Application of Lotus NnSAUR36 Gene in Improving Plant Height, Stem Strength and Biomass

Abstract

The present invention discloses the application of the lotus NnSAUR36 gene in improving plant height, stem strength and biomass, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the lotus NnSAUR36 gene of the present invention is shown in SEQ ID NO.4. The present invention finds that the lotus NnSAUR36 gene (Nn1g01094.1) can positively regulate plant height, stem strength and plant biomass. By overexpressing the gene, the lodging resistance of the plant can be significantly improved, the plant type can be improved and the plant biomass can be increased. The gene has important application prospects and provides a new way for molecular breeding and genetic improvement of lotus and other plants.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of lotus NnSAUR36 gene in improving plant height, stem strength and biomass. Background Art

[0002] Lotus (Nelumbo nucifera G.) is a perennial aquatic herbaceous plant of the genus Nelumbo in the family Nelumbonaceae. It is the only aquatic flower among the ten traditional famous flowers in China and an ancient dicotyledonous plant. It is an excellent wetland construction and family garden flower with diverse plant types, flower colors and flower shapes. The morphological construction of its plants is closely related to its ornamental characteristics and market applications. Different plant types have different application attributes. Lotus is a potential fresh cut flower resource, and the plant type and cut flower stem quality are closely related to the development of the stem. However, due to the high heterozygosity of the lotus genome, the current research on lotus mainly focuses on the collection of germplasm resources, the cultivation of new varieties, systematic development and classification, medicinal and edible functions, the mechanism of ornamental traits, and the creation of regeneration and genetic transformation systems. There are few reports on the research on lotus plant type.

[0003] Auxin is one of the most important signaling molecules among plant hormones and an important regulator of plant growth and development. It plays an important role in the entire life cycle of plants. Auxin can regulate cell division and differentiation, cell elongation, early embryogenesis, establishment of polarity, differentiation of vascular tissue, development of inflorescence and phyllotaxy, formation of apical hooks, apical dominance, establishment of root structure, fruit ripening, and response to light and gravity, thereby affecting the organogenesis of the entire plant. This is mainly manifested in the shaping of the morphology of plant roots, stems, leaves, flowers and fruits by auxin, thereby forming the morphology of the entire plant. Plant auxin response genes refer to genes whose expression changes in plants under the regulation of auxin. There are many types of auxin response genes, involving many aspects of plant growth and development. Common auxin response gene families include the auxin response factor ARF family, the AUX / IAA family, the GH3 gene family and the SAUR gene family. Screening more auxin response gene families and conducting in-depth research 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

[0004] The purpose of the present invention is to provide an application of the lotus NnSAUR36 gene in increasing plant height, stem strength and biomass, so as to solve the problems existing in the above-mentioned prior art. The present invention finds that the lotus NnSAUR36 gene (Nn1g01094.1) can positively regulate plant height, stem strength and plant biomass. By overexpressing the gene, the lodging resistance of the plant can be significantly improved, the plant type can be improved and the plant biomass can be increased. It has important application prospects and provides a new way for molecular breeding and genetic improvement of lotus and other plants.

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

[0006] The present invention provides applications of the lotus NnSAUR36 gene or related biological materials, including any of the following applications:

[0007] A1. Increase plant height;

[0008] A2. Improve the strength of plant stems;

[0009] A3. Increase plant biomass;

[0010] A4. Cultivate lodging-resistant plant strains;

[0011] The nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4.

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

[0013] B1, an expression cassette comprising the lotus NnSAUR36 gene;

[0014] B2, a recombinant vector comprising the lotus NnSAUR36 gene;

[0015] B3. A recombinant microorganism comprising the lotus NnSAUR36 gene.

[0016] The present invention also provides a recombinant vector, which contains the lotus NnSAUR36 gene; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4.

[0017] The present invention also provides a recombinant microorganism, comprising the lotus NnSAUR36 gene; or, comprising the above-mentioned recombinant vector; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4.

[0018] The present invention also provides a method for increasing plant height, comprising using genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in the plant body to increase the plant height; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4.

[0019] The present invention also provides a method for improving plant stem strength, comprising using genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in the plant body to improve the plant stem strength; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4.

[0020] The present invention also provides a method for increasing plant biomass, comprising using genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in the plant body to increase the plant biomass; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4.

