Application of lotus NnMUR3.5 gene in regulating plant flower development

Abstract

The application discloses application of lotus NnMUR3.5 gene in regulation of plant flower development and belongs to the technical field of genetic engineering. The nucleotide sequence of the lotus NnMUR3.5 gene is shown as SEQ ID NO. 5. It is found that the NnMUR3.5 gene in the lotus is significantly related to the length of a plant spathode and flower color. By overexpressing the gene in wild tobacco, the length of a spathode of the transgenic tobacco can be significantly increased, and the flower color can be changed, which indicates that the NnMUR3.5 gene has good application effect in regulation of the length of a plant spathode and flower color, is helpful to cultivation of a variety of flower types and flower colors, provides new gene resources and a new research approach for molecular breeding and genetic improvement of new ornamental lotus and other ornamental plants, and has important practical guiding values in breeding practice, variety improvement and variety popularization of the lotus and other plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to the application of NnMUR3.5 gene in regulating plant flower development. BACKGROUND

[0002] Lotus is the gem of aquatic plants, also known as lotus. It is a perennial emergent plant of Nelumbonaceae and Nelumbo. Lotus has entered people's life with its practicality, and has also penetrated into people's spiritual world with its beautiful color and elegant appearance. According to different purposes of utilization, lotus can be divided into three categories: flower lotus, seed lotus and rhizome lotus. With the improvement of people's requirements for ornamental, more and more people hope to see lotus with different flower types and colors. Therefore, flower type and color have become important phenotypes for ornamental lotus and important basis for variety classification. In recent years, the research on flower type and color of lotus has been continuously deepened. With the rapid development of molecular biology technology, many genes related to flower development have been cloned. People have a more profound understanding of the mechanism of flower development. Therefore, it is of great significance to fully utilize genetic engineering technology, explore gene resources and apply them to the screening of lotus germplasm resources, which can increase lotus germplasm resources and further improve the added value of lotus.

[0003] The cell wall is a structure unique to plant cells, which is composed of middle lamella, primary wall and secondary wall. The primary wall contains cellulose, pectin, xyloglucan (XyG) and other components. XyG is the most abundant hemicellulose in the primary wall of dicotyledonous plants and non-grass monocotyledonous plants. The main chain is β-1, 4 linked glucan, and the side chain contains xylose and other modifications. Its structure in Arabidopsis thaliana mainly has XLFG, XXXG, XXFG, etc. It is found that there is a YXXG structure of galacturonic acid in root hair. The glucose main chain structure of xyloglucan is basically the same in different plants, but there are certain differences in side chain modification. The xyloglucan main chain is composed of a β-1, 4 linked glucan, and the typical repeating mode of xylose residues (Xyl) is connected to the O-6 position of glucose. The xylose on these side chains is further replaced by different monosaccharides, disaccharides or trisaccharides. It is generally believed that xyloglucan plays an important role in the growth of primary cell wall of plants, so the identification of xyloglucan biosynthesis enzyme gene has become an important research field.

[0004] Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds between specific glycosyl donors and acceptors. In plants, glycosyltransferases are widely involved in different life processes such as plant growth and development, flowering and fruiting, and have many functions such as synthesizing secondary metabolites, regulating plant hormone levels, participating in plant defense, enhancing plant detoxification ability, affecting the formation of various qualities in the fruit ripening process, etc. MUR3, XLT2 and XUT1 genes in Arabidopsis belong to the GT47 family and are involved in XyG side chain synthesis. Among them, MUR3 is a XyG galactosyltransferase that can convert XXXG to XXLG. In the studied mutant of the mutagenized xyloglucan transferase, although some mutants have defects in root hair growth and other aspects, they have little effect on the growth of the aboveground part of the plant. Only the phenotype of the MUR3 gene mutation is relatively complex. Overexpression of the homologous genes XST1 and XST2 of MUR3 and XLT2 in tomato can complement the growth defects of Arabidopsis mutants, indicating that the side chain glycosyl function is conservative.

