme KOD PCR Master Mix (Sylanbo Technology Co., Ltd.), 25 μL of forward and reverse primers, 2.5 μL of cDNA, and 15 μL of ddH2O. The reaction program was as follows: denaturation at 98℃ for 10 s, annealing at 60℃ for 5 s, extension at 68℃ for 20 s, 30 cycles, and a final extension at 72℃ for 5 min. PCR amplification was detected by agarose gel electrophoresis. Bands matching the target gene size were excised and recovered using a gel extraction kit. The resulting product was then reacted with standard Taq PCR enzyme at 72°C for 20 min, followed by the addition of A. The product was ligated into the pMDT18-T vector from Takara to obtain the pMDT18-CsNAC25 ligation product. This product was then heat-shocked into competent DH5α *E. coli* cells (Sylanbo Biotechnology Co., Ltd.), plated on 50 μg / mL ampicillin-resistant LB agar, and incubated upside down at 37°C for 12 h. Single colonies were picked, and positive clones were identified by PCR after shaking. These clones were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing using universal primers for the T vector.

[0025] The determined nucleotide sequence of the CsNAC25 gene is as follows:

[0026] ATGGGCGATAAGAATGTGAACCTCCCACCTGGGTTCCGATTCTACCCAACCGATGAAGAGCTCGTCGTCCATTTCCTCCATCGTAAGGCCGCTCTCTTACCTTGCCATCCCGATGTCATCCCTGATCTCGATCTCTATCCTTATGATCCCTGGGATTTAGACGGTAAAGCTATGGCGGAGGGGAACAAATGGTACTTCTTTAGCCGGAAAACACAAAACCGGGTTACCGGAAATGGGTATTGGAAGCCATTGGGATTGGATGAACCCATATTCAGCAGTGGTGCTAGCAAGAAAGTTGCAGTTGGTATGAAGAAATATTATGTGTTCTATGTTGGAGAAGCTTCGGCCGGCGGGGCCAAAACGAACTGGATAATGCAGGAGTACCGGCTCTCAGACTCTGGTTCTAGTAGTAGTAGTAGATCATCTAAAAGAAGAGGAAATCCAAAAATAGATTATAGTAAATGGGTAGTATGTCGAGTGTACGAACGCAACTGTGAAGACGACGACGACAACGATGGGACGGAGCTTTCATGCTTGGATGAAGTTTTCTTAGCTTTGGATGATCTTGACGAAATTAGTCTACCAAATTAG(SEQ IDNO.1)。

[0027] The amino acid sequence of the protein encoded by the CsNAC25 gene is as follows:

[0028] MGDKNVNLPPGFRFYPTDEELVVHFLHRKAALLPCHPDVIPDLDLYPYDPWDLDGKAMAEGNKWYFFSRKTQNRVTGNGY

[0029] WKPLGLDEPIFSSGASKKVAVGMKKYYVFYVGEASAGGAKTNWIMQEYRLSDSGSSSSSRSSKRRGNPKIDYSKWVVCRV YERNCEDDDDNDGTELSCLDEVFLALDDLDEISLPN*(SEQ ID NO.2)。

[0030] Example 2: Analysis of the expression pattern of the CsNAC25 gene under low temperature stress

[0031] Healthy, disease-free Qiancha No. 1 tea seedlings with uniform growth were selected for low-temperature treatment. They were placed in a low-temperature incubator at 4℃, with a light intensity of approximately 6000 lux, a photoperiod of 16 hours, and a humidity of approximately 75%. At the corresponding treatment times (0h, 3h, and 24h), one bud and two leaves were harvested from each treated plant, with three biological replicates for each. The replicates were cryopreserved in liquid nitrogen at -80℃ for CsNAC25 gene expression analysis. RNA extraction and cDNA reverse transcription were performed using the same methods. Quantitative real-time PCR was performed using the Genstar premix kit from Guizhou Xibao Biotechnology Co., Ltd., and the target gene expression level was detected using a Bio-Rad Life Sciences quantitative PCR instrument (CFX Connect™). Data from the quantitative real-time PCR were calculated using a 2-1... –ΔΔCt The values ​​were calculated and organized using the value method. The upstream primer for quantitative fluorescence is (5'-TGGTGCTAGCAAGAAAGTTGC-3'), and the downstream primer is (5'-GTCGTCGTCTTCACAGTTGC-3'). Figure 1 The expression pattern of CsNAC25 under low temperature stress was investigated. The results showed that the expression of CsNAC25 gene was significantly downregulated after low temperature treatment in tea plants, suggesting that CsNAC25 may be involved in the low temperature stress response process of tea plants.

