Application of lotus NnXTH2 gene in promotion of dwarfing and compactness of plant type and enhancement of rachis mechanics

Abstract

The invention discloses application of a lotus NnXTH2 gene to promotion of dwarfing and compactness of plant types and enhancement of rachis mechanics, and belongs to the technical field of biology. And the NnXTH2 gene is used for coding a protein as shown in SEQ ID NO. 4. The research finds that the rachis mechanics of the transgenic tobacco strain overexpressing the NnXTH2 gene is obviously enhanced, the plant height of the plant is reduced, the internode spacing is shortened, and the plant type of the plant is dwarfed and compact. The result shows that overexpression of the NnXTH2 gene can significantly enhance the rachis mechanics of the plant, shorten the plant height and internode spacing of the plant, make the plant type dwarf and compact, and better meet the plant type and rachis quality requirements of fresh cut flowers. According to the invention, the function of the NnXTH2 gene in regulation and control of plant type and rachis mechanics is defined for the first time, and new genetic resources and theoretical basis are provided for cultivating high-quality cut flower plants with rachis not easy to break and higher ornamental value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of Nelumbo nucifera NnXTH2 gene in promoting plant dwarfing and compacting and enhancing the mechanical properties of flower stem. BACKGROUND

[0002] Nelumbo nucifera belongs to Nelumbonaceae and Nelumbo, and has important landscape, ecological and economic values as it has the functions of food, medicine and ornamental. Lotus cut flower has high ornamental value and broad market prospect. With the improvement of people's living standards, the market potential of lotus cut flower is increasingly significant. In the largest fresh cut flower trading market in Asia, Kunming Dounan Flower Market, lotus cut flower is deeply loved by people from June to July every year, and there are many varieties such as 'Zhen Guo Huang' and 'Jinling Rainbow'. The flower color is rich, and the flower type is mainly heavy-petaled varieties. However, there is a problem of easy breaking of flower stem during the vase insertion process of lotus cut flower. Therefore, how to solve the problem of flower stem breaking during the vase insertion process of lotus cut flower has become a research hotspot. Therefore, it is of important scientific significance and application value to excavate key gene resources and deeply study the molecular mechanism of lotus flower stem quality formation, which will improve the quality of lotus flower stem and promote the development of lotus in the flower market.

[0003] Plant cell wall maintains cell shape and structural stability, plays a crucial role in improving stem mechanical force, when the structure and composition of cell wall changes, it will directly affect the mechanical force of flower stem, the stem mechanical force is closely related to cell wall remodeling, which is closely related to plant cell wall remodeling enzyme xyloglucan endotransglucosylase / hydrolase (XTH). Xyloglucan is an important structural material of cell wall, and is the most abundant component of hemicellulose in the primary wall of dicotyledonous plants and non-grass monocotyledonous plants, which plays a mechanical support role. Xyloglucan endotransglycosylase / hydrolase has two functions of catalyzing glycosyl transfer and hydrolysis. Among them, xyloglucan endotransglycosylase (XET) takes xyloglucan as the substrate, XET catalyzes the 1,4-β-D-glycosidic bond in xyloglucan to generate a sugar chain which is delivered to the non-reducing end of other xyloglucan or oligosaccharide. Xyloglucan endohydrolase (XEH) specifically hydrolyzes 1,4-β-D-glycosidic bond in xyloglucan to make it crack. The catalytic reaction mechanism of this enzyme ensures efficient change and reconstruction of xyloglucan molecules. Previous studies have shown that DlXTH22 can promote the elongation of longan hairy roots, increase the thickness of cell wall and the content of hemicellulose. Overexpression of PtrtXET16 in poplar can increase the content of xyloglucan in the primary wall. Arabidopsis AtXTH31 enhances aluminum tolerance by increasing the content of xyloglucan, while the content of xyloglucan in mutant atxth31 is reduced, which reduces the absorption capacity of Arabidopsis to aluminum ions. Arabidopsis AtXTH21 regulates the growth of primary roots by changing the deposition of cellulose and the extension of cell wall. These results indicate that XTH family genes can change the thickness and content of cell wall components to improve the potential of plant flower stem, but there is no report on the involvement of NnXTH2 gene in plant flower stem quality. SUMMARY

