Application of ZaNAC25 gene in regulating Zanthoxylum bungeanum peel prick character

Abstract

The present application relates to the field of plant genetic engineering technology, and particularly relates to application of Zanthoxylum bungeanum Nac25 gene in regulating plant thorn. The application of ZaNAC25 gene with the nucleotide sequence shown in SEQ ID NO. 1 in regulating the thorn character of Zanthoxylum bungeanum: at least one thorn character is produced by overexpressing the ZaNAC25 gene, including 1) significantly reduced thorn number; 2) shortened thorn length; and 3) partially non-lignified sharp end, so that the touch is soft. The present application uses ZaNAC25 gene as a negative regulation factor of Zanthoxylum bungeanum thorn development, reduces the thorn character by regulating the synthesis pathway of gibberellin GA, specifically: the thorn number of the new organ of Zanthoxylum bungeanum is significantly reduced, the thorn length is shortened, the sharp end is partially non-lignified, and the touch is soft, so that the phenotype of Zanthoxylum bungeanum thorn is improved.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the ZaNAC25 gene in regulating the prickly trait of Sichuan pepper. Background Technology

[0002] Sichuan pepper (Zanthoxylum armatum), a specialty economic crop belonging to the Rutaceae family and the Zanthoxylum genus, is a core raw material in the fields of condiments, food processing, and traditional Chinese medicine due to its unique numbing and fragrant flavor, leading to a year-on-year expansion of the industry. However, the stems, leaves, and fruit clusters of Sichuan pepper plants are densely covered with sharp and hard thorns, posing multiple obstacles to actual production. For example, during the seedling stage, the thorns easily scratch seedlings and increase transplant losses; in field management, the efficiency of pruning and pest control is significantly reduced, and the risk of injury to operators is significantly increased; the degree of mechanization in the harvesting process is lower than that of other thornless crops, with most harvesting done manually, directly driving up production costs. Against the backdrop of continuously rising labor costs in recent years, some producing areas have even seen a phenomenon of "pepper farmers abandoning cultivation," seriously restricting the large-scale and intensive development of the Sichuan pepper industry. Therefore, analyzing the molecular regulatory mechanism of thorn development and selectively breeding low-thorn / thornless Sichuan pepper varieties has become an urgent need for industrial upgrading.

[0003] Plant spines are defensive structures formed by the specialization of epidermal or cortical cells and are not directly connected to vascular tissues. At present, research on plant spines is still in its early stages, with only a few regulatory genes such as LOG, WOX3, NAC93, and MYB86 identified. Most studies focus on herbaceous model plants, while the analysis of related mechanisms in woody economic crops is relatively scarce.

[0004] Both trichomes and spines are epidermal appendages of plants, and their developmental regulatory mechanisms have been partially elucidated. For example, the NAC and MYB transcription factor families play a central role in the initiation and elongation of trichomes—in Arabidopsis, NAC transcription factors regulate cell differentiation direction by binding to the promoter regions of trichome development genes; in cotton, the MYB gene directly participates in the secondary wall thickening process of fibers (specialized trichomes). However, the morphogenesis and cell differentiation pathways of trichomes and spines are fundamentally different, and existing trichome-related regulatory mechanisms cannot be directly extrapolated to spine development. Furthermore, in current spine regulation research, the function of NAC transcription factors has not been systematically explored in Sichuan pepper, and the association between NAC family members and spine development is rarely reported. This research gap results in a lack of precise gene resources for improving the spine trait in Sichuan pepper and also limits the molecular breeding process for spineless or low-spine Sichuan pepper varieties.

[0005] Therefore, we propose the application of the Nac25 gene regulation of Sichuan pepper in this invention to improve the thorn trait of Sichuan pepper and provide a new research direction for molecular breeding of thornless or less thorny Sichuan pepper varieties. Summary of the Invention

[0006] The purpose of this invention is to provide an application of the Nac25 gene in regulating the prickles of Sichuan pepper for improving the prickle trait of Sichuan pepper, and to provide a research direction for molecular breeding of thornless or less prickly varieties of Sichuan pepper.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of the ZaNAC25 gene, whose nucleotide sequence is shown in SEQ ID NO.1, in regulating the prickles of Sichuan pepper.