[0021] The present invention also provides a method for cultivating a lodging-resistant plant strain, comprising using genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in the plant body to cultivate a lodging-resistant plant strain; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4.

[0022] The present invention discloses the following technical effects:

[0023] The present invention finds that the NnSAUR36 gene (Nn1g01094.1) in lotus is significantly positively correlated with plant height, biomass and stem mechanical strength. By cloning the gene and overexpressing it in wild tobacco (K326), the strain with the highest expression level is selected for high-generation breeding to T2 generation homozygous strain, and the plant height, stem mechanical force and fresh weight of the whole plant, stem, leaf, flower and fruit are measured and analyzed. It is found that compared with the wild type, the plant height of the transgenic tobacco increases by 26%-59%, the stem piercing force and breaking force increase by 109%-146% and 383%-584% respectively, the whole plant fresh weight increases by 291%-495%, and the fresh weight of flowers and fruits increases by 331%-664%, indicating that the NnSAUR36 gene has important application prospects in improving plant lodging resistance, improving plant type and increasing plant biomass, and provides a new way for molecular breeding and genetic improvement of lotus and other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 The amino acid sequence comparison result of lotus NnSAUR36 gene and known Arabidopsis AtSAUR36 gene;

[0026] Figure 2 Schematic diagram of the construction process of pGWB418 (4 × Myc) -NnSAUR36-NOS vector; A is the map of pGWB418 (4 × Myc) -NnSAUR36-NOS vector, B is the fusion cloning flow chart, and C is the structure of NnSAUR36 gene; LB: T-DNA left border; 35S: Cauliflower virus 35S promoter; NOS: terminator; RB: T-DNA right border;

[0027] Figure 3 This is a schematic diagram of the tobacco leaf disc genetic transformation system mediated by Agrobacterium;

[0028] Figure 4 The results of identification and expression analysis of transgenic tobacco plants; A is the electrophoresis result of PCR products of positive transgenic tobacco plant seedlings, the target band is 765bp, M is DL 2000 DNA marker, and WT is wild type; B is the detection result of NnSAUR36 expression level in positive transgenic tobacco plant seedlings;

[0029] Figure 5 A is a graph showing the plant height measurement results of wild-type tobacco (WT) and transgenic tobacco T2 generation strains; A is a graph showing the mature stems of wild-type tobacco (WT) and transgenic tobacco T2 generation strains; B is a comparative analysis result of the plant height of wild-type tobacco (WT) and transgenic tobacco T2 generation strains;

[0030] Figure 6 A is a graph showing the results of measuring the mechanical force of the stems of wild-type tobacco (WT) and transgenic tobacco T2 generation strains; A is a graph showing the results of measuring the breaking force of the stems of wild-type tobacco (WT) and transgenic tobacco T2 generation strains; B is a graph showing the results of measuring the puncture force of the stems of wild-type tobacco (WT) and transgenic tobacco T2 generation strains;

[0031] Figure 7A is a graph showing the biomass measurement results of wild-type tobacco (WT) and transgenic tobacco T2 generation strains; A is an observation graph of the whole plant, leaves, flowers, and fruits of the wild-type tobacco (WT) and transgenic tobacco T2 generation strains; B is a comparative analysis result of the fresh weight of the whole plant, stems, leaves, flowers, and fruits of the wild-type tobacco (WT) and transgenic tobacco T2 generation strains. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Isolation and cloning of NnSAUR36 gene

[0038] (1) Isolation and cloning of NnSAUR36 gene

[0039] The lotus variety 'Dazu Honglian' (provided by the National Lotus Germplasm Resource Bank of Southwest Forestry University) was used as the experimental material. The total RNA of the petiole of lotus 'Dazu Honglian' variety was extracted using 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 The ratio is between 1.9 and 2.1, RNA 260 / 230 RNA with a ratio greater than 2.0 and a concentration greater than 500 ng / μL was used for the next step of analysis. IIQRT SuperMix for qRNA (+gDNA wiper) kit (purchased from Vazyme, China), with 1 μg of total RNA as a template, mixed with 4 μL 4×gDNA wiperMix, DEPC-water, the total volume was 16 μL, incubated at 42°C for 2 min, and chilled on ice for 3 min; then 5×Hiscript II qRT Super Mix II 4 μL was added and mixed, the total volume was 20 μL; then incubated at 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.