[0004] The application aims to provide an application of lotus NnXTH2 gene in promoting plant type dwarfing and compactness and enhancing flower stem mechanics, so as to solve the problems in the prior art. The application research finds that the flower stem mechanics of a transgenic tobacco strain overexpressing NnXTH2 gene is significantly enhanced, the plant height of the plant is reduced, the internode distance is shortened, the plant type is dwarfed and compacted. This shows that overexpression of NnXTH2 gene can significantly enhance the flower stem mechanics of the plant, shorten the plant height and internode distance, and make the plant type dwarfed and compacted, which is more in line with the plant type and flower stem quality requirements of fresh-cut flowers. The application first determines the function of NnXTH2 gene in regulating plant type and flower stem mechanics, and provides a new genetic resource and theoretical basis for breeding high-quality cut flower plants with flower stems not easy to break and higher ornamental performance.

[0005] To achieve the above object, the application provides the following scheme:

[0006] The application provides an application of NnXTH2 gene, including the application of any one of the following:

[0007] A1, enhancing the flower stem mechanics of the plant;

[0008] A2, promoting plant type dwarfing and compactness;

[0009] A3, breeding of a plant strain 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 application also provides an application of an expression cassette containing NnXTH2 gene, including the application of any one of the following:

[0013] A1, enhancing the flower stem mechanics of the plant;

[0014] A2, promoting plant type dwarfing and compactness;

[0015] A3, breeding of a plant strain with high cut flower quality;

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

[0017] The application also provides an application of a recombinant vector containing NnXTH2 gene, including the application of any one of the following:

[0018] A1, enhancing the flower stem mechanics of the plant;

[0019] A2, promoting plant type dwarfing and compactness;

[0020] A3, breeding of plant line with high cut flower quality;

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

[0022] The application further provides use of the recombinant microorganism containing the NnXTH2 gene, including use according to any one of the following:

[0023] A1, enhancing flower stem mechanics of plants;

[0024] A2, promoting dwarf and compact plant type;

[0025] A3, breeding of plant line with high cut flower quality;

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

[0027] Further, the promoting dwarf and compact plant type refers to reducing plant height and shortening plant internode distance.

[0028] Optionally, the plant is lotus or tobacco.

[0029] The application further provides a method for enhancing flower stem mechanics of plants, including the steps of introducing the NnXTH2 gene into plants by genetic transformation technology, stably overexpressing the NnXTH2 gene, and enhancing flower stem mechanics of plants.

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

[0031] The application further provides a method for promoting dwarf and compact plant type, including the steps of introducing the NnXTH2 gene into plants by genetic transformation technology, stably overexpressing the NnXTH2 gene, reducing plant height, and shortening plant internode distance.

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

[0033] The application further provides a method for breeding plant line with high cut flower quality, introducing the NnXTH2 gene into plants by genetic transformation technology, and obtaining transgenic plant line with stably overexpressed NnXTH2 gene; the flower stem of the transgenic plant line is not easy to break.

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

[0035] Optionally, the plant is lotus or tobacco.

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

[0037] The present application clones NnXTH2 gene from Nelumbo nucifera, transforms tobacco plants by constructing overexpression vector, and studies the function of NnXTH2 gene. It is found that the flower stem mechanics of transgenic tobacco lines overexpressing NnXTH2 gene is significantly enhanced, specifically, the stem mechanical force is improved, the plant height is reduced, and the internode distance is shortened, so that the plant type is dwarfed and compact. This shows that overexpression of NnXTH2 gene can significantly enhance the flower stem mechanics of plants, shorten the plant height and internode distance, and make the plant type dwarfed and compact, which is more in line with the requirements of plant type and flower stem quality of fresh-cut flowers. The present application first determines the function of NnXTH2 gene in regulating plant type and flower stem mechanics, and provides new genetic resources and theoretical basis for cultivating high-quality cut flower plants with higher ornamental performance and less broken flower stems. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0039] Figure 1 Nelumbo nucifera NnXTH2 gene and Arabidopsis AtXTH2 gene amino acid sequence alignment results;