[0009] At least one of the following skin spike traits can be produced by overexpressing the ZaNAC25 gene:

[0010] 1) The number of thorns is significantly reduced;

[0011] 2) The length of the thorns is shortened;

[0012] 3) Some of the tips are not woody, making them soft to the touch.

[0013] Application of ZaNAC25 protein with amino acid sequence as shown in SEQ ID NO.2 in regulating the prickles of Sichuan pepper.

[0014] At least one of the following spiky traits can be produced by promoting the synthesis of ZaNAC25 protein:

[0015] 1) The number of thorns is significantly reduced;

[0016] 2) The length of the thorns is shortened;

[0017] 3) Some of the tips are not woody, making them soft to the touch.

[0018] Any of the following applications of the ZaNAC25 gene with the nucleotide sequence shown in SEQ ID NO.1:

[0019] 1) Activate the transcriptional activity of the ZaGA20ox1 gene;

[0020] 2) Upregulates gibberellin levels in Sichuan pepper.

[0021] This invention has at least the following beneficial effects:

[0022] This invention utilizes the ZaNAC25 gene as a negative regulator of the development of prickles in Sichuan pepper. By regulating the synthesis pathway of gibberellin (GA), the shape of the prickles is reduced. Specifically, this leads to a significant reduction in the number of prickles on newly formed organs of Sichuan pepper, a shortening of prickle length, non-lignification of some tips, and a softer touch, thereby improving the phenotype of Sichuan pepper prickles. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The expression pattern of ZaNAC25 in different tissues of Sichuan pepper;

[0025] Figure 2 The correlation between ZaNAC25 expression level and the density of prickles in Sichuan pepper;

[0026] Figure 3 Subcellular localization of ZaNAC25;

[0027] Figure 4 Phenotypic features of the front and back of untreated leaves of wild-type (WT) Sichuan pepper;

[0028] Figure 5 The phenotype of Sichuan pepper treated with ZaNAC25 overexpression;

[0029] Figure 6 The number of thorns (including the upper surface of petiole, the upper surface of veins, the lower surface of petiole, and the lower surface of veins) of wild-type (WT) and ZaNAC25 overexpressed Sichuan pepper (OX) materials were statistically analyzed.

[0030] Figure 7 The spine length of wild-type (WT) and ZaNAC25-overexpressing Sichuan pepper (OX) materials was statistically analyzed (including the upper surface of the petiole, the upper surface of the vein, the lower surface of the petiole, and the lower surface of the vein).

[0031] Figure 8 EMSA verification of the ZaNAC25 transcription factor and the ZaGA20ox1 promoter;

[0032] Figure 9 Construction and validation of a dual-luciferase reporter system vector;

[0033] Figure 10 The number of thorns in plants that were not treated with gibberellin and plants that received exogenous gibberellin treatment is statistically analyzed.

[0034] Figure 11 The effect of exogenous gibberellin application on the development of prickles in Sichuan pepper. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] The core working principle of this invention is to use the ZaNAC25 gene as a negative regulator of the development of prickles in Sichuan pepper, thereby reducing the number and length of prickles by regulating the synthesis pathway of gibberellin (GA).

[0037] Example 1: This example mainly provides the sequences of the ZaNAC25 gene (SEQ ID NO.1) and ZaNAC25 protein (SEQ ID NO.2).