[0040] In-fusion cloning primers NnSAUR36-418F and NnSAUR36-418R 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, 31 cycles; 72℃ extension for 5min. The PCR product was then cloned into the pGWB418 vector (Wuhan Puint Biotechnology Co., Ltd.). The recovery of the target fragment was performed with reference to the UNIQ-10 column DNA gel recovery kit (Shanghai Biotechnology Co., Ltd.). The connection system between the target fragment and the pGWAB418 linearized vector was: 4.5μL target fragment, 0.5μL pGWB418 linearized vector, 5μL SolutionⅠ (Takara Biotechnology (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 NnSAUR36-418R. The positive colony was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0041] Primer information is as follows:

[0042] NnSAUR36-418F: 5'-GACTTGAACGGTAGCGCTGCCAATTACAACAATATCCGTAG-3' (SEQ IDNO.1);

[0043] NnSAUR36-418R: 5'-TCGGGGAAATTCGAGCTCCAACTAAATCCTCCATTACACCC-3' (SEQ IDNO.2);

[0044] 418F: 5'-TCGGGGAAATTCGAGCTC-3' (SEQ ID NO. 3).

[0045] (2) NnSAUR36 gene sequence analysis

[0046] After sequencing, the nucleotide sequence of the gene was 765 bp in length (SEQ ID NO.4), containing an open reading frame ORF of 501 bp in length (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. The results showed that the target band was homologous to AtSAUR36 of Arabidopsis thaliana at the amino acid level (similarity 43.35%), which was consistent with the results of the alignment on NCBI ( Figure 1 ). The amino acid sequence encoded by the NnSAUR36 gene is shown in SEQ ID NO.6.

[0047] SEQ ID NO.4:

[0048] GCCTTTCACCTAAACAAAGCCAATTACAACAATATCCGTAGATAGTCTACCTCCCATTCTCTCTCGCTGTCTGTCTCATCTTTCTCTTCACTCACTGCACACATTATGAGGAAGACGAGAGGTTTCAAGTTGGGCCGCAAACTGGTTAAGGTGTTTAGATGGGTAGTCAGGCCGAATCGAAAGCCAGCTGGCTACCGCCGATTGGAGTTGGATCCTCCGAGCTGCAAAAGCAAGGCCATATCCAAAATTTGCGACTGGGGTAATTGCCTGAAGCGAGGGGCGAAGGGACTCTGCTCTTCCAACAAGTCATCAGCCTCCGGCTACAGTCGCTGTGATCGGGAGGCATTGGAAGATAAACCGGTGCAAGTGCCGAAAGGACACATGGCGGTATACGTGGGTCAGAAAGACGACGGTCTTCATCGGTTCTTGGTGCCGGTTATCTACTTCAATCACCCCTTATTCGCAGACTTGTTGAGGGAGGCCGAGGAGGAGTATGGGTTCCACCACCCAGGTGGGATTACGATACCATGCCGGATAACTGAGTTCGAGAGCGTACAAACGAAGATCGCCGCCGGCGTGAAATCGACGTGGAAACGTCGCTTGTGAATTGTCACTGATTACAGTGTTGATGATGATGATGAAAAGTGAAAACCATGATTAAAGATAGATGATTAGATATACCCCTTTTTTTTCCATTTCTTTTGTTTCAA TTTTTGTTAATACTGGGTGTAATGGAGGATTTAGTTGTGAAATCAAAAGGATGTG;

[0049] SEQ ID NO.5:

[0050] ATGAGGAAGACGAGAGGTTTCAAGTTGGGCCGCAAACTGGTTAAGGTGTTTAGATGGGTAGTCAGGCCGAATCGAAAGCCAGCTGGCTACCGCCGATTGGAGTTGGATCCTCCGAGCTGCAAAAGCAAGGCCATATCCAAAATTTGCGACTGGGGTAATTGCCTGAAGCGAGGGGCGAAGGGACTCTGCTCTTCCAACAAGTCATCAGCCTCCGGCTACAGTCGCTGTGATCGGGAGGCATTGGAAGATAAACCGGTGCAAGTGCCGAAAGGACACATGGCGGTATACGTGGGTCAGAAAGACGACGGTCTTCATCGGTTCTTGGTGCCGGTTATCTACTTCAATCACCCCTTATTCGCAGACTTGTTGAGGGAGGCCGAGGAGGAGTATGGGTTCCACCACCCAGGTGGGATTACGATACCATGCCGGATAACTGAGTTCGAGAGCGTACAAACGAAGATCGCCGCCGGCGTGAAATCGACGTGGAAACGTCGCTTGTGA;

