A banana MaSVP3 gene and its application in delaying plant flowering

Abstract

The present invention relates to the field of genetic engineering, specifically a banana MaSVP3 gene and its use in delaying plant flowering. The nucleotide sequence of the banana MaSVP3 gene is shown in SEQ ID No. 1. The banana MaSVP3 gene shown in SEQ ID No. 1 is cloned and ligated into a vector. Finally, Arabidopsis thaliana is infected via Agrobacterium-mediated transformation to produce transgenic Arabidopsis thaliana plants with delayed flowering. Compared to the prior art, the present invention constructs MaSVP3 transgenic Arabidopsis thaliana and studies its reproductive developmental phenotype. Based on observations of the banana MaSVP3 gene in the model plant Arabidopsis thaliana, the present invention demonstrates that the banana MaSVP3 gene can delay flowering.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to a banana MaSVP3 gene and its application in delaying plant flowering. Background Art

[0002] The flowering process in plants is regulated by a variety of endogenous and exogenous signals. In addition to the traditional flowering induction mechanism, the JA signaling pathway is also involved in the flowering induction process, with short vegetative phases (SVPs) playing a key role. As a key regulatory factor that inhibits flowering in plants, SVP belongs to the MADS-box gene family and plays a crucial role in regulating flowering timing, flower development, and dormancy. The SVP gene is involved in the formation of the floral meristem. SVP dimers with the FLOWERING LOCUS C (FLC) protein directly target the promoter sequences of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FLOWERING LOCUS T (FT). This mechanism effectively inhibits flowering by reducing the expression levels of SOC1 and FT genes. This mechanism reflects the complex genetic strategy used by plants to precisely control flowering time. The molecular mechanisms by which SVPs regulate flowering in plants are complex and require further investigation. Existing literature reports that research on the function of SVPs genes mainly focuses on model species such as Arabidopsis and rice, while the function of SVPs in banana has not been reported. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a banana MaSVP3 gene and its application in delaying plant flowering.

[0004] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0005] A use of a banana MaSVP3 gene in delaying plant flowering. The nucleotide sequence of the banana MaSVP3 gene is shown in SEQ ID No. 1.

[0006] Furthermore, the plant is Arabidopsis thaliana.

[0007] Specifically, the application of the banana MaSVP3 gene in delaying plant flowering includes: cloning the banana MaSVP3 gene as shown in SEQ ID No. 1, connecting it to a vector, and finally infecting Arabidopsis thaliana through Agrobacterium-mediated transformation to obtain transgenic Arabidopsis thaliana plants with delayed flowering.

[0008] Preferably, the primers used to clone the banana MaSVP3 gene as shown in SEQ ID No. 1 include:

[0009] Forward primer: 5′-ATGGCGAGGGAGAAGATACAGA-3′;

[0010] Reverse primer: 5'-TCACACCCACTTTGACACTGGC-3'.

[0011] Compared with the existing technology, the present invention constructs MaSVP3 transgenic Arabidopsis to study its phenotype in reproductive development. Based on the observation of banana MaSVP3 gene in the model plant Arabidopsis, it is concluded that the application of banana MaSVP3 gene can delay flowering. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 For the extraction of total RNA from different banana tissues (X1-X6 are leaves, roots, pseudostems, bulbs, mixed samples of flower organs and fruits respectively).

[0013] Figure 2 This is the electrophoresis test result after gene cloning of banana MaSVP3.

[0014] Figure 3 The purpose is to screen the resistance of banana MaSVP3 transgenic Arabidopsis and detect the positive Arabidopsis seedlings by PCR followed by electrophoresis.

[0015] Figure 4 The phenotypes of WT (wild type) Arabidopsis and MaSVP3 transgenic Arabidopsis at 6-7 weeks.

