Osmanthus fragrans gene OfBBX24 for promoting plant adventitious bud differentiation as well as expression protein and application of osmanthus fragrans gene OfBBX24

Abstract

The invention discloses an osmanthus fragrans gene OfBBX24 for promoting plant adventitious bud differentiation as well as an expression protein and application thereof, and belongs to the field of plant genetic engineering. The amino acid sequence of the OfBBX24 protein provided by the invention is as shown in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene of the OfBBX24 protein is as shown in SEQ ID NO. 1. According to the invention, a transgenic plant is obtained by cloning the gene, constructing a plant overexpression vector of the gene and transforming tobacco grandiflorum. Experimental results show that overexpression of the OfBBX24 can significantly shorten the differentiation time of the adventitious buds of the transgenic tobacco leaves and improve the differentiation efficiency to 70% or above, which proves that the gene has a strong function of promoting in-vitro regeneration. The invention provides a key gene resource for solving the bottleneck problem that the callus is difficult to differentiate in a woody plant (especially sweet osmanthus) genetic transformation system, and has an important application prospect in forest molecular breeding and efficient propagation technologies.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, specifically relating to the Osmanthus gene OfBBX24 that promotes the differentiation of adventitious buds in plants, its expressed protein, and its applications. Background Technology

[0002] In recent years, with the rapid development of molecular biology techniques, some key genes regulating important traits in plants have been discovered. Due to inherent biological characteristics, many plants (especially woody plants) have failed to establish efficient and stable genetic transformation systems, making it impossible to verify the function of these genes in their native habitat. One of the most challenging aspects is adventitious bud differentiation. Therefore, finding genes regulating adventitious bud differentiation in plants is urgently needed.

[0003] BBX (B-box) genes are zinc finger protein transcription factors containing one or two B-box domains at the N-terminus. Some BBX proteins also have a CCT domain at the C-terminus, consisting of five subgroups. BBX genes play important roles in plant growth and development, including hormone signaling, photomorphogenesis, and flowering. Osmanthus fragrans is an excellent garden tree species with significant economic and ecological value and is widely used. Researchers studying Osmanthus have screened out many key genes that regulate important traits such as leaf color, flower color, and fragrance. Currently, a stable genetic transformation system for Osmanthus has not yet been successfully constructed, mainly because callus tissue is difficult to differentiate into adventitious buds. Therefore, identifying functional genes with the potential to regulate the differentiation of adventitious buds in Osmanthus will not only help establish a stable intrinsic genetic transformation system but also provide theoretical basis and technical reference for the genetic improvement and propagation research of other woody plants. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the primary technical problem solved by this invention is to provide an expression protein of the Osmanthus gene OfBBX24 that promotes adventitious bud differentiation in plants. The secondary technical problem to be solved by this invention is to provide a nucleic acid molecule encoding the aforementioned protein. The final technical problem to be solved by this invention is to provide specific applications of the aforementioned protein and nucleic acid molecule.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A protein of osmanthus of BBX24, which promotes the differentiation of adventitious buds in plants, and its amino acid sequence is shown in SEQ ID NO. 2.

[0007] An isolated nucleic acid molecule encoding the Osmanthus OfBBX24 protein.

[0008] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.1.

[0009] A recombinant vector comprising the aforementioned nucleic acid molecule.

[0010] A recombinant microorganism comprising the nucleic acid molecule or the recombinant vector.

[0011] A transgenic plant cell, tissue, or organ comprising the aforementioned nucleic acid molecule or the aforementioned recombinant vector.

[0012] A method for promoting adventitious bud differentiation in plants by overexpressing the Osmanthus ofBBX24 protein in plants.

[0013] In some embodiments, the method includes: constructing the nucleic acid molecule into an expression vector to obtain a recombinant expression vector; introducing the recombinant expression vector into plant tissue; and cultivating and screening transgenic plants with enhanced adventitious shoot differentiation ability.

[0014] In some embodiments, the plant is large-flowered tobacco or osmanthus.

[0015] The application of the described protein or nucleic acid molecule in promoting the differentiation of adventitious buds in plants.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] This application discloses the OfBBX24 gene of Osmanthus fragrans by cloning, and then constructs its overexpression vector pSuper1300-OfBBX24, which is stably transformed into Nicotiana macrophylla to obtain transgenic plants. Experiments show that overexpression of this gene in Nicotiana macrophylla significantly advances the time of adventitious bud differentiation in tissue culture of transgenic plants, with the differentiation efficiency increasing dramatically from almost zero in the control to nearly 77%. This application discloses for the first time that the OfBBX24 gene possesses a core function of strongly promoting in vitro regeneration of plants, providing an effective gene resource and solution for overcoming the long-standing technical bottleneck of callus regeneration difficulties in the genetic transformation system of woody plants, especially Osmanthus fragrans.