[0051] SEQ ID NO.6:

[0052] MRKTRGFKLGRKLVKVFRWVVRPNRKPAGYRRLELDPPSCKSKAISKICDWGNCLKR GAKGLCSSNKSSASGYSRCDREALEDKPVQVPKGHMAVYVGQKDDGLHRFLVPVIYFNHP LFADLLREAEEEYGFHHPGGITIPCRITEFESVQTKIAAGVKSTWKRRL。

[0053] Example 2 Obtaining of NnSAUR36 Overexpressing Transgenic Tobacco Plants

[0054] (1) Construction of Plant Overexpression Vector

[0055] AfeI and SacI restriction sites were added to both ends of the cloning amplification primers, 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℃ overnight. After 1% agarose gel electrophoresis, a single linearized vector fragment was recovered and named pGWB418 (4×Myc) vector.

[0056] The PCR product obtained by the above amplification was connected into the pGWB418 (4×Myc) vector using In-fusion enzyme, and the positive clones were screened and sequenced to obtain the lotus NnSAUR36 transgenic overexpression vector pGWB418 (4×Myc)-NnSAUR36-NOS, which contained a kanamycin-resistant gene sequence in its T-DNA region. The promoter of NnSAUR36 overexpression was the D35s promoter (see Figure 2 ).

[0057] (2) Genetic transformation of tobacco (K326)

[0058] The recombinant plasmid (i.e., overexpression vector) pGWB418 (4×Myc)-NnSAUR36-NOS was transferred into Agrobacterium GV3101 by conventional leaf disc method, and the tissue was infected and screened and differentiated into seedlings. The specific steps are as follows ( Figure 3 ):

[0059] a. Activation of Agrobacterium: Take the Agrobacterium strain GV3101 stored at -80℃ and streak it on LB solid medium containing rifampicin (50mg / mL), gentamicin (30mg / mL) and spectinomycin (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 (30mg / mL) and spectinomycin (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 12h, and multiply the bacteria until the OD value is 0.8.

[0060] b. Take young and tender top leaves, healthy leaves without wounds, and rinse them with running water for 20 minutes to clean them.

[0061] c. Immerse the leaves in 75% alcohol for 1 minute for disinfection, then place the leaves in 0.1% mercuric chloride solution, immerse them completely for 10 minutes, and immediately rinse them with sterile distilled water 2-3 times.

[0062] d. Use an alcohol lamp to sterilize the knife and tweezers, and leave them to cool for later use. Use filter paper to absorb excess water, cut off the tobacco leaf edge and main vein, cut the leaf into 1cm×1cm size, and put it into the shaken bacterial solution for infection for 10 minutes.

[0063] e. After the infection is completed, carefully pick up the leaves, spread them flat on sterilized filter paper, turn them upside down to dry, so that most of the infection liquid is filtered out. After the excess bacterial liquid is absorbed, spread the four corners of the leaves flat and insert them into the co-culture dish (MS (inorganic salts and trace elements) 4.404g / L, sucrose 30g / L, agar 6.8g / L, 6-BA 2.25mg / L, NAA 0.3mg / L formulated in 1962, adjust the pH of the culture medium to 5.8-6.0, and sterilize at 121℃ for 20min), with the front of the leaves facing up, seal the envelope, and place in the culture room for dark culture for 3 days.

[0064] f. After 3 days, transfer to differentiation medium (MS (inorganic salts and trace elements) 4.404 g / L, sucrose 30 g / L, agar 6.8 g / L, 6-BA 2.25 mg / L, NAA 0.3 mg / L of the 1962 formula, adjust the pH of the medium to 5.8-6.0, sterilize at 121°C for 20 min, add Kana 50 mg / L, Cef 400 mg / L in a sterile operating bench after sterilization).

[0065] g. After two weeks, the nutrients and antibiotics in the culture dish have been exhausted and the cells are transferred to a new dish (differentiation medium).