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

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

[0042] Figure 4 Agrobacterium-mediated tobacco leaf disc genetic transformation flowchart;

[0043] Figure 5 Tobacco NnXTH2 overexpression transgenic line positive seedling identification results and expression amount analysis results; wherein, A is the NnXTH2 overexpression transgenic positive seedling PCR amplification product electrophoresis map; B is the detection result of NnXTH2 expression level in NnXTH2 overexpression transgenic positive seedling;

[0044] Figure 6The results of plant type and stem mechanical detection of NnXTH2 overexpression transgenic T2 generation strain; wherein, A is the plant type observation figure of wild type (WT) and NnXTH2 (OE) strain; B is the stem mechanical detection result of wild type WT and NnCIGR1 (OE) strain; C is the plant height detection result of wild type WT and NnCIGR1 (OE) strain. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not restrictive of the application. It will be appreciated that the detailed description is not intended to limit the scope of the application, but rather to provide a more thorough description of certain aspects, features and embodiments of the application.

[0046] It should be understood that the terms used herein are merely descriptive, but are not intended to limit the application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each smaller range within any stated range or within any stated intermediate value is also encompassed within the scope of the application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict, the content of the present specification will control.

[0048] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0049] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0050] The experimental methods in the following examples are all routine methods unless otherwise specified. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are all purchased from routine biochemical reagent stores unless otherwise specified.

[0051] Example 1 Isolation and cloning of NnXTH2 gene

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

[0053] The lotus cultivar 'Shennuwei' (provided by the National Lotus Germplasm Repository of Southwest Forestry University) was selected as the experimental material. The Eastep® Super total RNA extraction kit (purchased from Promega Company, USA) was used to extract the total RNA of lotus flower organs. After RNA extraction, DNase I (purchased from Promega Company kit) was used for treatment. The integrity of RNA was detected by 1% (w / v) agarose gel (EtBr) electrophoresis (5 V / cm). The determination of nucleic acid concentration was carried out on IMPLEN Nano Photometer-N50 series ultramicro UV spectrophotometer (Germany). The RNA 260 / 280 ratio was between 1.9 and 2.1, the 260 / 230 ratio was greater than 2.0, and the concentration of RNA was greater than 500 ng / μL, which was used for the next step of analysis. The synthesis of cDNA was carried out using Hiscript® II QRT SuperMix for qRNA (+gDNA wiper) kit (purchased from Vazyme Company, China). 1 μg of total RNA was used as a template, mixed with 4 μL of 4×gDNA wiper Mix, DEPC-water, total volume 16 μL, 42 ℃ 2 min, placed on ice for 2-3 min; then 5×Hiscript II qRT Super Mix II 4 μL was mixed, the total volume was 20 μL; then 50 ℃ 15 min, 85 ℃ 5 sec, each cDNA was diluted to 200 μL and stored at -20 ℃ for use.

[0054] The target band was amplified using In-fusion cloning primers NnXTH2-418F and NnXTH2-418R, and amplified by TransTaq HiFi DNA Polymerase (Beijing Zixingjin Biological Technology Co., Ltd.). The PCR reaction conditions were as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ for 30 sec, 58 ℃ for 30 sec, 72 ℃ for 1 min 30 sec, 32 cycles; and 72 ℃ extension for 5 min. Then the PCR product was cloned into the pGWB418 vector. The recovery of the target fragment was performed according to the UNIQ-10 column DNA gel recovery kit (Shanghai Genechem Co., Ltd.). The ligation system of the target fragment and the linearized pGWAB418 vector was as follows: 4.5 μL of the target fragment, 0.5 μL of the pGWB418 vector, and 5 μL of Solution I (Bao Biological Engineering (Dalian) Co., Ltd.) were connected at 16 ℃ overnight. The ligation product was transformed into DH5α competent cells by heat shock method (after ice bath for 30 min, heat shock at 42 ℃ for 90 sec), and then the bacterial liquid was uniformly coated on an LB solid plate containing 100 mg / L Kan, and after about 10-12 h of culture, regular single colonies were selected, and colony PCR identification was performed using universal primers 418F / R. The colonies verified as positive by agarose gel electrophoresis were sent to Shanghai Genechem Co., Ltd. for sequencing, and the integrity of the target fragment was finally confirmed by sequence alignment. The positive colonies were sent to Shanghai Genechem 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, and 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, and non-underlined is the R primer of the gene, wherein the R primer is reverse complementary: GGTGGTGATTCCTTATTGG).