[0038] The nucleotide sequence of the ZaNAC25 gene is shown in SEQ ID NO.1 below:

[0039] SEQ ID NO.1:

[0040]

[0041] The amino acid sequence of the ZaNAC25 protein is shown in SEQ ID NO.2 below:

[0042] SEQ ID NO.2:

[0043] MESTDSSTSSQHPQLPPGFRFHPTDEELVVHYLKKKAASAPLPVAIIAEVDLYKFDPWELPSKATFGEQEWYFFSPRDRKYPNGARPNRAATSGYWKATGTDKPILTSNGTNQKVGVKKALVFYGGKPPKGIKTNWIMHEYRLIDNNSAAKPQVPDLVPNKKNSLRLDEWVLCRI YKKNNAQRPMERDREDCFEGMYVTLQQQTSSHQNPKSPTGKNISYGSLFEVHDEHFFEGMLSGEALQQQNSCVSQLASSSTKPAANMSIVSDPSTTNTVTAPIKRAMPPQQFWNEASSSGKRFHGDIISGSTATADNNNNSFVSLLNHLPQSVQFNLGDGTTLRQQFQLPSMNWNS

[0044] Example 2: This example discloses the identification and functional verification of the ZaNAC25 gene. Through qRT-PCR detection, it was found that the ZaNAC25 gene was differentially expressed in different tissues of Sichuan pepper, and its expression level was significantly negatively correlated with the density of thorns, directly proving that ZaNAC25 is a negative regulator of thorn development in Sichuan pepper.

[0045] 1. Tissue Expression Pattern: qRT-PCR analysis revealed that ZaNAC25 expression was highest in male flowers of Sichuan pepper, followed by leaves, and lowest in stems. Combined with the characteristic that spines are mainly distributed in stems and leaves, this suggests that ZaNAC25 participates in tissue-specific regulation of spine development, as detailed below:

[0046] 1.1 Experimental Materials

[0047] The test materials were 3-year-old clonal Zanthoxylum armatum var. novemfolius plants grown in the Zanthoxylum germplasm resource nursery of Chongqing Yongchuan College of Arts and Sciences. Three healthy individual plants with the same height, crown width, and growth vigor were selected (biological replication). During the flowering period (mid-March 2024), on a sunny morning between 9:00 and 10:00, male flower buds (unopened, 2.0±0.2 mm in diameter), functional leaves (3rd-4th leaves of new shoots), and stem cortex tissue (middle section of new shoots, 0.5±0.05 cm in diameter) were collected. All tissue samples were immediately flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0048] 1.2 Experimental Methods

[0049] Total RNA was extracted from each tissue using the Trizol method. After DNase I digestion to remove genomic DNA contamination, RNA purity and integrity were verified by UV spectrophotometry and agarose gel electrophoresis. Two μg of total RNA was used to synthesize the first strand of cDNA using the PrimeScript™ RT kit. Using the UBC gene of Sichuan pepper as an internal control (primer sequences: F: 5'-GGCAGCATCTCAAGCAAGCC-3'; R: 5'-GTCCACCAACCCAGCCGAAA-3'), qRT-PCR specific primers for ZaNAC25 were designed (F: 5'-CAGAGGCCGATGGAGAGGGA-3'; R: 5'-GCTTGGTGCTCGAAGAGGCT-3'). Amplification was performed on a CFX96 real-time quantitative PCR instrument using the SYBR® Green I fluorescent dye method. The reaction volume was 20 μL (containing 2 μL cDNA template, 0.8 μL each of forward and reverse primers, and 10 μL SYBR Mix). The program was: 95℃ pre-denaturation for 3 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles. Melting curve analysis was used to verify the amplification specificity. Each sample was tested in triplicate. -ΔΔCt The method calculates the relative expression level of genes.

[0050] 1.3 Results and Analysis

[0051] See Figure 1 qRT-PCR results showed that ZaNAC25 expression differed significantly among different tissues of Sichuan pepper: the highest relative expression level was found in male flower buds, followed by leaves, and the lowest expression level was found in stem cortex. Based on the phenotypic characteristic that Sichuan pepper spines are mainly distributed in stems and leaves, it is speculated that ZaNAC25 participates in the tissue-specific regulation of spine development.

