Application of brassica napus BnaC09.FUL transcription factor in promoting flowering of brassica napus

Abstract

The invention discloses application of a brassica napus BnaC09.FUL transcription factor in promoting flowering of brassica napus, and belongs to the technical field of plant genetic engineering application. According to the invention, a gene sequence of BnaC09.FUL in a variety of brassica napus' Westar 'is cloned; by utilizing a transgenic technology, the sequence is over-expressed under the background of brassica napus' Westar ', so that flowering of a transgenic line is remarkably promoted; the brassica napus BnaC09.FUL gene disclosed by the invention provides a new gene resource and a new breeding idea for promoting flowering of brassica napus through molecular design breeding, and has a good application prospect in the aspect of regulating and controlling the flowering time of brassica napus.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and relates to the function and application method of a Brassica napus transcription factor BnaC09.FUL, and specifically to the application of the Brassica napus transcription factor BnaC09.FUL in promoting flowering of Brassica napus. Background Art

[0002] Rapeseed (Brassica napus L.) is an important oil crop that not only provides edible oil, but also the cake after oil extraction can be used as animal feed. In addition, in order to make full use of the value of rapeseed, the development of functional rapeseed has emerged, such as rapeseed as a vegetable, rapeseed flowers as a source of nectar, rapeseed as green manure, and rapeseed as an ornamental plant (Wang Hanzhong. 2018. Rapeseed Industry Development Strategy Guided by New Demands, 40(05): 613-617.). Flowering period is an important agronomic trait. Studies have shown that timely flowering is crucial to plant yield. In order to better adapt to the local environment and changing climatic conditions in production, changing or fine-tuning the flowering period of plants is the main goal of breeding (Cao S, Luo X, Xu D, et al. Genetic architecture underlying light and temperaturemediated flowering in Arabidopsis, rice, and temperate cereals, The Newphytologist, 230(5): 1731–1745.). The main rapeseed production areas in my country are mainly distributed in the Yangtze River Basin. There is a crop rotation contradiction between rapeseed and other crops such as rice (Zhang Yaofeng, Yu Huasheng, Zeng Xiaoyuan, et al. 2019. Research progress and application of early-maturing cabbage rapeseed. Journal of Plant Genetic Resources, 20(02): 258-266.). In addition, the temperature in the Yangtze River Basin rises rapidly from April to May every year. The increase in temperature accelerates the maturity of rapeseed seeds, affecting the thousand-grain weight and oil content of seeds (Wang Biqing, Wang Guohuai. 2021. Research progress on physiological and biochemical characteristics of early-maturing rapeseed. Crop Research, 25(03): 269-271). Studies have shown that the flowering period and maturity period of rapeseed are strongly correlated (Zhou Q, Han D, Mason AS, et al. Earliness traits in rapeseed (Brassica napus): SNP loci and candidate genes identified by genome-wide association analysis. DNA research, 25(3): 229–244.). Therefore, carrying out early flowering and early maturity breeding can not only solve the contradiction of crop rotation, but also solve the adverse effects caused by high temperature forced ripening of rapeseed during the maturity period.

