Application of NnXTH23 gene in regulating plant height and stem thickness

Abstract

The present invention provides an application of the NnXTH23 gene in regulating plant height and stem thickness, and belongs to the field of genetic engineering technology. The application of the NnXTH23 gene or its related biomaterials of the present invention includes any of the following applications: A1, increasing plant height and increasing plant stem thickness; A2, breeding of large plant strains; A3, breeding of lodging-resistant plant strains; the nucleotide sequence of the NnXTH23 gene is shown in SEQ ID NO.4. The present invention finds that the NnXTH23 (Nn2g12677.1) gene is involved in regulating plant stem development, and overexpressing the NnXTH23 gene in rapeseed can significantly increase the plant height and stem thickness of rapeseed, providing new technical guidance for plant type improvement and functional improvement.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of NnXTH23 gene in regulating plant height and stem thickness. Background Art

[0002] Lotus (Nelumbo nucifera G.) is a perennial aquatic herb in the genus Nelumbo of the family Nelumbonaceae. Lotus is an excellent wetland construction and home garden flower for its edible, medicinal and ornamental uses. The morphological construction of its plants is closely related to its ornamental characteristics and market applications. Among them, different plant types have different application attributes. For example, large-plant lotus is widely used in wetland construction and landscaping, which is of great significance to improving water quality and beautifying the environment; while small-plant types such as bowl lotus can be used in home gardening and interior decoration; at the same time, lotus is also an emerging cut flower variety, which is deeply loved by the public. Both plant type and cut flower are closely related to the development of lotus stems. However, due to its high genomic heterozygosity, the current research on lotus at home and abroad mainly focuses on the origin and evolution of lotus, breeding and cultivation, classification and culture, and there are few studies on the molecular mechanism of lotus morphological construction.

[0003] The cell wall is the most important organelle that distinguishes plant cells from animal cells. It is wrapped around the outside of the plasma membrane and has both rigidity and flexibility. It is a complex structure that determines the shape and size of the cell. Plant growth is based on cell division, elongation and differentiation, all of which require the continuous reconstruction of the cell wall. The secondary wall is an important determinant of increasing the mechanical support of the cell wall. When cell elongation stops, the secondary wall begins to deposit on the inside of the primary wall and has rigidity. The primary wall is synthesized during cell division and elongation, has softness and plasticity, and plays a decisive role in cell size. Xyloglucan is the main hemicellulose in the primary cell wall of dicotyledons and non-gramineous monocotyledons. Xyloglucan endotransglycosidase / hydrolase (XTH) is widely present in various tissues and cells of plants. It modifies the cellulose-xyloglucan complex structure of the plant cell wall by catalyzing the breakage and reconnection of xyloglucan molecules, thereby achieving cell wall remodeling. It is one of the key enzymes in the process of plant cell wall reconstruction. During the growth of plant cells, XTH protein can not only relax cell walls, participate in cell wall synthesis, and strengthen cell walls, but also has the function of degrading cell walls.

[0004] Throughout the growth and development of plants, the cutting and reconnection of xyloglucan by XTH plays a very important role in cell wall remodeling. Studies have shown that the Arabidopsis AtXTH21 gene plays a major role in the growth of the taproot by changing the weight of xyloglucan and the deposition of cellulose, loosening and elongating the cell wall. Arabidopsis AtXTH9 is expressed in the flower buds and branches of the shoot apical meristem, and its physiological role is related to the elongation growth of the stems and branches. The deletion mutation of this gene causes the plant to have a short internode phenotype. Overexpression of the rapeseed BcXTH1 gene leads to elongation of Arabidopsis stems and a significant increase in plant height, indicating that it is involved in cell expansion and elongation.

