Application of a xyloglucan endotransglucosylase / hydrolase gene

Abstract

The present invention provides an application of a xyloglucan endotransglucosylase / hydrolase gene, belonging to the technical field of genetic engineering. The application of the xyloglucan endotransglucosylase / hydrolase gene or its related biological material of the present invention includes any one of the following applications: A1. Increasing plant height and increasing plant stem thickness; A2. Cultivating large-plant-type plant lines; A3. Cultivating lodging-resistant plant lines; the nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3. The present invention discovers that the xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674) in lotus participates in regulating plant stem development. Overexpressing this gene in rapeseed can significantly increase the plant height and stem thickness of rapeseed, providing new technical guidance for the improvement of plant type and function of lotus and other plants.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to the application of a xyloglucan endotransglucosylase / hydrolase gene. Background Art

[0002] Lotus (Nelumbo nucifera Gaertn.) is a perennial aquatic herb of the genus Nelumbo in the family Nelumbonaceae. It is the only aquatic flower among the top ten traditional famous flowers in China, with different colors, scents, postures, and rhythms, and has extremely high ornamental value. Lotus is divided into three major series: Chinese lotus series, American lotus series, and Sino-US hybrid lotus series. According to the lotus variety classification standard, the plant type size (including the flower diameter size that is positively correlated with it) is listed as a secondary standard to specifically classify the plant type and flower diameter. It is stipulated that the standing leaf height is more than 50 cm, the leaf diameter is more than 30 cm, and the flower diameter is more than 18 cm for large plant type or large flower type varieties. Any variety with a lower value than any of these indicators is classified as a medium or small variety. Large plant type varieties and medium plant type varieties are usually planted in groups on the water bank for garden and courtyard landscaping and cut flower viewing, such as 'Yiliang Qianban' (N.nucifera 'Yiliang Qianban'), 'Youyi Mudan' (N.nucifera 'Youyi Mudan'), etc., while small varieties are planted in small containers for home gardening viewing, such as 'Xinghuo' (N.nucifera 'Xinghuo'), 'Yanyu' (N.nucifera 'Yanyu'), etc. With the development of genetic engineering and molecular biotechnology, through the excavation and functional analysis of special plant type genes, molecular breeding has become a shortcut to quickly obtain varieties with ideal plant types.

[0003] As the most abundant biomass repository on Earth, the plant cell wall has the functions of support and protection, determines the cell morphology and mechanical characteristics, and simultaneously senses extracellular signals, which is crucial for plant growth and development. Its main components include structural polysaccharides such as cellulose, hemicellulose, and pectin, lignin, and a small amount of proteins and minerals. The proteins encoded by XTH genes catalyze the hydrolysis and synthesis of xyloglucan molecules, which are components of hemicellulose, and participate in the elongation and extension of the plant cell wall. They have the activities of two enzymes: one is xyloglucan endotransglucosylase (XET), which mainly acts on the β-1,4 glycosidic bond of xyloglucan to catalyze its cleavage, and the generated glycosyl groups combine with other oligosaccharide molecules; the activity of the other enzyme is hydrolase activity (XEH), which participates in catalyzing the hydrolysis of xyloglucan molecules. Studies have shown that XTH is involved in the life activities of various plants and is closely related to plant growth and development. For example, in the model plant Arabidopsis thaliana, AtXTH9 is specifically expressed in the shoot apical meristems of flower buds and flower stalks, and it is speculated that it is related to the elongation of stems and flower stalks. Further studies have found that the deletion mutation of this gene will cause the internodes of the plant to become shorter. Experiments on pea stem segments have proved that xyloglucan endotransglucosylase integrates xyloglucan to inhibit the elongation of the stem cell wall, while the addition of oligosaccharides can promote the elongation of the stem. The expression profiles of two adzuki bean XTH genes (VaXTH1 and VaXTH2) were studied, and it was found that the VaXTH1 gene was highly expressed at the base of the stem internodes, while VaXTH2 was highly expressed at the top of the internodes, and both of them responded to the internode elongation process induced by auxin. It is speculated that these two XTH genes play a role in stem elongation. Overexpression of the BcXTH1 gene promotes the elongation of the flower stalks of transgenic Arabidopsis thaliana, and the plant height increases significantly. In summary, XTH plays an important role in plant growth and development, especially in stem elongation and plant height increase.