[0066] h. After adventitious buds grow, cut off the callus tissue, otherwise it will slow down the rooting process, and transfer to the bud-strengthening medium (MS (inorganic salts and trace elements) 4.404 g / L, sucrose 30 g / L, agar 6.8 g / L, 6-BA 0.1 mg / L, NAA 0.1 mg / L, adjust the pH of the medium to 5.8-6.0, sterilize at 121°C for 20 min, add Kana 50 mg / L, Cef 400 mg / L in a sterile operating table after sterilization).

[0067] i. Half a month later, the healthy buds were transferred to rooting culture bottles (MS (inorganic salts and trace elements) 4.404g / L, sucrose 30g / L, agar 6.8g / L, NAA 0.1mg / L, the pH of the culture medium was adjusted to 5.8-6.0, sterilized at 121℃ for 20min, and after sterilization, Kana 50mg / L, Cef 400mg / L were added in a sterile operating table) to promote rooting.

[0068] j. Wait until the root system is strong and solid, and after hardening the seedlings for a week, move them to culture soil for cultivation.

[0069] (3) Identification of transgenic plants

[0070] a. Extraction of genomic DNA from tobacco leaves

[0071] The conventional CTAB method was used for DNA extraction. The specific steps are as follows: take young tobacco 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 them gently every 10 minutes, cool them to room temperature, add 700 μL of chloroform:isoamyl alcohol with a volume ratio of 24:1, repeatedly invert and mix, and then shake and mix for 1-2 minutes. Centrifuge at 10000 rpm at room temperature for 5 minutes. Take about 600 μL of the supernatant, add an equal volume of 24:1 chloroform:isoamyl alcohol, shake vigorously for 1-2 minutes, and centrifuge at 10000 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 10000 rpm at room temperature for 10-15 minutes to obtain a white precipitate. Slowly pour off the supernatant, take 800-900 μL 75% anhydrous ethanol to wash and blow the precipitate repeatedly, wash twice and pour off the supernatant, centrifuge at 10000rpm for 3min, use a pipette to absorb the excess alcohol, air dry for 10-15min, and add 50 μL ddH 2 O dissolution. The extracted tobacco leaf genomic DNA was tested for concentration using Nano drop micro-nucleic acid detector.

[0072] b. Detection of positive transgenic plants

[0073] The genomic DNA extracted in step a was used as a template to amplify the NnSAUR36 gene by PCR technology for positive seedling identification. The primers used were NnSAUR36-418F and NnSAUR36-418R. The control was a non-transgenic wild-type tobacco plant (WT). The test results showed that ( Figure 4 A), the 8 transformed plants numbered 1, 2, 6, 8, 10, 11, 12, and 13 were able to amplify the expected size of electrophoresis bands (765bp), while the wild-type control had no electrophoresis bands, indicating that the genome of the above-mentioned positive transgenic tobacco plants already contained foreign gene DNA fragments. The transgenic positive plant numbers obtained were prefixed with OE.

[0074] (4) qRT-PCR identification of transgenic tobacco overexpressing NnSAUR36

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

[0076] use Super total RNA extraction kit (purchased from Promega, USA) was used to extract total RNA from leaves of WT and transgenic positive plants identified in step (3). RNA was reverse transcribed into cDNA using IIQ RT SuperMix for qRNA (+gDNAwiper) Kit (Vazyme, China).

[0077] b. Real-time fluorescence quantitative PCR

[0078] In order to determine whether NnSAUR36 is overexpressed in tobacco, the real-time fluorescence quantitative PCR (qRT-PCR) method was used to analyze the expression level of NnSAUR36 gene in the transgenic positive plants identified in step (3). Green Realtime PCR Master Mix-Plus-Kit (Takara Biotech (Dalian) Co., Ltd.), using tobacco housekeeping gene NtRNR2 (GeneBank: X69885.1) as internal reference gene, and its primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0079] The qRT-PCR primers for NtRNR2 gene are:

[0080] NtRNR2-F: 5'-TACCCGTCACTGGGAAAC-3' (SEQ ID NO.7);

[0081] NtRNR2-R: 5'-GGAAGCCGTAGAAAGCAC-3' (SEQ ID NO. 8).

[0082] The qRT-PCR primers for NnSAUR36 gene are:

[0083] NnSAUR36-F: 5'-AGACGAGAGGTTTCAAGTTGGG-3' (SEQ ID NO. 9);

[0084] Q-NnSAUR36-R: 5'-CAGGCAATTACCCCAGTCGC-3' (SEQ ID NO. 10).

