A method for precisely regulating wax synthesis based on light-controlled chloride channel protein

Abstract

The application discloses a method for precisely regulating wax synthesis of plant leaves based on light-controlled chloride ion channel protein and belongs to the technical field of genetic engineering. The specific process is as follows: the light-controlled chloride ion channel protein opens or closes the ion channel under irradiation of different light, and the extracellular flow of chloride ions is promoted or terminated, so that the effect of promoting or terminating the wax synthesis is achieved. According to the principle of optogenetics, the application realizes the precise, reversible and space-time controllable regulation of the wax synthesis of the plant leaves, and assists in mining more genes related to the wax synthesis of the plants, thereby providing technical support for improving the adaptation ability of the plants to adversity.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for precisely regulating wax synthesis based on light-controlled chloride ion channel proteins. Background Technology

[0002] Plant leaf wax is a hydrophobic layer covering the plant epidermis, mainly composed of long-chain fatty acids and their derivatives (such as alkanes, aldehydes, alcohols, and esters). It plays an important role in reducing water loss, resisting pathogen invasion, and preventing ultraviolet damage. With global climate change and environmental degradation, enhancing plant resistance has become a key direction in agricultural and ecological research, and artificially regulating the synthesis of plant leaf wax is an important way to enhance plant resistance.

[0003] Optogenetics is a cutting-edge technology that uses light-sensitive proteins to precisely control cellular activities. In plant research, optogenetics has been successfully applied to regulate physiological processes such as stomatal movement and pollen tube growth, but there is still no relevant research in the area of ​​regulating wax synthesis in plant leaves.

[0004] Currently, the regulation of plant wax synthesis mainly relies on two methods: chemical regulation and genetic engineering regulation.

[0005] (1) Chemical regulation: By applying exogenous plant hormones (such as abscisic acid ABA), growth regulators (such as paclobutrazol), or other chemical substances (such as methyl jasmonate), the expression of genes related to plant wax synthesis is induced, thereby increasing the wax content. For example, in wheat research, exogenous application of ABA can upregulate the expression of the wax synthesis gene CER1, but this method has obvious shortcomings. First, the absorption, transport, and metabolism of chemical substances in plants are complex, making it difficult to accurately control the timing and dosage, resulting in poor timeliness; second, chemical substances may affect other physiological processes in plants, producing non-specific effects, such as inhibiting plant growth and altering flowering time.

[0006] (2) Genetic engineering regulation: Using gene editing technologies (such as CRISPR-Cas9), gene overexpression, or gene silencing, key genes for wax synthesis (such as CER1, KCS6, and ABCG11) are manipulated to alter the plant's wax synthesis capacity. For example, overexpression of the KCS6 gene in Arabidopsis thaliana can significantly increase leaf wax content. However, this method is a permanent modification of the plant genome, and once the gene is changed, it is difficult to dynamically adjust the wax synthesis level according to environmental changes. In addition, genetic engineering operations may cause unpredictable biosafety issues. Summary of the Invention

[0007] To address the aforementioned shortcomings in existing technologies, this invention provides a method for precisely regulating wax synthesis based on light-controlled chloride ion channel proteins. This invention aims to solve the problems of imprecise and non-dynamic regulation of plant leaf wax synthesis in existing technologies. Through the principles of optogenetics, it achieves precise, reversible, and spatiotemporally controllable regulation of plant leaf wax synthesis, and assists in the discovery of more genes related to plant wax synthesis, providing technical support for improving the plant's adaptability to adversity.

[0008] This invention is based on the principles of optogenetics and utilizes the light-controlled chloride ion channel protein ACR1 2.0's specific response to light to achieve the control of chloride ions (Cl... - Precise regulation of transmembrane efflux, thereby regulating the synthesis of leaf waxes in plants. Light-controlled chloride channel proteins open under white light irradiation, promoting the release of chloride ions... - From inside the cell to outside; when switched to red light irradiation, the protein returns to its initial conformation, the channel closes, and Cl... - Outflow has stopped. - Efflux alters intracellular ion balance and membrane potential, triggers downstream signal transduction pathways, regulates the expression of key genes in wax synthesis, promotes the synthesis, transport, and deposition of wax precursors, and ultimately achieves precise regulation of wax synthesis in plant leaves.

[0009] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0010] The purpose of this invention is to provide a method for precisely regulating wax synthesis based on light-controlled chloride ion channel protein. The specific process is as follows: the light-controlled chloride ion channel protein opens or closes the ion channel under different light irradiation, thereby promoting or terminating the outflow of chloride ions from the cell, thus promoting or terminating wax synthesis.

[0011] Furthermore, light-controlled chloride ion channel proteins open ion channels under white light irradiation, causing chloride ions to flow from inside the cell to outside the cell, thereby promoting wax synthesis.