[0016] Figure 5 The phenotypes of WT (wild type) Arabidopsis and MaSVP3 transgenic Arabidopsis at week 8. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0018] Experimental materials used in the following examples: Banana variety "Musa spp. AAA Cavendish subgroup cv 'baxi'" was obtained from the Guangzhou Branch of the National Banana Improvement Center, Fruit Research Institute, Guangdong Academy of Agricultural Sciences. Arabidopsis seeds used were the Columbia ecotype (Columbia-0).

[0019] The experimental reagents and instruments used include: RNA extraction kit purchased from Aikerui; reverse transcription kit purchased from TaKaRa; plasmid extraction kit and DNA gel recovery kit are both TRANSGEN brand, purchased from Beijing Quanshijin Biotechnology Co., Ltd.; PCR instrument is Bio-RAD C1000 TouchTM Thermal Cycler PCR instrument; ultra-clean workbench purchased from Suzhou Purification Technology Co., Ltd.; pBWA(V)HS-ccdb-GLosgfp expression vector is a laboratory storage material.

[0020] Unless otherwise specified, other raw materials, reagents and equipment were purchased from the market.

[0021] Example 1: Cloning of the banana MaSVP3 gene

[0022] 1. Total RNA extraction from Brazilian banana leaves, roots, pseudostems, bulbs, mixed flower organs and fruits

[0023] Total RNA from leaf samples was extracted using the Ecoray MiniBEST Plant RNA Extraction Kit. The steps are as follows:

[0024] (1) Transfer the fresh banana tissue sample into a liquid nitrogen pre-cooled mortar and pestle and grind the tissue into powder;

[0025] (2) Place 60 mg of powdered sample in a 1.5 mL enzyme-free centrifuge tube containing 500 μL of lysis buffer RLS Buffer (make sure 50× DTT solution has been added). Quickly vortex mix or pipette repeatedly until the sample is completely lysed (no visible precipitation).

[0026] (3) After the lysate was allowed to stand at room temperature for 2 minutes, it was centrifuged at 12,000 rpm at 4°C for 5 minutes;

[0027] (4) Transfer the supernatant to a new 1.5 mL enzyme-free centrifuge tube;

[0028] (5) Add half volume of anhydrous ethanol to the lysate;

[0029] (6) Transfer the mixture and precipitate to the Plant RNA Mini Column, centrifuge at 12,000 rpm for 2 minutes at room temperature, and discard the filtrate (if the mixture exceeds 700 μL, add it in batches);

[0030] (7) Add 500 μL RWA Buffer to the Plant RNA Mini Column tube, centrifuge at 13,000 rpm for 3 minutes, and discard the filtrate;

[0031] (8) Add 650 μL of RWB Buffer to the above tube, centrifuge at 13,000 rpm for 3 minutes, and discard the filtrate.

[0032] (9) Repeat step (8);

[0033] (10) Place a new 2 mL collection tube into the Plant RNA Mini Column tube and centrifuge at 13,000 rpm for 5 minutes;

[0034] (11) Place a new enzyme-free centrifuge tube in the Plant RNA Mini Column tube, add 70 μL of enzyme-free sterilized water to the center of the filter membrane, place it at 25°C for 7 minutes, and then centrifuge it at 13,000 rpm for 4 minutes at 25°C to elute the RNA to obtain purified RNA; if not used immediately, the dissolved RNA should be stored at -80°C.

[0035] The results of 1% agarose gel electrophoresis were shown in Figure 1 , with 28S RNA and 18S rRNA bands, indicating that the extracted total RNA was intact and largely undegraded. Furthermore, the absorbance of six RNA samples at 260 nm and 280 nm was measured using a Thermo NANODROP 2000 UV spectrophotometer. The A260nm / 280nm ratios were between 2.0 and 2.2, indicating that the total RNA was essentially free of contamination by carbohydrates, EDTA, phenol, creatine salts, or proteins, meeting the requirements for subsequent molecular experiments.

