Application of lotus NnXTH2 gene in promoting plant dwarf compact and enhancing flower stem mechanics

Abstract

This invention discloses the application of the lotus NnXTH2 gene in promoting dwarfing and compact plant growth and enhancing flower stem mechanical properties, belonging to the field of biotechnology. The NnXTH2 gene encodes the protein shown in SEQ ID NO.4. This invention found that transgenic tobacco lines overexpressing the NnXTH2 gene exhibited significantly enhanced flower stem mechanical properties, decreased plant height, and shortened internode distance, resulting in a dwarfing and compact plant growth. This indicates that overexpression of the NnXTH2 gene can significantly enhance flower stem mechanical properties, shorten plant height and internode distance, resulting in a dwarfing and compact plant growth, which better meets the plant growth and flower stem quality requirements of cut flowers. This invention clarifies for the first time the function of the NnXTH2 gene in regulating plant growth and flower stem mechanical properties, providing new genetic resources and theoretical basis for cultivating high-quality cut flower plants with less prone to flower stem breakage and higher ornamental value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the lotus NnXTH2 gene in promoting dwarfing and compact plant growth and enhancing the mechanical properties of flower stems. Background Technology

[0002] Lotus (Nelumbo nucifera) belongs to the genus Nelumbo in the family Nelumbonaceae. It is valued for its edible, medicinal, and ornamental uses, possessing significant landscape, ecological, and economic value. Cut lotus flowers have extremely high ornamental value and a broad market prospect. With the improvement of people's living standards, the market potential of cut lotus flowers is increasingly significant. In the Kunming Dounan Flower Market, Asia's largest cut flower trading market, cut lotus flowers are very popular from June to July each year. Numerous varieties, such as 'Zhenguo Huang' and 'Jinling Rainbow,' offer rich colors and are predominantly double-petaled. However, cut lotus flowers suffer from stem breakage during vase arrangement. Therefore, solving this problem has become a current research hotspot. Thus, exploring key gene resources and conducting in-depth research on the molecular mechanisms of lotus flower stem quality formation has significant scientific and application value, which will improve lotus flower stem quality and promote the development of lotus in the flower market.

[0003] Plant cell walls maintain cell shape and structural stability, playing a crucial role in enhancing stem mechanical strength. Changes in cell wall structure and composition directly affect stem mechanical strength, which is closely related to cell wall remodeling. This process is closely linked to xyloglucan endotransglucosylase / hydrolase (XTH), a plant cell wall remodeling enzyme. Xyloglucan is an important structural substance in cell walls and is the most abundant component of hemicellulose in the primary cell wall of dicotyledonous plants and non-grass monocotyledonous plants, playing a role in mechanical support. Xyloglucan endoglycosyltransferases / hydrolases have two main functions: catalyzing glycosyltransferase and hydrolysis. Xyloglucan endotransglycosylase (XET), using xyloglucan as a substrate, catalyzes the cleavage of the 1,4-β-D-glycosidic bond in xyloglucan, delivering the resulting sugar chain to the non-reducing end of other xyloglucans or oligosaccharides. Xyloglucan endohydrolase (XEH), on the other hand, specifically hydrolyzes the 1,4-β-D-glycosidic chain in xyloglucan, causing its cleavage. This enzymatic catalytic mechanism ensures efficient alteration and reconstruction of the xyloglucan molecule. Previous studies have shown that DlXTH22 can promote the elongation of hairy roots in longan, increase cell wall thickness, and increase hemicellulose content. Overexpression of poplar PtrtXET16 can increase the xyloglucan content in the primary cell wall. Arabidopsis thaliana AtXTH31 enhances aluminum tolerance by increasing xylglucan content, while the mutant atxthth31 exhibits decreased xylglucan content, leading to reduced aluminum ion uptake. Arabidopsis thaliana AtXTH21 regulates primary root growth by altering cellulose deposition and cell wall extension. These results suggest that XTH family genes can modify cell wall thickness and composition, potentially improving plant flower stem quality. However, there are currently no reports on the lotus NnXTH2 gene's involvement in plant flower stem quality. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the lotus NnXTH2 gene in promoting dwarfing and compact plant growth and enhancing flower stem mechanical properties, thereby addressing the problems existing in the prior art. This invention has found that transgenic tobacco lines overexpressing the NnXTH2 gene exhibit significantly enhanced flower stem mechanical properties, decreased plant height, and shortened internode distance, resulting in a dwarfing and compact plant growth. This indicates that overexpression of the NnXTH2 gene can significantly enhance flower stem mechanical properties, shorten plant height and internode distance, and produce a dwarfing and compact plant growth, better meeting the plant growth and flower stem quality requirements of cut flowers. This invention clarifies for the first time the function of the NnXTH2 gene in regulating plant growth and flower stem mechanical properties, providing new genetic resources and theoretical basis for cultivating high-quality cut flower plants with less prone to flower stem breakage and higher ornamental value.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides applications of the NnXTH2 gene, including any of the following applications:

[0007] A1. Enhance the mechanical properties of plant flower stems;

[0008] A2. Promotes dwarfing and compact plant growth;

[0009] A3. Breeding of plant strains with high cut flower quality;

[0010] The NnXTH2 gene encodes a protein as shown in SEQ ID NO.4.

[0011] The nucleotide sequence of the NnXTH2 gene is shown in SEQ ID NO.3.

[0012] The present invention also provides applications of expression cassettes containing the NnXTH2 gene, including any of the following applications:

[0013] A1. Enhance the mechanical properties of plant flower stems;

[0014] A2. Promotes dwarfing and compact plant growth;

[0015] A3. Breeding of plant strains with high cut flower quality;

[0016] The NnXTH2 gene encodes a protein as shown in SEQ ID NO.4.

[0017] The present invention also provides applications of recombinant vectors containing the NnXTH2 gene, including any of the following applications:

[0018] A1. Enhance the mechanical properties of plant flower stems;

[0019] A2. Promotes dwarfing and compact plant growth;

[0020] A3. Breeding of plant strains with high cut flower quality;

[0021] The NnXTH2 gene encodes a protein as shown in SEQ ID NO.4.

[0022] This invention also provides applications of recombinant microorganisms containing the NnXTH2 gene, including any of the following applications:

[0023] A1. Enhance the mechanical properties of plant flower stems;

[0024] A2. Promotes dwarfing and compact plant growth;

[0025] A3. Breeding of plant strains with high cut flower quality;

[0026] The NnXTH2 gene encodes a protein as shown in SEQ ID NO.4.

[0027] Furthermore, promoting a dwarf and compact plant structure refers to reducing plant height and shortening internode spacing.

[0028] Optionally, the plant may be lotus or tobacco.

[0029] The present invention also provides a method for enhancing the mechanical properties of plant flower stems, including the steps of introducing the NnXTH2 gene into the plant using genetic transformation technology, thereby achieving stable overexpression of the NnXTH2 gene and enhancing the mechanical properties of the plant flower stems.

[0030] The NnXTH2 gene encodes a protein as shown in SEQ ID NO.4.

[0031] The present invention also provides a method for promoting dwarfing and compact plant type, including the steps of introducing the NnXTH2 gene into the plant using genetic transformation technology, so as to stably overexpress the NnXTH2 gene, reduce the plant height, and shorten the internode distance.

[0032] The NnXTH2 gene encodes a protein as shown in SEQ ID NO.4.

[0033] This invention also provides a method for cultivating plant lines with high cut flower quality. Using genetic transformation technology, the NnXTH2 gene is introduced into a plant to obtain a transgenic plant line with stable overexpression of the NnXTH2 gene; the flower stems of the transgenic plant line are not easily broken.

[0034] The NnXTH2 gene encodes a protein as shown in SEQ ID NO.4.

[0035] Optionally, the plant may be lotus or tobacco.