[0012] Furthermore, the white light wavelength is 400–700 nm, and the illuminance is 50–200 μmol·m⁻¹. -2 ·s -1 .

[0013] Furthermore, the light-controlled chloride ion channel protein closes the ion channel under red light irradiation, terminating the flow of chloride ions from inside the cell to outside the cell and stopping the synthesis of waxes.

[0014] Furthermore, the red light wavelength is 650 nm, and the illumination intensity is 50 - 200 μmol·m. -2 ·s -1 .

[0015] Furthermore, the light-controlled chloride ion channel protein is ACR1 2.0.

[0016] Another object of the present invention is to provide the use of light-controlled chloride channel proteins in regulating plant wax synthesis.

[0017] Furthermore, the light-controlled chloride channel protein is ACR1 2.0, which promotes the flow of chloride ions from inside the cell to outside the cell under white light irradiation at a wavelength of 400~700 nm, thereby promoting the synthesis of waxes.

[0018] Furthermore, the light-controlled chloride channel protein ACR1 2.0 promotes the synthesis of ultra-long chain fatty acids under white light irradiation, while inhibiting the expression of the LOC107783985 gene, reducing fatty acid diversion, and promoting wax synthesis.

[0019] Furthermore, the light-controlled chloride channel protein is ACR1 2.0, which terminates the flow of chloride ions from inside the cell to outside the cell and terminates wax synthesis when irradiated with red light at a wavelength of 650 nm.

[0020] The beneficial effects of this invention are:

[0021] 1. Precise spatiotemporal control: Existing chemical regulation methods, such as using abscisic acid (ABA) to induce wax synthesis, cannot precisely control the timing and location of action. However, this invention utilizes photocontrolled chloride ion channel proteins. By using white and red light of specific wavelengths, the outflow of chloride ions can be precisely controlled at the cellular level, thereby precisely regulating the initiation, intensity, and termination of wax synthesis, achieving precise spatiotemporal control of wax synthesis in plant leaves.

[0022] 2. Dynamic and Reversible Regulation: Genetic engineering regulation permanently alters the plant genome, making it difficult to adapt to environmental changes. This invention establishes a dual-wavelength light regulation mode: white light irradiation promotes wax synthesis, while red light irradiation stops it. The level of wax synthesis can be flexibly adjusted according to actual environmental needs, achieving dynamic and reversible regulation.

[0023] 3. Low side effects: Exogenous chemical substances used in chemical regulation may have non-specific effects on plant growth and development. This invention is based on the plant's own physiological signal transduction mechanism and regulates through light-controlled ion channels, resulting in fewer side effects on the plant and making it more conducive to healthy plant growth. Attached Figure Description

[0024] Figure 1 To ensure stable expression of ACR1 2.0 in transgenic tobacco ( Nt Growth under different light conditions;

[0025] Figure 2 Electron micrograph of the ultrastructure of transgenic tobacco leaf surface after light exposure to stably express ACR1 2.0;

[0026] Figure 3 Detection of waxy components on the surface of transgenic tobacco leaves stably expressing ACR1 2.0;

[0027] Figure 4 To detect differentially accumulated metabolites between transgenic tobacco and wild-type groups that stably express ACR1 2.0;

[0028] Figure 5 Functional characterization of differentially accumulated metabolites between transgenic tobacco and wild-type groups that stably express ACR1 2.0;

[0029] Figure 6 To detect the expression of differentially expressed genes enriched in the heavy α-linolenic acid metabolic pathway in transgenic tobacco with stable ACR1 2.0 expression and wild-type tobacco. Detailed Implementation

[0030] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0031] Example 1

[0032] 1. Carrier Construction

[0033] The light-controlled chloride channel protein gene ACR1 2.0 was constructed into the plant expression vector pCAMBIA3300. A constitutive strong promoter, UBQ10, was selected to ensure stable and efficient gene expression on the plant cell membrane. Simultaneously, a suitable signal peptide was added to ensure the protein's localized expression on the cell membrane. Finally, an enhanced yellow fluorescent marker gene, eYFP, was ligated into the vector to facilitate subsequent screening of transgenic plants.

[0034] 2. Transformed plants

[0035] Using Agrobacterium-mediated transformation, the constructed expression vector was transferred into tobacco cells, thereby integrating the target gene into the plant genome. After tissue culture and screening, transgenic plants stably expressing the light-controlled chloride channel protein ACR1 2.0 were obtained.

