BHLH090 gene promoter and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to a bHLH090 gene promoter and application in a method for detecting and counting the number of arabidopsis myrosinase heteromorph, and the sequence of the bHLH090 gene promoter is shown as SEQ ID No.1. The bHLH090 gene promoter is selected from bHLH090 genes specifically expressed in the arabidopsis myrosinase heteromorph, and the bHLH090 gene promoter can be used for detecting and counting the number of the arabidopsis myrosinase heteromorph in the arabidopsis myrosinase heteromorph. A nuclear localization fusion expression vector of a specific expression gene bHLH090 is constructed, the bHLH090 gene specifically expressed by myrosinase cells is cut in, and a marker originally expressed in cytoplasm is localized to a cell nucleus through related construction and connection operations, so that accurate marking and localization of myrosinase heteromorphic cells are realized, and the accuracy of the myrosinase heteromorphic cells is improved. Specific marking and living body observation of myrosinase heteromorphic cells are achieved, and the lethal problem of a traditional dyeing method is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a bHLH090 gene promoter and its application in a method for detecting and counting the number of Arabidopsis myrosinase heteromorphic cells. Background Art

[0002] Myrosinase heterocysts are a type of cell unique to crucifers, and they play an important role in the chemical defense system of plants. This system is called the myrosinase-glucosinolate (GS) two-component system, which is a defense mechanism unique to crucifers. In this system, glucosinolate (GSL) and myrosinase (Myrosinase) are synthesized and accumulated in different cells. When a plant is attacked by a predator, GSL is released from its storage cells, comes into contact with myrosinase and catalyzes its hydrolysis, producing compounds that are toxic to both insects and microorganisms, forming the so-called "mustard oil bomb."

[0003] However, myrosinase cells are deeply buried in mesophyll cells and are difficult to observe. They can only be observed by staining or expressing genes in myrosinase heterocysts and fusion expression materials with reporter genes. Staining is lethal to the material itself, and it is impossible to track and observe the material in vivo. The method of fusion expression of genes and reporter genes can observe myrosinase cells in vivo, but there are problems such as irregular shapes of myrosinase cells and difficulty in counting their numbers.

[0004] There are currently three ways to observe myrosinase cells:

[0005] 1. Coomassie Brilliant Blue Staining Method: Boil the 17-day-old plants in Coomassie Brilliant Blue solution for 3 minutes. After incubation in chloral hydrate solution (chloral hydrate: water: glycerol, weight / volume / volume ratio of 8:2:1) for 3-5 hours, fix the plants on a slide with 60% glycerol and observe them with an optical microscope and a stereo microscope. (Myrosinase cells will be stained blue by Coomassie Brilliant Blue) Count the blue-stained myrosinase cells in the selected field of view.

[0006] 2. GUS staining method: Cultivate Arabidopsis plants with good growth status, select plants in the seedling stage, carefully remove the leaves with tweezers and scissors, and place them in a small culture dish; add an appropriate amount of fixative (a mixture of 90% glacial ethanol and 5% glacial acetic acid) to the culture dish, fix for 1-2 hours to keep the cells stable in morphology and structure. After fixation, rinse the tissue with sodium phosphate buffer for 3-5 times, each time for about 5 minutes, to remove the fixative; put the fixed tissue into an EP tube, add an appropriate amount of GUS staining solution (X-Gluc is used as a substrate and dissolved in sodium phosphate buffer (pH 7.0-7.5) to make staining solution), make sure the tissue is completely immersed in the staining solution, then cover the lid and incubate in a 37℃ incubator for 1 hour to overnight (until a clear blue color appears); transfer the stained tissue to 70% or 95% ethanol for decolorization 2-3 times, soaking for about 10-15 minutes each time, until the negative control material turns white, the purpose is to remove background pigments such as chlorophyll, so as to observe the blue GUS staining site more clearly; put the decolorized tissue on a microscope slide, add a small amount of water or glycerol, cover with a coverslip, and make a temporary slide specimen. Under an optical microscope, myrosinase cells with GUS activity will appear blue or blue spots, so as to count the number of cells and analyze the cell distribution.