[0005] In summary, the XTH family plays an important role in promoting plant growth and development. Screening more XTH family genes and conducting in-depth studies on the functions and regulatory mechanisms of related genes are of great significance to the development and utilization of plant germplasm resources. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the NnXTH23 gene in regulating plant height and stem thickness to solve the problems existing in the above-mentioned prior art. The present invention finds that the NnXTH23 (Nn2g12677.1) gene is involved in regulating plant stem development, and overexpressing the NnXTH23 gene in rapeseed can significantly increase the plant height and stem thickness of rapeseed, providing new technical guidance for plant type improvement and function improvement.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an application of the NnXTH23 gene or its related biological materials, including any of the following applications:

[0009] A1. Increase plant height and plant stem thickness;

[0010] A2. Large plant strain breeding;

[0011] A3. Breeding of lodging-resistant plant strains;

[0012] The nucleotide sequence of the NnXTH23 gene is shown in SEQ ID NO.4.

[0013] Preferably, the relevant biological material is any one of the following B1 to B3:

[0014] B1, an expression cassette comprising the NnXTH23 gene;

[0015] B2, a recombinant vector comprising the NnXTH23 gene;

[0016] B3. A recombinant microorganism comprising the NnXTH23 gene.

[0017] The present invention provides a recombinant vector comprising the NnXTH23 gene with a sequence as shown in SEQ ID NO.4.

[0018] The present invention provides a recombinant microorganism comprising the NnXTH23 gene of the sequence shown in SEQ ID NO.4 or comprising the recombinant vector described above.

[0019] The present invention provides a method for increasing plant height and stem thickness, wherein the NnXTH23 gene or its related biological material is transduced into a plant body to stably overexpress the NnXTH23 gene, thereby increasing plant height and stem thickness;

[0020] The nucleotide sequence of the NnXTH23 gene is shown in SEQ ID NO.4; the related biological material is any one of the following B1 to B3:

[0021] B1, an expression cassette comprising the NnXTH23 gene;

[0022] B2, a recombinant vector comprising the NnXTH23 gene;

[0023] B3. A recombinant microorganism comprising the NnXTH23 gene.

[0024] The present invention provides a method for cultivating a large plant strain, wherein the NnXTH23 gene or related biological materials are transduced into a plant body to stably overexpress the NnXTH23 gene, thereby cultivating a large plant strain;

[0025] The nucleotide sequence of the NnXTH23 gene is shown in SEQ ID NO.4; the related biological material is any one of the following B1 to B3:

[0026] B1, an expression cassette comprising the NnXTH23 gene;

[0027] B2, a recombinant vector comprising the NnXTH23 gene;

[0028] B3. A recombinant microorganism comprising the NnXTH23 gene.

[0029] The present invention provides a method for cultivating a lodging-resistant plant strain, wherein the NnXTH23 gene or a related biological material is transduced into a plant body, so that the NnXTH23 gene is stably overexpressed, and a lodging-resistant plant strain is cultivated;

[0030] The nucleotide sequence of the NnXTH23 gene is shown in SEQ ID NO.4; the related biological material is any one of the following B1 to B3:

[0031] B1, an expression cassette comprising the NnXTH23 gene;

[0032] B2, a recombinant vector comprising the NnXTH23 gene;

[0033] B3. A recombinant microorganism comprising the NnXTH23 gene.

[0034] The present invention discloses the following technical effects:

[0035] The present invention found that the NnXTH23 (Nn2g12677.1) gene was significantly upregulated in the stems of large-plant lotus varieties, and it was speculated that the gene played a vital role in regulating the development of plant stems. Through sequence comparison, it was found that the nucleotide sequence similarity between the gene and the model plant Arabidopsis AtXTH23 gene was 68.26%, and the protein sequence similarity was 70.00%. The present invention obtained an overexpression strain after transforming rapeseed with the overexpression vector pGWB418 (4×Myc)-NnXTH23-NOS constructed by heterologous transformation, which significantly improved the plant height and stem thickness of the overexpressed rapeseed; the overexpression strain was propagated in high generations, and the phenotype of the offspring was observed, and it was found that the gene could be stably inherited to the offspring, providing new genetic resources and guiding directions for the improvement of ornamental plant type and lodging-resistant plant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1 The figure is the result of amino acid sequence comparison between NnXTH23 gene and known Arabidopsis thaliana AtXTH23 gene;