[0004] At present, plant type breeding is one of the important directions in lotus breeding. The main means of traditional lotus plant type breeding are achieved through hybridization techniques or seedling selection breeding. It has the disadvantages of long cycle, low efficiency, and high uncertainty. The emergence of genetic engineering technology can quickly obtain ideal plant type varieties through the excavation and functional analysis of special plant type genes. At present, there is no report on the participation of lotus XTH family genes in lotus plant type formation. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of xyloglucan endotransglucosylase / hydrolase genes to solve the problems existing in the above-mentioned prior art. The present invention discovers that the xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674) in lotus participates in regulating plant stem development. Overexpressing this gene in rapeseed can significantly increase the plant height and stem thickness of rapeseed, providing new technical guidance for the improvement of lotus and other plant types and functions.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides the application of the xyloglucan endotransglucosylase / hydrolase gene or its related biological materials, including any of the following applications:

[0008] A1. Increasing the plant height and the stem thickness of plants;

[0009] A2. Cultivating plant lines with large plant types;

[0010] A3. Cultivating lodging-resistant plant lines;

[0011] The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3.

[0012] Preferably, the related biological material is any one of the following B1 to B3:

[0013] B1. An expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene;

[0014] B2. A recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene;

[0015] B3. A recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene.

[0016] The present invention provides a recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene with the sequence as shown in SEQ ID NO.3.

[0017] The present invention provides a recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene with the sequence as shown in SEQ ID NO.3, or containing the above-mentioned recombinant vector.

[0018] The present invention provides a method for increasing the plant height and the stem thickness of plants, which comprises transferring the xyloglucan endotransglucosylase / hydrolase gene or its related biological materials into plants to enable the stable overexpression of the xyloglucan endotransglucosylase / hydrolase gene, thereby increasing the plant height and the stem thickness of plants;

[0019] The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3; the related biological material is any one of the following B1 to B3:

[0020] B1. An expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene;

[0021] B2. A recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene;

[0022] B3. A recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene.

[0023] The present invention provides a method for cultivating large plant lines. The xyloglucan endotransglucosylase / hydrolase gene or its related biological materials are transferred into a plant body, so that the xyloglucan endotransglucosylase / hydrolase gene is stably overexpressed, and large plant lines are cultivated;

[0024] The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3; the related biological materials are any one of the following B1 to B3:

[0025] B1, an expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene;

[0026] B2, a recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene;

[0027] B3, a recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene.

[0028] The present invention provides a method for cultivating lodging-resistant plant lines. The xyloglucan endotransglucosylase / hydrolase gene or its related biological materials are transferred into a plant body, so that the xyloglucan endotransglucosylase / hydrolase gene is stably overexpressed, and lodging-resistant plant lines are cultivated;

[0029] The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3; the related biological materials are any one of the following B1 to B3:

[0030] B1, an expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene;

[0031] B2, a recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene;

[0032] B3, a recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene.

[0033] The present invention discloses the following technical effects:

[0034] The present invention cloned the xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674) from a large lotus variety, constructed an overexpression vector pGWB418(4×Myc)-NnXTH23-NOS of this gene and transformed rapeseed, and conducted a series of studies on the overexpressed rapeseed lines in terms of plant height increase, stem diameter increase, etc. It fully proves that the xyloglucan endotransglucosylase / hydrolase gene is involved in the development of plant stems and plays an important role. By regulating the expression of the gene and increasing the plant height and stem diameter, it is of great significance for clarifying the biological function of the gene.

[0035] Breeding plant materials that can regulate the plant height and stem diameter of lotus has always been highly regarded by lotus breeders. The gene cloned in this invention will contribute to solving the problem of lotus plant type improvement. The cloning of the gene will help cultivate large and medium-sized lotus varieties, and also provide theoretical and practical basis for its application in plant trait improvement; it has important practical guiding value in the breeding practice, variety improvement and variety promotion of lotus, and also increases genetic resources for the plant type breeding of other plants. Brief Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0037] Figure 1 Alignment result of the amino acid sequences of the xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674) and the known Arabidopsis thaliana AtXTH23 gene;