[0036] The present invention discloses the following technical effects:

[0037] This invention cloned the NnXTH2 gene from lotus, constructed an overexpression vector, transformed tobacco plants, and studied the function of the NnXTH2 gene. The study found that transgenic tobacco lines overexpressing the NnXTH2 gene exhibited significantly enhanced stem mechanical properties, specifically increased stem mechanical strength, decreased plant height, and shortened internode distance, resulting in a dwarfed and compact plant type. This indicates that overexpression of the NnXTH2 gene can significantly enhance stem mechanical properties, shorten plant height and internode distance, and produce a dwarfed and compact plant type, better meeting the plant type and stem quality requirements of cut flowers. This invention clarifies for the first time the function of the NnXTH2 gene in regulating plant type and stem mechanical properties, providing new genetic resources and theoretical basis for cultivating high-quality cut flower plants with less broken stems and higher ornamental value. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The amino acid sequence alignment results of the lotus NnXTH2 gene and the Arabidopsis AtXTH2 gene;

[0040] Figure 2 The construction flowchart of the pGWB418(4xMyc)-NnXTH2-NOS vector;

[0041] Figure 3 A schematic diagram of the pGWB418(4xMyc)-NnXTH2-NOS vector structure;

[0042] Figure 4 A flowchart illustrating Agrobacterium-mediated genetic transformation of tobacco leaf discs;

[0043] Figure 5 The results show the identification and expression level analysis of positive seedlings of tobacco NnXTH2 overexpressing transgenic lines; where A is the electrophoresis image of PCR amplification products of positive seedlings of NnXTH2 overexpressing transgenic lines; and B is the detection result of NnXTH2 expression level in positive seedlings of NnXTH2 overexpressing transgenic lines.

[0044] Figure 6The results show the plant architecture and stem mechanical properties of the T2 generation transgenic NnXTH2 overexpression lines. Among them, A is the plant architecture observation diagram of wild-type (WT) and NnXTH2 (OE) lines; B is the stem mechanical force test result of wild-type WT and NnCIGR1 (OE) lines; C is the plant height test result of wild-type WT and NnCIGR1 (OE) lines. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0051] Example 1: Isolation and Cloning of the NnXTH2 Gene

[0052] (1) Isolation and cloning of the NnXTH2 gene

[0053] The lotus variety 'Shennüzi' (provided by the National Lotus Germplasm Resource Bank of Southwest Forestry University) was selected as the experimental material. Total RNA was extracted from various tissues of lotus flower organs using the Eastep® Super Total RNA Extraction Kit (purchased from Promega, USA). After RNA extraction, the RNA was treated with DNase I (purchased from Promega). RNA integrity was detected by 1% (w / v) agarose gel (EtBr) electrophoresis (5V / cm). Nucleic acid concentration was determined using an IMPLEN Nano Photometer-N50 series ultraviolet spectrophotometer (Germany). RNA with a 260 / 280 ratio between 1.9 and 2.1, a 260 / 230 ratio greater than 2.0, and a concentration greater than 500 ng / μL was used for further analysis. cDNA synthesis was performed using the Hiscript® II QRT SuperMix for qRNA (+gDNA wiper) kit (purchased from Vazyme, China). Using 1 μg total RNA as a template, it was mixed with 4 μL of 4×gDNA wiper Mix in DEPC-water to a total volume of 16 μL. The mixture was incubated at 42 ℃ for 2 min, then quenched on ice for 2-3 min. Next, 4 μL of 5×Hiscript II qRT Super Mix II was added and mixed to a total volume of 20 μL. The mixture was then incubated at 50 ℃ for 15 min, followed by 85 ℃ for 5 sec. Each cDNA sample was diluted to 200 μL and stored at -20 ℃ for later use.