[0036] 2. First-strand cDNA synthesis

[0037] The purified RNA was synthesized into cDNA using the TAKARA reverse transcription kit PrimeScript™ IIlst Strand cDNA Synthesis Kit. The details are as follows:

[0038] (1) Prepare the following reverse transcription mixture on ice. The reaction system is as follows:

[0039]

[0040]

[0041] (2) After gentle mixing, react at 30°C for 10 minutes;

[0042] (3) 95°C for 5 minutes to inactivate the enzyme, then cool on ice;

[0043] (4) Take out and store in a -20℃ refrigerator.

[0044] 3. Primer design for gene sequences

[0045] The CDS gene sequence of MaSVP3 (Ma10_t19310.2) was downloaded from the banana genome database (https: / / banana-genome-hub.southgreen.fr / data_search / organism):

[0046] (See SEQ ID No. 1).

[0047] Primers were designed using PrimerPrimer 5.0 software and commissioned to Beijing Qingke Biotechnology Co., Ltd.

[0048] Forward primer: 5′-ATGGCGAGGGAGAAGATACAGA-3′;

[0049] Reverse primer: 5'-TCACACCCACTTTGACACTGGC-3'.

[0050] 4. Banana MaSVP3 specific amplification

[0051] Using banana cDNA as a template, the full-length sequence of the MaSVP3 gene was amplified using 2×Phanta Max Master Mix high-fidelity enzyme and primers MaSVP3-F / MaSVP3-R. The PCR reaction system is as follows:

[0052]

[0053] PCR amplification conditions: ① pre-denaturation at 95°C for 3 minutes; ② denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 1 minute for 30 cycles; ③ final extension at 72°C for 5 minutes.

[0054] 5. Agarose gel electrophoresis detection and gel recovery

[0055] The PCR reaction products were subjected to 1% agarose gel electrophoresis using 1x TAE (40 mM Tris-HCl (pH 8.3), 1 mM EDTA, 20 mM glacial acetic acid) as the electrophoresis buffer and Gel Red (Biotium) as the nucleic acid dye. The electrophoresis results were detected by ultraviolet light and the PCR products were recovered using the TRANSGEN DNA gel recovery kit. The specific steps are as follows:

[0056] (1) After DNA electrophoresis is complete, quickly cut the agarose gel region containing the target DNA fragment under UV light. It is recommended to use absorbent paper towels to remove the liquid on the surface of the gel, then cut the gel into small pieces and remove as much excess agarose as possible. Measure the weight of the gel (minus the weight of the empty tube) and calculate the total volume of the gel as 100 mg of gel corresponds to 100 μL.

[0057] (2) Add an equal volume of Buffer GDP to the gel. Heat the mixture in a 50-55°C water bath for 7-10 minutes, adjusting the heating time according to the size of the gel pieces, until the gel is completely dissolved. Invert the mixture while in the water bath to accelerate gel dissolution.

[0058] (3) Briefly centrifuge to recover any remaining liquid from the tube walls. Place the FastPure DNA MiniColumns-G column in a 2 mL collection tube, transfer no more than 700 μL of gel lysate to the column, and centrifuge at 12,000 rpm for 50 seconds. If the volume of gel lysate exceeds 700 μL, transfer the remaining lysate to the column in batches and centrifuge again.

[0059] (4) Pour off the filtrate in the tube and place the adsorption column back into the tube. Add 300 μL of Buffer GDP to the adsorption column, let it stand at room temperature for 1 minute, and then centrifuge at 12,000 rpm for 1 minute.

[0060] (5) Pour off the filtrate again, add 700 μL of Buffer GW to the adsorption column, and centrifuge at 12,000 rpm for 1 minute.

[0061] (6) Repeat step (5).

[0062] (7) After discarding the filtrate, centrifuge again at 13,000 rpm for 3 minutes.