[0007] 3. Fluorescent protein labeling method: The E1728 promoter sequence of the myrosinase gene is amplified from the Arabidopsis genome, and the promoter of the myrosinase gene is connected to the fluorescent protein gene YFP / GFP to construct a fusion gene expression vector E1728-erYFP, so that the fluorescent protein can be specifically expressed in myrosinase cells. The constructed fusion gene expression vector E1728-erYFP is introduced into Agrobacterium, and the Arabidopsis plants are infected with Agrobacterium to integrate the fusion gene into the Arabidopsis genome. The transgenic Arabidopsis plants with successful integration of the fusion gene are screened by antibiotic resistance, and the screened transgenic Arabidopsis seeds are sown in a suitable culture medium or soil, and cultured under suitable light, temperature and humidity conditions to allow them to grow and develop. When the transgenic Arabidopsis grows to an appropriate stage, the leaves are taken to make temporary slide specimens, and under a fluorescence microscope, multiple representative fields of view are selected to count the fluorescent myrosinase cells, and the total number of myrosinase cells in the entire tissue is estimated based on the ratio of the field area to the total tissue area.

[0008] Disadvantages of existing technology

[0009] (1) Limitations of the direct staining method with Coomassie Brilliant Blue: poor specificity, easy to non-specifically bind to other components in the cell, making it difficult to accurately distinguish myrosinase cells; the staining depth is affected by multiple factors and has poor stability, which is not conducive to observation and counting under unified standards; the staining process may also damage cell activity, limiting the study of living organisms and cell dynamics; the above problems make quantitative analysis difficult, making it difficult to establish an accurate standard curve to measure the number of cells, and the reliability and accuracy of the results are low.

[0010] (2) Limitations of GUS staining: Background substances can easily interfere with staining, and non-specific reactions can make the background colored, affecting the accuracy of cell identification and counting; differences in cell expression levels make it difficult to distinguish the depth of staining, which is not conducive to accurate counting; insufficient promoter specificity can easily lead to false positives; staining time and conditions are difficult to control and cannot be dynamically monitored; inhibitors in tissues can affect the results; and GUS staining materials are lethal and cannot be used for observation of living materials. These all seriously restrict the accuracy and comprehensiveness of the counting of Arabidopsis myrosinase cells.

[0011] (3) Limitations of fluorescent protein as a reporter gene: When the promoter is fused with fluorescent protein expression material, the expressed fluorescence is not localized in the nucleus. Therefore, the fluorescent protein is distributed throughout the cytoplasm of the cell. Therefore, it is difficult to distinguish two adjacent cells marked with this protein.

[0012] Therefore, a method to effectively observe and count the number of myrosinase heterocysts in living Arabidopsis materials is of great significance for exploring the chemical defense system of Cruciferae plants. Summary of the invention

[0013] The purpose of the present invention is to provide a bHLH090 gene promoter and its application in the method for detecting and counting the number of Arabidopsis myrosinase heteromorphic cells in view of the problems existing in the prior art.

[0014] In view of the irregular morphology and continuity of myrosinase heterocysts, it is impossible to clearly distinguish between the two cells. The method of the present invention selects the bHLH090 gene that is specifically expressed only in myrosinase cells, PCR amplifies its promoter sequence, such as SEQID No.1, and constructs a fusion expression system of the bHLH090 gene and a nuclear localization signal reporter gene for the detection and counting of the number of myrosinase heterocysts in Arabidopsis thaliana.

[0015] The second object of the present invention is to provide a PCR primer for amplifying the bHLH090 gene as shown in SEQ ID No. 1, and the sequence of the PCR primer is shown in SEQ ID No. 2-3.

[0016] The third object of the present invention is to provide a kit for detecting and counting the number of Arabidopsis myrosinase heterocysts, wherein the kit contains the bHLH090 gene promoter as shown in SEQ ID No. 1 or the PCR primers as shown in SEQ ID No. 2-3.

[0017] The fourth object of the present invention is to provide an expression cassette comprising the bHLH090 gene promoter as shown in SEQ ID No.1.

[0018] The fifth object of the present invention is to provide a vector comprising the bHLH090 gene promoter as shown in SEQ ID No. 1 or the aforementioned expression cassette.

