Application of Lotus NnXTH22 Gene in Increasing Plant Height and Stem Thickness

Abstract

The present invention provides an application of the lotus NnXTH22 gene in increasing plant height and stem thickness, and belongs to the field of genetic engineering technology. The application of the lotus NnXTH22 gene or its related biological materials of the present invention includes any of the following applications: A1, increasing plant height and increasing plant stem thickness; A2, cultivating large plant type plant strains; A3, cultivating lodging-resistant plant strains; the nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO.4. The present invention cloned the NnXTH22 (Nn2g12672.2) gene from the lotus, and verified the application of the gene in regulating plant stem growth through heterologous overexpression, providing new genetic resources for lotus and other plant type improvement and molecular breeding.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of lotus NnXTH22 gene in enhancing plant height and stem thickness. Background Art

[0002] Lotus (Nelumbo nucifera), Nymphaeaceae Salisb., Subfam. Nelumboideae, Genus Nelumbo (Nelumbo Adans.) plant. Also known as lotus, hibiscus, and water hibiscus, it is rich in resources, has a wide variety of varieties, and is widely cultivated. It is edible, medicinal, and ornamental. Due to the different purposes of long-term artificial breeding, lotus is divided into lotus root, seed lotus, and flower lotus. Its rich flower shapes, colors, and plant types determine the wide application value of lotus. Among them, different plant types have different application attributes. Large-plant lotus can be as high as two meters and can be used in wetland construction and landscaping; small-plant bowl lotus varieties are only a few dozen centimeters high and can be used in home gardening, interior decoration, etc. However, many large-plant varieties cannot be cultivated into small-plant varieties, and the unique flower shapes and colors of bowl lotus cannot be cultivated into large-plant varieties. At present, the research on lotus at home and abroad mainly includes phylogeny and evolution, genomics, genetics, breeding and medicinal uses, but the research on ornamental traits such as lotus plant shape is still insufficient. Therefore, discovering more genes involved in plant shape construction will help to assist in the selection of new lotus varieties with different plant shapes through molecular breeding methods.

[0003] During plant growth, the increase in cell volume is an important aspect of plant growth, and a basic requirement of this process is cell wall remodeling. Xyloglucan endotransglucosylase / hydrolase (XTH) can break and reconnect xyloglucan molecules, modify the composite structure formed by cellulose-xyloglucan, and thus achieve cell wall remodeling. It is related to the synthesis, relaxation and degradation of the cell wall and has a potential role in the development of plant type. Studies have shown that chickpea CaXTH1 is involved in regulating the elongation of epicotyl parenchyma cells and the development of vascular tissue, AtXTH9 is specifically expressed in the Arabidopsis shoot apex, and the mutant xth9 shows a significant dwarf phenotype. AtXTH10 is related to root development and vascular tissue by affecting cell wall structure. Exogenous BL can promote the growth of Arabidopsis stems by enhancing the gene expression of AtXTH3, 4, 5, 17, 22 and 23. AtXTH20 and AtXTH19 can be regulated by ANAC071 under auxin flow conditions and participate in the cell division of Arabidopsis inflorescence. In addition, Jia Xinlei et al. found that XTHs genes play a role in root and stem elongation and may be involved in the entire life cycle of the plant. These results indicate that XTH family genes have the potential to improve plant stems, but there are no reports on the involvement of lotus NnXTHs family genes in plant stem development. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the lotus NnXTH22 gene in increasing plant height and stem thickness to solve the problems of the above-mentioned prior art. The present invention cloned the NnXTH22 (Nn2g12672.2) gene from the lotus, and verified the application of the gene in regulating plant stem growth through heterologous overexpression, providing a new genetic resource for lotus and other plant type improvement and molecular breeding.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides applications of the lotus NnXTH22 gene or related biological materials, including any of the following applications:

[0007] A1. Increase plant height and plant stem thickness;

[0008] A2. Cultivate large plant strains;

[0009] A3. Cultivate lodging-resistant plant strains;

[0010] The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO.4.

