Application of CYP714A1 promoter in marking petal cells

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to application of a CYP714A1 promoter in marking petal cells, and the nucleotide sequence of the CYP714A1 promoter is as shown in SEQ ID NO. 1. According to the invention, the CYP714A1 promoter is used for marking and identifying a specific cell population of petals, and the CYP714A1 promoter is proved to be capable of identifying, positioning and identifying differentiated petal cells. The invention provides a method for cloning and applying a CYP714A1 promoter. The method comprises the following steps: operably connecting the CYP714A1 promoter to an expression vector, constructing a recombinant vector, introducing the recombinant vector into a plant, and starting downstream gene expression so as to track a claw cell population in Arabidopsis thaliana petals.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology and specifically relates to a CYP714A1 Application of promoter in marking petal cells. Background Art

[0002] Petals are a crucial component of floral organs and a core element of many crops and ornamental plants. Similar to leaves, petals follow a life cycle characterized by cell division, expansion and differentiation, aging, and apoptosis. Initially, cell division dominates organ development, manifesting as an increase in cell number. Then, as growth slows, cells undergo differentiation and expansion. At this point, cell expansion becomes the primary form of organ development, accompanied by cell differentiation and the development of certain unique structures. Organs have specific structures and functions. For example, the petals of the model plant Arabidopsis thaliana are divided into two distinct cell types: claws and blades. These cells develop from undifferentiated cells through three stages of cell division, differentiation, and expansion, exhibiting a high degree of spatiotemporal specificity. The development of these different cells determines the ultimate fate of floral organs. Understanding how these cells differentiate and the underlying genetic regulatory networks is a core issue in biotechnology and engineering. In particular, uncovering the molecular regulatory architecture and cellular evolutionary logic of these two processes is a crucial challenge currently under investigation in biology. Among them, using appropriate marker genes to accurately identify and classify the diverse cell types within a plant is a major challenge facing genetic engineering technology in petal research. The lack of effective marker genes to distinguish different cell types has plagued scientific research and production applications of petals, and practical applications also face technical barriers. Plant petal cells undergo differentiation to form different cell types, but how to distinguish these cells and their differentiation process remains a major challenge. Summary of the Invention

[0003] The purpose of the present invention is to provide a CYP714A1 The promoter is used to mark petal cells and distinguish different cell populations in Arabidopsis petals.

[0004] The technical solution adopted in the present invention is: The present invention provides a CYP714A1 Application of the promoter in marking petal cells, the CYP714A1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0005] Preferably, the construct comprises CYP714A1 The recombinant vector containing the promoter is introduced into the plant to prepare a transgenic plant to mark the petal cells.

[0006] Preferably, the recombinant vector further comprises a reporter gene.

[0007] Preferably, the reporter genes are β-glucuronidase gene and green fluorescent protein gene.

[0008] Preferably, by CYP714A1 After the promoter marks the petal cells, the specific cell population is identified; The identification method is any one of the following: 1) 5-Bromo-4-chloro-3-indole-β-glucuronide reacts with β-glucuronidase as a substrate to produce a blue product. The location of the blue product marks a specific cell population in the petal. 2) CYP714A1 The promoter controls the transcription of downstream green fluorescent protein, producing green fluorescent protein, and tracking the position of green fluorescence. CYP714A1 Promoter-labeled specific cell populations.

[0009] Preferably, the method for preparing the recombinant vector comprises the following steps: A pair of primers were synthesized, the primer sequences were shown as SEQ ID NO.2 and SEQ ID NO.3; Extract genomic DNA from Arabidopsis thaliana; Using the genomic DNA of Arabidopsis thaliana as a template, the primers were used to amplify the obtained CYP714A1 promoter; Will CYP714A1 The promoter is connected to the expression vector after enzyme digestion to obtain a recombinant vector.

[0010] Preferably, the starting vector of the expression vector is pLP100-KNAT3p-GUS or pMDC107-GFP .

[0011] Preferably, the restriction endonucleases used for enzymatic digestion of the expression vector are XbaI and KpnI.