[0038] Figure 2 Figure 1 is a schematic diagram of the construction process of the overexpression vector pGWB418 (4×Myc)-NnXTH23-NOS; A is a structural diagram of the vector pGWB418 (4×Myc)-NnXTH23-NOS; B is a schematic diagram of the vector construction process; C is the structure of the NnXTH23 (Nn2g12677.1) gene; LB: T-DNA left border; 35S: cauliflower virus 35S promoter; NOS: terminator; RB: T-DNA right border;

[0039] Figure 3 The flowchart of Agrobacterium-mediated genetic transformation of rapeseed hypocotyls;

[0040] Figure 4 A is the identification and expression analysis results of NnXTH23 overexpressing transgenic rapeseed positive plants; A is the electrophoresis identification result of NnXTH23 overexpressing transgenic rapeseed positive plants; B is the detection result of NnXTH23 expression level in NnXTH23 overexpressing transgenic rapeseed positive plants;

[0041] Figure 5 A is a graph showing the results of stem thickness detection of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants; A is a cross-sectional view and a side view of the stem of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants at the mature stage; B is a statistical result of the measured values ​​of stem thickness of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants at the mature stage;

[0042] Figure 6 A is a graph showing the plant height detection results of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants; A is a phenotypic graph of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants; B is a statistical result of plant height measurement values ​​of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] Example 1 Extraction, cloning and sequencing of NnXTH23 gene

[0049] (1) Isolation and cloning of NnXTH23 gene

[0050] 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 from the petioles of the lotus 'Gele Lingyin' variety 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 260 / 280 ratios between 1.9 and 2.1, 260 / 230 ratios greater than 2.0, and concentrations greater than 500ng / μL can be used for the next analysis. cDNA synthesis was performed using IIQ RT SuperMix for qRNA (+gDNAwiper) kit (purchased from Vazyme, China), with 1 μg of total RNA as a template, mixed with 4 μL 4×gDNAwiper Mix, DEPC-water, a total volume of 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.

[0051] In-fusion cloning primers NnXTH23-418F and NnXTH23-418R 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°C pre-denaturation for 1 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 31 cycles; 72°C extension for 5 min. The PCR product was recovered by gel recovery, and the recovery method was operated according to the UNIQ-10 column DNA gel recovery kit (Shanghai Sangon Biotechnology Co., Ltd.).

[0052] The above PCR products were cloned into the pGWB418 vector (Wuhan Puint Bioengineering Co., Ltd.). First, 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, sterile water was added to the final volume to 50μL, and the digestion was carried out at 37°C overnight. After 1% agarose gel electrophoresis, the single linearized vector fragment was recovered. The connection system of the PCR product and the pGWAB418 linearized vector was: 4.5μL PCR product, 0.5μL pGWB418 linearized vector, 5μL SolutionⅠ (Bao Bioengineering (Dalian) Co., Ltd.) was connected at 16°C 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 NnXTH23-418R. The positive colony was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0053] Primer information is as follows:

[0054] NnXTH23-418F: 5'-GACTTGAACGGTAGCGCTAATGGCTTATTCTTCCTCTACTTC-3' (SEQ IDNO.1);

[0055] NnXTH23-418R: 5'-TCGGGGAAATTCGAGCTCAATTTGTGAAATGCTTGGTATAGG-3' (SEQ IDNO.2);

[0056] 418F: 5'-TCGGGGAAATTCGAGCTC-3' (SEQ ID NO. 3).

[0057] (2) NnXTH23 gene sequence analysis

[0058] After sequencing, the amplified gene nucleotide sequence was 945 bp in length (SEQ ID NO.4), which contained an open reading frame ORF of 903 bp in length (SEQ ID NO.5). The amino acid sequence was compared 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 Acids Research, 1997, 25: 4876-82.) program. It was found that the gene was highly homologous to AtXTH23 of Arabidopsis thaliana at the amino acid level (similarity 70.00%), which was consistent with the results of the comparison on NCBI (such as Figure 1 ). The amino acid sequence encoded by the NnXTH23 gene is shown in SEQ ID NO.6.