[0036] Example 2

[0037] The transgenic plants were placed in a controlled artificial climate chamber. The light control parameters were as follows:

[0038] 1. Promote wax synthesis: When it is necessary to increase the synthesis of wax in plant leaves, provide white light irradiation with a wavelength of 400-700 nm and a light intensity of 50-200 μmol·m⁻¹. -2 ·s -1 Under these light conditions, light-controlled chloride ion channels open, promoting the release of intracellular chloride ions. - Outflow mediates the production of more wax in plant leaves.

[0039] 2. Stop wax synthesis: When it is necessary to stop wax synthesis, switch to red light irradiation with a wavelength of 650 nm and a light intensity of 50-200 μmol·m⁻¹. -2 ·s -1 Under red light, the light-controlled chloride ion channel protein returns to its initial conformation, the channel closes, and Cl... - Outflow has stopped.

[0040] Example 3

[0041] 1. Transgenic lines 1 (#1) and 2 (#2) stably expressing the light-controlled chloride channel protein ACR1 2.0 were cultured under different light conditions. Their growth was observed, and ultrastructural images of the leaf surface were taken. The waxy components of the plant surface were detected. The results are shown in […]. Figures 1-3 .

[0042] Figure 1 In the middle, a&d, under red light (650 nm, 16 h red light / 8 h dark, 200 μmol·m -2 ·s -1 (a) Growth of WT and tobacco expressing eYFP and ACR1 2.0 after 39 days of growth; (b) and (a) growth of tobacco grown under red light for 39 days, then transferred to white light (16 h white light / 8 h darkness, 400 μmol·m⁻¹). -2 ·s -1 The growth of WT (Wood Witch) and tobacco expressing eYFP and ACR1 2.0, respectively, after 8 days of growth under white light (c, b) followed by 17 days of growth under red light (c, b) (e and d ...

[0043] Figure 2 The upper image shows the red light (650 nm, 16 h red light / 8 h darkness, 200 μmol·m⁻¹) setting. -2 ·s-1 Representative 200× electron microscope images (n = 3) of WT tobacco plants grown for (39+8) days and expressing eYFP and ACR1 2.0, respectively; the image below shows red light (650nm, 16 h red light / 8 h dark, 200 μmol·m⁻¹). -2 ·s -1 After growing under light for 39 days, the cells were then transferred to white light (16 h white light / 8 h darkness, 400 μmol·m⁻¹). -2 ·s -1 Representative electron micrographs (n = 3) of WT and tobacco expressing eYFP and ACR1 2.0, respectively, after 8 days of growth under the control of WT; scale bar 50 μm, #1 represents transgenic line 1.

[0044] Figure 3 For the detection of plant surface wax components, A2-0-L1, A2-0-L2, and A2-0-L3 are three wax samples from transgenic line 2 stably expressing ACR12.0 after 0 days of white light irradiation; W-8-L1, W-8-L2, and W-8-L3 are three wax samples from wild-type tobacco (WT) after 8 days of white light irradiation; Y2-8-L1, Y2-8-L2, and Y2-8-L3 are three wax samples from transgenic line 2 stably expressing eYFP after 8 days of white light irradiation; and A2-8-L1, A2-8-L2, and A2-8-L3 are three wax samples from transgenic line 2 stably expressing ACR1 2.0 after 8 days of white light irradiation.

[0045] like Figures 1-3 As shown, transgenic lines stably expressing ACR1 2.0 effectively promoted wax synthesis under white light irradiation.

[0046] 2. Transgenic lines that stably express the light-controlled chloride channel protein ACR1 2.0 were cultured and then exposed to light at a wavelength of 400–700 nm and an intensity of 50–200 μmol·m⁻¹. -2 ·s -1 They were cultured under white light, and their differential metabolites compared to the wild type were detected. The results are shown in […]. Figure 4 and Figure 5 .

[0047] Figure 4In Figure a, a volcano plot shows the differential metabolite volcano plot of WT tobacco exposed to white light for 8 days versus ACR1 2.0 tobacco exposed to white light for 8 days; in figure b, a volcano plot shows the differential metabolite volcano plot of ACR1 2.0 tobacco exposed to white light for 0 days versus ACR1 2.0 tobacco exposed to white light for 8 days. A larger absolute value of the log2 Fold Change on the horizontal axis indicates a greater difference in metabolite expression levels; a larger value of the -log10 (P value) on the vertical axis indicates a more significant difference. c, Venn diagram of upregulated differential metabolites in WT tobacco exposed to white light for 8 days vs. ACR1 2.0 tobacco exposed to white light for 8 days and ACR1 2.0 tobacco exposed to white light for 0 days vs. ACR1 2.0 tobacco exposed to white light for 8 days; d, Venn diagram of downregulated differential metabolites in WT tobacco exposed to white light for 8 days vs. ACR1 2.0 tobacco exposed to white light for 8 days and ACR1 2.0 tobacco exposed to white light for 0 days vs. ACR1 2.0 tobacco exposed to white light for 8 days.