[0032] Example 3: Overexpression of CsNAC25 in tobacco weakens the cold resistance of transgenic tobacco.

[0033] The pMDT18-CsNAC25 positive clone plasmid obtained in Example 1 was digested with Xba I and Sal I. The target fragment of approximately 591 bp was recovered and ligated with the pSuper1300-GFP overexpression vector, which was also digested and gel-cleaved using the same method. The ligation was performed using T4 ligase, followed by heat shock transformation and screening with kanamycin plates. After identifying positive clones through double enzyme digestion, Agrobacterium was transformed into *Agrobacterium*, and then genetic transformation using the *Agrobacterium*-mediated tobacco leaf disc method was used to obtain transgenic tobacco overexpressing CsNAC25. First, *Agrobacterium* strains confirmed to have been transformed with CsNAC25 were co-cultured for 3 days on a co-culture medium (MS + 30 g / L sucrose + 6 g / L agar + 1 mg / L 6-BA + 0.2 mg / L NAA + 100 μmol / L AS, pH 5.8). Then, the cultures were transferred to a selection medium (MS + 30 g / L sucrose + 6 g / L agar + 1 mg / L 6-BA + 0.2 mg / L NAA + 20 mg / L Hyg + 100 mg / L Tim, pH 5.8) to induce callus and resistant shoot formation. The resistant shoots were further screened for resistance on a rooting medium (1 / 2 MS + 20 g / L sucrose + 6 g / L agar + 0.1 mg / L IBA + 25 mg / L Hyg + 100 mg / L Tim, pH 5.8). Transgenic tobacco overexpressing the CsNAC25 gene was identified using the quantitative real-time primers described in Example 2. Phenotypic observations were conducted on control (CK) and CsNAC25-overexpressing transgenic tobacco plants (OE1, OE2, and OE3) after cold stress treatment, and physiological indicators such as relative conductivity, soluble sugar content, and superoxide dismutase activity were measured. Figure 2 As shown in Figure A, after 24 hours of low-temperature treatment at 4℃, the transgenic plants overexpressing CsNAC25 exhibited more severe leaf wilting under cold stress compared to the control plants. Correspondingly, the relative electrical conductivity of the transgenic plants overexpressing CsNAC25 under cold stress was significantly higher than that of the control. Figure 2 The presence of B indicates that the cell membrane is more damaged and the cold resistance is weaker at low temperatures. Furthermore, the superoxide dismutase activity of transgenic tobacco overexpressing CsNAC25 decreased after cold stress treatment. Figure 2 In the middle (C), the content of soluble sugars, which are osmotic regulators, decreases. Figure 2 (D). These results suggest that overexpression of CsNAC25 may affect the accumulation of osmotic regulators and antioxidant enzyme activity under cold stress, thereby weakening the cold resistance of transgenic tobacco.

[0034] Example 4: Silent CsNAC25 enhances the cold resistance of tea plants.