[0012] Preferably, the method for constructing the transgenic plant comprises the following steps: Transforming the recombinant vector into Agrobacterium competent cells to obtain recombinant Agrobacterium, expanding the culture, and preparing an infection solution; The Arabidopsis thaliana was infected with the infection solution and then cultured to obtain transgenic plants.

[0013] Preferably, the preparation method of the infection solution is as follows: The recombinant Agrobacterium was cultured to OD 600 The concentration of sucrose is 0.8, the bacteria are collected, sucrose solution and surfactant are added to obtain the infection solution.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a CYP714A1 Application of the promoter in marking petal cells, the CYP714A1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1. CYP714A1 The promoter marks and identifies petal cells, demonstrating CYP714A1 The promoter can identify and locate the differentiated petal cells. The present invention provides a nucleotide sequence that can be specifically transcribed in a specific cell group in the petals of plants, and provides cloning and application CYP714A1 Promoter method.

[0015] Application of the present invention in plants CYP714A1 The method of promoter comprises: CYP714A1 The promoter is operably connected to an expression vector to construct a recombinant vector and initiate downstream gene expression to track claw cell populations in Arabidopsis petals.

[0016] The present invention proves CYP714A1 The promoter can specifically mark the claw cell population of Arabidopsis petals, so the CYP714A1 The promoter was used as a marker gene to screen claw cells and further applied to single-cell sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A is the map of the TDNA region of the recombinant vector. pHY-CYP714A1-GUS ; B: pMDC107-CYP714A1-GFP .

[0018] Figure 2 for CYP714A1 Promoter function identification results. A shows the distribution of the regions indicated by claw and blade in wild-type Arabidopsis; B shows GUS staining of transgenic Arabidopsis petals at stage 10 of development; C shows a picture of the GFP gene expression region at stage 10 of development of transgenic Arabidopsis petals. DETAILED DESCRIPTION

[0019] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0020] The inventive concept of the present invention is as follows: By linking exogenous DNA sequences to specific promoters, gene activity can be stimulated within plants. The choice of promoter determines the timing and location of gene expression. Expression control elements specific to specific cell populations or tissue locations, known as tissue cell-specific promoters, ensure that gene expression occurs only in specific locations. Based on this principle, by finding the appropriate promoter sequence and combining it with an exogenous gene, the expression of the exogenous gene in different types of petal cells can be achieved. Based on the location of exogenous gene expression, the target cell type within the petals can be identified.

[0021] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0022] The promoter nucleotide sequence in the embodiment of the present invention is a sequence isolated from Arabidopsis thaliana.

[0023] In this invention, the term "promoter" specifically refers to the DNA sequence within the gene regulatory region, which contains a crucial TATA box responsible for guiding RNA polymerase II to precisely locate the transcription start site to initiate transcription of the specified gene sequence. Furthermore, additional recognition signal sequences located upstream of the TATA box contribute to the fine-tuning of transcription efficiency. The nucleotide sequence of the promoter region disclosed in this invention specifically contains additional recognition sequences upstream of the promoter region.

[0024] "Specific expression" in the present invention refers to the expression of the target gene at a specific time or in a specific tissue or organ. The promoter expressed in a specific cell population in plant petals in the present invention refers to a promoter specifically expressed in a specific cell population in plant petals.

[0025] Generally speaking, if the mRNA abundance of a particular gene in a particular tissue or organ is at least fivefold higher, and ideally tenfold or even a hundredfold higher, than in other tissues or organs, the regulatory sequences driving its expression are considered to exhibit tissue or organ specificity. The activity and strength of a promoter can be quantified by the level of mRNA it drives.

[0026] The embodiments of the present invention relate to a DNA construct comprising a foreign nucleotide sequence operably linked to a promoter, wherein the promoter carries the genetic code disclosed in the present invention and can promote the transcription of the heterologous sequence in a plant cell environment. The so-called "operably linked" means to build a structure that allows the heterologous nucleotide sequence to be controlled by the promoter effect, while ensuring that the coding parts of the two nucleotide sequences after splicing remain within the correct coding frame. "Heterologous nucleotide sequence" is defined as a sequence that has not been linked to the promoter sequence described in the present invention in its natural state. CYP714A1 A promoter is any sequence to which it is operably linked.