[0059] SEQ ID NO.4:

[0060] AATGGCTTATTCTTCCTCTACTTCTCCTGGGCTCTCCTTAAAGTTTCTGTCTTGTCTACTGGTTTTGGGCTCTTTGGCTATTGTTGCTTCGGCTAGTAGTTTTTACCAAGATTTCGATGTCACATGGGGCGATGGTCGAGCAAAGATACTCAACACTGGGGAGCTTCTGACTCTGTCCCTTGACAAGGCCTCTGGCTCCGGCTTCCAATCCAAGCACGAGTACCTCTTCGGTAAGATAGACATGCAGCTCAAGCTTGTCCCCGGCAACTCAGCCGGCACGGTCACGGCCTACTACTTGTCATCTCAGGGGCCGACCCATGACGAGATTGACTTCGAATTCCTCGGCAACCTCAGCGGCGACCCTTATATTCTCCATACAAATGTCTTCAGTCAGGGAAAGGGAAACAGGGAGCAACAATTCTATCTCTGGTTCGATCCTACAGCAGACTTCCACACCTACTCCATCCTTTGGAACCCCCGACGCATAATCTTCTCTGTAGATGGAACGCCCATCAGAGAATTCAAGAACTCAGAATCCATTGGAGTTCCATTCCCCAAGAACCAGCCCATGAGGATCTACTCCAGCCTATGGAATGCTGATGACTGGGCCACCAGGGGTGGCCTTGTCAAGACCGATTGGACAAAGGCACCATTTACAGCCTCCTACAGAAACTTCAATGCCAATAACGCCTGCATCTGGTCTTCAGGCTCCTCTTCTTGCGACTCCAAATCTCCCACTTCAACGACAAATGATTCTCAATGGCTCTCGCAGGAGCTAGATTCAGCTGGTCAAGAGAAGATGAGATGGGTGCAAAAGAATTACATGATCTACAATTACTGCACGGATACAAAGCGCTTCCCCCGGGGCCTCCCTTCCGAATGCGCCAACGTTAACCTTTCTTAATTCTTAGAGCAACTTGTCCTATACCAAGCATTTCACAAATT;

[0061] SEQ ID NO.5:

[0062] ATGGCTTATTCTTCCTCTACTTCTCCTGGGCTCTCCTTAAAGTTTCTGTCTTGTCTACTGGTTTTGGGCTCTTTGGCTATTGTTGCTTCGGCTAGTAGTTTTTACCAAGATTTCGATGTCACATGGGGCGATGGTCGAGCAAAGATACTCAACACTGGGGAGCTTCTGACTCTGTCCCTTGACAAGGCCTCTGGCTCCGGCTTCCAATCCAAGCACGAGTACCTCTTCGGTAAGATAGACATGCAGCTCAAGCTTGTCCCCGGCAACTCAGCCGGCACGGTCACGGCCTACTACTTGTCATCTCAGGGGCCGACCCATGACGAGATTGACTTCGAATTCCTCGGCAACCTCAGCGGCGACCCTTATATTCTCCATACAAATGTCTTCAGTCAGGGAAAGGGAAACAGGGAGCAACAATTCTATCTCTGGTTCGATCCTACAGCAGACTTCCACACCTACTCCATCCTTTGGAACCCCCGACGCATAATCTTCTCTGTAGATGGAACGCCCATCAGAGAATTCAAGAACTCAGAATCCATTGGAGTTCCATTCCCCAAGAACCAGCCCATGAGGATCTACTCCAGCCTATGGAATGCTGATGACTGGGCCACCAGGGGTGGCCTTGTCAAGACCGATTGGACAAAGGCACCATTTACAGCCTCCTACAGAAACTTCAATGCCAATAACGCCTGCATCTGGTCTTCAGGCTCCTCTTCTTGCGACTCCAAATCTCCCACTTCAACGACAAATGATTCTCAATGGCTCTCGCAGGAGCTAGATTCAGCTGGTCAAGAGAAGATGAGATGGGTGCAAAAGAATTACATGATCTACAATTACTGCACGGATACAAAGCGCTTCCCCCGGGGCCTCCCTTCCGAATGCGCCAACGTTAACCTTTCTTAA;