[0035] (1) Construction of a VIGS vector for silencing the CsNAC25 gene in tea plants using VIGS technology: The CsNAC25 target gene fragment was ligated to the pTRV2 vector using homologous recombination. The pTRV2 vector was provided by Transduction Biotechnology Co., Ltd. First, restriction enzyme sites were selected to linearize the pTRV2 vector. After gel recovery, homologous arms were designed based on the pGBKT7 vector. Using cDNA from leaves of Qiancha No. 1 as a template, the conserved fragment interfering with the CsNAC25 gene (28-384 bp of the gene) was amplified. The sequence is as follows:

[0036] CCTGGGTCCGATTCTACCCAACCGATGAAGAGCTCGTCGTCCATTTCCTCCATCGTAAGGCCGCTCTCTTACCTTGCCA

[0037] TCCCGATGTCATCCCTGATCTCGATCTCTATCCTTATGATCCCTGGGATTTAGACGGTAAAGCTATGGCGGAGGGGAACA

[0038] AATGGTACTTCTTTAGCCGGAAAACACAAAACCGGGTTACCGGAAATGGGTATTGGAAGCCATTGGGATTGGATGAACCC

[0039] ATATTCAGCAGTGGTGCTAGCAAGAAAGTTGCAGTTGGTATGAAGAAATATTATGTGTTCTATGTTGGAGAAGCTTCGGC

[0040] CGGCGGGGCCAAAACGAACTGGATAATGCAGGAGTAC (SEQ ID NO.3). The CsNAC25 interference fragment was constructed into the pTRV2 vector using the seamless cloning kit from Yisheng Biotechnology Co., Ltd., and then transformed into E. coli. PCR verification and sequencing identification were subsequently performed.

[0041] (2) VIGS Experimental Procedure: (1) Transform Agrobacterium GV3101 competent cells with the recombinant plasmid. Pick a single colony that has been correctly identified and add it to 5 mL of YEP (50 mg / L Kan, 20 mg / L Rif) liquid medium. Then place it on a shaker (28°C, 200 r / min) and shake overnight. Then add 2 mL of bacterial solution to 150 mL of YEP (50 mg / L Kan, 20 mg / L Rif) medium and continue to culture until the OD600 is about 1.2. Use 50 mL centrifuge tubes to dispense the bacterial solution and centrifuge at 5000 r / min for 10 min to obtain the bacterial cells. Discard the medium and add the pre-prepared transformation solution to resuspend the bacterial cells until the OD600 is about 1.2. Mix pTRV1 and pTRV2 (empty control) and pTRV1 and pTRV2-CsNAC25 resuspension in equal proportions and incubate on a shaker (28°C, 200 r / min) for 1 h. (2) Prune well-grown tea branches into 8-10cm lengths, retaining two fully developed mature leaves on each cutting. Place the cuttings in a bottle containing a suspension solution, ensuring the suspension submerges the leaves. Let stand for 15 minutes, then use a vacuum pump to create a vacuum for 5 minutes at a pressure of 0.8 kPa. After soaking, allow the branches to air dry at room temperature to remove any remaining bacterial solution. Then, insert the cuttings into a nutrient solution and culture them in the dark in a 24℃ artificial climate chamber for 3 days. Afterward, place them in a 24℃ environment with a light / dark cycle of 16h / 8h for normal cultivation, changing the nutrient solution every two weeks. Observe the growth of new buds after they emerge.

[0042] (3) Analysis of CsNAC25 gene expression after silencing CsNAC25: As described above, RNA was extracted from tea plants containing the empty vector control TRV-EV and the silenced lines. After reverse transcription into cDNA, the gene expression level of CsNAC25 was detected using the quantitative fluorescent primers in Example 2. Figure 3 The displayed lines are the control (tea plants infected with the empty vector EV-TRV) and two independently constructed lines infected with pTRV2-CsNAC25 to silence the CsNAC25 gene. Figure 3 It is evident that, among the three groups of tea cuttings, the transcription level of CsNAC25 in the TRV2-CsNAC25-1 and TRV2-CsNAC25-2 lines was significantly lower than that in the control.