[0003] The MADS-box transcription factor family is one of the most important transcription factor families regulating flowering in plants (Parenicová L, de Folter S, Kieffer M, et al. 2003. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. The Plant cell, 15(7): 1538-1551.). AtFUL is a MIKC cType MADS-box (Smaczniak C, Immink RG, Angenent GC, Kaufmann K. 2012. Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies. Development. 139 (17): 3081-3098.), has been shown to play an important role in regulating the flowering time and floral organ development of Arabidopsis. AtFUL forms a heterodimer with another MADS transcription factor AtSOC1, and jointly regulates the maturation of axillary meristems by directly inhibiting the expression of AtAT-HOOK MOTIF NUCLEAR LOCALIZED 15 (Karami O, Rahimi A, Khan M, et al. 2020. A suppressor of axillary meristem maturation promotes longevity in flowering plants. Nature plants, 6 (4): 368-376.). AtFUL also positively regulates flowering time by directly activating AtSUPPRESSOR OF OVEREXPRESSION OF CO 1 expression and counteracting the repressive effects of AtFLOWERING LOCUS C (AtFLC) and AtSHORT VEGETATIVE PHASE (AtSVP) in Arabidopsis (Balanza V, Martínez-Fernández I, Ferrándiz C. 2014. Sequential action of FRUITFULL as a modulator of the activity of the floral regulators SVP and SOC1. Journal of experimental botany, 65(4): 1193–1203.) (Balanza et al., 2014).In addition, AtFUL redundantly represses inflorescence meristem identity with AtAP1 and AtCAL, and the ful ap1 cal triple mutant exhibits a complete loss of flowering phenotype (Ferrándiz C, Gu Q, Martienssen R, Yanofsky MF. 2000. Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER. Development, 127(4): 725–734.). AtFUL directly represses AtAP2 expression and maintains AtWUSCHEL expression in the shoot apical meristem to promote meristem arrest (Balanzà V, Martínez-Fernández I, Sato S, et al. Genetic control of meristemarrest and life span in Arabidopsis by a FRUITFULL-APETALA2 pathway. 2018. Nature communications, 9(1): 565.). In addition, AtFUL reduces branch angle by repressing the expression of AtSMALL AUXINUPREGULATED RNA 10, but increases branch number and shortens branch internodes by degrading cytokinins (Bemer M, van Mourik H,. JM, et al. 2017. FRUITFULL controls SAUR10expression and regulates Arabidopsis growth and architecture. Journal of experimental botany, 68(13):3391–3403.). In addition, FUL is found in chrysanthemum (Zhao, K, Ding, L, Xia, W, et al. 2020. Characterization of an APETALA1 and a FRUITFUL-like homolog inchrysanthemum. Scientia Horticulturae, 272:109518.), Brachypodium (Li Q, Wang Y, Wang F, et al. 2016. Functional conservation and diversification of APETALA1 / FRUITFULLgenes in Brachypodium distachyon. Physiologia plantarum, 157(4):507–518.), soybean (Jia Z, Jiang B, Gao X, et al. 2015. GmFULa, a FRUITFULL homolog, functions in the flowering and maturation of soybean. Plant cell reports,34(1):121-132.) and tomatoes (Jiang X, Lubini G, Hernandes-Lopes J, et al. 2022. FRUITFULL-like genes regulate flowering time and inflorescence architecture in tomato. The Plant cell, 34(3): 1002-1019.) and other plants have the function of promoting flowering.

[0004] Although researchers have isolated the FUL gene from a variety of plants, the function of the BnaC09.FUL transcription factor in regulating flowering time is still unclear. Therefore, exploring the function of the BnaC09.FUL transcription factor in regulating flowering time is of great significance for breeding new early-flowering varieties of BnaC09.FUL. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the transcription factor BnaC09.FUL of Brassica napus in promoting flowering of Brassica napus, so as to provide valuable gene resources and theoretical basis for breeding of Brassica napus.

[0006] To achieve the above tasks, the present invention adopts the following technical solutions:

[0007] The present invention successfully cloned the BnaC09.FUL gene from the Brassica napus "Westar" variety, connected the full-length CDS sequence of BnaC09.FUL to an overexpression vector with a 35S promoter, and transformed the Brassica napus variety "Westar" using Agrobacterium to further verify the function of the Brassica napus BnaC09.FUL transcription factor in regulating the flowering time of Brassica napus, and observed that the flowering time of the transgenic line 35S:BnaC09.FUL-GFP was significantly earlier than that of the wild type (Westar). Therefore, it is believed that BnaC09.FUL promotes the flowering of Brassica napus. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Screening and identification of 35S:BnaC09.FUL–GFP transgenic plants.

[0009] in,

[0010] A is a schematic diagram of the structure of the BnaC09.FUL gene in the binary overexpression vector pGreen 2×35S for plant transformation; RB indicates the right border; LB indicates the left border; NOS-pro indicates the promoter; NOS-ter indicates the terminator; Basta indicates glufosinate-ammonium; 35S indicates the 35S strong promoter; B is the PCR identification of 35S:BnaC09.FUL-GFP transgenic plants using specific primers; Const indicates plasmid; C is the expression of BnaC09.FUL in the transgenic plants 35S:BnaC09.FUL–GFP transgenic plants analyzed by fluorescence quantitative PCR technology, and the expression of BnaC09.FUL in each transgenic line was normalized with reference to the Westar line;

[0011] Figure 2 Observation and analysis of flowering time of wild type (Westar) and transgenic plant 35S:BnaC09.FUL-GFP. Scale bar is 10 cm.

[0012] in,

[0013] A is the phenotypic observation of flowering time of the wild type (Westar) and transgenic plants OE#5 and OE#10; B is the statistics of bolting time of the wild type (Westar) and transgenic plants OE#5 and OE#10; C is the statistics of flowering time of the wild type (Westar) and transgenic plants OE#5 and OE#10; OE#5 refers to the 35S:BnaC09.FUL-GFP#5 transgenic line, and OE#10 refers to the 35S:BnaC09.FUL-GFP#10 transgenic line. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0015] First of all, it should be noted that in the following examples, the reagents used are all from commercial channels unless otherwise specified, and the test methods, detection methods, etc. involved, unless otherwise specified, are all conventional test methods and detection methods already available in the prior art.