[0005] The expression patterns of MUR3-like genes (AtGT11-AtGT20) in Arabidopsis are diverse, some of which are widely expressed in seedlings, inflorescences and other tissues, and some of which are limited to roots or flower organs (such as AtGT19 in pollen grains and AtGT14 in stamens and carpels). Studies have shown that MUR3 and its homologous genes play an important role in plant vegetative growth, hypocotyl elongation and flower development by regulating XyG structure. However, the specific function of MUR3 gene in plant flower development is still unclear. Exploring the molecular mechanism of NnMUR3.5 affecting lotus flower development can provide a new theoretical basis for lotus flower type and color varieties. SUMMARY

[0006] The purpose of the present application is to provide the application of lotus NnMUR3.5 gene in regulating plant flower development to solve the problems existing in the prior art. The present application finds that the NnMUR3.5 gene in lotus is significantly related to the length of the plant flower tube and the color of the flower. By overexpressing the gene in tobacco, the length of the flower tube of the transgenic tobacco can be significantly increased, and the flower color can be changed, indicating that the NnMUR3.5 gene has good application effect in regulating the length of the plant flower tube and the color of the flower, which is helpful for cultivating diverse flower type and color varieties, and provides a new gene resource and a new research approach for the molecular breeding and genetic improvement of new ornamental lotus and other ornamental plants, and has important practical guiding value in the breeding practice, variety improvement and variety popularization of lotus and other plants.

[0007] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0008] The present application provides the application of lotus NnMUR3.5 gene in regulating plant flower development, and the regulation of plant flower development is to increase the length of the plant flower tube or change the color of the plant flower.

[0009] The nucleotide sequence of the lotus NnMUR3.5 gene is shown as SEQ ID NO. 5.

[0010] The application also provides application of the expression cassette containing the lotus NnMUR3.5 gene in regulating flower development of a plant, and the regulation of the flower development of the plant is to increase the length of a flower tube of the plant or to change the color of a flower of the plant.

[0011] The nucleotide sequence of the lotus NnMUR3.5 gene is shown as SEQ ID NO. 5.

[0012] The application also provides application of the recombinant vector containing the lotus NnMUR3.5 gene in regulating flower development of a plant, and the regulation of the flower development of the plant is to increase the length of a flower tube of the plant or to change the color of a flower of the plant.

[0013] The nucleotide sequence of the lotus NnMUR3.5 gene is shown as SEQ ID NO. 5.

[0014] The application also provides application of the recombinant microorganism containing the lotus NnMUR3.5 gene in regulating flower development of a plant, and the regulation of the flower development of the plant is to increase the length of a flower tube of the plant or to change the color of a flower of the plant.

[0015] The nucleotide sequence of the lotus NnMUR3.5 gene is shown as SEQ ID NO. 5.

[0016] Optionally, the plant is a lotus or a tobacco.

[0017] The application also provides a method for increasing the length of a flower tube of a plant, which comprises stably overexpressing the lotus NnMUR3.5 gene in the plant by using a genetic transformation technology, so as to increase the length of the flower tube of the plant; and the nucleotide sequence of the lotus NnMUR3.5 gene is shown as SEQ ID NO. 5.

[0018] The application also provides a method for changing the color of a flower of a plant, which comprises stably overexpressing the lotus NnMUR3.5 gene in the plant by using a genetic transformation technology, so as to change the color of the flower of the plant; and the nucleotide sequence of the lotus NnMUR3.5 gene is shown as SEQ ID NO. 5.

[0019] Optionally, the plant is a lotus or a tobacco.