[0018] In summary, this application provides important technical means for molecular breeding and efficient propagation of Osmanthus fragrans and other non-renewable woody plants, and has significant theoretical value and broad application prospects. Attached Figure Description

[0019] Figure 1 The image shows the callus tissue of Osmanthus fragrans, with a scale bar of 5 mm.

[0020] Figure 2 Image showing the cloning results of the OfBBX24 gene;

[0021] Figure 3 Image showing the results of GV3101 bacterial transfection;

[0022] Figure 4 Figure showing the quantitative detection results of OfBBX24 stably transformed large-flowered tobacco lines; Note: EV indicates injection of pSuper1300 empty vector bacterial solution; Lines 1-9 represent the obtained OfBBX24 transgenic tobacco lines;

[0023] Figure 5 Comparison of adventitious bud differentiation in leaves of two OfBBX24 positive lines and EV plants; Note: EV indicates the injection of pSuper1300 empty bacterial culture; Line 5 and Line 7 represent OfBBX24 transgenic tobacco lines. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] The material used in this application is callus induced from leaves of *Osmanthus fragrans* grown in the National Germplasm Resource Bank of Osmanthus fragrans. The callus tissue (…) was… Figure 1 The seedlings were placed into sterilized centrifuge tubes, immediately flash-frozen in liquid nitrogen, and then stored at -80°C. The large-flowered tobacco seedlings used were provided by the research group of Wang Lianggui at Nanjing Forestry University.

[0026] In this embodiment, total RNA was extracted from plants using the TIANGEN Plant RNA Extraction Kit (DP432). The extracted RNA was reverse transcribed into cDNA using the TaKaRa PrimeScript™ RT Master Mix (Perfect Real Time) Reverse Transcription Kit. The resulting cDNA was then diluted 10-fold with water and stored at -20°C.

[0027] Example 1: Construction of an overexpression vector for the OfBBX24 gene

[0028] (1) Obtaining the target gene

[0029] Based on the Osmanthus whole genome database previously published by the research group, one gene sequence was screened and named OfBBX24.

[0030] (3) Design primers

[0031] The full-length nucleotide sequence of the gene was analyzed using BioXM software to identify restriction enzyme sites. Pst I and SmaI were selected as the two restriction endonucleases. Primers were designed using CE design software. The required information was filled in, including the sequence near the restriction sites on the vector, the full length of the target gene, and the two restriction sites (5' and 3' ends) in sequence. The designed primers were then sent to Jereh Biotechnology Co., Ltd. for synthesis: Forward Primer: gactctagaaagcttctgcagATGAAGATTCAGTGTGATGTGTGTGA, Reverse Primer: tatttaaatgtcgaccccgggTCAACCAAGATCAGGCACGG.

[0032] (3) Vector double enzyme digestion

[0033] The pSuper1300 vector was activated and cultured after being removed from the -80℃ ultra-low temperature freezer. The pSuper1300 vector plasmid was extracted according to the kit, followed by a double enzyme digestion experiment. The system is as follows:

[0034] Restriction endonuclease 1 1 μL, restriction endonuclease 2 1 μL, buffer 2 μL, vector plasmid X μL, ddH2O 6 μL, total volume 20 μL.

[0035] Where X (µL) = 1000 ng / vector plasmid concentration (ng / µL). Gently shake the centrifuge tube to mix, centrifuge briefly for 6 seconds, and incubate in a 37°C water bath for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector, and then use a kit for gel extraction and recovery.

[0036] (4) Target gene amplification

[0037] Using cDNA diluted 10-fold as a template, PCR amplification was performed in the following system:

[0038] The total volume of the following components is 20 μL: Forward Primer 1 μL, Reverse Primer 1 μL, cDNA 1 μL, Prime STAR 10 μL, and ddH2O 7 μL.

[0039] Three 20 μL systems were prepared for each gene. The reaction conditions were: denaturation at 98 °C for 10 s; annealing at 58 °C for 15 s; extension at 72 °C for 1 min, 35 cycles; total extension at 72 °C for 10 min; and termination of the reaction at 16 °C. The obtained amplification products were subjected to agarose gel electrophoresis and then recovered by gel cutting using a kit.

[0040] (5) Connection transformation

[0041] The connection system is as follows:

[0042] 200 ng of target gene recovery product, 100 ng of plasmid double enzyme digestion recovery product, 2 μL of ligase, 4 μL of buffer, and ddH2O added to 20 μL, for a total volume of 20 μL.

[0043] Gently shake the centrifuge tube to mix it, centrifuge briefly for 6 seconds, incubate in a 37°C water bath for 60 minutes, and then on ice for 2 minutes.