[0011] Preferably, the relevant biological material is any one of the following B1 to B3:

[0012] B1, an expression cassette comprising the lotus NnXTH22 gene;

[0013] B2, a recombinant vector comprising the lotus NnXTH22 gene;

[0014] B3. A recombinant microorganism comprising the lotus NnXTH22 gene.

[0015] The present invention provides a recombinant vector comprising a lotus NnXTH22 gene having a sequence as shown in SEQ ID NO.4.

[0016] The present invention provides a recombinant microorganism comprising a lotus NnXTH22 gene having a sequence as shown in SEQ ID NO.4, or comprising the recombinant vector described above.

[0017] The present invention provides a method for increasing plant height and stem thickness, wherein a lotus NnXTH22 gene or a related biological material thereof is transduced into a plant body, so that the lotus NnXTH22 gene is stably overexpressed, thereby increasing plant height and stem thickness;

[0018] The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO.4; the related biological material is any one of the following B1 to B3:

[0019] B1, an expression cassette comprising the lotus NnXTH22 gene;

[0020] B2, a recombinant vector comprising the lotus NnXTH22 gene;

[0021] B3. A recombinant microorganism comprising the lotus NnXTH22 gene.

[0022] The present invention provides a method for cultivating a large-scale plant strain, wherein a lotus NnXTH22 gene or a related biological material is transduced into a plant body, so that the lotus NnXTH22 gene is stably overexpressed, and a large-scale plant strain is cultivated;

[0023] The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO.4; the related biological material is any one of the following B1 to B3:

[0024] B1, an expression cassette comprising the lotus NnXTH22 gene;

[0025] B2, a recombinant vector comprising the lotus NnXTH22 gene;

[0026] B3. A recombinant microorganism comprising the lotus NnXTH22 gene.

[0027] The present invention provides a method for cultivating a lodging-resistant plant strain, wherein a lotus NnXTH22 gene or a related biological material thereof is transduced into a plant body, so that the lotus NnXTH22 gene is stably overexpressed, and a lodging-resistant plant strain is cultivated;

[0028] The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO.4; the related biological material is any one of the following B1 to B3:

[0029] B1, an expression cassette comprising the lotus NnXTH22 gene;

[0030] B2, a recombinant vector comprising the lotus NnXTH22 gene;

[0031] B3. A recombinant microorganism comprising the lotus NnXTH22 gene.

[0032] The present invention discloses the following technical effects:

[0033] The present invention cloned the NnXTH22 (Nn2g12672.2) gene from lotus, enriching the research on XTH family genes in lotus. By constructing an overexpression vector pGWB418 (4×Myc)-NnXTH22-NOS, the NnXTH22 overexpression strain was obtained after transforming rapeseed, and the function acquisition of NnXTH22 was studied, and the role of the gene in regulating plant stem growth was verified, providing raw materials for the research on the regulating stem development function of NnXTH22, which has good application value. By carrying out high-generation reproduction of the NnXTH22 overexpression strain and observing its phenotype, it is of great significance to clarify the biological function of the NnXTH22 gene.

[0034] The discovery of NnXTH22, a special gene for lotus plant type, provides important theoretical guidance for discovering and identifying plant types and in-depth exploration of the molecular action mechanism of plant type improvement genes; it also provides new genetic resources for molecular breeding, genetic improvement and ideal plant type breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1The amino acid sequence alignment result of NnXTH22 (Nn2g12672.2) gene and the known Arabidopsis AtXTH22 gene;

[0037] Figure 2 Schematic diagram of the construction process of pGWB418 (4×Myc)-NnXTH22-NOS vector; A is a schematic diagram of the pGWB418 (4×Myc)-NnXTH22-NOS vector structure, B is the fusion cloning method, and C is the NnXTH22 (Nn2g12672.2) gene structure; LB: T-DNA left border; 35S: Cauliflower virus 35S promoter; NOS: terminator; RB: T-DNA right border;

[0038] Figure 3 The flowchart of Agrobacterium-mediated genetic transformation of rapeseed hypocotyls;