[0020] The application discloses the following technical effects:

[0021] The present application finds that the NnMUR3.5 gene in lotus is significantly related to the length and color of the flower tube of the plant. By cloning the gene and overexpressing it in wild tobacco, it is found through the determination of the length and color of the flower tube of the transgenic strain that the length of the flower tube of the transgenic tobacco can be increased by 15.54%-19.55% compared with the wild type, and the color of the flower tube can be changed from Deep Purplish Pink to Strong Purplish Red, indicating that the NnMUR3.5 gene has good application effect in regulating the length and color of the flower tube of the plant, and is helpful for cultivating diverse flower types and color varieties, providing new gene resources and new research approaches for the molecular breeding and genetic improvement of new ornamental lotus and other ornamental plants, and having important practical guiding value for the breeding practice, variety improvement and variety popularization of lotus and other plants. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0023] Figure 1 NnMUR3.5 gene of lotus and known amino acid sequence alignment results of Arabidopsis thaliana AtMUR3 gene;

[0024] Figure 2 pGWB418-NnMUR3.5 vector construction process schematic diagram;

[0025] Figure 3 pGWB418-NnMUR3.5 vector structure schematic diagram;

[0026] Figure 4 Agrobacterium-mediated tobacco leaf disc genetic transformation process schematic diagram;

[0027] Figure 5 Transgenic tobacco plant identification and expression level analysis results; wherein, A is the electrophoresis result of the PCR product of the positive transgenic plant, and the target band is 1546bp; B is the detection result of the expression level of NnMUR3.5 in the positive transgenic plant;

[0028] Figure 6 Wild type tobacco (WT) and transgenic tobacco T3 generation strain flower tube length measurement result graph; wherein, A is the flower tube morphology graph of wild type tobacco (WT) and transgenic tobacco T3 generation strain, and the scale is 1cm; B is the comparison analysis result of the flower tube length of wild type tobacco (WT) and transgenic tobacco T3 generation strain;

[0029] Figure 7 Figure 6 is a chart of flower color assay results for wild type tobacco (WT) and transgenic tobacco T3 generation lines; wherein A is a chart of flower color assay results for wild type tobacco (WT); B is a chart of flower color assay results for transgenic tobacco OE-6 T3 generation line; C is a chart of flower color assay results for transgenic tobacco OE-9 T3 generation line; D is a chart of flower color assay results for transgenic tobacco OE-13 T3 generation line; E is a chart of flower color assay results for transgenic tobacco OE-14 T3 generation line; F is a chart of flower color assay results for transgenic tobacco OE-16 T3 generation line; all scales are 1 cm. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present application will now be described in detail, with reference being made to the figures. In this context, it is to be understood that the description of the present application is not to be construed as a limitation on the application but merely as a description of certain aspects, features and embodiments of the application.

[0031] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where particular ranges of values are given, it is to be understood that each intervening value, to the upper or lower limit of the ranges is also specifically included. The aforementioned

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0033] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0034] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0035] Example 1 Isolation and cloning of NnMUR3.5 gene

[0036] (1) Isolation and cloning of the NnMUR3.5 gene

[0037] The lotus variety 'Dazu Honglian' (provided by the National Lotus Germplasm Resource Bank of Southwest Forestry University) was selected as the experimental material, and the following methods were adopted. Total RNA was extracted from various tissues of lotus flower using the Super Total RNA Extraction Kit (purchased from Promega, USA). After extraction, RNA was treated with DNase I (purchased from Promega). RNA integrity was assessed by 1.2% (w / v) agarose gel (EtBr) electrophoresis (5V / cm). Nucleic acid concentrations were determined using an IMPLEN Nano Photometer-N50 series ultra-micro UV spectrophotometer (Germany). RNA with a 260 / 280 ratio between 1.9 and 2.1, a 260 / 230 ratio greater than 2.0, and a concentration greater than 500 ng / mL was used for further analysis. cDNA synthesis was performed using… The IIQ RT SuperMix for qRNA (+gDNAwiper) kit (purchased from Vazyme, China) was used. 1 μg total RNA was used as a template and mixed with 4 μL of 4×gDNAwiperMix and DEPC-water to a total volume of 16 μL. The mixture was incubated at 42°C for 2 min, then quenched on ice for 2-3 min. Next, 4 μL of 5×Hiscript II qRT Super Mix II was added and mixed to a total volume of 20 μL. The mixture was then incubated at 50°C for 15 min, followed by 85°C for 5 sec. Each cDNA sample was diluted to 200 μL and stored at -20°C until use.