[0016] Example:

[0017] 1. Acquisition, cloning and vector construction of Brassica napus gene BnaC09.FUL

[0018] 1.1 Obtaining genes from Brassica napus

[0019] The protein sequence encoded by the AtFUL (AT5G60910) gene was found using the Arabidopsis database Tair website (https: / / www.arabidopsis.org), and the Brassica napus Westar genome was obtained from the BnIR database (https: / / yanglab.hzau.edu.cn / BnIR). The homologous sequences of FUL in Brassica napus were obtained by local Blast, and the homologous sequences of FUL on the C09 staining were selected and named BnaC09.FUL (BnaC09G0073800WE).

[0020] The CDS sequence of the Brassica napus gene BnaC09.FUL is shown in SEQ ID NO.1, which is 729 bp in length and encodes 242 amino acids. The encoded amino acid sequence is shown in SEQ ID NO.2.

[0021] 1.2 Cloning and vector construction of Brassica napus genes

[0022] 1.2.1 RNA extraction and cDNA synthesis

[0023] Total RNA from different tissues of Brassica napus “Westar” was extracted using the SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology, Changsha, China, Code No. AG21019). One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit (TransGen, Beijing, China, Code No.AE311) was used to synthesize cDNA. The specific steps are as follows:

[0024] (1) Transfer the plant tissue sample stored at -80°C into a 1.5 mL RNase free centrifuge tube and grind it with a grinding rod until the plant tissue is powdery.

[0025] (2) Add 500 μL of lysis buffer PPS to the above centrifuge tube and immediately pipette repeatedly until there is no obvious precipitation in the sample.

[0026] (3) Centrifuge the above lysate at 12000 rpm and 4°C for 5 minutes. Transfer the supernatant to a new 1.5 mL RNase free centrifuge tube. Add 1 / 10 volume of Buffer PA-2 to the supernatant and vortex to mix 10 times. Centrifuge again at 12000 rpm and 4°C for 5 minutes.

[0027] (4) Transfer the supernatant to a new 2.0 mL RNase free centrifuge tube. Add 500 μL of lysis buffer Buffer RLS (add 50×DTT Solution in proportion before use), and immediately pipette repeatedly to vortex until there is no obvious precipitation, so that the sample is fully lysed.

[0028] (5) Add 1 / 2 volume of anhydrous ethanol to the above lysate and mix well by pipetting. If obvious sticky matter or precipitation appears, pipette several times to break up the precipitate.

[0029] (6) Transfer the mixed solution and precipitate to the Plant RNA Mini Column, centrifuge at 12,000 rpm for 2 minutes at room temperature, and discard the filtrate.

[0030] (7) Add 600 μL of Buffer RWA to the Plant RNA Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate.

[0031] (8) Add 750 μL of Buffer RWB (add the specified volume of anhydrous ethanol before use) to the Plant RNA Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate.

[0032] (9) Prepare DNase I reaction solution by mixing 4 μL of DNase I (RNase free), 5 μL of 10× DNase I Buffer, and 41 μL of RNase free water. Add 50 μL of DNase I reaction solution to the center of the Plant RNA Mini Column membrane and let stand at room temperature for 15 minutes.

[0033] (10) Add 350 μL of Buffer RWB to the center of the Plant RNA Mini Column membrane, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0034] (11) Add 750 μL of Buffer RWB to the Plant RNA Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate.

[0035] (12) Place the adsorption column of the Plant RNA Mini Column on a new 2.0 mL Collection Tube and centrifuge at 12,000 rpm for 2 minutes at room temperature.

[0036] (13) Place the adsorption column of the Plant RNA Mini Column in a new 1.5 mL RNase Free centrifuge tube, add 50 μL RNase Free Water to the center of the adsorption column membrane, let it stand at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 2 minutes at room temperature to elute the RNA.

[0037] (14) Use a reverse transcription kit to synthesize cDNA. Take 1 μL RNA, 1 μL Anchored Oligo(dT)18 Primer, 1 μL 2×ES Reaction Mix, The reaction mixture was prepared with 1 μL of RT / RI Enzyme Mix, 1 μL of gDNA Remover, and 6 μL of RNase free water. The reaction conditions were incubation at 42°C for 15 minutes and heating at 85°C for 5 seconds. The obtained cDNA was stored at -20°C.