[0039] Figure 4 The results of identification and expression analysis of NnXTH22 overexpressing transgenic rapeseed positive plants; A is the identification result of NnXTH22 overexpressing transgenic rapeseed positive plants, PCR amplification of Kan gene fragments on the vector, the target size is 289bp; B is the detection result of NnXTH22 expression level in NnXTH22 overexpressing transgenic rapeseed positive plants;

[0040] Figure 5 A is a graph showing the plant height detection results of wild-type rapeseed (WT) and NnXTH22 (OE) overexpressing plants; A is a phenotypic graph of wild-type rapeseed (WT) and NnXTH22 (OE) overexpressing plants, with a scale of 20 cm; B is a statistical result of the plant height measurement values ​​of wild-type rapeseed (WT) and NnXTH22 (OE) overexpressing plants;

[0041] Figure 6 A is a graph showing the results of stem thickness detection of wild-type rapeseed (WT) and NnXTH22 (OE) overexpressing plants; A is a cross-sectional view of the stems of wild-type rapeseed (WT) and NnXTH22 (OE) overexpressing plants at the mature stage, with a scale of 2 cm; B is a side view of the stems of wild-type rapeseed (WT) and NnXTH22 (OE) overexpressing plants at the mature stage, with a scale of 20 cm; C is the statistical results of the measured values ​​of stem thickness of wild-type rapeseed (WT) and NnXTH22 (OE) overexpressing plants at the mature stage. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] Example 1 Extraction, cloning and sequencing of NnXTH22 (Nn2g12672.2) gene

[0048] (1) The lotus variety 'Gele Lingyin' (provided by the National Lotus Germplasm Resource Bank of Southwest Forestry University) was used as the experimental material. The total RNA of the petioles of the lotus variety 'Gele Lingyin' was extracted using the Super total RNA extraction kit (purchased from Promega, USA). After RNA extraction, it was treated with DNaseI (purchased from Promega kit), and the RNA integrity was detected by 1.2% (w / v) agarose gel (EtBr) electrophoresis (5V / cm). The nucleic acid concentration was determined on an IMPLEN NanoPhotometer-N50 series ultra-micro UV 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 500ng / μl was used for the next analysis. cDNA synthesis was performed using IIQ RT SuperMix for qRNA (+gDNA wiper) kit (purchased from Vazyme, China), with 1 μg of total RNA as template, mixed with 4 μL 4×gDNA wiper Mix, DEPC-water, total volume 16 μL, 42°C for 2 min, and chilled on ice for 2-3 min; then 5×Hiscript II qRT Super Mix II 4 μL was added and mixed, the total volume was 20 μL; then 50°C for 15 min, 85°C for 5 sec, each cDNA was diluted to 200 μL and stored at -20°C for later use.

[0049] In-fusion cloning primers NnXTH22-418F and NnXTH22-418R were used to amplify the target band, and TransTaq HiFi DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.) was used for amplification. The PCR reaction conditions were: 94℃ pre-denaturation for 1min; 94℃30sec, 58℃30sec, 72℃1min 20sec, 31 cycles; 72℃ extension for 5min. The PCR product was then cloned into the pGWB418 vector. The recovery of the target fragment was performed with reference to the UNIQ-10 column DNA gel recovery kit (Shanghai Bioengineering Co., Ltd.). The connection system between the target fragment and the pGWAB418 linearized vector was: 4.5μL target fragment, 0.5μL pGWB418 vector (Wuhan Puint Bioengineering Co., Ltd.), 5μL SolutionⅠ (Bao Bioengineering (Dalian) Co., Ltd.) at 16℃ overnight. The ligation product was transformed into DH5α competent cells by heat stimulation, and the bacterial solution was applied to LB solid plates containing 100 mg / L Kan antibiotics. After about 10-12 hours of growth, a single colony was selected for colony PCR, and the primers were 418F and NnXTH22-418R. The positive colony was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0050] Primer information is as follows:

[0051] NnXTH22-418F: 5'-GACTTGAACGGTAGCGCTCTTAGTACATAGGCATAGCAATAT GG-3' (SEQ ID NO. 1);

[0052] NnXTH22-418R: 5'-TCGGGGAAATTCGAGCTCTTTAAGAGGTATGGTGGTATTAGA GC-3' (SEQ ID NO. 2);

[0053] 418F: 5'-TCGGGGAAATTCGAGCTC-3' (SEQ ID NO. 3).