[0052] 2. Correlation analysis between ZaNAC25 expression level and spine density: Analysis of Sichuan pepper materials with different spine densities showed that materials with fewer spines exhibited higher ZaNAC25 expression levels, while materials with more spines showed lower expression levels, directly demonstrating a negative correlation between ZaNAC25 and spine development. Details are as follows:

[0053] 2.1 Experimental Materials

[0054] Three Sichuan pepper germplasm resources with significant differences in prickle density were selected (see Table 1). Stem tissue (0.5 ± 0.05 cm in diameter, 5 cm in length) from the middle section of new shoots was collected during the peak vegetative growth period (July 2024). Three healthy single plants were selected from each germplasm as biological replicates, and the sample treatment was the same as in 1.1. At the same time, the number of prickles on the leaves was counted, and varieties with few prickles (Lessprickles), medium prickles (Medium prickles), and more prickles (More prickles) were distinguished according to the number of prickles.

[0055] Table 1

[0056] 2.2 Experimental Methods

[0057] RNA extraction, cDNA synthesis, and qRT-PCR detection methods are the same as in 1.2.

[0058] 2.3 Results and Analysis

[0059] See Figure 2 The results showed that the relative expression level of ZaNAC25 was significantly negatively correlated with the spine density: the highest expression level of ZaNAC25 (1.0) was observed in germplasm S1, which had the lowest spine density, while the lowest expression level (0.2) was observed in germplasm S3, which had the highest spine density. This result directly demonstrates that the expression level of ZaNAC25 is negatively correlated with the degree of spine development in Sichuan pepper, suggesting that it may act as a negative regulator in the spine formation process.

[0060] Example 3 discloses the subcellular localization of ZaNAC25: The PGreen-ZaNAC25-GFP vector was constructed, and localization was observed using a plant protoplast transient transformation system. The results showed that ZaNAC25 is a nuclear localized protein, consistent with the functional localization characteristics of NAC family transcription factors.

[0061] 3. Carrier Construction

[0062] Using seamless cloning technology, the CDS sequence of ZaNAC25 (with its natural stop codon removed) was fused with the C-terminus of the GFP tag's coding frame to construct the recombinant expression vector pGreen-ZaNAC25-GFP for subcellular localization. The recombinant expression plasmid pGreen-NAC25-GFP was finally obtained by transforming E. coli.

[0063] 4. Tobacco suspension cell culture and transformation

[0064] 4.1 Take 2 g of tobacco suspension cells BY2 cultured for about 8 days, centrifuge at 3500 g for 15 min, and wash the collected cells twice with Tris-MES buffer;

[0065] 4.2 Cells were digested at 37 °C for 1 h using Tris-MES buffer containing 1% caylase, 0.2% Y-23 dissociation enzyme, and 1% BSA.

[0066] 4.3 The obtained protoplasts were washed twice with W5 buffer, filtered through two layers of nylon cloth, and the protoplasts were washed with W5 buffer.

[0067] 4.4 Under a microscope, the final concentration of protoplasts was adjusted to 10⁶ ml⁻¹ using a hemocytometer;

[0068] 4.5 Mix 0.2 ml of protoplast cell suspension with 50 µg of salmon sperm DNA and 30 µg of plasmid, and add PEG to make the final concentration 20%. Incubate at room temperature for 1 h.

[0069] 4.6 After centrifuging the protoplast mixture at 1000 rpm for 8 min, resuspend it in W5 buffer and incubate it on a constant temperature shaker at 25 ℃ and 50 rpm for at least 16 h.

[0070] 4.7 The transformed protoplasts were observed and photographed under a laser confocal microscope (Note: The composition of the culture medium used for the preparation and transformation of tobacco suspension cells is shown in the following tables (Tables 2-5).

[0071] Table 2. Basic culture medium for tobacco suspension cells

[0072] Note: Adjust the pH to 5.8.

[0073] Table 3. Preparation method of 40% (m / v) PEG solution

[0074] Note: Adjust the pH to 6.5 using a 1M potassium hydroxide solution.

[0075] Table 4 Tris-MES Buffer

[0076] Note: Adjust the pH to 5.6 using 1 M Tris base.

[0077] Table 5 W5 Cleaning Buffer

[0078] Note: Adjust the pH of the solution to 5.8 using KOH.