[0038] Figure 2 Schematic diagram of the construction process of the pGWB418(4×Myc)-NnXTH23-NOS vector; where, LB: left border of T-DNA; 35S: cauliflower mosaic virus 35S promoter; NOS: terminator; RB: right border of T-DNA;

[0039] Figure 3 Flow chart of Agrobacterium-mediated genetic transformation of rapeseed hypocotyls;

[0040] Figure 4 Identification and expression level analysis results of overexpressing transgenic rapeseed positive plants; where, A is the identification result of overexpressing transgenic rapeseed positive plants, PCR amplifies the Kan gene fragment on the vector, and the target size is 289bp; B is the detection result of the expression level in overexpressing transgenic rapeseed positive plants;

[0041] Figure 5 Plant height detection result diagram of wild-type rapeseed (WT) and overexpressing (OE) plants; A is the phenotype diagram of wild-type rapeseed (WT) and overexpressing (OE) plants, and the scale is 20cm; B is the statistical result of the plant height measurement values of wild-type rapeseed (WT) and overexpressing (OE) plants;

[0042] Figure 6Detection result diagram of the stem thickness of wild - type rapeseed (WT) and over - expressing plants (OE); A is the side view of the stems of wild - type rapeseed (WT) and over - expressing plants (OE) at the mature stage, and the scale bar is 10 cm; B is the cross - sectional view of the stems of wild - type rapeseed (WT) and over - expressing plants (OE) at the mature stage, and the scale bar is 2 cm; C is the statistical result of the measured values of the stem thickness of wild - type rapeseed (WT) and over - expressing plants (OE) at the mature stage. Detailed implementation manners

[0043] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0044] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0045] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0046] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0047] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open - ended terms, meaning including but not limited to.

[0048] Example 1 Extraction, cloning, and sequencing of the xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674)

[0049] (1) Isolation and cloning of the xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674)

[0050] The large lotus cultivar 'Gele Lingyin' (provided by the National Lotus Germplasm Resource Bank of Southwest Forestry University) was selected as the experimental material. The total RNA of lotus stems was extracted using Super Total RNA Extraction Kit (purchased from Promega Corporation, USA). After RNA extraction, it was treated with DNase I (purchased from the Promega kit). The integrity of RNA was detected by electrophoresis on a 1.2% (w / v) agarose gel (EtBr) at 5 V / cm. The determination of nucleic acid concentration was carried out on an IMPLEN Nano Photometer-N50 series ultra-micro ultraviolet spectrophotometer (Germany). RNA with a 260 / 280 ratio between 1.9 and 2.1, a 260 / 230 ratio greater than 2.0, and a concentration greater than 500 ng / μL could be used for the next analysis. The synthesis of cDNA was carried out using IIQRT SuperMix for qRNA(+gDNA wiper) Kit (purchased from Vazyme Biotech Co., Ltd., China). Using 1 μg of total RNA as a template, it was mixed with 4 μL of 4×gDNAwiper Mix and DEPC-water, with a total volume of 16 μL. Incubate at 42°C for 2 min and then quickly cool on ice for 2 - 3 min; then add 4 μL of 5×Hiscript II qRT Super Mix II and mix well, with a total volume of 20 μL; then incubate at 50°C for 15 min and 85°C for 5 sec. After each cDNA was diluted to 200 μL, it was stored at -20°C for later use.

[0051] In-fusion cloning primers Nn2g12674-418F and Nn2g12674-418R were used to amplify the target band, and TransTaq HiFi DNA Polymerase (TransGen Biotech Co., Ltd., Beijing) was used for amplification. The PCR reaction conditions were: pre-denaturation at 94°C for 3 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min 30 sec, for 32 cycles; extension at 72°C for 5 min. After electrophoresis on a 1% agarose gel, the PCR products were recovered, and the recovery method was carried out according to the UNIQ-10 Column DNA Gel Extraction Kit (Sangon Biotech Co., Ltd., Shanghai).