[0058] (2) Sequence analysis of NnXTH2 gene

[0059] The sequenced gene was found to have a nucleotide sequence of 1020 bp. Further analysis found that the gene was NnXTH2 gene, which had a nucleotide sequence of 1020 bp (SEQ ID NO. 3), containing an open reading frame of 876 bp in length (positions 68-943 of SEQ ID NO. 3). Using the 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 Acids Research, 1997, 25: 4876-82.) program to align the amino acid sequences, it was found that the target band was highly homologous to AtXTH2 of Arabidopsis thaliana at the amino acid level (similarity of 40.54 %), which was consistent with the alignment results on NCBI. 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] MAFSNGVSVLLIALVMSSLMAACAGSFYQDFDLTWGDHRAKIFNGGQLLSLSLDRTSGSGFQSKKEYLFGRIDMQLKLVAGNSAGTVTAYYLSSQGPTHDEIDFEFLGNLSGDPYILHTNVFTQGKGNREQQFYLWFDPTRNFHTYSIVWNPQRIIFLVDNVPIREFSNAESIGVPFPKNQPMRIYSSLWNADDWATRGGLVKTDWSKAPFTAYYRNFNANACVWSSGSSSCSSRSTNSFKDSDWQTQELDGWSRRRLRWVQKYYMIYNYCTDLKRFPQGLPPECRRSRFL.

[0064] Example 2 Obtaining of tobacco plants overexpressing NnXTH2 transgene

[0065] (1) Construction of plant overexpression vector

[0066] The cloning primers were added with Afe I and Sac I restriction sites at both ends, i.e. primers NnXTH2-418F / NnXTH2-418R, and the PCR product was obtained according to the method of Example 1. The pGWB418 vector was double-digested with Afe I and Sac I, and the enzyme digestion system was as follows: pGWB418 (1700 ng / μL) 15 μL, rCutsmartt Buffer 5 μL, Afe I (10000 U / L) 1 μL, Sac I (20000 U / L) 1 μL, and sterile water was added to make up the final volume to 50 μL, and the enzyme digestion was carried out at 37 ℃ overnight. After 1 % agarose gel electrophoresis, the single linearized vector fragment was recovered.

[0067] According to the flowchart shown in Figure 2 , the above-mentioned PCR product obtained by amplification was connected into the pGWB418 (4xMyc) vector by In-fusion enzyme, positive clones were screened and identified by sequencing, thereby obtaining the lotus NnXTH2 transgenic overexpression vector pGWB418 (4xMyc)-NnXTH2-NOS, which contains the kanamycin-resistant gene sequence in the T-DNA region, and the NnXTH2 overexpression promoter is 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 introduced into Agrobacterium GV3101 by conventional leaf disc infection method, and the specific steps are as follows (the specific steps are as follows) Figure 4

[0070] a. Infection: wash the young top leaves of wild tobacco with 75% alcohol for 1 min, then wash the leaves with 0.1% mercuric chloride solution for 5 min, and finally wash the leaves with sterile water for 3 times; use sterilized forceps and scissors to cut off the leaf edges and main veins, and cut the leaves to 1 cm x 1 cm in size, and then put them into the pre-shaken bacterial solution for 10 min. After the infection, carefully clamp the leaves and lay them on sterilized filter paper, and turn them up and down to filter out most of the infection solution. After the excess bacterial solution is absorbed, the leaves are inserted into their culture dishes with the four corners, with the front of the leaves facing up, and dark culture for 3 days.

[0071] b. Differentiation: transfer the explants after 3 days of dark culture to the differentiation medium (1962 formula MS inorganic salt and trace element 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, after sterilization and cooling, add Kana 50 mg / L, Cef 400 mg / L antibiotics), 24 ℃, 16 h light / 8 h dark culture.