[0079] The recombinant expression plasmid pGreen-NAC25-GFP was transformed into BY2 tobacco suspension cells, and its cellular localization was observed using a laser confocal microscope. In the experimental group, green fluorescence was concentrated only in the nucleus region (see...). Figure 3 ZaNAC25 is a nuclear localized protein, consistent with the subcellular localization characteristics of NAC family transcription factors, which "exert transcriptional regulatory functions in the cell nucleus".

[0080] Example 4 discloses the regulation of the spine phenotype by ZaNAC25 overexpression. Using the established aseptic rapid propagation system of Sichuan pepper, stable overexpression of the ZaNAC25 gene was achieved through Agrobacterium-mediated stem segment infection, efficiently obtaining regenerated plants overexpressing ZaNAC25. It was observed that the number and length of spines in the new organs were significantly reduced, and some tips were non-lignified and soft to the touch.

[0081] 5. Carrier Construction

[0082] The ZaNAC25 gene was ligated into the intermediate vector 8GWN using the Takara In Fusion kit, followed by sequencing. The correctly sequenced 8GWN-ZaNAC25 plasmid was then recombined with the plant overexpression vector CaMV35S-K303 using the Gateway LR Clonase II kit, successfully constructing the overexpression vector K303-ZaNAC25. The specific steps are as follows:

[0083] 5.1 Using the TAKARA In-Fusion® HD Cloning Kit, the fragment amplified using amplification primers ZaNAC25-F (5'-ATACTTCCAACTAGTGCGGCCGCATGGAGAGCACCGATTCATCGA-3') and ZaNAC25-R (5'-ATGGTCATCCCGGGACCTGCAGGTTATGAATTCCAATTCATGCTAGGAAG-3') was ligated into the intermediate vector 8GWN, which had been double-digested with NotⅠ and Sbfi. The ligation was then transformed into DH5α, plated on plates containing 100 μg / mL spectinomycin, and single-clone strains were selected for colony PCR. Strains that amplified the target fragment were sent for sequencing. Correct sequencing results indicated a positive strain containing the 8GWN-ZaNAC25 plasmid.

[0084] 5.2 Plasmids were extracted from positive strains by shaking. Using this plasmid as a template, amplification was performed using amplification primers ZaNAC25-F and ZaNAC25-R. The plasmid with the correct amplified band was then subjected to homologous recombination with the plant expression vector CaMV35S-K303 using a Gateway LR Clonase II kit. The product was transformed into DH5α and plated on a plate containing 100 μg / mL kanamycin for screening. Single clones were selected and amplified using amplification primers K303-F1 (5'- GGCTTGTCCCGCGTCATCGGCGGG-3')) and ZaNAC25-R, followed by colony PCR. When the amplified target band size matched the expectation and was verified by sequencing, the strain was identified as positive, and the overexpression vector K303-ZaNAC25 was obtained.

[0085] 5.3 Transform the overexpression vector containing the ZaNAC25 gene into the microbial transformant (Agrobacterium tumefaciens GV3101) to prepare engineered bacteria; specifically: shake the obtained positive strain (overexpression vector K303-ZaNAC25) to extract the plasmid, which is the K303-NAC25 plasmid of the overexpression vector, transform it into Agrobacterium tumefaciens GV3101, screen on plates containing 100 μg / mL kanamycin and 50 μg / mL rifampin, select single clone strains for colony PCR verification, and the strains that pass the verification are the engineered bacteria containing the overexpression vector K303-ZaNAC25.

[0086] 6. Genetic transformation to obtain overexpression lines

[0087] 6.1 Pretreatment of plants before transformation

[0088] Before the transformation operation, all pepper plants were subjected to dark treatment for a period of time to enhance the transformation sensitivity of the explants and to elongate the internodes to facilitate the acquisition of explant stem segments.

[0089] 6.2 Explant preparation and pre-culture

[0090] Cut the Sichuan pepper explants into stem segments (selecting parts with longer bud intervals), and make two cross-sectional cuts on each stem segment (without cutting it off completely). Place the treated stem segments on a pre-culture / co-culture medium and put them in a 25℃ light incubator for one day of pre-culture in the dark.