[0038] The target band was amplified using In-fusion cloning primers NnMUR3.5-418F / NnMUR3.5-418R, and was amplified using TransTaq HiFi DNA Polymerase (Beijing Zixiaojin Biotech Co., Ltd.), with the PCR reaction conditions being: 94°C pre-denaturation for 3 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min 30 sec, 32 cycles; and 72°C extension for 5 min. Then the PCR product was cloned into a pGWB418 vector. The recovery of the target fragment was performed according to the UNIQ-10 column DNA gel recovery kit (Shanghai Genechem Co., Ltd.). The connection system of the target fragment and the linearized pGWAB418 vector was: 4.5 μL of the target fragment, 0.5 μL of the pGWB418 vector, and 5 μL of Solution I (Bao Biotech (Dalian) Co., Ltd.), which was connected at 16°C overnight. The connection product was transformed into DH5α competent cells by heat shock, and the bacterial liquid was spread on an LB solid plate containing 100 mg / L Kan antibiotic. After about 10-12 h of growth, a single colony was selected for colony PCR, with the primers being universal primers 418F / R. The positive colony was sent to Shanghai Genechem Co., Ltd. for sequencing.

[0039] The primer sequences are as follows:

[0040] NnMUR3.5-418F:

[0041] 5'-GACTTGAACGGTAGCGCTGATAATGGGAGTTTGGCTGAT-3' (SEQ ID NO. 1);

[0042] NnMUR3.5-418R:

[0043] 5'-TCGGGGAAATTCGAGCTCTCCACTGTCCCAAACAAATT-3' (SEQ ID NO. 2);

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

[0045] 418R: 5'-TCGGGGAAATTCGAGCTC-3' (SEQ ID NO. 4).

[0046] (2) NnMUR3.5 gene sequence analysis

[0047] The sequencing showed that the amplified nucleotide sequence of the gene was 1766 bp in length, containing an open reading frame (SEQ ID NO. 5) of 1461 bp in length. The alignment of the amino acid sequence 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 showed that the target band was homologous to MUR3 of Arabidopsis thaliana at the amino acid level (43.87% similarity), which was consistent with the result of alignment on NCBI. Figure 1 The amino acid sequence encoded by the NnMUR3.5 gene is shown as SEQ ID NO. 6.

[0048] SEQ ID NO. 5:

[0049]

[0050] SEQ ID NO.6:

[0051] MGSGLEPAVSQLDVSGNGSEYNNHVNSRLPDDNASSATNTIDNGSLADSNSCSGRYIHMYDLPIRFNDDVIKDCRSLNVWYDMCRPLTNRGLGPQLINCNKVFSDKGWYETDQFMLEIIFHERMKRYECLTNDSSLASAHYVPFYAGFDVSRYLWGFNTSIRDATPLALINWLRERSEWKVLGGRDHFLVAGRTTWEFRRLTEEDSDWGNRLMVLPEGKNMTMLVIESSPWHRNDFAIPYPTYFHPSTDDEVFQWQNRIRRLRRRYLFSFAGAPRPNISGSVRGQIIEQCQASNKCRLLDCSTKSNCQNPSNVMKLFQTSAFCLQPPGDSYTRRSAFDSMLAGCIPVFFHPGSAYVQYLWHLPKNYTKYSVFISEDDVKAGNVSIEEKLRRIPKEEVKAMREQVIQLIPGLIYANPSSTLETVEDAFDLAVRGVIDRVNRIRKEIKDGVNSDLSYDEIDSWKYNLFGTVEKHEWDSFFYKWKYTKS.

[0052] Example 2 Obtaining of NnMUR3.5 transgenic tobacco plants overexpressing

[0053] (1) Construction of plant overexpression vector

[0054] The NnMUR3.5-418F / NnMUR3.5-418R primers were cloned and amplified at both ends of the primers with Afe I and Sac I restriction sites, respectively, and the PCR product was obtained according to the method of Example 1. The pGWB418 vector was double-digested with Afe I and Sac I, and the enzyme digestion system was as follows: pGWB418 (1700 ng / μL) 15 μL, rCutsmartt Buffer 5 μL, Afe I (10000 U / L) 1 μL, Sac I (20000 U / L) 1 μL, sterile water to make up the final volume to 50 μL, 37°C enzyme digestion overnight. After 1% agarose gel electrophoresis, the single linearized vector fragment was recovered and named as pGWB418(4×Myc) vector.