[0085] (This pair of primers is a qRT-PCR specific primer designed based on the nucleotide sequence sequenced in Example 1)

[0086] PCR program: 95℃ pre-denaturation for 30 sec, then 40 cycles (95℃ for 15 sec, 60℃ for 30 sec, 72℃ for 15 sec). Figure 4As shown in Figure B, the expression levels of NnSAUR36 in OE-1, OE-2, OE-6, OE-8, OE-10, OE-11, OE-12 and OE-13 strains were significantly higher than the non-transgenic wild-type (WT) control, and the expression levels of OE-1, OE-2, OE-8 and OE-10 strains were more than 300 times that of the wild-type strains, the expression levels of OE-11 and OE-13 strains were more than 120 times, and the expression levels of OE-6 and OE-12 strains were about 40 times that of the wild-type. This indicates that these strains are independent NnSAUR36 overexpression transgenic strains.

[0087] Example 3 Increase in plant height, stalk mechanical force and biomass of NnSAUR36 overexpressing transgenic tobacco T2 generation strains

[0088] (1) Determination of plant height of T2 generation of NnSAUR36 overexpressing transgenic tobacco

[0089] The plant height of wild tobacco (WT) and NnSAUR36 (OE) overexpressing plants with fully mature fruits was measured. The measurement method was to select T2 plants of three independent strains, OE-1, OE-8 and OE-10, and measure their plant height using a 3-meter telescopic measuring ruler. The measurement started from the bottom of the cultivation pot to the highest point of the main inflorescence. Figure 5 As shown, the data showed that the average heights of the three transgenic lines were significantly higher than that of the WT.

[0090] (2) Determination of mechanical force of stems of NnSAUR36 overexpressing transgenic tobacco T2 lines

[0091] The mechanical force of the stems of wild tobacco (WT) and NnSAUR36 (OE) overexpressing plants with fully mature fruits was measured. The main stems of the T2 generation plants of three independent strains, OE-1, OE-8 and OE-10, were collected, with 8 plants in each strain. The stems were evenly divided into three sections: upper, middle and lower. The stem breaking force and puncture force were measured using a stolon tester (DIK7401, Japan). The results are as follows: Figure 6 As shown in A and B, the data showed that the stem breaking force and puncture force of the three transgenic lines were significantly higher than those of the WT.

[0092] (3) Determination of fresh weight of T2 generation of NnSAUR36 overexpressing transgenic tobacco lines

[0093] The fresh weight of the whole plant, stem, leaf, flower and fruit of wild tobacco (WT) and NnSAUR36 (OE) overexpression strain with fully mature fruit was weighed using an electronic balance. Figure 7 As shown, the data showed that the fresh weights of plants, stems, leaves, flowers and fruits of the three transgenic lines were significantly higher than those of WT.

[0094] Therefore, it can be shown that the NnSAUR36 gene can positively regulate plant height and increase stem mechanical force and biomass.

[0095] 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 NnSAUR36 gene or its related biological materials, characterized in that: For any of the following applications: A1. Increase plant height; A2. Improve the strength of plant stems; A3. Increase plant biomass; A4. Cultivate lodging-resistant plant strains; The nucleotide sequence of the lotus NnSAUR36 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 NnSAUR36 gene; B2, a recombinant vector comprising the lotus NnSAUR36 gene; B3, a recombinant microorganism comprising the lotus NnSAUR36 gene; The plant is tobacco.

2. A method for increasing plant height, characterized in that: The method comprises utilizing genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in a plant to increase the plant height; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4; and the plant is tobacco.

3. A method for improving the strength of plant stems, characterized in that: The method comprises utilizing genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in a plant body to improve the plant stem strength; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4; and the plant is tobacco.

4. A method for increasing plant biomass, characterized in that: The method comprises utilizing genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in a plant to increase plant biomass; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQID NO.4; and the plant is tobacco.

5. A method for cultivating lodging-resistant plant strains, characterized in that: The method comprises utilizing genetic transformation technology to stably overexpress the lotus NnSAUR36 gene in a plant body to cultivate a lodging-resistant plant strain; the nucleotide sequence of the lotus NnSAUR36 gene is shown in SEQ ID NO.4; and the plant is tobacco.

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