A ProWUS promoter specifically expressed in the apical meristem of hybrid tulip tree and its applications

Abstract

This invention discloses a promoter, ProWUS, specifically for the apical meristem of hybrid tulip tree and its applications, belonging to the field of plant genetic engineering technology. Based on the genome sequence information of Chinese tulip tree, the ProWUS promoter sequence was cloned, and its nucleotide sequence is shown in SEQ ID NO.1. Driven by the cloned ProWUS promoter, the reporter gene can be specifically expressed at the apical meristem of the plant. Using the promoter provided by this invention, the specific expression of the corresponding target gene in the apical meristem can be induced in plant genetic engineering, which not only enriches the molecular biological theoretical basis of plant shoot tips but also has important application value in the development of plant apical meristems and the maintenance of stem cells.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a ProWUS promoter specifically expressed in the apical meristem of hybrid tulip tree and its application. Background Technology

[0002] The hybrid tulip tree (Liriodendron sino-americanum PCYieh ex Shang et Z.R.Wang) is an interspecific hybrid bred by Professor Ye Peizhong, a renowned Chinese forestry breeder, through artificial hybridization of the Chinese tulip tree (Liriodendron chinense (Hemsl.) Sarg.) and the North American tulip tree (Liriodendron tulipifera L.). The hybrid tulip tree grows faster than the Chinese tulip tree, exhibiting significant hybrid vigor. It boasts an attractive tree shape, leaves resembling a traditional Chinese jacket, and large flowers with bright yellow outer petals, making it highly ornamental and a suitable tree species for various purposes such as garden cultivation, roadside greening, and afforestation of barren hills and wastelands. However, despite its many advantages, hybrid seed production is limited by the season, and its reproductive capacity is restricted, directly impacting the promotion and application of this tree species. Currently, hybrid tulip trees are mainly propagated through sexual reproduction via artificial hybridization, as well as traditional methods such as cuttings and grafting. However, hybrid seed production is inefficient and yields few seeds. The germination rate of F1 generation seeds is very low. Furthermore, the difficulty in rooting cuttings and the low propagation coefficient of grafting greatly limit the rapid propagation and utilization of superior hybrids.

[0003] The complete genome sequencing of *Liriodendron tulipifera* has been completed, making it possible to identify, clone, and validate genes related to its growth and development. The WOX gene family plays a particularly important role in the maintenance of plant stem cells, apical meristem, and root meristem development. In the apical meristem, WUS activates CLV3 expression, but nucellus cells expressing WUS do not show CLV3 expression. When WUS is expressed ectopically in flowers and leaves, the cells in these areas do not sense the presence of ectopically expressed WUS, but when STM is also present in such leaves, the CLV3 gene is expressed at a high level. When WUS is expressed in root tip cells, the presence of auxin can induce the expression of a series of embryonic marker genes. Regardless, cells in different environments exhibit varying responsiveness to WUS signals. The presence of WUS induces cellular pluripotency, and these pluripotent cells differentiate in different directions based on their surrounding environment: leaf formation is initiated at the shoot tip during vegetative growth, flower bud formation at the shoot tip during reproductive growth, floral organ formation within the flower bud, induction of integument formation by chalaza cells in the ovule, and leaf primordia, somatic embryos (in the presence of exogenous auxin), and flower buds (in the presence of LFY) in the root. Multiple factors regulate WUS expression, but they all converge within a 57-base region of its regulatory domain, which provides essential elements for spatiotemporal WUS expression. Furthermore, WUS expression is indirectly regulated by microRNAs, thereby altering meristem size. Therefore, identifying and cloning apical meristem-specific expression promoters in hybrid tulip trees not only enriches the molecular biological foundation of plant shoot tips but also has significant application value in plant apical meristem development and stem cell maintenance.

[0004] In plant genetic engineering, promoters are commonly classified into three types: constitutive promoters, inducible promoters, and tissue-specific promoters. Tissue-specific promoters are those under which gene transcription generally occurs only in certain specific organs or tissues. Tissue-specific promoters typically contain several elements controlling tissue-specific expression; the type, number, and relative position of these elements collectively determine their specificity. There are currently no reports of identifying and cloning apical meristem-specific expression promoters in hybrid tulip trees. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by this invention is to provide a ProWUS promoter specifically expressed in the apical meristem of hybrid tulip tree and its applications. To solve the above technical problem, the technical solution adopted by this invention is as follows:

[0006] A promoter ProWUS, specifically expressed in the apical meristem of hybrid tulip tree, has the nucleotide sequence shown in SEQ ID NO.1.