[0063] (8) Transfer the adsorption column to a 1.5 mL sterile centrifuge tube, add 20 to 30 μL of elution solution to the center of the adsorption column, let it stand for 2 minutes to fully elute the DNA, and then centrifuge at 12,000 rpm for 1 minute to complete the elution. Discard the adsorption column and store the collected DNA sample at -20°C for future use; connect the collected DNA sample to the pUC19 vector and send it to Shanghai Bioengineering Company for sequencing verification. The correctly sequenced fragment was detected by 1% agarose gel electrophoresis and found to be consistent with the gene fragment length predicted on the banana genome website. The length of the MaSVP3 gene fragment is 687 bp (see Figure 2 ).

[0064] Example 2: Application of banana MaSVP3 gene to delay plant flowering

[0065] 1. Ligation of target fragments and transformation of competent E. coli

[0066] (1) Target fragment ligation system:

[0067]

[0068]

[0069] Reaction conditions: 26°C for 5 minutes.

[0070] (2) Transformation of target fragment ligation system

[0071] Take 100 μL of Escherichia coli DH5α competent cells (Qingke Biotechnology) and add 10 μL of the ligation product, mix well, and place on ice for 25 minutes; heat shock in a 42°C metal bath for two minutes, then place on ice for 3 minutes; add 700 μL of LB solution to a 2 mL centrifuge tube and resuscitate on a shaker at 210 rpm at 37°C for 1 hour; centrifuge at 4500 rpm for 6 minutes, retain 200 μL of the supernatant in the centrifuge tube, transfer the supernatant to an LB plate containing Amp, evenly spread the plate, and incubate at 37°C for about 10 hours; pick a single colony and perform PCR amplification with universal primers (forward primer: 5'-ATGGCGAGGGAGAAGATACAGA-3'; reverse primer: 5'-TCACACCCACTTTGACACTGGC-3') to determine whether the target fragment is successfully connected.

[0072] 2. Screening and identification of recombinants

[0073] Single colonies were plated in 1 mL of LB medium containing Amp-resistant bacteria and incubated at 37°C on a shaker for 10 hours. An appropriate amount of the bacterial culture was then amplified using Taq enzyme for PCR. The specific PCR reaction configuration is shown in Table 2-6. Bacteria containing PCR products that meet the required band size were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing confirmation. Bacteria with confirmed sequencing results were expanded and stored at -80°C until further use.

[0074] 3. Extraction of recombinant plasmid

[0075] Extract plasmid using the TRANSGEN EasyPure Plasmid MiniPrep Kit:

[0076] (1) Column equilibration: 50 μL of equilibration buffer BL was added to the CP3 in the tube, and the mixture was centrifuged at 13,000 rpm for 1 min. After discarding the waste liquid, the adsorption column was returned to the collection tube for subsequent use.

[0077] (2) Place 3 mL of bacterial suspension in a centrifuge tube and centrifuge at 13,000 rpm for 3 minutes;

[0078] (3) Add 300 μL of solution P1 to the above centrifuge tube;

[0079] (4) Add 300 μL of solution P2 to a 2 mL centrifuge tube and shake gently to mix;

[0080] (5) Add 450 μL of solution P3 to the above centrifuge tube and centrifuge at 13,000 rpm for 12 minutes;

[0081] (6) Transfer the supernatant after centrifugation to CP3, centrifuge at 13,000 rpm for 2 minutes, discard the supernatant, and place the CP3 into the collection tube again;

[0082] (7) Add 600 μL of rinse buffer PW to CP3, centrifuge at 12000 rpm (approximately 13400 g) for 3 minutes, and discard the supernatant;

[0083] (8) Same as step (7);

[0084] (9) Place CP3 in a tube and centrifuge at 13,000 rpm for 5 minutes;

[0085] (10) Place CP3 in a new 2 mL enzyme-free centrifuge tube, add 60 μL of deionized water to the center of the adsorption membrane, let it stand at 25°C for 5 minutes, then centrifuge at 13,000 rpm for 5 minutes to collect it and store it at -20°C.