[0054] (2) NnXTH22 gene sequence analysis

[0055] After sequencing, it was found that the full length of the nucleotide sequence amplified from this gene was 920 bp (SEQ ID NO.4), which contained an open reading frame ORF of 873 bp (SEQ ID NO.5). The amino acid sequence was aligned 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, and it was found that this target band was highly homologous to AtXTH22 of Arabidopsis thaliana at the amino acid level (similarity 68.94%), which was consistent with the results of the alignment on NCBI ( Figure 1 ). The amino acid sequence encoded by the NnXTH22 gene is shown in SEQ ID NO.6.

[0056] SEQ ID NO.4:

[0057] CTTAGTACATAGGCATAGCAATATGGCTTTTTCTGAAAGAGCATGGTTCAGCTTTTGGGCTTGCCTAGTGATTTTAAGCTCTTTAACCGTGTCCGCTTTGTCTGGTAATTTCTACCAAGATTTTGATATCACATGGGGCGATGGTCGGGCAAAGATACTCAACAACGGGGAACTCCTTACTCTCTCCCTAGACAAGGCCTCTGGCTCTGGCTTTCAGTCCAAGAACGAGTTTCTATTCGGGAAGATTGATATGCAGCTCAAGCTCGTCCCTGGCAACTCTGCTGGCACCGTTACTGCCTACTACTTGTCATCTCAGGGACCAACCCACGATGAGATTGACTTCGAATTCCTTGGCAATCTCAGTGGTGACCCCTACATCCTCCATACTAACGTCTTCAGCCAAGGCAAAGGTAACAGAGAGCAGCAGTTCTACCTTTGGTTCGACCCAACTGCTGATTTTCACACTTACTCCATTTTGTGGAATCCCCAACGTATTGTCTTCTCTGTTGATGGAATTCCGATCAGGGAATTCAACAACTCGGAATCAATTGGAGTTCCATTCCCCAAGAGCCAACCCATGAGGATATACTCCAGCCTCTGGAACGCTGATGACTGGGCCACAAGGGGTGGCCTTGTCAAAACCGACTGGACACAAGCACCTTTCACGGCCTCCTACAGAAACTTCAAGGCCGATGCTTGTATTTGGTCCTCTGGTGCTTCTTCTTGCGACTCTAGATCTCCCTCCTCGACCTCTAACGCCAACGCCTGGTGGCTCTCACAGGAGCTGGACTCAACAAGCCAAGAGAGAATGGGATGGGTTCAGAAGAACTACATGATATACAATTACTGCACAGATACAAAGCGCTTCCCTCAAGGCCTCCCTCTGGAATGCTAGCTCTAATACCACCATACCTCTTAAA;

[0058] SEQ ID NO.5:

[0059] ATGGCTTTTTCTGAAAGAGCATGGTTCAGCTTTTGGGCTTGCCTAGTGATTTTAAGCTCTTTAACCGTGTCCGCTTTGTCTGGTAATTTCTACCAAGATTTTGATATCACATGGGGCGATGGTCGGGCAAAGATACTCAACAACGGGGAACTCCTTACTCTCTCCCTAGACAAGGCCTCTGGCTCTGGCTTTCAGTCCAAGAACGAGTTTCTATTCGGGAAGATTGATATGCAGCTCAAGCTCGTCCCTGGCAACTCTGCTGGCACCGTTACTGCCTACTACTTGTCATCTCAGGGACCAACCCACGATGAGATTGACTTCGAATTCCTTGGCAATCTCAGTGGTGACCCCTACATCCTCCATACTAACGTCTTCAGCCAAGGCAAAGGTAACAGAGAGCAGCAGTTCTACCTTTGGTTCGACCCAACTGCTGATTTTCACACTTACTCCATTTTGTGGAATCCCCAACGTATTGTCTTCTCTGTTGATGGAATTCCGATCAGGGAATTCAACAACTCGGAATCAATTGGAGTTCCATTCCCCAAGAGCCAACCCATGAGGATATACTCCAGCCTCTGGAACGCTGATGACTGGGCCACAAGGGGTGGCCTTGTCAAAACCGACTGGACACAAGCACCTTTCACGGCCTCCTACAGAAACTTCAAGGCCGATGCTTGTATTTGGTCCTCTGGTGCTTCTTCTTGCGACTCTAGATCTCCCTCCTCGACCTCTAACGCCAACGCCTGGTGGCTCTCACAGGAGCTGGACTCAACAAGCCAAGAGAGAATGGGATGGGTTCAGAAGAACTACATGATATACAATTACTGCACAGATACAAAGCGCTTCCCTCAAGGCCTCCCTCTGGAATGCTAG;