[0027] The present invention discloses CYP714A1 Promoters Floral organ-specific promoters can be used in plant genetic engineering, for example to mark and obtain petal-specific cell types.

[0028] "Transformed plants" or "transgenic plants" of the present invention refer to plants that contain a heterologous nucleotide sequence within their genome. This heterologous nucleotide sequence is stably inherited to the next generation and exists within the genome along with the recombinant DNA construct. "Transgenic events" are obtained by transforming plant cells with an exogenous DNA construct and then screening for the desired positive transgenic lines based on the inserted exogenous gene.

[0029] The "plant" of the present invention includes whole plants, plant tissues and organs, such as leaves, roots, stems, seeds, plant cells, and their offspring. In the embodiments, the partial plant of the transgenic plant should be understood to include the petals and plant cells of the transgenic plant or its offspring.

[0030] The methods used in the following examples are all conventional methods unless otherwise specified. Primers and sequencing were all performed by Shanghai Shenggong Company. PCR kits and endonucleases used in vector construction were purchased from Bao Bioengineering Co., Ltd., and T4 DNA ligase was purchased from Promega. The methods were all carried out according to the methods provided in the kits. pHY-GUS Modified from this experiment.

[0031] The Chinese-English comparison table of the present invention is shown in Table 1.

[0032] Table 1 Chinese-English comparison table

[0033] Example 1 CYP714A1 The application of the promoter in marking petal cells is as follows: CYP714A1 The nucleotide sequence of the promoter was isolated from Arabidopsis thaliana, totaling 1695 bp, and the sequence information is shown in SEQ ID NO.1.

[0034] SEQ ID NO.1:

[0035] 1. Acquisition CYP714A1 promoter and construct a recombinant vector.

[0036] Design and synthesize clones CYP714A1 The forward primer PF1-Xba I and the reverse primer PR1 required for the promoter - The sequence information of KpnI, PF1-XbaI and PR1-KpnI is shown in SEQ ID NO.2 and SEQ ID NO.3.

[0037] PF1-Xba I, SEQ ID NO. 2: GC TCTAGA AGAAACAGCAAGAGCTTTTATAAGG.

[0038] PR1-Kpn I, SEQ ID NO. 3: GG GGTACC TTTCTTATCTTTCTTTTTCTTAGAA.

[0039] The underlined TCTAGA in SEQ ID NO.2 is the restriction enzyme cleavage site of Xba I; the underlined GGTACC in SEQ ID NO.3 is the restriction enzyme cleavage site of Kpn I.

[0040] use CYP714A1 The forward and reverse primers of the promoter were used to perform PCR amplification using Arabidopsis genomic DNA as a template to obtain PCR products. The reaction conditions are shown in Table 2. The PCR products were ligated into different expression vectors to obtain recombinant vectors. The method is as follows: 1) pLP100-KNAT3p-GUS The KNAT3 promoter on the vector was removed and digested with XbaI and KpnI to obtain the expression vector. pHY-GUS The enzyme digestion conditions were 37℃ for 1h and then inactivated at 80℃ for 15min.

[0041] After the PCR reaction, the PCR product was recovered by 1% agarose gel electrophoresis, and the product was double-digested with XbaI and KpnI and ligated into the expression vector that was also double-digested with XbaI and KpnI. pHY-GUS Then transform and culture, pick the colonies with positive PCR results for sequencing, and after sequencing verification, extract the corresponding positive clone plasmid and name it as the recombinant vector pHY-CYP714A1-GUS .

[0042] 2) Use XbaI and KpnI to pMDC107-GFP The expression vector was digested with enzymes at 37°C for 1 h and then inactivated at 80°C for 15 min.