[0072] c. Strong bud: after the adventitious buds grow out, try to remove the callus and transfer it to the strong bud medium (1962 formula MS inorganic salt and trace element 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, after sterilization and cooling, add Kana 50 mg / L, Cef 400 mg / L antibiotics), and culture for 30 days under the conditions of 24 ℃, 16 h light / 8 h dark culture.

[0073] d. Rooting culture: when the root system is strong and healthy, transfer it to the rooting solid medium (1962 formula MS inorganic salt and trace element 4.404 g / L, sucrose 30 g / L, agar 6.8 g / L, after sterilization and cooling, add Kana 50 mg / L, Cef 400 mg / L antibiotics) for rooting.

[0074] (3) Identification of transgenic plants

[0075] a. Extraction of tobacco leaf genomic DNA

[0076] ​DNA extraction was performed by using the conventional CTAB method. The specific steps were as follows: 1-2 cm long tender wild-type tobacco leaves were placed in a pre-cooled mortar, and liquid nitrogen was added for 2-3 times of grinding until the slurry was fine, then transferred to a centrifuge tube, and 700 μL of 2x CTAB solution was added. Incubate at 70°C for 30 min, shake gently once every 6 min, incubate at 70°C for 30 min, shake gently once every 10 min. Cool to room temperature, add 700 μL of Tris-saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1, mix well by repeatedly inverting, and then shake gently for about 40 times. Centrifuge at 3100 rpm, room temperature for 15 min. About 500 μL of supernatant was taken, and an equal volume of 24:1 chloroform: isoamyl alcohol was added. Shake well and centrifuge at 3100 rpm, room temperature for 15 min. Discard the supernatant and add 1 mL of anhydrous ethanol at -20°C. After 30 min in a -20°C ice bath, centrifuge at 12000 rpm, room temperature for 10 min. Wash the precipitate with 75% alcohol and blow it repeatedly for 3 min to remove salt. Discard the alcohol, dry in the air, and then add 30-50 μL of ddH2O to each sample for dissolution. The extracted tobacco genomic DNA was detected by Nanodrop micro-nucleic acid detector.

[0077] b. Positive transgenic plant detection

[0078] Tender leaves of the transformed plants were taken, and DNA was extracted by the CTAB method. The pGWB418-NnXTH2 gene on the vector was amplified by PCR for positive plant identification, and the control was a non-transgenic wild-type plant (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 detection results show that (Fig. Figure 5 A), among the 5 transformed plants, the expected size of the electrophoretic band can be amplified, while the wild type and ddH2O control have no electrophoretic band, indicating that the transgenic tobacco genome contains foreign gene DNA fragments.

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

[0083] a. Extraction of tobacco leaf genomic RNA

[0084] Total RNA of wild type plant (WT) and NnXTH2 overexpression positive lines identified in step (3) were extracted by using Eastep® Super Total RNA Extraction Kit (purchased from Promega Corporation, USA). And the RNA was reversely transcribed into cDNA by using Hiscript® II Q RT SuperMix for qRNA (+gDNA wiper) Kit (purchased from Vazyme Company, China).

[0085] b. Real-time fluorescent quantitative PCR

[0086] In order to determine whether NnXTH2 is overexpressed in tobacco, the transgenic plants were analyzed by real-time fluorescent quantitative PCR (qRT-PCR) method. qRT-PCR used SYBR® Green Realtime PCR Master Mix-Plus-Kit (Bao Biological Engineering (Dalian) Co., Ltd.), and the primers were synthesized by Nanjing Kingsway Biological Technology 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: 95 ℃ pre-denaturation for 30 sec, and then 40 cycles of 95 ℃ for 10 sec, 60 ℃ for 10 sec, and 72 ℃ for 26 sec. The numbers of NnXTH2 overexpression positive lines identified in step (3) were added with OE (the abbreviation of overexpression) in front.

[0091] The detection results showed that Figure 5 B), the NnXTH2 expression levels of OE-7, OE-9, OE-12, OE-17 and OE-23 lines were significantly higher than that of wild type plant, and the most significant was OE-9 line, whose expression amount increased by 562.46% compared with wild type, indicating that these lines were independent NnXTH2 overexpression transgenic lines.