[0060] SEQ ID NO.6:

[0061] MAFSERAWFSFWACLVILSSLTVSALSGNFYQDFDITWGDGRAKILNNGELLTLSLDKASGSGFQSKNEFLFGKIDMQLKLVPGNSAGTVTAYYLSSQGPTHDEIDFEFLGNLSGDPYILHTNVFSQGKGNREQQFYLWFDPPTAD FHTYSILWNPQRIVFSVDGIPIREFNNSESIGVPFPKSQPMRIYSSLWNADDWATRGGLVKTDWTQAPFTASYRNFKADACIWSSGASSCDSRSPSSTSNANAWWLSQELDSTSQERMGWVQKNYMIYNYCTDTKRFPQGLPLEC.

[0062] Example 2 Obtaining NnXTH22 (Nn2g12672.2) overexpressing transgenic rapeseed plants

[0063] (1) Construction of plant overexpression vector

[0064] Afel and Sac1 restriction sites were added to both ends of the cloning amplification primers NnXTH22-418F and NnXTH22-418R, and the pGWB418 vector was double-digested with AfeI and SacI. The digestion system was: pGWB418 (1700ng / μL) 15μL, rCutsmartt Buffer 5μL, AfeI (10000U / L) 1μL, SacI (20000U / L) 1μL, and sterile water was added to the final volume of 50μL. The digestion was carried out at 37°C overnight. After 1% agarose gel electrophoresis, a single linearized vector fragment was recovered and named pGWB418 (4×Myc) linearized vector.

[0065] The PCR product obtained by the above amplification was connected to the pGWB418 (4×Myc) linearized vector using In-fusion enzyme, and the positive clones were screened and sequenced to obtain the lotus NnXTH22 transgenic overexpression vector, named pGWB418 (4×Myc)-NnXTH22-NOS. The construction process is as follows Figure 2 As shown, the T-DNA region of the vector contains a gene sequence for resistance to kanamycin, and the promoter for overexpression of NnXTH22 is the D35s promoter.

[0066] (2) Genetic transformation of rapeseed

[0067] The recombinant plasmid (i.e., overexpression vector) pGWB418(4×Myc)-NnXTH22-NOS was transformed into Agrobacterium GV3101 by conventional hypocotyl infection method, and then screened and differentiated into seedlings. The specific steps are as follows (transformation process see Figure 3 ):

[0068] a. Sowing: Wash the westar seeds of Brassica napus with 75% alcohol for 1 minute, then wash the seeds with 0.1% mercuric chloride solution for 5 minutes, and finally wash the seeds with sterile water for 5 times; use sterilized tweezers to put the seeds into M0 solid culture medium (MS inorganic salts and trace elements 4.404g / L formulated in 1962, adjusted to pH 5.8-5.9, added with 7g / L agar, sterilized by conventional high-pressure steam sterilization method), and culture the inoculated seeds in the dark at 24°C for 5 days.