[0054] The target band was amplified using in-fusion cloning primers NnXTH2-418F and NnXTH2-418R, amplified using TransTaq HiFi DNA Polymerase (Beijing TransGen Biotech Co., Ltd.). PCR reaction conditions were: 94 ℃ pre-denaturation for 3 min; 32 cycles of 94 ℃ for 30 sec, 58 ℃ for 30 sec, and 72 ℃ for 1 min 30 sec; extension at 72 ℃ for 5 min. The PCR product was then cloned into the pGWB418 vector. Recovery of the target fragment was performed using the UNIQ-10 column DNA gel extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). The ligation system for the target fragment and the pGWB418 linearized vector was: 4.5 μL target fragment, 0.5 μL pGWB418 vector, and 5 μL Solution I (Takara Bio Engineering (Dalian) Co., Ltd.), ligated overnight at 16 ℃. The ligation product was transformed into DH5α competent cells using a heat shock method (30 min on ice followed by 90 sec heat shock at 42 °C). The bacterial culture was then evenly spread on LB agar plates containing 100 mg / L Kan and incubated for approximately 10-12 h. Regularly shaped single colonies were selected and identified by colony PCR using universal primers 418F / R. Colonies confirmed as positive by agarose gel electrophoresis were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The integrity of the target fragment was finally confirmed by sequence alignment. Positive colonies were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0055] The primer sequences are as follows:

[0056] NnXTH2-418F:5' GACTTGAACGGTAGCGCT AACCCAACTCCACTCAACA 3', SEQ ID NO.1 (underlined is the 418 adapter, non-underlined is the F primer of the gene).

[0057] NnXTH2-418R:5' TCGGGGAAATTCGAGCTC CCAATAAGGAATCACCACC 3', SEQ ID NO.2 (underlined is the 418 adapter, non-underlined is the R primer of the gene, where the R primer is reverse complementary: GGTGGTGATTCCTTATTGG).

[0058] (2) NnXTH2 gene sequence analysis

[0059] Sequencing revealed that the amplified nucleotide sequence of this gene was 1020 bp in length. Further analysis identified the gene as NnXTH2, with a full-length nucleotide sequence of 1020 bp (SEQ ID NO.3), containing an 876 bp open reading frame (positions 68-943 of SEQ ID NO.3). Amino acid sequence alignment using ClustalX (Thompson JD, Gibson TJ, Plewniak F, et al. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic AcidsResearch, 1997, 25: 4876-82.) showed that this target band was highly homologous to Arabidopsis thaliana's AtXTH2 at the amino acid level (40.54% similarity), consistent with the results obtained from NCBI alignment. Figure 1 The amino acid sequence encoded by the NnXTH2 gene is shown in SEQ ID NO.4.

[0060] SEQ ID NO.3:

[0061]

[0062] SEQ ID NO.4:

[0063] MAFSNGVSVLLIALVMSSLMAACAGSFYQDFDLTWGDHRAKIFNGGQLLSLSLDRTSGSGFQSKKEYLFGRIDMQLKLVAGNSAGTVTAYYLSSQGPTHDEIDFEFLGNLSGDPYILHTNVFTQGKGNREQQFYLWFDPTRNFHTY SIVWNPQRIIFLVDNVPIREFSNAESIGVPFPKNQPMRIYSSLWNADDWATRGGLVKTDWSKAPFTAYYRNFNANACVWSSGSSSCSSRSTNSFKDSDWQTQELDGWSRRRLRWVQKYYMIYNYCTDLKRFPQGLPPECRRSRFL.

[0064] Example 2: Obtaining transgenic tobacco plants overexpressing NnXTH2

[0065] (1) Construction of plant overexpression vectors

[0066] AfeI and SacI restriction sites were added to both ends of the cloning primers, resulting in primers NnXTH2-418F / NnXTH2-418R. PCR products were obtained according to the method in Example 1. The pGWB418 vector was double-digested with AfeI and SacI. The digestion system was as follows: 15 μL pGWB418 (1700 ng / μL), 5 μL rCutsmartt Buffer, 1 μL AfeI (10000 U / L), 1 μL SacI (20000 U / L), and sterile water to a final volume of 50 μL. Digestion was performed overnight at 37 °C. After 1% agarose gel electrophoresis, the single linearized vector fragment was recovered.

[0067] according to Figure 2 The procedure shown involves using an in-fusion enzyme to ligate the PCR product obtained from the above amplification into the pGWB418(4xMyc) vector. Positive clones are screened and identified by sequencing, thereby obtaining the lotus NnXTH2 transgenic overexpression vector pGWB418(4xMyc)-NnXTH2-NOS. Its T-DNA region contains a kanamycin-resistant gene sequence, and the NnXTH2 overexpression promoter is the 35S promoter (see...). Figure 3 ).