[0007] Vectors or host bacteria containing the ProWUS promoter, which is specifically expressed in the apical meristem of the hybrid tulip tree.

[0008] Furthermore, the vector is a plant recombinant expression vector.

[0009] Furthermore, the target gene is linked to the 3′ end of the ProWUS promoter in the vector.

[0010] Furthermore, the host bacterium is Escherichia coli or Agrobacterium.

[0011] The application of the ProWUS promoter, which regulates the specific expression of the target gene in the apical meristem of the hybrid tulip tree, to achieve this.

[0012] Furthermore, the application specifically involves: linking the target gene downstream of the promoter ProWUS to construct a plant recombinant expression vector, then introducing the recombinant plant expression vector into a plant or plant cell to cultivate a transgenic plant in which the target gene is specifically expressed in the apical meristem.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] Based on the genome sequence information of *Liriodendron tulipifera*, this invention designs specific primers to amplify the ProWUS promoter sequence. A plant promoter expression vector, p2300-ProWUS:H2B-mCherry, is constructed using homologous recombination cloning technology. The 3' end of the ProWUS promoter in the vector is ligated with the histone H2B gene, followed by the mCherry reporter gene. The ProWUS promoter is transformed into hybrid *Liriodendron tulipifera* plants via Agrobacterium-mediated transformation. Fluorescence microscopy observation of the *Liriodendron tulipifera* plants transformed with the ProWUS promoter vector shows that the reporter gene mCherry is specifically expressed in the apical meristem under the drive of the ProWUS promoter. Therefore, the promoter provided by this invention can induce specific expression of the corresponding target gene in the apical meristem in plant genetic engineering experiments, which not only enriches the molecular biological theoretical basis of plant shoot tips but also has important application value in plant apical meristem development and stem cell maintenance. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the ProWUS:H2B-mCherry expression vector.

[0016] Figure 2 This is a fluorescence micrograph of a plant transplanted with the ProWUS promoter. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores.

[0018] The main reagent kits and reagents used in the following examples are: High-efficiency plant genomic DNA extraction kit (TIANGEN). Max Super-Fidelity DNA Polymerase (Vazyme), TSINGKE DNA Gel Extraction Kit (TSINGKE), Trelief™ 5α (TSINGKE TSC01), Plasmid Mini-Prep Kit (TIANGEN), Restriction Endonucleases (New England Biolabs), Agrobacterium strain EHA105 (Weidi), and all primer synthesis and sequencing were performed by Nanjing Qingke Biotechnology Co., Ltd.

[0019] Example 1: Cloning of the ProWUS promoter of hybrid tulip tree

[0020] 1. Total RNA extraction from hybrid tulip tree leaves

[0021] Take 100 mg of fresh leaf tissue from hybrid tulip tree, add liquid nitrogen and grind thoroughly. Extract DNA from the hybrid tulip tree using the TIANGEN High-Performance Plant Genomic DNA Extraction Kit. The steps are as follows: 1) Add 400 μL of buffer FGA and 6 μL of LNase A, vortex for 1 min, and incubate at room temperature for 10 min; 2) Add 130 μL of buffer LP2, mix thoroughly, and vortex for 1 min; 3) Centrifuge at 12000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube; 4) Add 1.5 times the volume of buffer LP3, and immediately vortex thoroughly for 15 sec; 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in a collection tube), centrifuge at 12000 rpm for 30 sec, discard the waste liquid, and place the adsorption column CB3 in a collection tube; 6) Add... 7) Add 600 μL of wash buffer PW, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube; 8) Repeat the previous operation steps; 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 minutes, and discard the waste liquid; 10) Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual wash buffer in the adsorption material; 11) Transfer the adsorption column CB3 into a clean centrifuge tube, add 100 μL of elution buffer TB dropwise to the middle of the adsorption membrane, place at room temperature for 5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution into the centrifuge tube. The solution is the hybridized tulip tree DNA.

[0022] 2. Cloning of the ProWUS promoter of hybrid tulip tree

[0023] Using hybrid tulip tree DNA as material and referencing the genome sequence information of Chinese tulip tree, specific primers were designed to amplify the ProWUS promoter sequence. The primer sequences are as follows:

[0024] ProWUS-F: 5′-GAAAAGTGAAAAAGCAAAATAGAAAATTG-3′,

[0025] ProWUS-R: 5′-GGGGGTTTGGGTTTGC-3′.