[0069] b. Activation of Agrobacterium: 3 days after sowing, take the Agrobacterium strain GV3101 stored at -80℃ and streak it on LB solid medium containing rifampicin (50mg / mL), gentamicin (50mg / mL) and kanamycin (50mg / mL), and culture it at 28℃ for 16h. Pick a single colony and place it in 5mL LB liquid medium containing rifampicin (50mg / mL), gentamicin (50mg / mL) and kanamycin (50mg / mL), and culture it at 28℃, 200rpm shaking for 20-24h to grow Agrobacterium to the logarithmic phase. Take 500μL of the cultured bacterial solution and expand it in 50mL LB liquid medium (containing three antibiotics) for about 12h.

[0070] c. Preparation of infection solution: Pour the activated Agrobacterium solution into two 50mL sterile centrifuge tubes, centrifuge at 3500rpm for 15min, remove the supernatant, place on ice, and wash the solution with 1mL DM liquid culture medium (MS inorganic salts and trace elements 4.404g / L, sucrose 30g / L, pH of the culture medium adjusted to 5.8-5.9, sterilize at 121℃ for 20min, add 2,4-D 1mg / L, AS 100mmol / L, KT 0.3mg / L in a sterile operating table after sterilization), centrifuge, remove the supernatant, add 2-3mL DM liquid culture medium to resuspend the bacteria, and adjust the infection solution OD to 5.8-5.9. 600 It is about 0.6.

[0071] d. Infecting explants: Place the infection solution in step c on ice. At the same time, use a scalpel to cut the hypocotyls of the dark-cultured Brassica napus seedlings in step a (the length of each hypocotyl should be 0.8-1.0 cm). Use sterile tweezers to transfer the cut hypocotyls into a plate containing the infection solution. Infect for 15-30 minutes depending on the concentration of the solution (shake every 3 minutes).

[0072] e. Co-cultivation: Transfer the infected explants to a plate containing filter paper (sterilized in advance), dry the infection fluid visible on the surface of the explants, and then transfer to M1 solid culture medium (MS inorganic salts and trace elements 4.404g / L, mannitol 18g / L, sucrose 30g / L, 2,4-D 2mg / L, KT 0.3mg / L formulated in 1962, adjust the pH of the culture medium to 5.8-5.9, add 7g / L agar, add 100mmol / LAS after sterilization), and culture in the dark at 24℃ for 40-48h.

[0073] f. Screening: Transfer the explants after co-cultivation to M2 solid culture medium (MS inorganic salts and trace elements 4.404 g / L, mannitol 18 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, KT 0.3 mg / L, adjust the pH of the culture medium to 5.8-5.9, add agar 7 g / L) for 20 days of screening culture at 24°C, 16 h light / 8 h dark culture.

[0074] g. Differentiation culture: The screened explants were transferred to M3 solid culture medium (MS inorganic salts and trace elements 4.404 g / L formulated in 1962, glucose 10 g / L, xylose 0.25 g / L, yeast extract (MES) 0.6 g / L, the pH of the culture medium was adjusted to 5.8-5.9, agar 7 g / L was added, and after sterilization, zeatin (ZT) 2 mg / L, IAA 0.1 mg / L, TMT 250 mg / L, Kan 25 mg / L) were added to start differentiation culture, and subcultured every 15-20 days until differentiation and budding (the culture conditions were consistent with the screening conditions in step f).

[0075] h. Rooting culture: When obvious growth points can be found on the differentiated seedlings, use a scalpel to carefully cut the seedlings along the junction of the callus tissue and the bud (be careful not to damage the growth point during the operation), and then transfer the seedlings to M4 solid culture medium (MS inorganic salts and trace elements 2.202g, sucrose 10g / L, IBA 0.5mg / L formulated in 1962, adjust the pH of the culture medium to 5.8-5.9, add 7g / L agar, and add TMT 250mg / L and Kan 25mg / L after sterilization) for rooting.

[0076] (3) Identification of transgenic plants

[0077] Take young leaves of transformed plants, use conventional CTAB method to extract DNA, and use PCR to amplify Kan gene on the vector for positive seedling identification. The primers used are:

[0078] Bn-Kan-F: 5'ACTGGGCACAACAGACAATCG3' (SEQ ID NO.7);

[0079] Bn-Kan-R: 5'GCATCAGCCATGATGGATACTTT3' (SEQ ID NO. 8).