[0068] (2) Genetic transformation of tobacco (K326)

[0069] The recombinant plasmid pGWB418(4xMyc)-NnXTH2-NOS (i.e., overexpression vector) was transformed into Agrobacterium GV3101 using the conventional leaf disc infection method. Seedlings were then screened and differentiated. The specific steps are as follows: Figure 4 ):

[0070] a. Infection: Wash the tender terminal leaves of wild tobacco shoots with 75% alcohol for 1 min, then wash the leaves with 0.1% mercuric chloride solution for 5 min, and finally wash three times with sterile water. Use sterile tweezers and scissors to remove the leaf margins and midribs, cutting the leaves to a size of 1 cm × 1 cm. Place the leaves in the pre-shaken bacterial solution for 10 min. After infection, carefully remove the leaves and lay them flat on sterilized filter paper, turning them over to allow most of the infection solution to be filtered out. After the excess bacterial solution is absorbed, place the leaves flat at the four corners on a petri dish, with the leaf surface facing up, and incubate in the dark for 3 days.

[0071] b. Differentiation: After 3 days of dark culture, the explants were transferred to differentiation medium (MS formulation of 1962, 4.404 g / L inorganic salts and trace elements, 30 g / L sucrose, 6.8 g / L Agar, 2.25 mg / L 6-BA, 0.3 mg / L NAA, pH adjusted to 5.8-5.9, sterilized and cooled, then Kana 50 mg / L and Cef 400 mg / L antibiotics were added). The medium was cultured at 24 ℃ for 16 h light / 8 h dark.

[0072] c. Bud strengthening: After adventitious buds emerge, remove as much callus tissue as possible and transfer the bud strengthening medium (MS inorganic salts and trace elements 4.404 g / L, sucrose 30 g / L, Agar 6.8 g / L, 6-BA 2.25 mg / L, NAA 0.3 mg / L, adjust the pH of the medium to 5.8-5.9, sterilize and cool, then add Kana 50 mg / L and Cef 400 mg / L antibiotics) for 30 days of culture under the following conditions: 24 ℃, 16 h light / 8 h dark culture.

[0073] d. Rooting culture: Once the roots are robust and healthy, transfer them to a solid rooting medium (MS formula from 1962, containing 4.404 g / L inorganic salts and trace elements, 30 g / L sucrose, 6.8 g / L Agar, sterilized and cooled, then adding 50 mg / L Kana and 400 mg / L Cef antibiotics) to promote rooting.

[0074] (3) Identification of transgenic plants

[0075] a. Extraction of genomic DNA from tobacco leaves

[0076] DNA extraction was performed using the standard CTAB method. The specific steps were as follows: Young wild-type tobacco leaves (1-2 cm in length) were placed in a pre-cooled mortar and ground with liquid nitrogen 2-3 times until a fine paste was formed. The paste was then transferred to a centrifuge tube, and 700 μL of 2x CTAB solution was added. The mixture was incubated at 70°C for 30 min, with gentle shaking every 6 min, followed by another 30 min incubation at 70°C, with gentle shaking every 10 min. After cooling to room temperature, 700 μL of a 25:24:1 Tris-saturated phenol:chloroform:isoamyl alcohol solution was added. The mixture was repeatedly inverted and gently shaken approximately 40 times. The mixture was centrifuged at 3100 rpm for 15 min at room temperature. Approximately 500 μL of the supernatant was collected, and an equal volume of a 24:1 chloroform:isoamyl alcohol solution was added. The mixture was then centrifuged at 3100 rpm for 15 min at room temperature. Discard the supernatant and add 1 mL of frozen -20°C anhydrous ethanol. After incubating at -20°C for 30 min, centrifuge at 12000 rpm for 10 min at room temperature. Wash with 75% ethanol and repeatedly pipette the precipitate for 3 min to remove salt. Discard the ethanol, air dry, and dissolve each sample in 30-50 μL of ddH2O. Analyze the concentration of extracted tobacco genomic DNA using a Nanodrop micro-nucleic acid analyzer.