[0052] Clone the above PCR products into the pGWB418 vector. First, double-digest the pGWB418 vector with AfeI and SacI. The digestion system is as follows: 15 μL of pGWB418 (1700 ng / μL), 5 μL of rCutsmart Buffer, 1 μL of AfeI (10000 U / L), 1 μL of SacI (20000 U / L), and make up the final volume to 50 μL with sterile water. Digest overnight at 37°C. After 1% agarose gel electrophoresis, recover the single linearized vector fragment. The ligation system of the PCR product and the linearized pGWAB418 vector is as follows: 4.5 μL of the target fragment, 0.5 μL of the pGWB418 vector, and 5 μL of SolutionⅠ (TaKaRa Bio Inc., Dalian). Ligate overnight at 16°C. Transform the ligation product into DH5α competent cells by heat shock. Spread the bacterial solution on an LB solid plate containing 100 mg / L Kanamycin antibiotic. After growing for about 10 - 12 h, pick single colonies for colony PCR. The primers are the universal primers 418F / R. Send the positive colonies to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0053] The primer information is as follows:

[0054] Nn2g12674-418F: 5’-GACTTGAACGGTAGCGCTCCAGTTCCTCATACTTCGAGAGTC-3’ (SEQ ID NO.1);

[0055] Nn2g12674-418R: 5’-TCGGGGAAATTCGAGCTCCTCTAAGATGTGTTGGTGTCGG-3’ (SEQ ID NO.2).

[0056] (2) Sequence analysis of the xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674)

[0057] Sequencing showed that the full length of the gene nucleotide sequence amplified by the above primers was 949 bp (SEQ ID NO.3), which contained an open reading frame ORF of 891 bp in length (SEQ ID NO.4). 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 was used for amino acid sequence alignment and it was found that this target band was highly homologous to AtXTH23 of Arabidopsis thaliana at the amino acid level (72.3% similarity), which was consistent with the result of alignment on NCBI( Figure 1 ). The amino acid sequence of this gene is shown in SEQ ID NO.5.

[0058] SEQ ID NO.3:

[0059] CCAGTTCCTCATACTTCGAGAGTCCTACTATCCAAATCTCCGCTCAACAATTACCTATGGCTTCTTCTCATGGGTTTTTCTTAAAATCTCTGTTTTGTTTTCTGGTTTTGATATCTTTGGCCATTGCTACCGTTTCAGCTAGTAGTTTCAACCAAGACTTCGACATCACATGGGGCGATGGTCGAGCAAAGATACTCAACAATGGGGATCTTCTCACTCTGTCCCTTGACAAAGCCTCTGGCTCTGGCTTCCAATCCAGGCACGAGTACCTCTTTGGTAAGATAGACATGCAGCTCAAGCTCGTCGCCGGCAACTCAGCCGGCACGGTCACGGCCTACTACTTATCATCTCAGGGTCCGACCCATGACGAGATTGACTTCGAATTTCTCGGCAACCTCAGCGGTGATCCCTATATTCTACACACTAATGTCTTCAGCCAGGGGAAGGGAAACAGGGAGCAACAGTTCTATCTCTGGTTCGATCCTACAGCAGACTTCCACACTTATTCCATTCTCTGGAACCCCCAACGCATTATCTTCTCTGTAGATGGAATACCCATAAGAGAATTCAAGAACTCAGAATCCATTGGAGTTCCATTCCCCAAGAATCAGGCCATGAGGATCTACTCCAGCCTATGGAATGCTGATGATTGGGCCACAAGGGGTGGCCTTGTCAAGACCGATTGGACAAAAGCACCATTTACAGCTTCCTATAGAAACTTCAACGCCAACGCCTGCATCATGTCTTCAGGCTCCTCTTCTTGTGACTCCAAATCTCCCACTTCAACAACTGACAATCTCCAATGGCTCTCGCAAGAGCTGGACTCAGCTGGTCAAGAGAGGATGAAATGGGTGCAGAACAATTATATGATCTACAATTACTGCACAGATACAAAGCGTTTTCCTCAGGGCCTCCCTCCTGAGTGCTCCGACACCAACACATCTTAGAG。

[0060] SEQ ID NO.4:

[0061] ATGGCTTCTTCTCATGGGTTTTTCTTAAAATCTCTGTTTTGTTTTCTGGTTTTGATATCTTTGGCCATTGCTACCGTTTCAGCTAGTAGTTTCAACCAAGACTTCGACATCACATGGGGCGATGGTCGAGCAAAGATACTCAACAATGGGGATCTTCTCACTCTGTCCCTTGACAAAGCCTCTGGCTCTGGCTTCCAATCCAGGCACGAGTACCTCTTTGGTAAGATAGACATGCAGCTCAAGCTCGTCGCCGGCAACTCAGCCGGCACGGTCACGGCCTACTACTTATCATCTCAGGGTCCGACCCATGACGAGATTGACTTCGAATTTCTCGGCAACCTCAGCGGTGATCCCTATATTCTACACACTAATGTCTTCAGCCAGGGGAAGGGAAACAGGGAGCAACAGTTCTATCTCTGGTTCGATCCTACAGCAGACTTCCACACTTATTCCATTCTCTGGAACCCCCAACGCATTATCTTCTCTGTAGATGGAATACCCATAAGAGAATTCAAGAACTCAGAATCCATTGGAGTTCCATTCCCCAAGAATCAGGCCATGAGGATCTACTCCAGCCTATGGAATGCTGATGATTGGGCCACAAGGGGTGGCCTTGTCAAGACCGATTGGACAAAAGCACCATTTACAGCTTCCTATAGAAACTTCAACGCCAACGCCTGCATCATGTCTTCAGGCTCCTCTTCTTGTGACTCCAAATCTCCCACTTCAACAACTGACAATCTCCAATGGCTCTCGCAAGAGCTGGACTCAGCTGGTCAAGAGAGGATGAAATGGGTGCAGAACAATTATATGATCTACAATTACTGCACAGATACAAAGCGTTTTCCTCAGGGCCTCCCTCCTGAGTGCTCCGACACCAACACATCTTAG。

[0062] SEQ ID NO.5:

[0063] MASSHGFFLKSLFCFLVLISLAIATVSASSFNQDFDITWGDGRAKILNNGDLLTLSLDKASGSGFQSRHEYLFGKIDMQLKLVAGNSAGTVTAYYLSSQGPTHDEIDFEFLGNLSGDPYILHTNVFSQGKGNREQQFYLWFDPTADFHTYSILWNPQRIIFSVDGIPIREFKNSESIGVPFPKNQAMRIYSSLWNADDWATRGGLVKTDWTKAPFTASYRNFNANACIMSSGSSSCDSKSPTSTTDNLQWLSQELDSAGQERMKWVQNNYMIYNYCTDTKRFPQGLPPECSDTNTS。

[0064] Example 2 Obtaining of Transgenic Rapeseed Plants with Overexpression of Xyloglucan Endotransglucosylase / Hydrolase Gene (Nn2g12674)

[0065] (1) Construction of Plant Overexpression Vector

[0066] Add Afel and Sacl restriction enzyme sites to both ends of the cloning and amplification primers respectively. Name the designed primers Nn2g12674-418F (SEQ ID NO.1) and Nn2g12674-418R (SEQ ID NO.2). Amplify the PCR product according to the method of Example 1. Digest the pGWB418 vector with AfeI and SacI. The digestion system is: pGWB418 (1700 ng / μL) 15 μL, rCutsmartt Buffer 5 μL, AfeI (10000 U / L) 1 μL, SacI (20000 U / L) 1 μL, make up the final volume to 50 μL with sterile water, and digest overnight at 37°C. After 1% agarose gel electrophoresis, recover the single linearized vector fragment and name it pGWB418(4×Myc) linearized vector.

[0067] Use In-fusion enzyme to ligate the above amplified PCR product into the pGWB418(4×Myc) linearized vector, screen positive clones and identify them by sequencing, so as to obtain the transgenic overexpression vector of lotus, and this vector is named pGWB418(4×Myc)-NnXTH23-NOS. The construction process is as Figure 2 shown. The gene sequence of kanamycin resistance is contained in the T-DNA region of this vector, and the promoter for overexpression is the D35s promoter.

[0068] (2) Genetic Transformation of Rapeseed

[0069] The recombinant plasmid (i.e., overexpression vector) pGWB418(4×Myc)-NnXTH23-NOS was transferred into Agrobacterium tumefaciens GV3101 by the conventional hypocotyl infection method, and then screened and differentiated into seedlings. The specific steps are as follows (for the transformation process, see Figure 3 ):

[0070] a. Sowing: Wash Brassica napus westar seeds with 75% alcohol for 1 min, then wash the seeds with 0.1% mercuric chloride solution for 5 min, and finally wash the seeds 5 times with sterile water; Use sterilized forceps to put the seeds into M0 solid medium (MS inorganic salts and trace elements of the 1962 formula, 4.404 g / L, adjust the pH to 5.8 - 5.9, add agar 7 g / L, sterilize according to the conventional autoclaving method), and incubate the inoculated seeds in the dark at 24 °C for 5 days.