[0080] The control was non-transgenic wild-type plants (WT).

[0081] The specific steps of the conventional CTAB method are as follows: take young and tender cabbage rapeseed leaves with a length of 1 to 2 cm, place them in a pre-cooled mortar, add liquid nitrogen 2 to 3 times during the process to grind them into a fine slurry, transfer them to a 1.5 mL centrifuge tube, and add 700 μL of 2x CTAB solution. Incubate at 70°C for 30 minutes, shake gently every 6 minutes, and incubate at 70°C for 30 minutes, shake gently every 10 minutes. Cool to room temperature, add 700 μL of Tris saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1, repeatedly invert and mix, and gently shake about 40 times. Centrifuge at 3100 rpm at room temperature for 15 minutes. Take about 500 μL of the supernatant and add an equal volume of 24:1 chloroform: isoamyl alcohol. After shaking, centrifuge at 3100 rpm at room temperature for 15 minutes. Discard the supernatant, add 1mL of frozen -20℃ anhydrous ethanol, place in an ice bath at -20℃ for 30min, and centrifuge at 12000rpm for 10min at room temperature. Soak and wash with 75% alcohol and repeatedly blow and beat the precipitate for 3min to remove salt. Pour away the alcohol, air dry, and add 30-50μLddH2O to each sample to dissolve. The extracted rapeseed genomic DNA was tested for concentration using Nanodrop micro-nucleic acid detector.

[0082] Test results such as Figure 4 As shown in A (bars are mean ± SD, n>3 biological independent samples, showing single data points; two-tailed t-test was used to determine the significant differences between groups: ****P<0.0001, *P<0.05, ns no significant difference), 7 transformed plants 3, 4, 6, 14, 22, 34 and 44 were able to amplify the expected size of the electrophoresis band (289bp), while the wild-type control had no electrophoresis band, indicating that the transgenic rapeseed genome already contained exogenous gene DNA fragments. The transgenic positive plant number obtained by identification was preceded by OE.

[0083] (4) qRT-PCR identification of NnXTH22 overexpressing transgenic rapeseed

[0084] a. Extraction of genomic RNA from Brassica napus leaves

[0085] use Super total RNA extraction kit (purchased from Promega, USA) was used to extract total RNA from leaves of WT and NnXTH22 overexpression positive strains identified in step (3). RNA was reverse transcribed into cDNA using IIQ RT SuperMix for qRNA (+ gDNAwiper) kit (Vazyme, China).

[0086] b. Real-time fluorescence quantitative PCR

[0087] In order to determine whether NnXTH22 is overexpressed in rapeseed, the transgenic plants identified in step (3) were analyzed using the cDNA extracted as above as a template using quantitative real-time PCR (qRT-PCR). Green Realtime PCR Master Mix-Plus-Kit (Takara Biotech (Dalian) Co., Ltd.), using rape housekeeping gene BnActin7 as internal standard gene, and its primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The qRT-PCR primers for BnActin7 (LOC106418315) are:

[0088] BnActin7-F1: 5'-TCTTCCTCACGCTATCCCTCG-3' (SEQ ID NO.9);

[0089] BnActin7-R1: 5'-AGCCGTTCCAGCTCTTGC-3' (SEQ ID NO. 10).

[0090] The qRT-PCR primers for NnXTH22 gene are:

[0091] Q-NnXTH22-F: 5'-GCTGGACTCAACAAGCCAAG-3' (SEQ ID NO. 11);

[0092] Q-NnXTH22-R: 5'-CGAACGGTCTGTCAAAATTCC-3' (SEQ ID NO. 12).

[0093] (This pair of primers is a qRT-PCR specific primer designed based on the nucleotide sequence sequenced in Example 1)

[0094] PCR program: pre-denaturation at 95°C for 30 sec, followed by 40 cycles (95°C for 15 sec, 60°C for 30 sec, 72°C for 15 sec).