[0044] Transformation: In a clean bench, use a pipette to take 5 μL of the ligation product and add it to 50 μL of Trelief™ 5α competent cells. Gently mix, incubate on ice for 5 min, incubate in water at 42°C for 60 s, then incubate on ice for 2 min. Add 250 μL of LB liquid (without Kana) and incubate at 37°C and 200 rpm in a shaker for 30 min.

[0045] Spreading: Take 200 μL of the incubated bacterial solution, spread it evenly on LB solid medium (containing 50 mg / L Kana) with a sterile glass rod, and let it dry. After sealing with film, invert the plate and incubate in a 37°C constant temperature incubator for 12-14 h.

[0046] (6) Detection and sequencing of positive single colonies

[0047] After bacteria have grown on the culture medium, single colony detection is performed in a clean bench. Eight plump single colonies are selected for each gene and sequentially backed up on LB solid medium containing Kana resistance. The corresponding single colonies are then transferred to the following system for bacterial testing using a sterile toothpick:

[0048] 35sF 1μL, Gene R 1μL, Green Mix 10μL, ddH2O 8μL, total volume 20μL.

[0049] The PCR reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ total extension for 10 min; and termination of the reaction at 16℃. The obtained amplification products were subjected to agarose gel electrophoresis. Figure 2 Three correct positive colonies were selected and sent for testing. The nucleotide sequence of the OfBBX24 gene was determined as shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein was shown in SEQ ID NO.2.

[0050] (7) Double enzyme digestion verification

[0051] The plasmid with the correct sequence obtained from sequencing was verified by double enzyme digestion, as shown in the following system:

[0052] Restriction endonuclease 1 1 μL, restriction endonuclease 2 1 μL, buffer 2 μL, vector plasmid X μL, ddH2O 6 μL, total volume 20 μL.

[0053] Where X (µL) = 1000 ng / vector plasmid concentration (ng / µL). Gently shake the centrifuge tube to mix, centrifuge briefly for 6 seconds, and incubate in a water bath at 37°C for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector to detect the double digestion status.

[0054] Example 2: Transformation of Agrobacterium GV3101

[0055] (1) Take out the GV3101 competent cells stored in the -80℃ ultra-low temperature freezer and place them on ice to thaw. Add 1µL of plasmid to every 33µL of competent cells, mix well by pipetting, and then successively freeze in an ice bath for 20min, quick freeze in liquid ammonia for 5min, water bath at 37℃ for 5min, and ice bath for 5min.

[0056] (2) Add 500µL of non-resistant LB liquid medium and incubate at 28℃ and 200rppm on a shaker for 1h.

[0057] (3) After the culture is completed, centrifuge the bacterial solution at 6000r for 1min, discard part of the supernatant, and leave 100µL to spread evenly on LB solid medium (containing 50mg / LKana), seal with sealing film, and invert in an incubator at 28℃ for 40-48h.

[0058] (4) Microbial testing and backup: The target band in the microbial test is correct and the brightness is consistent. Figure 3 If the corresponding colonies from the backup plate are picked into LB liquid medium (containing 50 mg / L Kana) and shaken, the bacterial solution and 50% glycerol are mixed at a volume ratio of 3:7 to preserve the bacteria. After being quick-frozen in liquid nitrogen, the solution is stored in an ultra-low temperature freezer at -80°C.

[0059] Example 3: Infecting large-flowered tobacco and obtaining transgenic plants

[0060] (1) Shaking culture: Take out the pSuper1300 empty vector, the target gene-linked vector bacterial culture and the helper vector P19 and thaw them on ice. Use a pipette to add the bacterial culture to 20 mL of LB liquid medium (containing 50 mg / L Kana) and incubate in the dark on a shaker at 28 °C and 200 rppm until the bacterial culture OD. 600 = Between 0.6 and 0.7.

[0061] (2) Explant disinfection: After harvesting the tender leaves of Nicotiana macrophylla, the surface of the leaves was cleaned with detergent to remove the dust layer. After rinsing with running water for 30 minutes, the leaves were transferred to a clean bench for disinfection. First, 75% ethanol was poured into a beaker and shaken to ensure that the ethanol was in full contact with the surface of the tender leaves for 30 seconds. The leaves were then rinsed three times with sterile water. Next, the leaves were soaked in 5% NaCl for 10 minutes and rinsed four times with sterile water. The moisture on the surface of the leaves was then absorbed with sterile filter paper. After disinfection, the leaf edges and veins were removed with a sterile scalpel. The remaining leaves were then cut into small pieces of 0.5 × 0.5 cm for infection.

[0062] (3) Infection: Use sterile tweezers to transfer the cut tobacco leaves into the infection solution and infect for 10 minutes, shaking the conical flask once every 2 minutes.

