Use of cyp714a1 promoter in marking petal cells

Abstract

The application belongs to the field of plant biotechnology and particularly relates to a CYP714A1 application of a promoter in marking petal cells, the CYP714A1 nucleotide sequence of the promoter is shown in SEQ ID NO. 1. The application uses CYP714A1 the promoter to mark and identify specific cell populations of petals, which proves that CYP714A1 the promoter can identify and mark the differentiated petal cells. The application provides CYP714A1 a method for cloning and application of the promoter, comprising: CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP operably connecting the promoter to an expression vector to construct a recombinant vector, introducing the recombinant vector into a plant, starting expression of a downstream gene, and tracking the claw cell population in Arabidopsis petals.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, and specifically relates to a... CYP714A1 Application of promoters in labeling petal cells. Background Technology

[0002] Petals are an important component of plant floral organs and a core element of many crops and ornamental plants. Similar to leaves, the life cycle of petals follows a trajectory of cell division, volume expansion and differentiation, senescence, and apoptosis. Initially, cell division dominates the development of organ shape, manifested as an increase in cell number. Then, as the growth rate slows, cells differentiate and expand in size. At this point, cell volume expansion becomes the main form of organ development, accompanied by cell differentiation and the development of certain unique structures. Organs possess specific structures and functions. For example, the petals of the model plant Arabidopsis thaliana are divided into two different types of cells: the claw and the blade. These cells develop from undifferentiated cells through three stages: cell division, cell differentiation, and volume expansion, exhibiting high spatiotemporal specificity. The development of different cells determines the ultimate fate of the floral organ. A deep understanding of how these cells complete cell differentiation and the underlying gene regulatory network is a core issue in the fields of biotechnology and engineering. In particular, unraveling the molecular regulatory architecture and cellular evolutionary logic of these two processes is a crucial challenge that the biological community urgently needs to overcome. One of the major challenges facing the application of genetic engineering technology in petal research is the accurate identification and classification of diverse cell types within plants using appropriate marker genes. In both scientific research and production applications of petals, the lack of effective marker genes to distinguish between different cell types has led to significant obstacles in scientific exploration and technological barriers in practical applications. Plant petal cells differentiate into different cell types, but distinguishing these cells and their differentiation processes remains a major challenge. Summary of the Invention

[0003] The purpose of this invention is to provide a CYP714A1 The application of promoters in labeling petal cells to distinguish different cell populations in Arabidopsis petals.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides CYP714A1 The application of promoters in labeling petal cells, the CYP714A1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0006] Preferably, constructing includes CYP714A1 The promoter is a recombinant vector, which is introduced into the plant to prepare transgenic plants to label petal cells.

[0007] Preferably, the recombinant vector further includes a reporter gene.

[0008] Preferably, the reporter gene is a β-glucuronidase gene and a green fluorescent protein gene.

[0009] Preferably, by CYP714A1 After the promoter is labeled on petal cells, specific cell populations are identified.

[0010] The method of identification refers to any one of the following:

[0011] 1) 5-Bromo-4-chloro-3-indole-β-glucuronide is used as a substrate to react with β-glucuronidase to produce a blue product. The location of the blue color can be used to mark specific cell populations of petal cells.

[0012] 2) CYP714A1 The promoter controls the transcription of downstream green fluorescent protein, producing green fluorescent protein. The location of the green fluorescence can be used to trace the protein. CYP714A1 Specific cell populations marked by promoters.

[0013] Preferably, the method for preparing the recombinant vector includes the following steps:

[0014] A pair of primers were synthesized, and the primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3;

[0015] Genomic DNA was extracted from Arabidopsis thaliana.

[0016] Using Arabidopsis thaliana genomic DNA as a template, the primers were used to amplify the DNA by PCR. CYP714A1 promoter;

[0017] Will CYP714A1 The promoter is linked to the enzyme-digested expression vector to obtain the recombinant vector.

[0018] Preferably, the starting carrier of the expression vector is pLP100 - KNAT3p - GUS or pMDC107 - GFP .

[0019] Preferably, the restriction endonucleases used to digest the expression vector are XbaI and KpnI.

[0020] Preferably, the method for constructing the transgenic plant includes the following steps:

[0021] The recombinant vector was transferred into Agrobacterium competent cells to obtain recombinant Agrobacterium, which was then cultured on a large scale to prepare an infection solution.