[0019] Furthermore, the vector is probHLH090:nls-GFP.

[0020] The sixth object of the present invention is to provide a cell comprising the bHLH090 gene promoter as shown in SEQ ID No. 1, the expression cassette as described in claim 4 or the vector as described in claim 5.

[0021] The seventh object of the present invention is to provide a method for detecting and counting the number of Arabidopsis myrosinase heterocysts using the bHLH090 gene promoter, specifically comprising: cloning the bHLH090 gene, cutting the recombinant plasmid pOCA28:nls-GFP with the restriction endonuclease BamH1, linearizing it, recombining and connecting the target gene promoter with the linearized pOCA28:nls-GFP plasmid to obtain an expression vector target gene promoter:nls-GFP, transforming and amplifying with the help of Escherichia coli competent cells DH5α, and subjecting the expression vector to antibiotics (kan ＋ ) screening and PCR technology to screen the positive clones of probHLH090:nls-GFP, pick the single colony containing the recombinant vector, save and expand, extract the plasmid and sequence it; introduce the recombinant plasmid into Agrobacterium tumefaciens competent cells and culture it in LB / Kan ＋ / Rif ＋ Culture on solid medium for 1-2 days, pick a single colony and plate it in LB / Kan medium. ＋ / Rif ＋The cells were cultured on liquid culture medium, and then PCR was performed on the bacterial liquid. The size of the PCR bands was detected by gel electrophoresis. The obtained band colony PCR was further expanded and cultured, and the plasmid was extracted and sent out for sequencing. The obtained cDNA sequence was consistent with the expected sequence. The plasmid containing the target fragment was transformed into the Columbia ecotype Arabidopsis plants by Agrobacterium infection, and the transgenic offspring T0 generation seeds were harvested. The T0 generation seeds were disinfected and then spread on 1 / 2MS hygromycin plates to obtain plants that can grow on 1 / 2MS hygromycin plates. The vector was successfully transformed in Arabidopsis and the expression was observed using a fluorescence microscope. Image data processing was used to quickly count the number of myrosinase heterocysts.

[0022] Furthermore, the Agrobacterium infection is an inflorescence infection method.

[0023] The beneficial technical effect of the present invention is as follows: the present invention selects the bHLH090 gene specifically expressed in Arabidopsis myrosinase heterocysts, constructs a nuclear localization fusion expression vector of the specific expression gene bHLH090, cuts in the bHLH090 gene specifically expressed in myrosinase cells, and through related construction and connection operations, locates the marker originally expressed in the cytoplasm in the cell nucleus, thereby achieving accurate marking and positioning of myrosinase heterocysts, achieving specific marking and in vivo observation of myrosinase heterocysts, and avoiding the lethality problem of traditional staining methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0025] Figure 1 This is the result of bHLH090 promoter cloning detection in Example 1 of the present invention.

[0026] Figure 2 This is the positive clone detection result of Escherichia coli in Example 1 of the present invention.

[0027] Figure 3 This is the sequencing result of the positive clones in Example 1 of the present invention.

[0028] Figure 4 This is the positive clone seedling detection result of Example 1 of the present invention.

[0029] Figure 5 This is the fluorescence microscopic observation result of Example 1 of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] A method for detecting and counting the number of Arabidopsis myrosinase heterocysts, comprising the steps of:

[0033] 1. Extract genomic DNA from Arabidopsis leaves:

[0034] 2. Design restriction site primers based on the 2001bp DNA sequence before the start codon of the bHLH090 (AT1G10610) gene in the published complete gene sequence of Arabidopsis thaliana (shown in SEQ ID No.2-3), and amplify the promoter sequence of the bHLH090 gene (shown in SEQ No.1) using PCR technology.

[0035] F:5'- cgactctagaggatccccgggcgAGAAAGATTATCTCGATCATTTTGTG -3'(SEQNo.2),

[0036] R: 5'-ggaattaattccgctttatccatgCTCACTCTCTCTCTCTCTCTCTCTCAC-3' (SEQ No. 3).