[0063] (4) Co-culture: After the leaves have been infected, they are taken out and laid flat on sterile filter paper. After the bacterial solution has dried slightly, the leaves are laid flat on the symbiotic culture medium and incubated in the dark at 25°C for 3 days.

[0064] (5) Screening culture: After co-culturing for 3 days, the leaves are transferred to the screening medium for culture. The medium is replaced every 15 days until resistant callus and resistant buds grow.

[0065] (6) Rooting culture: When the length of the sprouted adventitious buds reaches more than 5cm, cut them off from the tissue and remove the callus tissue connected to the stem tissue. Then transfer them to the rooting culture medium to induce rooting.

[0066] (7) Hardening off and transplanting: When the taproot of the transgenic seedlings has grown to about 5 cm and 7-8 leaves have emerged, remove the tobacco seedlings from the culture medium. Without damaging the root system, rinse off the agar gel remaining between the root tissues and place them in a tissue culture bottle containing deionized water for acclimatization culture for 2 days. During acclimatization, change the water regularly to prevent contamination and damage to the tobacco seedlings. After acclimatization is complete, mark the tobacco seedlings with numbers and transplant them into sterilized substrate soil for continued cultivation.

[0067] (8) Screening of transgenic plants: When the transgenic tobacco seedlings were 30 days old, RNA was extracted from the leaves using a kit and reverse transcribed into cDNA. The cDNA was then diluted 10 times and semi-quantitative PCR was performed. After confirming that the cDNA quality was up to standard, qRT-PCR was performed to detect the gene expression level. Transgenic seedlings with high expression levels and consistent growth status were selected for subsequent functional verification. Figure 4 ).

[0068] Example 4: Observation of adventitious bud differentiation in leaves of OfBBX24 transgenic plants

[0069] (1) Explant disinfection: Leaves from two transgenic plants and EV plants were collected and cleaned of dust from the leaf surface with detergent. After rinsing with running water for 30 minutes, the leaves were transferred to a clean bench for disinfection. First, 75% ethanol was poured into a beaker and shaken to ensure full contact between the ethanol and the leaf surface for 30 seconds. The leaves were then rinsed three times with sterile water. Next, the leaves were soaked in 5% NaClO for 10 minutes and rinsed four times with sterile water. The leaves were then dried with sterile filter paper. After disinfection, the leaf edges and veins were removed with a sterile scalpel. The remaining leaves were then cut into 0.5×0.5cm pieces and placed on culture medium (MS + 2.25 mg / L 6-BA + 0.3 mg / L NAA).

[0070] (2) Observation of adventitious bud differentiation time and efficiency: After the leaves have grown on the culture medium, the differentiation of adventitious buds is observed every 3 days. For example Figure 5 As shown in Table 1, after 15 days of growth on the culture medium, no adventitious buds were observed in the EV plants, while obvious adventitious bud differentiation was observed in the leaves of the two transgenic plants. Furthermore, Table 1 shows that the adventitious bud differentiation efficiency of the two lines was significantly higher than that of the EV, approaching 77%. These results indicate that the OfBBX24 gene has the function of improving adventitious bud differentiation efficiency.

[0071] Table 1. Adventitious bud differentiation efficiency of OfBBX24 positive lines and EV plants after 15 days of leaf growth.

[0072]

[0073] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A protein of OfBBX24 from Osmanthus fragrans, characterized in that, The protein has the function of promoting the differentiation of adventitious buds of plants, and the amino acid sequence is shown as SEQ ID NO.

2.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the OfBBX24 protein of Osmanthus fragrans according to claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence is shown as SEQ ID NO.

1.

4. A recombinant vector, characterized in that, The nucleic acid molecule according to claim 2 or 3.

5. A recombinant microorganism, characterized in that, The recombinant vector according to claim 4.

6. A transgenic plant cell, tissue or organ, characterized in that, The recombinant vector according to claim 4.

7. A method for promoting adventitious shoot differentiation in plants, characterized by, The OfBBX24 protein of Osmanthus fragrans according to claim 1 is overexpressed in plants.

8. The method of claim 7, wherein, The method comprises the following steps: The nucleic acid molecule according to claim 2 or 3 is constructed into an expression vector to obtain a recombinant expression vector, the recombinant expression vector is introduced into plant tissues, and cultivation and screening are performed to obtain transgenic plants with enhanced differentiation ability of adventitious buds.

9. The method of claim 7, wherein, The plant is Nicotiana plumbaginifolia or Osmanthus fragrans.

10. The protein according to claim 1, or the nucleic acid molecule according to claim 2 or 3 is applied to promoting the differentiation of adventitious buds of plants.

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