[0063] SEQ ID NO.6:

[0064] MAYSSSSPGLSLKFLSCLLVLGSLAIVASASSFYQDFDVTWGDGRAKILNTGELLTLSLDKASGSGFQSKHEYLFGKIDMQLKLVPGNSAGTVTAYYLSSQGPTHDEIDFEFLGNLSGDPYILHTNVFSQGKGNREQQFYLWFDPTADF HTYSILWNPRRIIFSVDGTPIREFKNSESIGVPFPKNQPMRIYSSLWNADDWATRGGLVKTDWTKAPFTASYRNFNANNACIWSSGSSSCDSKSPTSTTNDSQWLSAGQEKMRWVQKNYMIYNYCTDTKRFPRGLPSECANVNLS.

[0065] Example 2 Obtaining NnXTH23 overexpressing transgenic rapeseed plants

[0066] (1) Construction of plant overexpression vector

[0067] AfeI and SacI restriction sites were added to both ends of the cloning primers NnXTH23-418F and NnXTH23-418R, respectively, and PCR products were obtained according to the method of Example 1. The pGWB418 vector was double-digested with AfeI and SacI, and the digestion system was: pGWB418 (1700ng / μL) 15μL, rCutsmartt Buffer 5μL, AfeI (10000U / L) 1μL, SacI (20000U / L) 1μL, sterile water was added to the final volume to 50μL, and 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.

[0068] The PCR product obtained by the above amplification was connected into the pGWB418 (4×Myc) linearized vector using In-fusion enzyme, and the positive clones were screened and sequenced to obtain the desired gene ORF, thereby obtaining the lotus NnXTH23 transgenic overexpression vector pGWB418 (4×Myc)-NnXTH23-NOS, whose T-DNA region contains the kanamycin resistance gene sequence, and the promoter of NnXTH23 overexpression is the D35s promoter. The structure of pGWB418 (4×Myc)-NnXTH23-NOS is shown in Figure 2 Middle A; see the above construction process Figure 2 B; the structure of NnXTH23 (Nn2g12677.1) gene is shown in Figure 2 Middle C.

[0069] (2) Genetic transformation of rapeseed

[0070] The recombinant plasmid (i.e., overexpression vector) pGWB418(4×Myc)-NnXTH23-NOS was transferred into Agrobacterium GV3101 by conventional hypocotyl infection method, and seedlings were selected and differentiated. The specific steps are as follows (the genetic transformation process of rapeseed hypocotyls is shown in Figure 3 ):

[0071] 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.

[0072] 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.

[0073] 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.

[0074] d. Infecting explants: Place the infection solution in step c on ice. At the same time, use a scalpel to cut the hypocotyls (length 0.8-1.0 cm) of the dark-cultured Brassica napus seedlings in step a, and 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, shaking every 3 minutes.

[0075] 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.

[0076] f. Screening: Transfer the co-cultivated explants 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, pH of the culture medium adjusted to 5.8-5.9, agar 7 g / L) for 20 days of screening culture at 24°C, 16 h light / 8 h dark culture.

[0077] 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, pH of the culture medium was adjusted to 5.8-5.9, agar 7 g / L was added, zeatin 2 mg / L, IAA 0.1 mg / L, timentin TMT 250 mg / L, Kan 25 mg / L were added after sterilization), and differentiation culture was started, with subculture every 15-20 days until differentiation and budding (culture conditions were consistent with the screening conditions in step f).