[0037] Use 1% agarose gel electrophoresis at 180V for 20 minutes. Figure 1 As shown, the PCR product at 2001 bp was cut out under ultraviolet light and recovered using the Novozyme Gel Recovery Kit. The recovery procedure is described in the kit instructions of the specific manufacturer. The recovered DNA (the recovered product is marked as: rDNAG1) was dissolved in 40 μL of ddH2O water and recombined with the vector after detection.

[0038] 3. Preparation of linearized vector: Use restriction endonuclease BamH1 (FlyCut ® ), the plasmid pOCA28:nls-GFP was digested at 37°C for 2h, and the circular plasmid was used as a control. The products after digestion were detected by 1% agarose gel electrophoresis at 180V for 20min to ensure that the plasmid was completely linearized; after confirming the linearization state of the plasmid, the linearized vector was purified using the Novozymes purification kit. For specific purification steps, please refer to the manufacturer's instructions.

[0039] 4. Recombination reaction

[0040] Using homologous recombination technology, the purified bHLH090 promoter DNA fragment and the linearized pOCA28:nls-GFP vector were added to the seamless cloning connection system (ClonExpress Ultra One Step Cloning Kit V3) at a mass ratio of 1:1 and reacted at 37°C for 30 minutes.

[0041] 5. Conversion

[0042] Mix 5-10 μL of the recombination reaction product with 50 μL of the transformed E. coli competent cells (DH5a), keep them on ice for 30 minutes, place them in a 42°C water bath for 45 seconds, and then place them on ice for 1 minute. Add 800 μL of LB liquid culture medium and culture them in a constant temperature shaker at 260 rpm and 37°C for 45 minutes. Centrifuge the expanded E. coli at 8000 rpm for 1 minute. Discard the supernatant and spread the mixed cells on a solid LB resistance culture medium containing spectinomycin, and culture them upside down at 37°C for 12 hours.

[0043] 6. Identification of positive clones by PCR

[0044] Fourteen single colonies were selected for PCR identification at the same time, with the same primers as above. The results are as follows: Figure 2 shown.

[0045] 7. Expansion of positive clones and plasmid extraction

[0046] The positive clones identified above were picked and placed in 50 ml of liquid LB resistance culture medium containing spectinomycin, and cultured in a constant temperature shaker at 37°C, 220 rpm for 12 hours.

[0047] The cultured positive cloned E. coli was collected into a centrifuge tube by centrifugation at 8000 rpm for 10 min, and the plasmid was extracted according to the operating procedures of the plasmid extraction kit (Quanshijin Company).

[0048] 8. Plasmid sequencing

[0049] The extracted plasmid DNA was sent to Shanghai Biotech Co., Ltd. for sequencing, and the sequencing results were compared with the original sequence to confirm the accuracy of the positive clones. Figure 3 shown.

[0050] 9. Transformation of Agrobacterium

[0051] The correctly sequenced probHLH090-pOCA28-nlsGFP nuclear localization expression vector was transformed into Agrobacterium tumefaciens (GV3100). For specific experimental steps, please refer to the standard method of Agrobacterium competent transformation.

[0052] 10. Transformation of Arabidopsis thaliana by Agrobacterium inflorescence infection

[0053] The Agrobacterium carrying the probHLH090-pOCA28-nlsGFP nuclear localization expression vector was transformed into Arabidopsis thaliana by the inflorescence infection method (for specific steps, see the inflorescence infection method). The specific method is as follows: the successfully transformed and propagated Agrobacterium tumefaciens was spread on LB medium containing rifampicin and hygromycin for cultivation. After two days of cultivation, a single colony was picked and placed in liquid LB medium containing rifampicin and hygromycin. The culture was shaken at 220 rpm, 28°C, and protected from light until the bacterial liquid reached OD 600 =0.5, that is, the Agrobacterium tumefaciens infection solution is prepared and can be used for infection.

[0054] Select Arabidopsis thaliana with good growth status, immerse its inflorescence in the bacterial solution for 5 seconds, and after all inflorescences are infected, place the Arabidopsis thaliana plants in the dark at 22°C for 16-24 hours. Repeat the infection once every week, and infect 3-4 times cumulatively. After the infection is completed, culture the Arabidopsis thaliana normally, and collect the seeds after all the fruit pods are mature and mark them as T0 generation.