[0043] After the PCR reaction, the PCR product was recovered by 1% agarose gel electrophoresis, and the product was double-digested with XbaI and KpnI and ligated into pMDC107-GFP Then transform and culture, pick the colonies with positive PCR results for sequencing, and after sequencing verification, extract the corresponding positive clone plasmid and name it as the recombinant vector pMDC107-CYP714A1-GFP .

[0044] The map of the TDNA region of the recombinant vector constructed by the present invention is as follows Figure 1 shown. Figure 1 In the figure, LB is the left border of TDNA, RB is the right border of TDNA; KanR represents the kanamycin resistance gene; HypR represents the hygromycin resistance gene; NOS promoter represents 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 the present invention. CYP714A1 promoter.

[0045] Table 2 PCR amplification reaction conditions

[0046] 2. Use recombinant vectors to construct transgenic plants.

[0047] 2.1. Heat shock method to recombinant vector pHY-CYP714A1-GUS Agrobacterium tumefaciens GV3101 strain was introduced, and infection solution was prepared to transform Arabidopsis thaliana. The specific method is as follows: Take 5 μL of recombinant vector pHY-CYP714A1-GUS Add to 100 μL GV3101 competent cells and flick to mix, let stand on ice for 30 minutes, put in liquid nitrogen for 5 minutes, 37 ° C water bath for 5 minutes, let stand on ice for 5 minutes, add 700 μL LB medium, 28 ° C, 180 rpm culture for 2 hours, then centrifuge at 5000 rpm for 2 minutes, discard 650 μL supernatant and spread the remaining liquid on LB medium with rifampicin and kanamycin double antibodies. Incubate at 28 ° C for 2 days, pick a single colony for colony PCR, and the positive colony is the recombinant Agrobacterium. The recombinant Agrobacterium is expanded and cultured to OD 600 The concentration of the recombinant Agrobacterium was 0.8. 200 mL of the recombinant Agrobacterium bacterial solution was centrifuged and the bacterial pellet was collected. 500 mL of 5% by mass sucrose solution was added, and then 100 μL of surfactant L-77 was added to obtain the infection solution.

[0048] 30-day-old Arabidopsis plants that had already grown siliques were pruned, watered thoroughly, and incubated for 12 hours. The plants were then immersed in the infecting solution and removed after 30 seconds, with the solution constantly agitated. The entire plant was then shaded for 16 hours. After shading, the plants were cultured normally, harvested, and selected with 100 mg / L hygromycin until homozygous T3 generation Arabidopsis was obtained to obtain transgenic Arabidopsis.

[0049] 2.2. Heat shock method to recombinant vector pMDC107-CYP714A1-GFP Transform Agrobacterium tumefaciens GV3101 strain into Arabidopsis thaliana, prepare infection solution, and transform Arabidopsis thaliana using the same method as described in 2.1.

[0050] 3. Functional identification.

[0051] 3.1. GUS activity detection.

[0052] Petals were isolated from transgenic Arabidopsis and GUS activity was detected using the following method: (1) Fix with 90% acetone by volume for 20 minutes at 4°C for 20 minutes.

[0053] (2) Add 1 mL of GUS solution to wash away the acetone and wash twice.

[0054] (3) Add GUS staining solution and vacuum on ice for 20 minutes.

[0055] (4) Place at 37°C and observe the dyeing effect. If it is dyed, proceed to the next step.

[0056] (5) Aspirate the GUS staining solution and add 1 mL of 70% ethanol to stop the staining reaction and decolorization.

[0057] (6) Replace the ethanol until the decolorization is complete. When decolorizing with ethanol, heat it in a metal bath at 40°C. The negative control material is completely decolorized when it turns white or light yellow.

[0058] (7) Add 200 μL of 50% glycerol to the slide, take out the decolorized petals with tweezers, flatten them in the glycerol, cover them with a coverslip, observe the petals with a dissecting microscope, observe the details with a microscope and take pictures.

[0059] The GUS gene generates β-glucuronidase, which hydrolyzes the X-Gluc substrate to form an insoluble blue precipitate. CYP714A1 When the promoter is activated, the GUS gene is strongly expressed, showing a strong blue color, which can be used to mark these special cell populations.