[0086] 4. Construction of plant expression vector

[0087] The expression vector pBWA(V)HS-ccdb-GLosgfp was double-digested with restriction endonucleases BsaI / Eco31I. The digestion system consisted of 4 μL of plasmid, 2 μL of 10× buffer, 1 μL each of BsaI / Eco31I, and 13 μL of ddH2O. Mix well and place in a 37°C waterbath for approximately 2 hours. The expression vector was then recovered by agarose gel electrophoresis (see 2.5 for recovery methods). The recovered vector and fragment were then subjected to homologous recombination using a ligation system consisting of 5 μL of the pBWA(V)HS-ccdb-GFP gel-recovered product, 5 μL of the MaSVP3 gel-recovered product, and 10 μL of Biorun 2 EasyClone Mix. The mixture was then incubated in a 37°C metal bath for 12 hours. The ligation system was then transformed into competent Escherichia coli DH5α (Qingke Biotechnology) and inverted for overnight culture. GFP-F / MaSVP3-GFP-R primers were used to detect bacterial colonies to determine whether the target fragment was inserted in the forward or reverse direction. The correct colonies were expanded and sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0088] 5. Transformation of recombinant plasmid into Agrobacterium and identification of positive clones

[0089] (1) GV3101 (Qingke Biotechnology) and EHA105 competent Agrobacterium (Qingke Biotechnology) stored at -80°C were brought to room temperature, partially thawed to an ice-water mixture, and then placed in an ice box;

[0090] (2) Take 30 μL of competent cell and add 1.5 μL of homologous recombination plasmid into a 1.5 mL sterile centrifuge tube. Manually tap the bottom of the tube to mix it evenly. Then, let it stand on ice for 5 minutes, treat it in liquid nitrogen for 5 minutes, keep it in a constant temperature metal bath at 37°C for 5 minutes, and finally cool it in an ice bath for 5 minutes.

[0091] (3) Add 700 μL of antibiotic-free LB liquid medium and then culture with shaking at 28°C for 2 to 3 hours;

[0092] (4) Centrifuge at 6000 rpm for 5 minutes, aspirate 600 μL of supernatant, and retain about 100 μL of supernatant, pipette and resuspend, and apply to a plate containing (30 μg / mL Rif, 50 μg / mL Kan). Invert the culture dish and place it in a 28°C constant temperature incubator for 2 to 3 days.

[0093] (5) Pick a single colony and use detection primers to detect whether it has been transformed into Agrobacterium competent cells GV3101 and EHA105 by PCR;

[0094] (6) The correctly screened colonies were cultured in LB liquid medium containing 30 μg / mL rifampicin and 50 μg / mL kanamycin, and then stored at -80°C.

[0095] 6. Agrobacterium tumefaciens GV3101-mediated transformation of Arabidopsis

[0096] The banana MaSVP3 gene was successfully transferred into Agrobacterium GV3101 (Qingke Biotech). Wild-type Arabidopsis thaliana from Columbia was planted in advance; WT (wild-type) Arabidopsis served as a control. When the Arabidopsis reached peak flowering, all fertilized, mature pods were carefully removed, leaving only those in or about to bloom. The plants were then watered thoroughly the day before infection. Using the inflorescence dip method, the Arabidopsis thaliana was infected with Agrobacterium carrying the target gene.

[0097] The specific operations are as follows:

[0098] (1) Weigh 15 g of sucrose into a 250 mL Erlenmeyer flask and dilute to 100 mL with distilled water. Then add 40 μL of Silwet-77 surfactant and place Agrobacterium tumefaciens GV3101 (Qingke Biotechnology) in the suspension to prepare an Agrobacterium suspension with an OD600 of 0.8-1.2.

[0099] (2) Immerse the inflorescence of Arabidopsis thaliana completely in the prepared bacterial solution for 2 to 3 seconds, then seal it with plastic wrap to maintain a humidity above 80%, and incubate it in the dark at 25°C for 24 hours. The entire immersion process lasts for 7 days, during which three immersion treatments are performed;

[0100] (3) Re-infection was performed according to the number of inflorescences, with a re-infection cycle of 7 days for a total of three rounds of treatment. After the infiltration was completed, the Arabidopsis plants were placed in a light incubator for normal growth, and watered in time according to the growth status of the plants until the fruit pods matured. The seeds were collected in time and the positive plants were screened.