[0077] b. Detection of positive transgenic plants

[0078] Young leaves of the transformed plants were collected, and DNA was extracted using the CTAB method. Positive seedlings were identified by PCR amplification of the pGWB418-NnXTH2 gene in the vector. The control group consisted of non-transgenic wild-type plants (K326). The primer sequences used are as follows:

[0079] pGWB418-F: 5' GACTTGAACGGTAGCGCT 3'; SEQ ID NO.5;

[0080] NnXTH2-418R: 5'TCGGGGAAATTCGAGCTCCCAATAAGGAATCACCACC 3'; SEQ ID NO. 2.

[0081] The test results show that ( Figure 5 In the five transformed plants (A), the expected-sized electrophoretic bands were amplified, while the wild-type and ddH2O control did not show any electrophoretic bands, indicating that the transgenic tobacco genome already contains exogenous gene DNA fragments.

[0082] (4) qRT-PCR identification of transgenic tobacco overexpressing NnXTH2

[0083] a. Extraction of genomic RNA from tobacco leaves

[0084] Total RNA was extracted from leaves of wild-type plants (WT) and NnXTH2-overexpressing positive lines identified in step (3) using the Eastep® Super Total RNA Extraction Kit (Promega, USA). The RNA was then reverse transcribed into cDNA using the Hiscript® IIQ RT SuperMix for qRNA (+gDNA wiper) Kit (Vazyme, China).

[0085] b. Real-time quantitative PCR

[0086] To determine whether NnXTH2 was overexpressed in tobacco, real-time quantitative PCR (qRT-PCR) was used to analyze transgenic plants. The qRT-PCR kit was SYBR® Green Realtime PCR Master Mix-Plus (Takara Bio Engineering (Dalian) Co., Ltd.), and primers were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0087] The qRT-PCR primer sequences are as follows:

[0088] Q-NnXTH2-F: 5' GTTCTCTAATGGCCGCCTGT 3'; SEQ ID NO.6;

[0089] Q-NnXTH2-R: 5' GGTCCTGTCGAGTGAAAGGG 3'; SEQ ID NO. 7.

[0090] The PCR program was as follows: pre-denaturation at 95 ℃ for 30 sec, followed by 40 cycles (95 ℃ for 10 sec, 60 ℃ for 10 sec, and 72 ℃ for 26 sec). The NnXTH2 overexpression positive lines identified in step (3) were numbered with OE (abbreviation for overexpression).

[0091] The test results show that ( Figure 5 The expression levels of NnXTH2 in the OE-7, OE-9, OE-12, OE-17 and OE-23 lines were significantly higher than those in the wild-type plants. The most significant increase was observed in the OE-9 line, which showed a 562.46% increase in expression compared to the wild-type, indicating that these lines are independent transgenic NnXTH2 overexpression lines.

[0092] Example 3: Plant type and flower stem mechanical analysis of NnXTH2 overexpression transgenic tobacco T2 generation

[0093] (1) Determination of the mechanical properties of the flower stems of the T2 generation tobacco line overexpressing NnXTH2

[0094] The mechanical force of the apical first node of the flower stalk of wild-type tobacco (WT) and its three transgenic lines (OE-7, OE-9, and OE-17) in their T2 generation was measured using a stem strength tester (YYD-1, Beijing Shunkeda Technology Co., Ltd.). The results showed that the mechanical strength of the flower stalks of the transgenic lines was significantly increased compared to WT: 11.54% for OE-7, 47.69% for OE-9, and 6.15% for OE-17. This indicates that the NnXTH2 gene has a good effect on regulating the mechanical quality of flower stalks. Figure 6 B).