[0055] The PCR product obtained from the above amplification was ligated into the pGWB418(4×Myc) vector using an in-fusion enzyme. Positive clones were screened and sequenced for identification to obtain the desired gene fragment (1461 bp, SEQ ID NO.5), thus obtaining the lotus NnMUR3.5 transgenic overexpression vector pGWB418-NnMUR3.5. Figure 2 The T-DNA region of NnMUR3.5 contains a gene sequence for kanamycin resistance, and the promoter for overexpression of NnMUR3.5 is the D35s promoter (see schematic diagram). Figure 3 ).

[0056] (2) Genetic transformation of tobacco (K326)

[0057] The recombinant plasmid (i.e., the overexpression vector) pGWB418-NnMUR3.5 was transformed into Agrobacterium GV3101 using the conventional leaf disc infection method. Seedlings were then selected and differentiated. The specific steps are as follows (see the flowchart). Figure 4 ):

[0058] a. Infection: Wash the tender terminal leaves of wild tobacco shoots with 75% alcohol for 1 minute, then wash with 0.1% mercuric chloride solution for 5 minutes, and finally wash three times with sterile water. Use sterile tweezers and scissors to remove the leaf margins and midribs, cutting the leaves to a size of 1×1cm. Place the leaves in the pre-shaken bacterial solution for 10 minutes. After infection, carefully remove the leaves and lay them flat on sterilized filter paper, turning them over to allow most of the infection solution to be filtered out. After the excess bacterial solution has been absorbed, place the leaves flat at the four corners on a petri dish, with the leaf surface facing up, and incubate in the dark for 3 days.

[0059] b. Differentiation: After 3 days of dark culture, the explants were transferred to differentiation medium (MS formulation of 1962, with 4.404 g / L inorganic salts and trace elements, 30 g / L sucrose, 6.8 g / L Agar, 2.25 mg / L 6-BA, 0.3 mg / L NAA, pH 5.8-5.9, sterilized and cooled, with 50 mg / L Knna and 400 mg / L Cef antibiotics added), and cultured at 24°C for 16 hours in the dark and 8 hours in the dark.

[0060] c. Bud strengthening: After adventitious buds emerge, remove as much callus tissue as possible and transfer the bud strengthening medium (MS formula from 1962, 4.404 g / L inorganic salts and trace elements, 30 g / L sucrose, 6.8 g / L Agar, 2.25 mg / L 6-BA, 0.3 mg / L NAA, pH 5.8-5.9, sterilized and cooled, then add 50 mg / L Knna and 400 mg / L Cef antibiotics) for 30 days of culture under the following conditions: 24℃, 16 h light, 8 h dark culture.

[0061] d. Rooting culture: when the roots are thick and strong, transfer to rooting solid medium (MS inorganic salt and trace element 4.404 g / L of 1962 formula, sucrose 30 g / L, agar 6.8 g / L, after sterilization and cooling, add Knna 50 mg / L, Cef 400 mg / L of antibiotics) to root.

[0062] (3) Identification of transgenic plants

[0063] a. Extraction of tobacco leaf genomic DNA

[0064] The conventional CTAB method was used for DNA extraction. The specific steps are as follows: take the length of 1-2 cm of tender tobacco leaves, place in the pre-cooled mortar, add 2-3 times of liquid nitrogen grinding to fine slurry, transfer to 1.5 mL centrifuge tube, add 700 μL of 2xCTAB solution. Incubate at 70°C for 30 min, shake gently once every 6 min, incubate at 70°C for 30 min, shake gently once every 10 min. Cool to room temperature, add 700 μL of Tris-saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1, mix well by inverting repeatedly, and shake gently for about 40 times. Centrifuge at 3100 rpm for 15 min at room temperature. Take 500 μL of supernatant, add equal volume of 24:1 chloroform: isoamyl alcohol. Shake well and centrifuge at 3100 rpm for 15 min at room temperature. Discard the supernatant, add 1 mL of anhydrous ethanol at -20°C, and incubate in a -20°C ice bath for 30 min, then centrifuge at 12000 rpm at room temperature for 10 min. Wash the precipitate with 75% alcohol and blow it repeatedly for 3 min to remove salt. Pour off the alcohol, air dry, and add 30-50 μL of ddH2O to each sample. Extract the genomic DNA, detect the concentration with Nanodrop micro nucleic acid detector, and store for later use.