[0071] b. Activation of Agrobacterium tumefaciens: Three days after sowing, streak the Agrobacterium tumefaciens strain GV3101 stored at -80 °C on LB solid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL), and culture at 28 °C for 16 h. Pick a single colony into 5 mL of LB liquid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL), and culture with shaking at 28 °C and 200 rpm for 20 - 24 h until the Agrobacterium grows to the logarithmic phase. Take 500 μL of the cultured bacterial solution and expand the culture in 50 mL of LB liquid medium (containing the three antibiotics) for about 12 h.

[0072] c. Preparation of infection bacterial solution: Pour the activated Agrobacterium liquid into 2 sterile 50 mL centrifuge tubes, centrifuge at 3500 rpm for 15 min, discard the supernatant, place on ice, wash the bacterial solution with 1 mL of DM liquid medium (MS inorganic salts and trace elements of the 1962 formula, 4.404 g / L, sucrose 30 g / L, adjust the pH of the medium to 5.8 - 5.9, sterilize at 121 °C for 20 min, add 2,4-D 1 mg / L, AS 100 mmol / L, KT 0.3 mg / L in the sterile operating table after sterilization), centrifuge, discard the supernatant, and resuspend the cells with 2 - 3 mL of DM liquid medium to make the OD600 of the infection bacterial solution about 0.6.

[0073] d. Infection of explants: Place the infection bacterial solution in step c on ice. At the same time, use a scalpel to cut the hypocotyls of the Brassica napus seedlings cultured in the dark in step a (the length of each hypocotyl section is preferably 0.8 - 1.0 cm), and use sterile forceps to transfer the cut hypocotyls into a petri dish containing the infection bacterial solution, and infect for 15 - 30 min according to the bacterial solution concentration (shake it every 3 min).

[0074] e. Co-culture: Transfer the infected explants to a petri dish filled with filter paper (which should be sterilized in advance), blot dry the visible infection fluid on the surface of the explants, and then transfer them to M1 solid medium (MS inorganic salts and trace elements formulated in 1962, 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 medium to 5.8 - 5.9, add agar 7 g / L, and add 100 mmol / L AS after sterilization), and culture in the dark at 24 °C for 40 - 48 h.

[0075] f. Screening: Transfer the co-cultured explants to M2 solid medium (MS inorganic salts and trace elements formulated in 1962, 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 medium to 5.8 - 5.9, add agar 7 g / L), and conduct screening culture for 20 days under the conditions of 24 °C, 16 h light / 8 h dark culture.

[0076] g. Differentiation culture: Transfer the screened explants to M3 solid medium (MS inorganic salts and trace elements formulated in 1962, 4.404 g / L, glucose 10 g / L, xylose 0.25 g / L, yeast extract (MES) 0.6 g / L, adjust the pH of the medium to 5.8 - 5.9, add agar 7 g / L, and add zeatin (ZT) 2 mg / L, IAA 0.1 mg / L, TMT 250 mg / L, Kan 25 mg / L after sterilization), start differentiation culture, and subculture every 15 - 20 days until buds are differentiated (the culture conditions are the same as the screening conditions in step f).

[0077] h. Rooting culture: After obvious growth points can be found on the seedlings with differentiated buds, use a scalpel to carefully cut the seedlings along the position where the callus and the buds are connected (be careful not to damage the growth points during the operation process), and then transfer the seedlings to M4 solid medium (MS inorganic salts and trace elements formulated in 1962, 2.202 g, sucrose 10 g / L, IBA 0.5 mg / L, adjust the pH of the medium to 5.8 - 5.9, add agar 7 g / L, and add TMT 250 mg / L, Kan 25 mg / L after sterilization) for rooting.