[0091] 6.3 Activation of Agrobacterium and Preparation of Infection Solution

[0092] The engineered bacteria containing the overexpression vector K303-ZaNAC25 obtained in 5.3 were subjected to the following operations:

[0093] (1) Primary activation: Add 100 μL of pre-preserved Agrobacterium bacterial suspension to 30 mL of LB liquid medium containing 30 μg kanamycin (Kan) + 30 μg rifampicin (Rif); place in a constant temperature shaker at 28℃ and incubate at 200 rpm in the dark for 24-36 h until the bacterial suspension is fully activated. Reached version 1.0.

[0094] (2) Secondary activation: Take the bacterial culture from the first activation and transfer it to LB liquid medium (which may not contain antibiotics), and continue to culture until... The bacterial culture was brought to a value of 1.0; then centrifuged at 6000 rpm and 4℃ for 10 min to collect the bacterial cells.

[0095] (3) Preparation of infection solution: Resuspend the bacterial cells in KCMS liquid medium (100ml MS + 100ml AS) and adjust the bacterial solution. The optimal infection concentration is 0.1.

[0096] 6.4 Explant Infection and Co-culture

[0097] Immerse the explants that have been pre-cultured for 24 hours in the prepared Agrobacterium infection solution for 8 minutes (they can be placed in a shaker for gentle shaking); after removing excess bacterial solution, transfer the explants to the pre-culture / co-culture medium and incubate in the dark for 2 days.

[0098] 6.5 Differentiation Culture and Rooting Culture

[0099] After co-culture, the explants were transferred to differentiation medium and placed in a light incubator. Once adventitious buds differentiated and seedlings formed, subsequent rooting culture was carried out.

[0100] 6.6 Blank Control Settings

[0101] A blank control group was set up, and the operation steps were exactly the same as those of the experimental group. This group was used to observe the growth status of explants and to compare and verify the transformation effect.

[0102] 7. Long-term qualitative observation of the dermal prickles phenotype (180 days after transformation)

[0103] The rooted transformation seedlings / control group seedlings were transplanted to a greenhouse for conventional cultivation for 6 months, and the morphological characteristics of the prickles on the newly formed mature leaves were observed:

[0104] WT group: The epidermis of the leaves (upper / lower petiole, veins) is densely covered with sharp, conical spines, with a length of 1mm-16mm, and feels hard to the touch. Figure 4 );

[0105] OX group: The spicules of the newly formed organs showed a significantly "sparse and short" phenotype—the number and length of spicules were reduced, some tips were non-lignified (height <1mm), the texture was soft, and there was no stinging sensation as in the WT group. Figure 5 ).

[0106] 8. Quantitative statistical analysis of dermatophyte phenotype (6 months post-injection)

[0107] The method of "random sampling + coordinate positioning and counting" was used to quantitatively analyze the prickles on both sides of the petiole (upper, middle and lower sections) and leaf veins (6 plants were counted in each group):

[0108] Quantitative statistics: The number of thorns in the OX group was significantly reduced compared to the WT group—0% reduction on the upper surface of the petiole, 23% reduction on the lower surface of the petiole, 90% reduction on the upper surface of the veins, and 21% reduction on the lower surface of the veins. Figure 6 );

[0109] Length statistics: The length of the spines in the OX group was significantly shorter than that in the WT group—the average length of spines on the upper surface of the petiole was 0.95 mm (1.35 mm in the WT group), a reduction of 30%; the average length of spines on the upper surface of the veins was 0.15 mm (0.45 mm in the WT group), a reduction of 67%; the average length of spines on the lower surface of the petiole was 0.32 mm (0.72 mm in the WT group), a reduction of 56%; and the average length of spines on the lower surface of the veins was 0.15 mm (0.24 mm in the WT group), a reduction of 38% (see...). Figure 7 );

[0110] The above results indicate that ZaNAC25 overexpression can stably and long-term inhibit the occurrence and elongation of spines in new organs of Sichuan pepper, clearly demonstrating that it is a negative regulator of spine development.