[0022] Transgenic plants were obtained by infecting Arabidopsis thaliana with the infection solution and then culturing them.

[0023] Preferably, the method for preparing the infiltration solution is as follows:

[0024] Recombinant Agrobacterium was cultured to OD. 600 The concentration was set to 0.8. The bacterial cells were collected, and sucrose solution and surfactant were added to obtain the infection solution.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention provides CYP714A1 The application of promoters in labeling petal cells, the CYP714A1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1. This invention utilizes... CYP714A1 Promoter labeling and recognition of petal cells proved CYP714A1 The promoter can identify and locate differentiated petal cells. This invention provides a nucleotide sequence that can be specifically transcribed in a specific cell population of plant petals, and provides cloning and application methods. CYP714A1 Methods for promoters.

[0027] Application of this invention in plants CYP714A1 Methods for promoters include: CYP714A1 The promoter is operatively linked to the expression vector to construct a recombinant vector and initiate downstream gene expression to track the claw cell population in Arabidopsis petals.

[0028] This invention confirms CYP714A1 The promoter can specifically label the claw cell population of Arabidopsis petals, thus enabling the application of the present invention. CYP714A1 The promoter is used as a marker gene to screen claw cells and is further applied to single-cell sequencing. Attached Figure Description

[0029] Figure 1 A: The TDNA region map of the recombinant vector. pHY - CYP714A1 - GUS B: pMDC107 - CYP714A1 - GFP .

[0030] Figure 2 for CYP714A1 Promoter function identification results: A shows the distribution of the regions indicated by the claw and blade in wild-type Arabidopsis thaliana; B shows GUS staining at stage 10 of petal development in transgenic Arabidopsis thaliana; C shows an image of the GFP gene expression region at stage 10 of petal development in transgenic Arabidopsis thaliana. Detailed Implementation

[0031] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0032] The inventive concept of this invention is as follows:

[0033] By linking a foreign DNA sequence to a specific promoter, gene activity can be activated within a plant, and the choice of promoter determines the timing and site of gene expression. Specific cell populations or tissue sites for gene expression control elements, i.e., tissue-cell-specific promoters, ensure that gene expression occurs only in specific locations. Based on this principle, by finding appropriate promoter sequences and combining them with foreign genes, it is possible to achieve the expression of foreign genes in different types of petal cells. The type of target cells within the petal can be identified based on the expression site of the foreign gene.

[0034] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0035] The promoter nucleotide sequence of this invention is a sequence isolated from Arabidopsis thaliana.

[0036] In this invention, the term "promoter" specifically refers to a DNA sequence within a gene regulatory region that contains a crucial TATA box responsible for guiding RNA polymerase II to precisely locate the transcription start site to initiate transcription of a specified gene sequence. Furthermore, an additional recognition signal sequence located upstream of the TATA box collaboratively participates in the fine-tuning of transcription efficiency. The nucleotide sequences of the promoter regions disclosed in this invention contain additional recognition sequences upstream of specific promoter regions.

[0037] In this invention, "specific expression" refers to the expression of a target gene at a specific time or in a specific tissue or organ. The promoters for specific cell populations in plant petals described in this invention refer to promoters specifically expressed in specific cell populations within plant petals.

[0038] Generally speaking, if the abundance of a specific gene's messenger RNA mRNA in a particular tissue or organ is at least five times higher than in other tissues or organs, and ideally ten times or even a hundred times higher, then the regulatory sequence that promotes its expression is considered to exhibit tissue or organ specificity. The activity and strength of a promoter can be quantified by the level of the mRNA it drives.

[0039] Embodiments of this invention relate to a DNA construct comprising a foreign nucleotide sequence operatively linked to a promoter carrying the genetic code disclosed herein and capable of facilitating transcription of the heterologous sequence in a plant cell environment. "Operational linking" refers to constructing a structure that allows the heterologous nucleotide sequence to be controlled by the promoter's effectiveness, while ensuring that the coding portions of the two nucleotide sequences remain within the correct coding frame after splicing. A "heterologous nucleotide sequence" is defined as a sequence that has not been encountered in its natural state with the promoter sequence described herein. CYP714A1 Any sequence in which the promoter has established an operational connection.

[0040] The invention disclosed CYP714A1 Flower organ-specific promoters can be used in plant genetic engineering, such as labeling and obtaining petal-specific cell types.