[0078] (3) Identification of overexpressed transgenic positive plants

[0079] Take the young leaves of the transformed plants, extract DNA using the CTAB method, and identify positive seedlings by PCR amplifying the Kan gene on the vector. The primer information used is as follows:

[0080] Bn-Kan-F: 5’ACTGGGCACAACAGACAATCG3’ (SEQ ID NO.6);

[0081] Bn-Kan-R: 5’GCATCAGCCATGATGGATACTTT3’ (SEQ ID NO.7).

[0082] The control was non-transgenic wild-type plants (WT).

[0083] The specific steps for DNA extraction by the CTAB method are as follows:

[0084] DNA was extracted using the conventional CTAB method. The specific steps were as follows: Take young Brassica napus leaves with a length of 1 - 2 cm and place them in a pre-cooled mortar. Add liquid nitrogen 2 - 3 times during the process and grind until it becomes a fine paste. Then transfer it to a 1.5 mL centrifuge tube and add 700 μL of 2×CTAB solution. Incubate at 70°C for 30 min, gently shake once every 6 min, and incubate at 70°C for another 30 min, gently shake once every 10 min. Cool to room temperature, then add 700 μL of Tris-saturated phenol:chloroform:isoamyl alcohol with a volume ratio of 25:24:1. Invert and mix repeatedly, and then gently shake about 40 times. Centrifuge at 3100 rpm at room temperature for 15 min. Pipette about 500 μL of the supernatant, add an equal volume of chloroform:isoamyl alcohol with a volume ratio of 24:1. After shaking well, centrifuge at 3100 rpm at room temperature for 15 min. Discard the supernatant, add 1 mL of frozen -20°C absolute ethanol, incubate in an ice bath at -20°C for 30 min, then centrifuge at 12000 rpm at room temperature for 10 min. Wash the precipitate with 75% alcohol and repeatedly blow and beat it for 3 min to remove salts. Pour out the alcohol, air-dry it, and dissolve each sample with 30 - 50 μL of ddH2O. The extracted genomic DNA of Brassica napus was detected for concentration using a Nanodrop micro nucleic acid detector.

[0085] The detection results are as Figure 4 shown in A below. For the 6 transgenic plants numbered 2, 3, 7, 8, 18, and 22, electrophoretic bands of the expected size (289 bp) could be amplified, while the wild-type and ddH2O controls did not show electrophoretic bands, indicating that the transgenic rapeseed genome already contained exogenous gene DNA fragments. Add OE in front of the numbers of the identified transgenic positive plants.

[0086] (4) qRT-PCR identification of overexpressing transgenic rapeseed

[0087] a. Extraction of genomic RNA from Brassica napus leaves

[0088] Using Super Total RNA Extraction Kit (purchased from Promega, USA), extract the total RNA from the leaves of WT and the overexpressing positive lines identified in step (3). And use IIQ RT SuperMix for qRNA(+gDNAwiper) Kit (Vazyme, China) to reverse transcribe RNA into cDNA.

[0089] b. Real-time fluorescence quantitative PCR

[0090] To determine whether it is overexpressed in rapeseed, using the above-extracted cDNA as a template, analyze the transgenic plants identified in step (3) by real-time fluorescence quantitative PCR (qRT-PCR). qRT-PCR uses Green Realtime PCR Master Mix-Plus- Kit (TaKaRa Bio Inc., Dalian, China), use the rapeseed housekeeping gene BnActin7 as an internal reference gene, and its primers are synthesized by Nanjing GenScript Biotech Co., Ltd. The qRT-PCR primer information of BnActin7 (GeneID:LOC106418315) is as follows:

[0091] BnActin7-F1: 5’-TCTTCCTCACGCTATCCTCCG-3’ (SEQ ID NO.8);

[0092] BnActin7-R1: 5’-AGCCGTCTCCAGCTCTTGC-3’ (SEQ ID NO.9).

[0093] The qRT-PCR primer information of the gene is as follows:

[0094] Q-Nn2g12674-F: 5’-ACAACCAGTTCCTCATACTTCG-3’ (SEQ ID NO.10);

[0095] Q-Nn2g12674-R: 5’-ATCTTTGCTCGACCATCGCC-3’ (SEQ ID NO.11).

[0096] (This pair of primers is a qRT-PCR specific primer designed based on the nucleotide sequence obtained by sequencing in Example 1)

[0097] PCR program: Pre-denature at 95°C for 30 sec, and then go through 40 cycles (95°C for 10 sec, 60°C for 10 sec, 72°C for 26 sec).