[0111] Example 5: This example discloses the molecular mechanism by which ZaNAC25 regulates spur development.

[0112] 9. Specific binding of ZaNAC25 to the promoter of the gibberellin synthesis gene ZaGA20ox1 (electrophoretic mobility shift analysis, EMSA)

[0113] Gibberellin (GA) is a key hormone regulating the development of epidermal protrusions in plants, and ZaGA20ox1 is the rate-limiting enzyme gene in the GA synthesis pathway of Sichuan pepper. To verify the transcriptional regulatory relationship between ZaNAC25 and ZaGA20ox1, an EMSA experiment was conducted:

[0114] Protein and probe preparation:

[0115] 9.1. Preparation of fusion protein: The prokaryotic expression vector pCold-NAC25 was constructed, transformed into Escherichia coli BL21(DE3) strain, and after expression induced by IPTG, His-ZaNAC25 fusion protein was obtained by purification by Ni-NTA affinity chromatography column;

[0116] 9.2. Probe Design and Synthesis:

[0117] For the ZaGA20ox1 promoter region (ATG upstream -55bp region), a biotin-labeled wild-type probe was synthesized with the sequence: 5'-GTGAAAACACAAAGAAAGAGAGGGAGAGTA-3' (containing the core binding sequence fragment of ZaNAC25 "AAACACAAAGAAAGAGAGGGA").

[0118] Synthetic biotin-labeled mutant probe: The “AAACACAAAGAAAGAGAGGGA” region of the wild-type probe is mutated to continuous guanine (G), with the sequence: 5'-GTGAGGGGGGGGGGGGGGGGGGGGGGAGTA-3';

[0119] An unlabeled wild-type probe (Cold probe) with the same sequence as the biotin-labeled wild-type probe was prepared as a specific competitive control.

[0120] 10. Experimental Groups and Results (see...) Figure 8 ):

[0121] Group 10.1 (His-tagged protein + wild-type probe): Only the free probe band is shown, with no protein-probe binding hysteresis band, indicating that the His tag itself does not bind to the ZaGA20ox1 promoter probe.

[0122] 10.2 Group 2 (His-ZaNAC25 + wild-type probe): A clear hysteresis band (bound probe) appeared, while the signal of the free probe band weakened, proving that His-ZaNAC25 can specifically bind to the wild-type probe of the ZaGA20ox1 promoter.

[0123] 10.3 Group 3 (His-ZaNAC25 + mutant probe): Only the free probe band is shown, with no hysteresis band, indicating that "AAACACAAAGAAAGAGAGGGA" is the core conserved sequence for His-ZaNAC25 to bind to the ZaGA20ox1 promoter, and the binding effect disappears after mutation in this region;

[0124] Group 4 (His-ZaNAC25 + wild-type probe + cold probe): The hysteresis band signal was significantly weakened, while the free probe band signal was enhanced, proving that the binding of His-ZaNAC25 to the wild-type probe is specific (the unlabeled probe competitively binds to His-ZaNAC25).

[0125] The above results confirm that ZaNAC25 can regulate the transcription of ZaGA20ox1 by recognizing and binding to the conserved sequence “AAACACAAAGAAAGAGAGGGA” in the ZaGA20ox1 promoter region.

[0126] 11. Transcriptional activation of ZaNAC25 by ZaGA20ox1 (dual-luciferase reporter system)

[0127] To clarify the direction of ZaNAC25's transcriptional regulation of ZaGA20ox1, the dual-luciferase reporter system (Dual-LUC) was used for validation:

[0128] Carrier construction:

[0129] Effector: The CDS fragment of ZaNAC25 is inserted into the pGreenII 62-SK vector, and expression is driven by the 35S promoter;

[0130] Reporter vector: The promoter fragment of ZaGA20ox1 (1000bp) is inserted into the pGreenII 0800-LUC vector, which drives the expression of the LUC (firefly luciferase) gene. The vector carries the REN (renaeus luciferase) gene as an internal control.