[0101] The seeds obtained for the first time belong to the T0 generation, and the T0 generation seeds are disinfected. First, disinfect with 95% ethanol for 10 minutes, then disinfect with 75% ethanol for 10 minutes, and rinse with sterile water 2 to 3 times, 1 minute each time. Afterwards, spread the seeds evenly on the screening culture medium containing hygromycin (Hyg), and place them at 4°C for 2 to 3 days to complete the dormancy treatment; then, move the culture dish to an environment of 23 to 25°C, adopt a photoperiod of 16 hours of light / 8 hours of darkness, and continue to culture for 10 to 14 days. After two weeks, depending on the growth of the plants, transfer the surviving seedlings selected to the nutrient soil and continue to culture at 23°C / 16 hours of light / 8 hours of darkness.

[0102] Filter results such as Figure 3 As shown in the figure: Arabidopsis thaliana was successfully genetically transformed using the inflorescence infection method, and then the T0 generation seeds were collected. In the process of screening positive plants of T0 generation Arabidopsis thaliana seeds, as shown in the figure: Figure 3 As shown in a in the figure, the seeds were cultured in MS medium containing 30 mg / L hygromycin (Hyg). Non-transgenic Arabidopsis seedlings showed signs of leaf yellowing and poor root development, which eventually led to plant apoptosis. In contrast, the transgenic Arabidopsis seedlings indicated by the red arrows showed healthy green leaves and normal root growth, and the leaves did not show yellowing in their later growth stages. Leaf DNA was extracted from T1 generation transgenic Arabidopsis plants and the control wild-type Arabidopsis, and PCR detection was performed using hygromycin (Hyg) universal primers to further confirm whether the target gene was successfully transferred into Arabidopsis. Wild-type Arabidopsis leaf DNA was used as a negative control, as shown in Figure 3 As shown in b, all positive seedling PCR products detected showed consistent specific bands, while no target specific bands were observed in the negative control wild-type plant samples. The results confirmed that the target gene had been successfully transferred into the transgenic Arabidopsis plants.

[0103] Then, homozygous transgenic Arabidopsis was obtained in the T2 generation, and its phenotype was observed. The growth comparison results of MaSVP3 transgenic Arabidopsis and WT (wild type) Arabidopsis during the 6th and 7th weeks were as follows: Figure 4 and Figure 5 As shown by Figure 4 It can be seen that there is little difference between MaSVP3 transgenic Arabidopsis and wild-type Arabidopsis. Figure 5 It can be seen that the growth and development of the transgenic line was significantly slower than that of the wild type at the 8th week of growth. Statistical data showed that the vine sprouting time of MaSVP3 transgenic Arabidopsis was about ten days later than that of the wild type.

[0104] In summary, the banana MaSVP3 gene can delay flowering in Arabidopsis.

[0105] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An application of the banana MaSVP3 gene in delaying flowering in Arabidopsis thaliana, characterized in that: The nucleotide sequence of the banana MaSVP3 gene is shown in SEQ ID No.

1.

2. The use of the banana MaSVP3 gene in delaying flowering of Arabidopsis thaliana according to claim 1, characterized in that: The banana MaSVP3 gene shown in SEQ ID No. 1 was cloned and connected to a vector, and finally Arabidopsis was infected by Agrobacterium-mediated transformation to obtain transgenic Arabidopsis plants with delayed flowering.

3. The use of the banana MaSVP3 gene in delaying flowering of Arabidopsis thaliana according to claim 1, characterized in that The primers used to clone the banana MaSVP3 gene shown in SEQ ID No. 1 include: Forward primer: 5′-ATGGCGAGGGAGAAGATACAGA-3′; Reverse primer: 5'-TCACACCCACTTTGACACTGGC-3'.

Images