[0026] The high-fidelity PCR reaction system is as follows: ddH2O 18μL; 2×PhantaMax Buffera 25μL; dNTP Mix (10mM each) 1μL; upstream primer (10μM) 2μL; downstream primer (10μM) 2μL; hybridized tulip tree DNA 1μL; PhantaMax Super-Fideelity DNA Polymerase 1μL.

[0027] PCR reaction program: pre-denaturation 95℃, 3 min; 95℃, 15 s, 56℃, 15 s, 72℃, 2 min, 35 cycles; 72℃, 5 min; 4℃, ∞.

[0028] The amplified product was sequenced, and the ProWUS promoter sequence of 3888 bp was obtained, as shown in SEQ ID NO.1.

[0029] Example 2: Construction of ProWUS promoter expression vector for hybrid tulip tree and tissue-specific expression analysis

[0030] 1. Construct expression vectors containing the ProWUS promoter using homologous recombination technology.

[0031] Using the gene fragment obtained in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0032] PLhwusH2B-F: 5′-CAGCTATGACCATGATTACGAATTCGAAAAGTGAAAAAGCAAAATAGAAAATTGAAATAC-3′,

[0033] PLhwusH2B.REV: 5′-CTGCCTTCGCCATGGTACCGGGGGTTTGGGTTTGC-3′.

[0034] The high-fidelity PCR reaction system is as follows: ddH2O 18μL; 2×PhantaMax Buffera 25μL; dNTP Mix (10mM each) 1μL; upstream primer (10μM) 2μL; downstream primer (10μM) 2μL; hybridized tulip tree DNA 1μL; PhantaMax Super-Fidelity DNA Polymerase 1μL.

[0035] PCR reaction program: pre-denaturation 95℃, 3 min; 95℃, 15 s, 56℃, 15 s, 72℃, 2 min, 35 cycles; 72℃, 5 min; 4℃, ∞.

[0036] The amplification products were detected by 1% agarose gel electrophoresis to obtain the target fragment.

[0037] The target fragment was recovered and purified using a DNA gel extraction kit from TSINGKE. The steps were as follows: 1) Add 250 μL of Buffer BL to the EC column and centrifuge at 12000g for 1 min to activate the silica membrane; 2) Under a 365nm UV lamp, use a clean blade to cut off the DNA band to be recovered, removing the gel without DNA, and place the gel containing the target DNA band into a 2ml centrifuge tube; 3) Add 500 μL of Buffer GL; 4) Incubate at 65℃ for 6 min, inverting and mixing every 2 min until the gel is completely dissolved and the solution is pale yellow; 5) Transfer the solution to the EC column, centrifuge at 12000g for 1 min, discard the waste liquid, and return the EC column to the empty collection tube; 6) Add 700 μL of Buffer BL to the EC column. 7) Centrifuge at 12000g for 1 min with BufferW2 and discard the waste liquid; repeat once; 8) Place the adsorption column EC back into the empty collection tube and centrifuge at 12000g for 2 min; 9) Remove the adsorption column EC and place it in a clean 1.5 mL centrifuge tube. Let it stand at 25°C for 2 min with the cap open. Add 40 μL of Eluent preheated at 65°C to the center of the adsorption column membrane. Let it stand at 25°C for 2 min and centrifuge at 12000g for 2 min. The filtrate is the recovered fragment product.

[0038] The p35S:H2B-mCherry empty vector plasmid was double-digested using restriction endonucleases from New England Biolabs. The double digestion reaction system was as follows: 3 μL p35S:H2B-mCherry plasmid; 1 μL EcoRI; 1 μL KpnI; 2 μL CutSmart Buffer; 13 μL ddH2O. The double digestion reaction was carried out at 37°C for 60 minutes, followed by heat inactivation.

[0039] The product after the enzymatic digestion reaction was completed was subjected to fragment recovery. The ethanol precipitation method for fragment recovery is as follows:

[0040] 1) Add 2 μL of 3M CH3COONa (pH 5.2) and mix well; 2) Add 50 μL of pre-cooled anhydrous ethanol, mix well, and place in a -20°C refrigerator for 60 minutes; 3) Centrifuge at 12000 rpm and 4°C for 10 minutes to recover the precipitate; 4) Wash the precipitate with 75% pre-cooled ethanol, centrifuge at 12000 rpm and 4°C for 4 minutes, discard the supernatant, and dry at room temperature; 5) Dissolve the precipitate in 20 μL of sterile water to obtain the linearized fragment.