[0038] 1.2.2 Cloning of BnaC09.FUL gene

[0039] The specific primers designed according to the CDS sequence of the BnaC09.FUL gene and the qRT-PCR primers used to detect each transgenic line are shown in Table 1:

[0040] The qRT-PCR primers used are shown in Table 1:

[0041] Table 1 BnaC09.FUL primer information

[0042]

[0043] Using the "Westar" cDNA sequence as a template and high-fidelity enzymes for PCR amplification, the reaction system is as shown in Table 2:

[0044] Table 2 PCR amplification of target fragments

[0045]

[0046] The PCR reaction conditions are shown in Table 3:

[0047] Table 3 High-fidelity PCR reaction conditions

[0048]

[0049] After the reaction, agarose gel electrophoresis was performed to determine whether the size of the target gene was correct based on the DNA marker; the band of the correct size was quickly cut, and the target gene fragment was purified and recovered according to the instructions of the universal DNA purification and recovery kit (TIANGEN DP214), and the quality and concentration of the target gene obtained after recovery and purification was detected using the NanoDrop ND-1000 instrument.

[0050] 1.2.3 Plant overexpression vector construction

[0051] (1) The plant binary expression vector was digested with restriction endonucleases in a 37°C water bath for 5-6 hours. The digested vector was recovered and purified according to the PCR product recovery and purification method described above, and the quality and concentration of the linearized vector obtained after digestion and purification was detected by NanoDrop ND-1000 instrument. The reaction system was prepared on ice according to the instructions for homologous recombination of ClonExpressMultiS One Step Cloning Kit, in which the purified target fragment and the linearized vector after digestion were mixed and connected at a molar ratio of 3:1, and the reaction procedure was to react at 37°C for 30 minutes to complete the recombination of the target fragment and the linearized vector.

[0052] (2) Heat shock transformation of E. coli and identification of expression vectors. The homologous recombination ligation products were added to the competent cells of E. coli DH5α, gently pipetted and mixed, and placed on ice for 20 minutes, in a 42°C water bath for 90 seconds, and quickly placed on ice for 2 minutes. 700 μL of LB culture medium was added, and the culture was placed in a 37°C shaker for 30 minutes. The bacterial solution was then spread on LB solid culture medium containing 50 μg / mL Kanamycin (Kan+), and inverted in a 37°C constant temperature incubator overnight. Ten monoclonal colonies were picked at a time and placed in 7 μL ddH2O and mixed evenly. 1 μL of bacterial solution was taken as a template, and the forward primer (35S) on the vector and the reverse primer of the target fragment were used to configure the reaction system for PCR amplification. The PCR products were subjected to agarose gel electrophoresis, and two monoclonal colonies with correct bands were selected and expanded in LB liquid culture medium containing 50 μg / mL Kan+. The bacterial solution was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were identical to the target sequence, indicating that the BnaC09.FUL gene in Brassica napus 'Westar' had been successfully cloned into the plant overexpression vector.

[0053] (3) Extract the recombinant plasmid. Take 500 μL of the bacterial solution whose sequencing results are consistent with the target fragment sequence and place it in a shaking tube that has been pre-added with 8 mL of LB culture medium containing resistance (50 μg mL–1, Kan+) for expansion culture. Then refer to the plasmid extraction kit (TIANGEN DP103) for plasmid extraction to obtain the 35S:BnaC09.FUL-GFP overexpression vector.

[0054] 2. Acquisition, screening and identification of transgenic plants

[0055] 2.1.1 Genetic transformation of Brassica napus ‘Westar’

[0056] (1) The successfully constructed 35S:BnaC09.FUL-GFP overexpression vector was transformed into Agrobacterium tumefaciens GV3101 cells, single clones were picked and verified by colony PCR using the vector forward primer (35S) and the reverse primer of the target fragment.

[0057] (2) Use sterile forceps and a scalpel to cut the hypocotyls of Westar seedlings 6 days after sowing, each with a length of 0.8-1 cm.

[0058] (3) Cultivate Agrobacterium to an OD of 0.8, centrifuge the cultured strain at 6000 rpm for 10 min, discard the supernatant, and resuspend in the same volume of DM as the bacterial solution (20 mL DM dilution, in a sterilized culture dish).

[0059] (4) Immerse the cut explants in the resuspension solution from the previous step and infect for 15-30 minutes, shaking 4-5 times in between.