[0043] (4) Silencing CsNAC25 enhanced the cold resistance of tea plants. Phenotypic observations were conducted on tea plants of both control and CsNAC25-silenced types after cold stress treatment, and physiological indicators such as relative ion permeability, soluble sugar content, and ethylene content were measured. Figure 4As shown in Figure A, after 24 hours of treatment at 4℃, the tea plants with silenced CsNAC25 exhibited less leaf wilting under cold stress compared to the control plants. Correspondingly, the relative electrical conductivity of plants with silenced CsNAC25 under cold stress was significantly lower than the control, indicating less cell membrane damage and stronger cold resistance at low temperatures. Figure 4 (B). Meanwhile, plants of the silent CsNAC25 group showed higher levels of osmotic regulators and soluble sugars after cold treatment. Figure 4 (C). Furthermore, tea plants silencing CsNAC25 reduced ethylene accumulation (C). Figure 4 (D). Further quantitative PCR analysis showed that in tea plants with silenced CsNAC25, cold-induced expression of CsCBF1 and CsCBF3 was significantly upregulated, while the expression of ethylene biosynthesis-related genes CsACS1 and CsACS2, and ethylene signal transduction-related genes CsERF1 and CsERF2 were significantly downregulated. Figure 5 These results indicate that silencing CsNAC25 activates the CBF signaling pathway in tea plants, thereby increasing the accumulation of osmotic regulators under cold stress and potentially enhancing the cold resistance of tea plants by inhibiting ethylene biosynthesis and signaling pathways at low temperatures.

[0044] In summary, CsNAC25 gene expression is downregulated by low temperature. Experiments in tobacco showed that overexpression of CsNAC25 weakened the cold resistance of transgenic plants, manifested as a significant increase in ion permeability, a decrease in the content of soluble sugars (osmotic regulators), and a decline in superoxide dismutase activity at low temperatures. In tea plants, silencing CsNAC25 expression using VIGS technology significantly improved cold resistance. Silencing CsNAC25 effectively reduced leaf wilting and ion leakage under low-temperature stress, while simultaneously activating the key CBF signaling pathway and promoting the accumulation of osmotic regulators. Furthermore, silencing CsNAC25 treatment also inhibited low-temperature-induced ethylene biosynthesis and its signal transduction. These results indicate that CsNAC25 plays an important negative regulatory role in the tea plant's response to low-temperature stress, with mechanisms involving CBF-dependent regulation and ethylene pathway inhibition. This application not only elucidates for the first time the negative regulatory role and molecular mechanism of CsNAC25 in the cold tolerance of tea plants, but also provides a theoretical basis for developing regulators targeting CsNAC25 (such as exogenous sprays) through genetic engineering. These regulators are expected to be applied to tea plants during cold snaps and other low-temperature disasters, thereby effectively improving their cold resistance. This has significant theoretical importance and broad application prospects for ensuring stable yield and quality in the tea industry.

[0045] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. Tea tree CsNAC25 Application of genes in negative regulation of cold resistance in plants, specifically in tea trees. CsNAC25 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is a tea plant or tobacco.

2. The application according to claim 1, wherein the plant is a tea tree.

3. The application according to claim 2, wherein the negative regulation is CsNAC25 Under cold stress, the gene participates in the regulation of ethylene biosynthesis through the CBF-dependent pathway, thereby negatively regulating the cold tolerance of tea trees.

4. Silence CsNAC25 The application of genes in positively enhancing the cold resistance of tea trees, the tea trees CsNAC25 The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing.

5. In the application according to claim 4, the silencing method is VIGS technology, antisense oligonucleotide gene silencing technology, nanomaterial delivery, or microRNA interference, utilizing Agrobacterium-mediated vacuum permeation, in vitro spraying of Agrobacterium-mediated liquid, or direct injection of Agrobacterium-mediated liquid. CsNAC25 Gene silencing.

6. In the application according to claim 5, the silencing method is VIGS technology: using homologous recombination to... CsNAC25 The interference fragment was constructed into the pTRV2 vector to obtain the recombinant plasmid pTRV2- CsNAC25 Then, containing pTRV2- CsNAC25 The Agrobacterium tumefaciens solution was introduced into the plant, causing the plant to... CsNAC25 Gene silencing expression.

7. The application according to claim 6, wherein CsNAC25 The interference fragment is positions 28-384, totaling 357 bp, as shown in SEQ ID NO.1.

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