[0041] The "transformed plant" or "transgenic plant" of this invention refers to a plant containing a heterologous nucleotide sequence in its genome. This heterologous nucleotide sequence can be stably inherited by the next generation and exists in the genome along with the recombinant DNA construct. A "transgenic event" is obtained through the following steps: transforming plant cells using a foreign DNA construct, and screening based on the inserted foreign gene to obtain the desired positive transgenic line.

[0042] The term "plant" in this invention includes the whole plant, plant tissues and organs such as leaves, roots, and stems, seeds, plant cells, and their offspring. In the embodiments, a portion of the transgenic plant should be understood as including petal-shaped plant cells of the transgenic plant or its offspring.

[0043] Unless otherwise specified, all methods used in the following examples are conventional methods. Primers and sequencing were performed by Shanghai Sangon Biotech Co., Ltd. PCR kits and endonucleases used in vector construction were purchased from Takara Bio Inc., and T4 DNA ligase was purchased from Promega. All methods were performed according to the instructions provided with the kits. The vectors used in the experiments... pHY - GUS Obtained by modifying this experiment.

[0044] The English-Chinese glossary of this invention is shown in Table 1.

[0045] Table 1. Chinese-English Translation Table

[0046]

[0047] Example 1

[0048] CYP714A1 The application of promoters in labeling petal cells is as follows:

[0049] CYP714A1The nucleotide sequence of the promoter was isolated from Arabidopsis thaliana, totaling 1695 bp, and the sequence information is shown in SEQ ID NO.1.

[0050] SEQ ID NO.1:

[0051]

[0052] 1. Obtain CYP714A1 Promoters and construct recombinant vectors.

[0053] Design and synthesize clones CYP714A1 The promoter requires the forward primer PF1-Xba I and the reverse primer PR1. - The sequence information of KpnI, PF1-Xba I and PR1-Kpn I is shown in SEQ ID NO.2 and SEQ ID NO.3.

[0054] PF1-Xba I, SEQ ID NO.2:

[0055] GC TCTAGA AGAAACAGCAAGAGCTTTTATAAGG.

[0056] PR1-Kpn I, SEQ ID NO.3:

[0057] GG GGTACC TTTCTTATCTTTCTTTTTCTTAGAA.

[0058] The underlined TCTAGA in SEQ ID NO.2 is the restriction site for Xba I; the underlined GGTACC in SEQ ID NO.3 is the restriction site for Kpn I.

[0059] use CYP714A1 The forward and reverse primers of the promoter were used to perform PCR amplification with Arabidopsis thaliana genomic DNA as a template to obtain PCR products. The reaction conditions are shown in Table 2. The PCR products were then ligated into different expression vectors to obtain recombinant vectors. The method is as follows:

[0060] 1) pLP100 - KNAT3p - GUS The KNAT3 promoter on the vector was removed, and the expression vector was obtained after digestion with XbaI and KpnI enzymes. pHY - GUS The enzyme digestion conditions were: first at 37℃ for 1 hour, then at 80℃ for 15 minutes for inactivation.

[0061] After the PCR reaction, the PCR products were recovered by 1% agarose gel electrophoresis. The products were then double-digested with XbaI and KpnI and ligated into an expression vector that had also been double-digested with XbaI and KpnI. pHY - GUS Subsequently, the colonies were transformed and cultured. Colonies with positive PCR results were selected for sequencing. After the sequencing confirmed the results were correct, the corresponding positive clone plasmids were extracted and named recombinant vectors. pHY - CYP714A1 - GUS .

[0062] 2) Using XbaI and KpnI to... pMDC107 - GFPThe expression vector was digested with enzymes under the following conditions: first at 37°C for 1 hour, then at 80°C for 15 minutes to inactivate it.

[0063] After the PCR reaction, the PCR products were recovered by 1% agarose gel electrophoresis. The products were then double-digested with XbaI and KpnI and ligated into ligation products that had also been double-digested with XbaI and KpnI. pMDC107 - GFP Subsequently, the colonies were transformed and cultured. Colonies with positive PCR results were selected for sequencing. After the sequencing confirmed the results were correct, the corresponding positive clone plasmids were extracted and named recombinant vectors. pMDC107 - CYP714A1 - GFP .