[0131] Transient transformation and detection: The effector vector and reporter vector were co-transfected into Nicotiana benthamiana leaves at a 1:1 ratio. The empty pGreenII 62-SK vector + reporter vector was used as a control. After 48 h of culture, the LUC / REN ratio was determined using a luciferase assay kit.

[0132] Results (see) Figure 9 The LUC / REN ratio in the ZaNAC25 effector vector group was 3.5 times that of the control group, indicating that ZaNAC25 can significantly activate the transcriptional activity of the ZaGA20ox1 promoter.

[0133] To verify the regulatory function of the "ZaNAC25-ZaGA20ox1-GA" pathway on spine development, an in vitro culture experiment was conducted using exogenous gibberellin applied to Sichuan pepper:

[0134] Experimental materials and grouping: Sterile Sichuan pepper seedlings (2-leaf-1-heart stage) with uniform growth were selected and divided into 2 groups of 10 seedlings each.

[0135] 1. Mock group: MS medium + 1 mg / L zeatin (ZT, to promote seedling vegetative growth);

[0136] 2. GT group: MS medium + 1 mg / L ZT + 50 mg / L GA3 (gibberellin);

[0137] Culture conditions: Incubated in a light incubator with an 8-hour light cycle every 16 hours, temperature 25℃±1℃, humidity 70%;

[0138] Phenotypic observation (see Figure 10 After 14 days of cultivation, the stems and leaves of the Mock group seedlings had dense, sharp, conical spines; the number of spines in the GT group seedlings was significantly reduced, with only sporadic distribution, and the remaining spines were short and blunt.

[0139] Quantitative statistics (see) Figure 11 The number of spurs in the three biological replicate groups (GT1, GT2, GT3) were 0%, 13%, and 26% of those in the Mock group, respectively, with an average reduction of 87%.

[0140] Therefore, combining the above, by overexpressing the ZaNAC25 gene, we can effectively reduce the number and length of thorns in Sichuan pepper, making them shorter, blunter, and softer to the touch, thus significantly improving the production management and harvesting efficiency of Sichuan pepper. This improved Sichuan pepper variety is particularly suitable for large-scale, intensive modern agricultural planting bases. In these bases, the processes of seedling raising, transplanting, field pruning, pest and disease control, and mechanized harvesting of Sichuan pepper will be greatly optimized. For example, in the seedling stage, thornless or low-thorn seedlings can effectively avoid mutual scratching, reduce transplanting losses, and improve the survival rate; in field management, operators can significantly reduce the risk of being scratched by thorns, improving the efficiency and safety of pruning and pest and disease control; in the harvesting stage, the thornless or low-thorn characteristics make mechanized harvesting possible, significantly reducing labor costs and improving harvesting efficiency, thereby promoting the large-scale and intensive development of the Sichuan pepper industry and solving the current difficulties faced by pepper farmers.

[0141] Therefore, we can conclude that the ZaNAC25 gene can have the following applications:

[0142] 1) Regulate the prickly properties of Sichuan pepper;

[0143] 2) Activate the transcriptional activity of ZaGA20ox1 and upregulate gibberellin synthesis.

[0144] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. Application of the ZaNAC25 gene, whose nucleotide sequence is shown in SEQ ID NO.1, in regulating the prickly trait of Sichuan pepper; The application method is as follows: at least one of the following skin spike traits is generated by overexpressing the ZaNAC25 gene: 1) The number of thorns is significantly reduced; 2) The length of the thorns is shortened; 3) Some of the tips are not woody, making them soft to the touch.

2. Application of ZaNAC25 protein with the amino acid sequence shown in SEQ ID NO.2 in regulating the prickles of Sichuan pepper; The application method is as follows: to generate at least one of the following spiky traits by promoting the synthesis of ZaNAC25 protein: 1) The number of thorns is significantly reduced; 2) The length of the thorns is shortened; 3) Some of the tips are not woody, making them soft to the touch.

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