[0065] b. Positive transgenic plant detection

[0066] Take the tender leaves of the transformed plants, extract DNA using the above steps, and identify positive plants by PCR amplification of the Kan gene on the vector. The primer sequences used are as follows:

[0067] Bn-Kan-F: 5'-GACTTGAACGGTAGCGCTGATAATGGGAGTTTGGCTGAT-3' (SEQ ID NO. 7);

[0068] Bn-Kan-R: 5'-TCGGGGAAATTCGAGCTCTCCACTGTCCCAAACAAATT-3' (SEQ ID NO. 8).

[0069] The amplification product was detected by electrophoresis. The detection results are as follows: Figure 5As shown in Figure A, transgenic plants OE-6, OE-9, OE-13, OE-14, OE-16, OE-23, and OE-27 were able to amplify electrophoretic bands of the expected size (1461 bp), while wild-type plants (WT) and the ddH2O control did not produce any electrophoretic bands. This indicates that the above plants are all positive transgenic plants, and their genomes already contain exogenous gene DNA fragments.

[0070] (4) qRT-PCR identification of transgenic tobacco overexpressing NnMUR3.5

[0071] a. Extraction of genomic RNA from tobacco leaves

[0072] use The Super Total RNA Extraction Kit (purchased from Promega, USA) was used to extract total RNA from the leaves of WT and NnMUR3.5 positive transgenic plants identified in step (3). The RNA was then extracted using... The IIQ RT SuperMix for qRNA (+gDNAwiper) kit (Vazyme, China) reverse transcribes RNA into cDNA.

[0073] b. Real-time quantitative PCR

[0074] To determine whether the NnMUR3.5 gene is overexpressed in tobacco, real-time quantitative PCR (qRT-PCR) was used to analyze the positive transgenic plants identified in step (3). qRT-PCR used... The GreenRealtime PCR Master Mix-Plus kit (Takara Bio Engineering (Dalian) Co., Ltd.) uses the following qRT-PCR primer sequences: (These primers are qRT-PCR specific primers designed based on the nucleotide sequences obtained from sequencing in Example 1)

[0075] NnMUR3.5-F: 5'-TGCTTCTCTGCCACCAACA-3' (SEQ ID NO.9);

[0076] Q-NnMUR3.5-R: 5'-CCTGTTTTGTCAATGGTCGGC-3' (SEQ ID NO. 10).

[0077] PCR program: 95℃ pre-denaturation for 30 seconds, followed by 40 cycles (95℃ for 10 seconds, 60℃ for 10 seconds, 72℃ for 26 seconds).

[0078] The results are as follows Figure 5The NnMUR3.5 gene expression levels of OE-6, OE-9, OE-13, OE-14, and OE-16 lines were significantly higher than that of the wild type control (WT), and the most significant were OE-13 and OE-16 lines, whose expression levels were increased by 134497.72% and 157091.21% compared with the wild type, respectively, indicating that these lines were independent NnMUR3.5 overexpression transgenic lines. The expression levels of OE-23 and OE-27 lines were lower than those of OE-6, OE-9, OE-13, OE-14, and OE-16 lines, so no data statistics were performed, and no subsequent experiments were performed to save experimental costs.