[0092] Example 3 NnXTH2 overexpression transgenic tobacco T2 generation plant type and flower stem mechanical detection

[0093] (1) Determination of T2 generation tobacco flower stem mechanics of NnXTH2 overexpression lines

[0094] The stem mechanical force of the middle part of the first node of the top flower stalk of the T2 generation of wild type tobacco (WT) and its three transgenic lines (OE-7, OE-9, and OE-17) at the flowering stage was measured by a stem strength tester (YYD-1, Beijing Shunkeda Technology Co., Ltd.). The results showed that the mechanical level of the flower stalk of the transgenic lines was significantly increased compared with WT: the OE-7 line increased by 11.54%, the OE-9 line increased by 47.69%, and the OE-17 line increased by 6.15%. This indicates that the application effect of NnXTH2 gene in the regulation of the mechanical quality of the flower stalk is good. Figure 6 B).

[0095] (12) T2 generation of NnXTH2 overexpression lines

[0096] The plant type of the T2 generation of wild type tobacco (WT) and its three transgenic lines (OE-7, OE-9, and OE-17) at the flowering stage was observed. The plant height was accurately measured by a telescopic measuring ruler. The specific measurement method was as follows: the starting point was set as the bottom of the cultivation pot, and the end point was the highest point of the main inflorescence of the plant. The vertical distance between the two points was the plant height. The results showed that compared with WT, the transgenic lines with overexpression of NnXTH2 gene showed significant changes in plant type: the plant height of the transgenic lines was significantly lower than that of the wild type, with a decrease of 26%-36% (C of Figure 6 A). This decrease in plant height led to a significant shortening of the internode distance, making the overall plant type more compact (A of Figure 6 A). This phenomenon fully demonstrates that overexpression of NnXTH2 gene can effectively promote plant dwarfing and shortening of internode distance.

[0097] In summary, overexpression of NnXTH2 gene can significantly enhance the mechanical properties of the flower stalk, shorten the plant height and internode distance, and make the plant dwarf, which is more in line with the requirements of the plant type and flower stalk quality of fresh-cut flowers.

[0098] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art should fall within the scope of protection determined by the claims of the present application.

Claims

1. Use of the NnXTH2 gene, characterized in that, Use of any one of the following: A1, enhancing the mechanical strength of flower stems of plants; A2, promoting dwarf and compact plant type; A3, breeding plant lines with high cut flower quality; The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4.

2. Use of an expression cassette comprising the NnXTH2 gene, characterized in that, Use of any one of the following: A1, enhancing the mechanical strength of flower stems of plants; A2, promoting dwarf and compact plant type; A3, breeding plant lines with high cut flower quality; The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4.

3. Use of a recombinant vector comprising the NnXTH2 gene, characterized in that, Use of any one of the following: A1, enhancing the mechanical strength of flower stems of plants; A2, promoting dwarf and compact plant type; A3, breeding plant lines with high cut flower quality; The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4.

4. Use of a recombinant microorganism comprising the NnXTH2 gene, characterized in that, Use of any one of the following: A1, enhancing the mechanical strength of flower stems of plants; A2, promoting dwarf and compact plant type; A3, breeding plant lines with high cut flower quality; The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4.

5. Use according to any one of claims 1 to 4, characterized in that, The promotion of dwarf and compact plant type refers to reducing plant height and shortening plant internode distance.

6. Use according to any one of claims 1 to 4, characterized in that, The plant is lotus or tobacco.

7. A method of enhancing the mechanical strength of a floral stem of a plant, characterized in that, The method comprises the following steps: The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4.

8. A method for promoting dwarf and compact plant architecture, characterized by, The method comprises the following steps: The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4.

9. A method for breeding a plant line with high cut flower quality, characterized in that, The method comprises the following steps: The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4.

10. The method according to any one of claims 7-9, characterized in that, The method comprises the following steps: The NnXTH2 gene encodes a protein as shown in SEQ ID NO.

4. The plant is lotus or tobacco.

Images