[0095] like Figure 4As shown in Figure B, the expression levels of NnXTH22 in OE-3, OE-4, OE-6, OE-14, OE-22, OE-34 and OE-44 strains were significantly higher than the wild-type (WT) control, the expression level of OE-3 strain was more than 30 times that of the wild-type strain, the expression levels of OE-14 and OE-34 strains were more than 20 times, the expression level of OE-44 strain was more than 10 times, and the expression levels of OE-4, OE-6 and OE-22 strains were about 1 times and 3 times that of the wild-type, indicating that these strains are independent NnXTH22 overexpression transgenic strains.

[0096] Example 3 Increased plant height and thickened stems of NnXTH22 (Nn2g12672.2) overexpressing transgenic T3 rapeseed lines

[0097] (1) Determination of plant height of T3 generation rapeseed plants overexpressing NnXTH22

[0098] The plant heights of WT and NnXTH22 overexpressing plants at the silique stage were measured. The measurement method was to wait until the plant height no longer increased significantly, then select the T3 strains of OE-3, OE-14 and OE-34 with the highest expression levels, and use a 3-meter telescopic measuring ruler to measure their plant heights. The measurement started from the bottom of the cultivation pot to the highest point of the main inflorescence. The results are shown in Figure 5 As described above, the average heights of the three overexpressing transgenic lines were significantly higher than that of WT.

[0099] (2) Determination of stem diameter of NnXTH22 overexpressing T3 rapeseed lines

[0100] The stem diameter of WT and NnXTH22 overexpressing plants at the silique stage was measured. The measurement method was to select the T3 lines of OE-3, OE-14 and OE-34 with the highest expression levels, and measure the diameter of the middle part of the internode from the penultimate node at the base of the plant with a digital vernier caliper. The results are shown in Figure 6 As shown, the average stem diameter of the three overexpressing transgenic lines was significantly higher than that of WT.

[0101] In summary, overexpression of the NnXTH22 gene can significantly enhance plant height and stem thickness.

[0102] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of lotus NnXTH22 gene or its related biological materials, characterized in that: An application that is any of the following: A1. Increase plant height and plant stem thickness; A2. Cultivate large plant strains; A3. Cultivate lodging-resistant plant strains; The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO. 4; The relevant biological material is any one of the following B1 to B3: B1, an expression cassette comprising the lotus NnXTH22 gene; B2, a recombinant vector comprising the lotus NnXTH22 gene; B3, a recombinant microorganism comprising the lotus NnXTH22 gene; The plant is rapeseed.

2. A method for increasing plant height and stem thickness, characterized in that: The lotus NnXTH22 gene or its related biological materials are introduced into the plant body to stably over-express the lotus NnXTH22 gene and increase the plant height and stem thickness; The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO. 4; the related biological material is any one of the following B1 to B3: B1, an expression cassette comprising the lotus NnXTH22 gene; B2, a recombinant vector comprising the lotus NnXTH22 gene; B3, a recombinant microorganism comprising the lotus NnXTH22 gene; The plant is rapeseed.

3. A method for cultivating a large plant strain, characterized in that: The lotus NnXTH22 gene or its related biological materials are introduced into the plant body to stably over-express the lotus NnXTH22 gene and cultivate a large plant strain; The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO. 4; the related biological material is any one of the following B1 to B3: B1, an expression cassette comprising the lotus NnXTH22 gene; B2, a recombinant vector comprising the lotus NnXTH22 gene; B3, a recombinant microorganism comprising the lotus NnXTH22 gene; The plant is rapeseed.

4. A method for cultivating lodging-resistant plant strains, characterized in that: The lotus NnXTH22 gene or its related biological materials are introduced into the plant body to stably over-express the lotus NnXTH22 gene, and cultivate a lodging-resistant plant strain; The nucleotide sequence of the lotus NnXTH22 gene is shown in SEQ ID NO. 4; the related biological material is any one of the following B1 to B3: B1, an expression cassette comprising the lotus NnXTH22 gene; B2, a recombinant vector comprising the lotus NnXTH22 gene; B3, a recombinant microorganism comprising the lotus NnXTH22 gene; The plant is rapeseed.

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