[0078] h. Rooting culture: When obvious growth points can be found on the differentiated seedlings, use a scalpel to carefully cut off 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.

[0079] (3) Identification of positive plants of NnXTH23 overexpressing transgenic rapeseed

[0080] The young leaves of the transformed plants were taken and DNA was extracted by CTAB method. The Kan gene on the vector was amplified by PCR for positive seedling identification. The primers used were Bn-Kan-F and Bn-Kan-R. The control was a non-transgenic wild-type plant (WT).

[0081] Primer information is as follows:

[0082] Bn-Kan-F: 5'-ACTGGGCACAACAGACAATCG-3' (SEQ ID NO.7);

[0083] Bn-Kan-R: 5'-GCATCAGCCATGATGGATACTTT-3' (SEQ ID NO. 8).

[0084] The specific steps of CTAB method for DNA extraction are as follows:

[0085] Take young leaves with a length of 1 to 2 cm, place them in an ice box prepared in advance, add 700 μL of 2×CTAB solution during the process, grind them into a fine slurry, transfer them to a 1.5 mL centrifuge tube, incubate at 70°C for 30 minutes, shake gently every 10 minutes, cool to room temperature, add 700 μL of chloroform:isoamyl alcohol with a volume ratio of 24:1, repeatedly invert and mix, and then shake for 1 to 2 minutes. Centrifuge at 10,000 rpm at room temperature for 5 minutes. Take about 600 μL of the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), shake vigorously for 1-2 minutes, and centrifuge at 10,000 rpm at room temperature for 5 minutes. Take about 500 μL of the supernatant and add an equal volume of isopropanol, mix gently, place at room temperature for 10-20 minutes, and centrifuge at 10,000 rpm at room temperature for 10-15 minutes to obtain a white precipitate. Slowly pour off the supernatant, take 800-900μL 75% anhydrous ethanol to wash and repeatedly blow the precipitate, wash twice and pour off the supernatant, centrifuge at 10000rpm for 3min, absorb the excess alcohol with a pipette, air dry for 10-15min, and add 30-50μL ddH2O to each sample to dissolve. The extracted rapeseed genomic DNA was tested for concentration using Nanodrop micro-nucleic acid detector.

[0086] Test results such as Figure 4 As shown in Figure A, the eight transformed plants, OE-1, OE-6, OE-18, OE-19, OE-25, OE-32, OE-35 and OE-38, were able to amplify electrophoresis bands of the expected size (289 bp), while the wild-type control had no electrophoresis bands, indicating that the transgenic rapeseed genome already contained exogenous gene DNA fragments.

[0087] (4) qRT-PCR identification of NnXTH23 overexpressing transgenic rapeseed

[0088] a. Extraction of genomic RNA from rapeseed leaves

[0089] use Super total RNA extraction kit (purchased from Promega, USA) was used to extract total RNA from leaves of WT and NnXTH23 overexpressing transgenic rapeseed positive plants (OE-1, OE-6, OE-18, OE-19, OE-25, OE-32, OE-35, OE-38) identified in step (3). RNA was reverse transcribed into cDNA using IIQ RT SuperMix for qRNA (+gDNAwiper) Kit (Vazyme, China).

[0090] b. Real-time fluorescence quantitative PCR

[0091] In order to determine whether NnXTH23 is overexpressed in rapeseed, the real-time fluorescence quantitative PCR (qRT-PCR) method was used to analyze the transgenic rapeseed positive plants identified in step (3) using the cDNA extracted as a template. Green Realtime PCR Master Mix-Plus-Kit (Takara Biotech (Dalian) Co., Ltd.), using the rape housekeeping gene BnActin7 (Brassica napus actin-7) as the internal reference gene, and its primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0092] The qRT-PCR primer information of BnActin7 (Gene ID: LOC106418315) is as follows:

[0093] BnActin7-F1: 5'-TCTTCCTCACGCTATCCCTCG-3' (SEQ ID NO.9);

[0094] BnActin7-R1: 5'-AGCCGTTCCAGCTCTTGC-3' (SEQ ID NO. 10).