[0064] The TDNA region map of the recombinant vector constructed in this invention is as follows: Figure 1 As shown. Figure 1 In the diagram, LB represents the left boundary of the TDNA, and RB represents the right boundary; KanR indicates the kanamycin resistance gene; HypR indicates the hygromycin resistance gene; NOS promoter indicates... nos Gene promoter; NOS terminator nos The terminator of the gene; GUS represents the β-glucuronidase gene; GFP represents the green fluorescent protein gene; the petal-specific cell promoter is the petal-specific cell expression promoter isolated and identified in this invention. CYP714A1 Promoter.

[0065] Table 2 PCR amplification reaction conditions

[0066]

[0067] 2. Construct transgenic plants using recombinant vectors.

[0068] 2.1. The recombinant vector is prepared by thermal shock. pHY - CYP714A1 - GUS Agrobacterium GV3101 strain was introduced to prepare an infection solution, which was then used to transform Arabidopsis thaliana. The specific method is as follows:

[0069] Take 5 μL of recombinant vector pHY - CYP714A1 - GUS Add 100 μL of GV3101 competent cells and gently mix. Incubate on ice for 30 min, then in liquid nitrogen for 5 min, incubate at 37°C for 5 min, and incubate on ice for another 5 min. Add 700 μL of LB medium and incubate at 28°C and 180 rpm for 2 h. Centrifuge at 5000 rpm for 2 min, discard 650 μL of supernatant, and spread the remaining liquid onto LB medium supplemented with rifampicin and kanamycin antibiotics. Incubate upside down at 28°C for 2 days. Pick single colonies for colony PCR; positive colonies are recombinant Agrobacterium. Expand the recombinant Agrobacterium and culture it to OD200. 600The concentration was 0.8. After centrifuging 200 mL of recombinant Agrobacterium bacterial culture, the bacterial precipitate was collected, 500 mL of 5% sucrose solution was added, and then 100 μL of surfactant L-77 was added to obtain the infection solution.

[0070] Cut off the inflorescences of 30-day-old Arabidopsis thaliana plants that had already developed siliques, water them thoroughly, and culture them for 12 hours. Then, immerse the Arabidopsis thaliana plants in the infection solution, removing them after 30 seconds while continuously stirring the solution. Next, treat the entire Arabidopsis thaliana plant in darkness for 16 hours, followed by normal culture. Harvest the seeds, and screen with 100 mg / L hygromycin until homozygous T3 generation Arabidopsis thaliana is obtained, thus acquiring transgenic Arabidopsis thaliana.

[0071] 2.2. The recombinant vector is recombined using a thermal shock method. pMDC107 - CYP714A1 - GFP Agrobacterium GV3101 strain was introduced to prepare an infection solution, which was then used to transform Arabidopsis thaliana, using the same method as in 2.1.

[0072] 3. Functional identification.

[0073] 3.1 GUS activity detection.

[0074] Petals were isolated from transgenic Arabidopsis thaliana and GUS activity was detected. The GUS activity detection method is as follows:

[0075] (1) Fix with 90% acetone for 20 min; the conditions for fixation are 4℃ for 20 min.

[0076] (2) Add 1 mL of GUS washing solution to wash away the acetone, and wash twice.

[0077] (3) Add GUS staining solution and vacuum on ice for 20 minutes.

[0078] (4) Place at 37°C and observe the staining effect during the period. Once stained, proceed to the next step.

[0079] (5) Aspirate the GUS staining solution, add 1 mL of 70% ethanol (by volume), and stop the staining reaction and decolorization.

[0080] (6) Replace the ethanol until decolorization is complete. When decolorizing with ethanol, it is necessary to heat it on a metal bath at 40°C. The negative control material is white or light yellow when the decolorization is complete.

[0081] (7) Add 200 μL of 50% glycerol to the slide, take out the decolorized petals with tweezers, spread them flat in the glycerol, cover with a coverslip, observe the petals with a dissecting microscope, observe the details and take pictures under a microscope.

[0082] The GUS gene produces β-glucuronidase, which hydrolyzes X-Gluc substrates, forming an insoluble blue precipitate. This precipitate is located at specific cellular groups in the petals. CYP714A1When the promoter is activated, the GUS gene is strongly expressed, showing a strong blue color, which can be used to mark these specific cell groups.

[0083] 3.2 Visualization and detection of GFP green fluorescent protein expression.