[0079] Example 3 Flower tube growth and flower color change of NnMUR3.5 overexpression transgenic tobacco T3 generation

[0080] (1) Measurement of T3 generation tobacco flower tube length of NnMUR3.5 overexpression lines

[0081] The T3 generation flower tube lengths of wild type tobacco (WT) and transgenic lines OE-6, OE-9, OE-13, OE-14, and OE-16 at the full flowering stage were measured, and at least 15 plants of each line were measured using a digital vernier caliper, and the results are shown in Figure 6 , the flower tube length of the OE-6 line was increased by 15.54% compared with WT, the flower tube length of the OE-9 line was increased by 18.44% compared with WT, the flower tube length of the OE-13 line was increased by 18.64% compared with WT, the flower tube length of the OE-14 line was increased by 19.55% compared with WT, and the flower tube length of the OE-16 line was increased by 16.68% compared with WT. It was shown that NnMUR3.5 gene had good application effect in regulating flower organ size and flower tube length.

[0082] (2) Comparison of T3 generation tobacco flower color of NnMUR3.5 overexpression lines

[0083] The T3 generation flower colors of wild type tobacco (WT) and transgenic lines OE-6, OE-9, OE-13, OE-14, and OE-16 at the full flowering stage were compared, and the comparison method was that at least 3 flowers were randomly selected from each plant of each line, and the Royal Horticultural Society (RHS) plant color chart was used. The results are shown in Figure 7 , the color range of the wild type tobacco was Deep Purplish Pink, and the color range of the transgenic lines was Strong Purplish Red, and the flower color changed obviously. It was shown that NnMUR3.5 gene played an important role in regulating flower color.

[0084] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. The application of the lotus NnMUR3.5 gene in regulating plant flower development, characterized by, The regulation of plant flower development is to increase the length of the plant flower tube or change the color of the plant flower. The nucleotide sequence of the lotus NnMUR3.5 gene is shown in SEQ ID NO.5; The plant in question is tobacco; The change in plant flower color refers to changing the flower color of tobacco from Deep Purplish Pink to Strong Purplish Red by overexpressing the lotus NnMUR3.5 gene in tobacco.

2. The application of an expression cassette containing the lotus NnMUR3.5 gene in regulating plant flower development, characterized in that... The regulation of plant flower development is to increase the length of the plant flower tube or change the color of the plant flower. The nucleotide sequence of the lotus NnMUR3.5 gene is shown in SEQ ID NO.5; The plant in question is tobacco; The change in plant flower color refers to changing the flower color of tobacco from Deep Purplish Pink to Strong Purplish Red by overexpressing the lotus NnMUR3.5 gene in tobacco.

3. The application of a recombinant vector containing the lotus NnMUR3.5 gene in regulating plant flower development, characterized by: The regulation of plant flower development is to increase the length of the plant flower tube or change the color of the plant flower. The nucleotide sequence of the lotus NnMUR3.5 gene is shown in SEQ ID NO.5; The plant in question is tobacco; The change in plant flower color refers to changing the flower color of tobacco from Deep Purplish Pink to Strong Purplish Red by overexpressing the lotus NnMUR3.5 gene in tobacco.

4. Application of recombinant microorganisms containing the lotus NnMUR3.5 gene in regulating plant flower development, characterized by: The regulation of plant flower development is to increase the length of the plant flower tube or change the color of the plant flower. The nucleotide sequence of the lotus NnMUR3.5 gene is shown in SEQ ID NO.5; The plant in question is tobacco; The change in plant flower color refers to changing the flower color of tobacco from Deep Purplish Pink to Strong Purplish Red by overexpressing the lotus NnMUR3.5 gene in tobacco.

5. A method for increasing the length of a plant flower tube, characterized in that, This includes using genetic transformation technology to stably overexpress the lotus NnMUR3.5 gene in plants, thereby increasing the length of the plant's flower tube; the nucleotide sequence of the lotus NnMUR3.5 gene is shown in SEQ ID NO.5; The plant in question is tobacco.

6. A method for changing the flower color of plants, characterized in that, This includes using genetic transformation technology to stably overexpress the lotus NnMUR3.5 gene in plants, thereby altering the flower color; the nucleotide sequence of the lotus NnMUR3.5 gene is shown in SEQ ID NO. 5; The plant in question is tobacco; The change in plant flower color refers to changing the flower color of tobacco from Deep Purplish Pink to Strong Purplish Red.

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