[0095] The qRT-PCR primer information of the NnXTH23 gene is as follows (the primer pair is a qRT-PCR specific primer designed based on the nucleotide sequence sequenced in Example 1):

[0096] NnXTH23-F: 5'-TCTTGCGACTCCAAATCTCC-3' (SEQ ID NO. 11);

[0097] Q-NnXTH23-R: 5'-TAAGAAAGGTTAACGTTGGCG-3' (SEQ ID NO. 12).

[0098] 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).

[0099] Test results such as Figure 4 As shown in B (bars are mean ± SD, n>3 biological independent samples, showing a single data point; two-tailed t-test was used to determine the significant difference between groups: ****P<0.0001, *P<0.05, ns no significant difference), the expression levels of NnXTH23 in OE-1, OE-6, OE-18, OE-19, OE-25, OE-32, OE-35, and OE-38 strains were significantly higher than the wild-type control, the expression levels of OE-1, OE-32, and OE-38 strains were more than 35 times that of the wild-type, the expression levels of OE-19 and OE-25 strains were more than 15 times, the expression level of OE-6 strain was about 10 times that of the wild-type, and the expression levels of OE-18 and OE-35 strains were not significantly different from the wild-type. This indicates that these strains are independent NnXTH23 overexpressing transgenic strains.

[0100] Example 3 Effect of NnXTH23 overexpression on plant stem thickening and rapeseed plant height increase

[0101] (1) Determination of stem thickness of NnXTH23 overexpressing T3 rapeseed lines

[0102] The stem thickness of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants with fully mature siliques was measured. The measurement method is as follows: a. Observe the stems of rapeseed plants with the naked eye; b. Select T3 plants of three independent strains, OE-19, OE-25 and OE-38, and measure the diameter of the middle part of the internode from the last node at the base of the plant with a digital vernier caliper. The results are shown in Figure 2. Figure 5 As shown, the stem thickness of the three transgenic lines was significantly higher than that of the wild type.

[0103] (2) Determination of plant height of NnXTH23 overexpressing T3 generation rapeseed lines

[0104] The plant heights of wild-type rapeseed (WT) and NnXTH23 (OE) overexpressing plants at the silique stage were measured. The measurement methods were as follows: a. Observe the plant height of the rapeseed plants with the naked eye; b. When the plant height no longer increases significantly, select T3 plants of three independent strains, OE-19, OE-25 and OE-38, and measure their plant heights using a 3-meter telescopic measuring ruler. The measurement starts from the bottom of the cultivation pot to the highest point of the main inflorescence. The results are shown in Figure 2. Figure 6 As shown, the average heights of the three transgenic lines were significantly higher than those of the wild type.

[0105] The above test results indicate that the NnXTH23 gene can positively regulate plant stem thickness and plant height.

[0106] 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. NnXTH23 The application of genes or related biological materials, characterized in that: The application is: Increase plant height and increase plant stem thickness; Said NnXTH23 The nucleotide sequence of the gene is shown in SEQ ID NO.4; The plant is rapeseed; The relevant biological material is any one of the following B1 to B3: B1. Contains the NnXTH23 expression cassette of the gene; B2. Contains the NnXTH23 Recombinant vectors of genes; B3. Contains the NnXTH23 Genetically recombinant microorganisms.

2. A method for increasing plant height and stem thickness, characterized in that: Will NnXTH23 Genes or related biological materials are introduced into plants to NnXTH23 Stable gene overexpression increases plant height and stem thickness; Said NnXTH23 The nucleotide sequence of the gene is shown in SEQ ID NO.4; the related biological material is any one of the following B1 to B3: B1. Contains the NnXTH23 expression cassette of the gene; B2. Contains the NnXTH23 Recombinant vectors of genes; B3. Contains the NnXTH23 Genetically recombinant microorganisms; The plant is rapeseed.

Images