[0055] 11. Screening of positive transgenic Arabidopsis seedlings

[0056] The collected T0 generation seeds were sterilized and then plated on 1 / 2MS kanamycin plates. After 10 days of culture, Figure 4 As shown, the positive clone seedlings that can grow normally are transplanted into the soil, cultured at 22°C to mature, and the mature seeds are collected and marked as the T1 generation. The collected T1 generation seeds are disinfected and spread on 1 / 2MS kanamycin plates. After culturing for 10 days, the positive clone seedlings that can grow normally are transplanted into the soil, cultured at 22°C to mature, and the mature seeds are collected and marked as the T2 generation. The T2 generation seeds obtained at this time can be used for microscopic observation and quantitative statistics of myrosinase heterocysts.

[0057] 12. Fluorescence microscope observation and photography

[0058] The screened T2 seeds were sterilized and planted on 1 / 2MS medium, cultured at 22°C for 14 days, and the true leaves were removed with pointed tweezers and placed on a glass slide to prepare a temporary water-sealed slide. The slide was then placed under a fluorescence microscope for microscopic observation and photographed.

[0059] 13. Statistical analysis of the number of myrosinase heterocysts

[0060] Take pictures using a fluorescence microscope (e.g. Figure 5 ), the spindle-shaped fluorescent marker marked by the red arrow is the nucleus of a myrosinase cell. Counting them can give the number of myrosinase cells per unit area or per leaf.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. The bHLH090 gene promoter as shown in SEQ ID No. 1 is used in the method for detecting and counting the number of Arabidopsis myrosinase heterocysts.

2. A PCR primer for amplifying the bHLH090 gene promoter according to claim 1, characterized in that: The PCR primer sequences are shown in SEQ ID No. 2-3.

3. A kit for detecting the number of Arabidopsis myrosinase heterocysts, characterized in that: The kit contains the bHLH090 gene promoter according to claim 1 or the PCR primer according to claim 2.

4. An expression cassette, characterized in that Comprising the bHLH090 gene promoter according to claim 1.

5. A carrier, characterized in that It comprises the bHLH090 gene promoter according to claim 1 or the expression cassette according to claim 4.

6. The carrier according to claim 5, characterized in that The vector is probHLH090:nls-GFP.

7. A cell, characterized in that It comprises the bHLH090 gene promoter according to claim 1, the expression cassette according to claim 4 or the vector according to claim 5.

8. A method for detecting and counting the number of myrosinase heterocysts in Arabidopsis thaliana using the bHLH090 gene promoter, characterized in that: The bHLH090 gene promoter was homologously cloned, and the recombinant plasmid pOCA28:nls-GFP was cut with restriction endonuclease BamH1 and linearized. The target gene promoter was recombined with the linearized pOCA28:nls-GFP plasmid to obtain the expression vector target gene promoter:nls-GFP, which was transformed and propagated with Escherichia coli competent cells DH5α and then treated with antibiotics (kan ＋ ) screening and PCR technology to screen the positive clones of probHLH090:nls-GFP, pick the single colony containing the recombinant vector, save and expand, extract the plasmid and sequence it; introduce the recombinant plasmid into Agrobacterium tumefaciens competent cells and culture it in LB / Kan ＋ / Rif ＋ Culture on solid medium for 1-2 days, pick a single colony and plate it in LB / Kan medium. ＋ / Rif ＋ The cells were cultured on liquid culture medium, and then PCR was performed on the bacterial liquid. The size of the PCR bands was detected by gel electrophoresis. The obtained band colony PCR was further expanded and cultured, and the plasmid was extracted and sent out for sequencing. The obtained cDNA sequence was consistent with the expected sequence. The plasmid containing the target fragment was transformed into Columbia-type Arabidopsis plants by Agrobacterium infection, and the transgenic offspring T0 generation seeds were harvested. The T0 generation seeds were disinfected and then spread on 1 / 2MS hygromycin plates to obtain plants that can grow on 1 / 2MS hygromycin plates. The vector was successfully transformed in Arabidopsis and the expression was observed using a fluorescence microscope. Image data processing was used to quickly count the number of myrosinase heterocysts.

9. The method according to claim 8, characterized in that: The Agrobacterium infection is an inflorescence infection method.

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