[0095] (12) Observation of the plant type of the T2 generation of NnXTH2 overexpression lines

[0096] The plant architecture of wild-type tobacco (WT) and its three transgenic lines (OE-7, OE-9, and OE-17) in their T2 generation during full-blooming period was observed. Plant height was precisely measured using a telescopic measuring ruler. Specifically, the measurement starting point was set at the bottom of the cultivation pot, and the ending point was the highest point of the main inflorescence; the vertical distance between these two points was the plant height. The results showed that, compared to WT, the transgenic lines overexpressing the NnXTH2 gene exhibited significant changes in plant architecture: the plant height of the transgenic lines was significantly reduced compared to the wild type, with a reduction ranging from 26% to 36%. Figure 6 (C). This reduction in plant height leads to a dwarfed plant shape and a significantly shortened internode distance, resulting in a more compact overall plant structure. Figure 6 (A). This phenomenon fully demonstrates that overexpression of the NnXTH2 gene can effectively promote dwarfing of plant structure and shortening of internode distance.

[0097] In summary, overexpression of the NnXTH2 gene can significantly enhance the mechanical properties of the flower stem, shorten the plant height and internode distance, and dwarf the plant, which is more in line with the plant shape and flower stem quality requirements of cut flower varieties.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. NnXTH2 The application of genes is characterized by, By stabilizing overexpression NnXTH2 genes to achieve the use according to any one of A1. Enhance the mechanical properties of plant flower stems; A2. Promotes dwarfing and compact plant growth; A3. Breeding of plant strains with high cut flower quality; The NnXTH2 The gene encodes a protein as shown in SEQ ID NO.4; The plant in question is either lotus or tobacco.

2. Includes NnXTH2 The application of gene expression cassettes is characterized by, Through stable overexpression NnXTH2 Genes can be used to achieve any of the following applications: A1. Enhance the mechanical properties of plant flower stems; A2. Promotes dwarfing and compact plant growth; A3. Breeding of plant strains with high cut flower quality; The NnXTH2 The gene encodes a protein as shown in SEQ ID NO.4; The plant in question is either lotus or tobacco.

3. Includes NnXTH2 The application of gene recombinant vectors is characterized by, Through stable overexpression NnXTH2 Genes can be used to achieve any of the following applications: A1. Enhance the mechanical properties of plant flower stems; A2. Promotes dwarfing and compact plant growth; A3. Breeding of plant strains with high cut flower quality; The NnXTH2 The gene encodes a protein as shown in SEQ ID NO.4; The plant in question is either lotus or tobacco.

4. Includes NnXTH2 The application of recombinant microorganisms is characterized by, Through stable overexpression NnXTH2 Genes can be used to achieve any of the following applications: A1. Enhance the mechanical properties of plant flower stems; A2. Promotes dwarfing and compact plant growth; A3. Breeding of plant strains with high cut flower quality; The NnXTH2 The gene encodes a protein as shown in SEQ ID NO.4; The plant in question is either lotus or tobacco.

5. The application according to any one of claims 1-4, characterized in that, Promoting a dwarf and compact plant structure refers to reducing plant height and shortening internode spacing.

6. A method for enhancing the mechanical properties of plant flower stems, characterized in that, This includes using genetic transformation technology to... NnXTH2 Genes are introduced into plants to enable NnXTH2 Stable overexpression of genes enhances the mechanical properties of plant flower stems; The NnXTH2 The gene encodes a protein as shown in SEQ ID NO.4; The plant in question is either lotus or tobacco.

7. A method for promoting dwarfing and compact plant growth, characterized in that, This includes using genetic transformation technology to... NnXTH2 Genes are introduced into plants to enable NnXTH2 Stable overexpression of genes reduces plant height and shortens internode distance. The NnXTH2 The gene encodes a protein as shown in SEQ ID NO.4; The plant in question is either lotus or tobacco.

8. A method for cultivating plant lines with high-quality cut flowers, characterized in that, Using genetic transformation technology, NnXTH2 Genes are introduced into plants to obtain NnXTH2 A transgenic plant line with stable gene overexpression; the flower stems of the transgenic plant line are not easily broken; The NnXTH2 The gene encodes a protein as shown in SEQ ID NO.4; The plant in question is either lotus or tobacco.

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