[0060] (5) Transfer to M1 medium, place 20-25 explants per dish, and place in the dark at 24°C.

[0061] (6) After 36-48 hours, transfer the cells to M2 and culture them in an artificial climate chamber (24°C, 16 hours day / 8 hours night).

[0062] (7) After three weeks, transfer the cells to M3 medium and subculture every 2-3 weeks until green shoots appear.

[0063] (8) Transfer to M4 for rooting, which takes 2-4 weeks until a relatively complete root system appears.

[0064] 2.1.2 Culture medium and formula used for genetic transformation:

[0065] (1) LB medium (1 L): peptone 10 g, yeast extract 5 g, sodium chloride 10 g, solid medium plus agar 15 g / L, autoclave for 20 min.

[0066] (2) DM (100 mL): MS 0.44 g, sucrose 3 g, adjust pH to 5.84-5.88 after volume adjustment, sterilize. Add 100 μL of acetosyringone (100 μM) when using.

[0067] (3) M0 (100 mL): MS 0.22 g, sucrose 3 g, adjust pH to 5.84-5.88 after volume adjustment, agar 0.7 g, sterilize.

[0068] (4) M1 (500 mL): MS 2.2 g, sucrose 15 g, mannitol 9 g, 2,4-dichlorophenoxyacetic acid (1 mg / L) 0.5 mL, furanaminopurine (0.3 mg / L) 0.5 mL, adjust the pH to 5.84-5.88 after volume adjustment, add agar 3.5 g, sterilize. Add 500 μL of acetosyringone when using.

[0069] (5) M2 (500 mL): MS 2.2 g, sucrose 15 g, mannitol 9 g, 2,4-D (1 mg / L) 0.5 mL, furanaminopurine (0.3 mg / L) 0.5 mL, adjust the pH to 5.84-5.88 after volume adjustment, add agar 3.5 g, and sterilize. When using, add Staurosporine 5 μL, Timentin (300 mg / mL) 0.5 mL, and Hygromycin B (25 mg / mL) 250 ul.

[0070] (6) M3 (500 mL): MS 2.2 g, glucose 5 g, xylose 0.125 g, MES 0.3 g, adjust pH to 5.84-5.88 after constant volume, add agar 3.5 g, sterilize. When using, add trans-Zeatin (2.0 mg / L) 0.5 mL, IAA (0.1 mg / L) 0.5 mL, Timentin (300 mg / L) 0.5 mL, AgNO3 75 μL, Hygromycin B (25 mg / mL) 250 μL.

[0071] (7) M4 (500 mL): MS 4.4 g, sucrose 5 g, adjust pH to 5.84-5.88 after volume adjustment, add agar 3.5 g, sterilize. When using, add Timentin (300 mg / L) 0.5 mL.

[0072] 2.2 Screening and identification of transgenic plants

[0073] The transformed T0 generation rapeseed seedlings were moved to an artificial climate incubator for normal culture. After four true leaves grew, they were sprayed with glufosinate-ammonium herbicide (at a concentration of 0.06% (v / v) every other day. ), spray 5 to 6 times. Take the cabbage rapeseed seedlings that can still grow normally after spraying Basta and move them to new nutrient soil. After the rosette leaves grow out, extract the genomic DNA, use the vector forward primer (35S) and the reverse primer of the target fragment to detect whether the target gene is successfully transferred, identify the correct T0 generation transgenic plants, and identify the plants that have successfully transferred the target gene as T0 generation transgenic plants. The seeds (T1 generation) harvested after maturity are spread on MS medium containing 10μg / mL glufosinate ammonium and vernalized at 4℃ for 2 to 3 days, and then continue to screen. Select the seedlings with normal leaf and root development and transfer them to nutrient soil. After normal growth and maturity, harvest the individual plants, thereby obtaining T2 generation homozygous transgenic seeds for subsequent experiments.

Claims

1. Application of the BnaC09.FUL transcription factor of Brassica napus to promote flowering of Brassica napus, characterized in that: The nucleotide sequence of the Brassica napus BnaC09.FUL gene is shown in SEQ ID NO.

1.

2. The use of the Brassica napus BnaC09.FUL transcription factor according to claim 1 in promoting flowering of Brassica napus, characterized in that: The BnaC09.FUL gene was transformed into Brassica napus "Westar" and a transgenic line 35S:BnaC09.FUL-GFP with significantly advanced flowering time was obtained.

3. The use of the Brassica napus BnaC09.FUL transcription factor in promoting flowering of Brassica napus according to claim 2, characterized in that: The flowering time of transgenic plants was significantly advanced.

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