[0041] Using Vazyme The recombination reaction was performed using the One Step Cloning Kit. The following reaction mixture was prepared on ice: p35S: 4 μL H2B-mCherry plasmid linearized fragment; 1 μL ProWUS promoter recovery fragment; 4 μL 5×CE II Buffer; 2 μL Exnase II; 9 μL ddH2O. The mixture was gently pipetted and the bottom of the tube was collected after brief centrifugation. The reaction was incubated at 37°C for 30 minutes (PCR instrument); after the reaction, the temperature was lowered to 4°C.

[0042] Transformation was performed using *E. coli* DH5α competent cells from TIANGEN: 1) Place competent cells in an ice bath; 2) Add 10 μL of recombinant product to the competent cell suspension, gently tumble to mix, and incubate on ice for 30 min; 3) Place the centrifuge tube in a 42°C water bath for 90 sec, then quickly transfer the tube to an ice bath to cool the cells for 3 min, without shaking the centrifuge tube during this process; 4) Add 900 μL of sterile LB medium (antibiotic-free) to the centrifuge tube, mix well, and incubate at 37°C and 150 rpm for 45 min to revive the cells; 5) Mix the contents of the centrifuge tube, centrifuge at 4000 rpm for 2 min, discard part of the culture medium, resuspend the bacterial suspension, spread it on LB solid agar medium containing kanamycin, place the plate at room temperature until the liquid is absorbed, invert the plate, and incubate at 37°C for 16 h.

[0043] Single colonies were picked from the screening plates for PCR detection and sequencing verification. The PCR reaction used Vazyme's 2×Rapid Taq Master Mix reagent, and the reaction system was as follows: ddH2O 8.5μL; 2×Rapid Taq Master Mix 12.5μL; ProWUS-F 1μL; ProWUS-R 1μL; downstream primer (10μM) 2μL; bacterial culture 2μL.

[0044] PCR reaction program: 95℃, 3 min; 95℃, 15 s, 56℃, 15 s, 72℃, 2 min, 35 cycles; 72℃, 5 min. Clones that tested positive by bacterial culture PCR were sent to Qingke Biotechnology Co., Ltd. (Nanjing) for sequencing and identification. The sequences were correct, yielding the expression vector p2300-ProWUS:H2B-mCherry containing the ProWUS promoter. Figure 1 The 3' end of this promoter is attached to the histone H2B gene, followed by the mCherry reporter gene. Driven by the ProWUS promoter, the mCherry reporter gene is specifically expressed at the apical meristem of the plant. The vector used is used to assemble the NPT II gene expression cassette, which serves as a selection marker for transgenic plants. G418 sulfate can be used for screening transgenic plants. Assembling the LB and RB sequences can promote the integration of the assembled ProWUS promoter expression framework and the selection marker gene NPTII into the genome of the plant recipient cell.

[0045] 2. Tissue-specific expression analysis of ProWUS promoter

[0046] The constructed p2300-ProWUS:H2B-mCherry expression vector was transformed into Agrobacterium strain EHA105 (Weidi) using a conventional liquid nitrogen freeze-thaw method. This plasmid carries an NPT II marker gene driven by a 35S promoter. The ProWUS promoter was then transformed into hybrid tulip tree callus tissue via Agrobacterium-mediated transformation. After selection on a medium containing G418 antibiotic, the hybrid tulip tree callus transformed with the ProWUS promoter vector was induced into adult embryos. The fluorescence microscopy results are shown in the figure (…). Figure 2 This indicates that mCherry, driven by the ProWUS promoter, can be specifically expressed in the apical meristem of plants.

Claims

1. A hybrid Liriodendron tulipifera terminal meristem-specific expression promoter ProWUS, the nucleotide sequence of which is shown as SEQ ID NO.

1.

2. An expression cassette, a vector, a transgenic cell line or a host bacterium containing the hybrid Liriodendron tulipifera terminal meristem-specific expression promoter ProWUS of claim 1.

3. The carrier of claim 2, wherein, The vector is a plant recombinant expression vector.

4. The carrier of claim 3, wherein, The 3' end of the promoter ProWUS is connected to a target gene.

5. The host cell of claim 2, wherein the host cell is a yeast cell. The host bacterium is Escherichia coli or Agrobacterium.

6. Use of the hybrid Liriodendron tulipifera terminal meristem-specific expression promoter ProWUS of claim 1 to regulate specific expression of a target gene in plant terminal meristem.

7. Use according to claim 6, characterized in that, The target gene is connected downstream of the promoter ProWUS to construct a plant recombinant expression vector, and then the recombinant plant expression vector is introduced into plant cells to cultivate transgenic plants in which the target gene is specifically expressed in plant terminal meristem.

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