[0084] This invention also used the GFP (green fluorescent protein) gene for visualization of target gene expression, with the GFP gene and... CYP714A1 After promoter fusion, the GFP-encoded fluorescent protein emits green fluorescence at a specific wavelength, reflecting the gene's activation state and expression location in real time, distinguishing it from other cell populations. The specific method is as follows:

[0085] Fluorescence imaging was performed using an FV3000 laser confocal microscope. A drop of water was placed on a slide, and a petal from a stage 10 transgenic Arabidopsis thaliana was taken. Water was added to completely cover the tissue, and a coverslip was placed on top. The slide was then inverted and placed on the microscope stage. Under bright field conditions, the focus was adjusted until the plant cells were clearly visible. The laser scanning was then switched, and the excitation wavelength of GFP was set to 488 nm.

[0086] The above technical solution involves designing and cloning a promoter, constructing a recombinant vector, performing Agrobacterium-mediated transformation, flower infection, and GUS staining identification. GFP Fluorescence imaging. The results are as follows:

[0087] GUS staining of Arabidopsis petals allows observation of the staining results. GUS Gene-specific expression in the claw region of the petal. Figure 2 B represents the petals of Arabidopsis thaliana from the 10th period; the blue area is... CYP714A1 The result of staining the promoter-activated GUS gene; the blue area shows the specific cells in the petals. Figure 2 C is CYP714A1 Startup driver GFP Gene expression is shown; the green areas represent petal-specific cells. The two verification methods demonstrate this. CYP714A1 The promoter binds to cells in the claw region, forming a clear distinction from cells in the blade region.

[0088] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. CYP714A1 The use of a promoter in a marker petal cell, characterized in that, The nucleotide sequence of the promoter is shown in SEQ ID NO.

1. CYP714A1 The nucleotide sequence of the promoter is shown in SEQ ID NO.

1. The marked petal cell refers to a claw cell group of a 10th stage Arabidopsis thaliana petal.

2. Use according to claim 1, wherein recombinant vectors comprising CYP714A1 promoters, introducing the recombinant vectors into the plants to make transgenic plants to mark petal cells.

3. Use according to claim 2, wherein the compound is ###0002### The recombinant vector further comprises a reporter gene.

4. Use according to claim 3, wherein the compound is ###0002### The reporter gene is a β-glucuronidase gene and a green fluorescent protein gene.

5. The use according to claim 4, wherein the compound is ###0002### By CYP714A1 After the promoter-labeled petal cells are initiated, identification of specific cell populations is performed; The identification method refers to any one of the following: 1) 5-bromo-4-chloro-3-indole-β-glucuronide as a substrate reacts with β-glucuronidase to produce a blue product, and through the position where the blue color appears, a specific cell population of the petal cell is marked; 2) CYP714A1 The promoter controls the transcription of the downstream green fluorescent protein, which produces green fluorescent protein. By the location where the green fluorescence appears, it is tracked to CYP714A1 The promoter marks a specific cell population.

6. The use according to claim 2, wherein The preparation method of the recombinant vector comprises the following steps: Synthesizing a pair of primers, and the primer sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3; Extracting genomic DNA of Arabidopsis thaliana; Using the genomic DNA of Arabidopsis thaliana as a template, the primer was used to obtain CYP714A1 Promoter; The promoter is ligated to the expression vector after the enzyme digestion, and a recombinant vector is obtained. CYP714A1 The promoter is ligated to the expression vector after the enzyme digestion, and a recombinant vector is obtained.

7. Use according to claim 6, wherein The starting vector of the expression vector is pLP100-KNAT3p- GUS or pMDC107-GFP .

8. The use according to claim 6, wherein the compound is ###0002### The restriction endonuclease used for enzyme digestion of the expression vector is XbaI and KpnI.

9. The use according to claim 2, wherein the compound is ###0005### The construction method of the transgenic plant comprises the following steps: Transferring the recombinant vector into an agrobacterium competent cell to obtain a recombinant agrobacterium, expanding culture, and preparing an infection solution; After using the infection solution to infect Arabidopsis thaliana, culture is performed to obtain a transgenic plant.

10. Use according to claim 9, wherein The preparation method of the infection solution is as follows: The recombinant Agrobacterium is cultured to OD 600 0.8, the bacterial bodies are collected, a sucrose solution and a surfactant are added, and the infection solution is obtained.

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