[0060] 3.2. GFP green fluorescent protein visual expression detection.

[0061] The present invention also uses the GFP green fluorescent protein gene to visualize the expression of the target gene. CYP714A1 After promoter fusion, the fluorescent protein encoded by GFP emits green fluorescence at a specific wavelength, reflecting the activation state and expression location of the gene in real time, distinguishing it from other cell groups. The specific method is as follows:

[0062] Fluorescence imaging was performed using an FV3000 laser confocal microscope. A drop of water was placed on a glass slide. A petal from a stage 10 transgenic Arabidopsis thaliana plant was taken and further water was added to completely cover the tissue. After applying a coverslip, the specimen was placed upside down on the microscope stage. Under brightfield conditions, the focus was adjusted to clearly visualize the plant cells. Laser scanning was switched to a 488nm wavelength for GFP excitation.

[0063] Through the above technical scheme, the promoter was designed and cloned - the recombinant vector was constructed - Agrobacterium was transformed - the flower was infected - GUS staining was used to identify and GFP Fluorescence imaging of the . The results are as follows: Take Arabidopsis petals for GUS staining, and you can see the GUS Gene expression is specific to the claw area of petals. Figure 2 B is the 10th stage Arabidopsis petals, the blue area is CYP714A1 The results of the staining process after the promoter activates the GUS gene. The blue area shows the specific cells in the petals. Figure 2 C is CYP714A1 Promoter drive GFP The expression of genes, the green area is the petal-specific cells. Two verification methods can be seen CYP714A1 The promoter binds to cells in the claw region, clearly distinguishing them from cells in the blade region.

[0064] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0065] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. CYP714A1 The use of a promoter in marking petal cells is characterized in that: described CYP714A1 The nucleotide sequence of the promoter is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that Build includes CYP714A1 The recombinant vector containing the promoter is introduced into the plant to prepare a transgenic plant to mark the petal cells.

3. The use according to claim 2, characterized in that The recombinant vector also includes a reporter gene.

4. The use according to claim 3, characterized in that The reporter genes are β-glucuronidase gene and green fluorescent protein gene.

5. The use according to claim 4, characterized in that pass CYP714A1 After the promoter marks the petal cells, the specific cell population is identified; The identification method is any one of the following: 1) 5-Bromo-4-chloro-3-indole-β-glucuronide reacts with β-glucuronidase as a substrate to produce a blue product. The location of the blue product marks a specific cell population in the petal. 2) CYP714A1 The promoter controls the transcription of downstream green fluorescent protein, producing green fluorescent protein, and tracking the position of green fluorescence. CYP714A1 Promoter-labeled specific cell populations.

6. The use according to claim 2, characterized in that The preparation method of the recombinant vector comprises the following steps: A pair of primers were synthesized, the primer sequences were shown as SEQ ID NO.2 and SEQ ID NO.3; Extract genomic DNA from Arabidopsis thaliana; Using the genomic DNA of Arabidopsis thaliana as a template, the primers were used to amplify the obtained CYP714A1 promoter; Will CYP714A1 The promoter is connected to the expression vector after enzyme digestion to obtain a recombinant vector.

7. The use according to claim 6, characterized in that The starting vector of the expression vector is pLP100-KNAT3p- GUS or pMDC107-GFP .

8. The use according to claim 6, characterized in that The restriction endonucleases used to digest the expression vector were XbaI and KpnI.

9. The use according to claim 2, characterized in that The method for constructing the transgenic plant comprises the following steps: Transforming the recombinant vector into Agrobacterium competent cells to obtain recombinant Agrobacterium, expanding the culture, and preparing an infection solution; The Arabidopsis thaliana was infected with the infection solution and then cultured to obtain transgenic plants.

10. The use according to claim 9, characterized in that The preparation method of the infection solution is as follows: The recombinant Agrobacterium was cultured to OD 600 The concentration of sucrose is 0.8, the bacteria are collected, sucrose solution and surfactant are added to obtain the infection solution.

Images