[0098] The detection results are as Figure 4Shown in B (columns are mean ± SD, n > 3 biologically independent samples, showing individual data points; two-tailed t-test was used to determine significant differences between groups: ****P < 0.0001, *P < 0.05, ns no significant difference). The expression levels of OE-2, OE-3, OE-7, OE-8, OE-18, and OE-22 lines were all significantly higher than those of the non-transgenic wild-type control. Among them, the most significant were OE-22 and OE-8 lines, and their expression levels increased by 5244.81% and 6509.38% respectively compared with the wild-type, indicating that these lines were independent overexpressing transgenic lines.

[0099] Example 3 Evaluation of the increase in plant height and stem thickness of T2 generation overexpressing transgenic rapeseed with xyloglucan endotransglucosylase / hydrolase gene (Nn2g12674)

[0100] (1) Measurement of plant height of T2 generation rapeseed in overexpressing lines

[0101] The plant heights of wild-type rapeseed (WT) and transgenic lines OE-3, OE-8, and OE-22 at the mature stage of silique development were measured. At least 3 plants of each line were measured, and the measurement height was from the seedling pot to the top of the highest inflorescence. The results are as Figure 5 shown. The plant height of OE-3 line increased by 37.89% compared with WT, the plant height of OE-8 line increased by 29.82% compared with WT, and the plant height of OE-22 line increased by 36.90% compared with WT, indicating that the xyloglucan endotransglucosylase / hydrolase gene had a good application effect in enhancing plant height.

[0102] (2) Measurement of stem thickness of T2 generation rapeseed in overexpressing lines

[0103] The stems of transgenic rapeseed at the mature stage of silique development were harvested. The measurement method was to randomly select at least 3 plants of each line and measure the diameter at the middle of the penultimate internode of the plant with a digital vernier caliper, and try to ensure that the measurement parts were consistent. The results are as Figure 6 shown. The stem thicknesses of OE-3, OE-8, and OE-22 lines all increased by more than 20% compared with the wild-type. Among them, the increase in stem thickness of OE-22 was the most significant, increasing by 35.08%, indicating that the xyloglucan endotransglucosylase / hydrolase gene played an important role in stem thickening.

[0104] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of xyloglucan endotransglucosylase / hydrolase gene or its related biological material, characterized in that, An application as described in any of the following: A1. Increasing plant height and increasing the stem thickness of plants; A2. Cultivating large-plant-type plant lines; A3. Cultivating lodging-resistant plant lines; The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3; The related biological material is any one of the following B1 - B3: B1. An expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene; B2. A recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene; B3. A recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene; The plant is rapeseed.

2. A method for increasing plant height and stem thickness, characterized in that, Transfer the xyloglucan endotransglucosylase / hydrolase gene or its related biological material into the plant body to stably overexpress the xyloglucan endotransglucosylase / hydrolase gene, and increase plant height and stem thickness; The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3; the related biological material is any one of the following B1 - B3: B1. An expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene; B2. A recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene; B3. A recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene; The plant is rapeseed.

3. A method for cultivating large plant strains, characterized in that, Transfer the xyloglucan endotransglucosylase / hydrolase gene or its related biological material into the plant body to stably overexpress the xyloglucan endotransglucosylase / hydrolase gene, and cultivate large plant lines; The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3; the related biological material is any one of the following B1 - B3: B1. An expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene; B2. A recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene; B3. A recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene; The plant is rapeseed.

4. A method for cultivating a lodging-resistant plant line, characterized in that, Transfer the xyloglucan endotransglucosylase / hydrolase gene or its related biological material into the plant body to stably overexpress the xyloglucan endotransglucosylase / hydrolase gene, and cultivate lodging-resistant plant lines; The nucleotide sequence of the xyloglucan endotransglucosylase / hydrolase gene is as shown in SEQ ID NO.3; the related biological material is any one of the following B1 - B3: B1. An expression cassette containing the xyloglucan endotransglucosylase / hydrolase gene; B2. A recombinant vector containing the xyloglucan endotransglucosylase / hydrolase gene; B3. A recombinant microorganism containing the xyloglucan endotransglucosylase / hydrolase gene; The plant is rapeseed.

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