[0048] Figure 5 Figure a shows the KEGG enrichment of differential metabolites in the WT group (8 days of white light irradiation) versus the ACR1 2.0 group (8 days of white light irradiation); figure b shows the KEGG enrichment of differential metabolites in the ACR1 2.0 group (0 days of white light irradiation) versus the ACR1 2.0 group (8 days of white light irradiation). In the figure, the horizontal axis represents the enrichment factor, which is the ratio of the number of differential metabolites to the total number of metabolites annotated to that pathway; a higher value indicates a higher degree of enrichment. The vertical axis represents the metabolic pathway. The p-value is color-coded, from green to red, indicating a decreasing p-value and a gradually increasing degree of enrichment. The size of the dot represents the number (Count) of differential metabolites enriched in that pathway.

[0049] 3. Under white light irradiation, the genes involved in the pathways affecting wax synthesis by ACR1 2.0 were detected and analyzed. The results are shown in […]. Figure 6 .

[0050] Figure 6 In Figure a, there is a heatmap showing the differentially expressed genes in the α-linolenic acid (ALA) metabolic pathway, comparing WT tobacco exposed to white light for 8 days versus ACR12.0 tobacco exposed to white light for 8 days. Figure b shows a heatmap showing the differentially expressed genes in the ALA metabolic pathway, comparing ACR12.0 tobacco exposed to white light for 0 days versus ACR12.0 tobacco exposed to white light for 8 days. The horizontal axis represents different samples, and the vertical axis represents different genes. Red indicates high gene expression levels, and blue indicates low gene expression levels.

[0051] Analysis of differentially expressed genes enriched in the α-linolenic acid metabolic pathway revealed that after white light irradiation of ACR1 2.0 plants, the genes LOC104117921, LOC107791409, LOC107806557, LOC104096874, LOC107788258, LOC104216671, LOC104084890, LOC109219413, LOC104115832, and LOC104116693 were significantly upregulated in both control groups, promoting wax synthesis. Furthermore, white light irradiation can activate the α-linolenic acid metabolic pathway. α-Linolenic acid is a C18 fatty acid, and C18 fatty acids can participate in the synthesis of very long chain fatty acids (VLCFAs). VLCFAs are wax precursors, forming wax components through a series of enzymatic reactions.

[0052] The genes LOC107770901, LOC104221778, LOC104086421, LOC107783985, LOC104111680, and LOC107829038 were all significantly downregulated in both comparison groups. Among them, LOC107783985 encodes allene oxide synthase (AOS), a key enzyme in the jasmonic acid synthesis pathway, and its expression was significantly downregulated after white light irradiation. This suggests that chloride ion efflux may reduce the diversion of fatty acids to the jasmonic acid pathway by inhibiting LOC107783985 expression, thus promoting more fatty acids to flow to the wax synthesis metabolic pathway and further promoting wax accumulation.

[0053] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for precisely regulating wax synthesis based on light-controlled chloride ion channel proteins, characterized in that, The specific process is as follows: the light-controlled chloride ion channel protein opens or closes the ion channel under different light irradiation, which promotes or terminates the outflow of chloride ions from the cell, thereby promoting or terminating the synthesis of waxes. The light-controlled chloride ion channel protein is ACR1 2.0, which opens the ion channel under white light irradiation, causing chloride ions to flow from inside the cell to outside the cell, thereby promoting wax synthesis. It closes ion channels under red light irradiation, stopping the flow of chloride ions from inside the cell to outside the cell and terminating wax synthesis.

2. The method according to claim 1, characterized in that, The wavelength of white light is 400~700 nm, and the illuminance is 50-200 μmol·m⁻¹. -2 ·s -1 .

3. The method according to claim 1, characterized in that, The red light has a wavelength of 650 nm and an illumination intensity of 50 - 200 μmol·m⁻¹. -2 ·s -1 .

4. The use of the light-controlled chloride channel protein as described in claim 1 in regulating plant wax synthesis.

5. The use according to claim 4, characterized in that, The light-controlled chloride channel protein ACR1 2.0 promotes the flow of chloride ions from inside the cell to outside the cell under white light irradiation at a wavelength of 400~700 nm, thereby promoting the synthesis of waxes.

6. The use according to claim 4, characterized in that, The light-controlled chloride channel protein ACR1 2.0 promotes the synthesis of ultra-long chain fatty acids under white light irradiation, while inhibiting the expression of the LOC107783985 gene, reducing fatty acid diversion, and promoting wax synthesis.

7. The use according to claim 4, characterized in that, The light-controlled chloride channel protein ACR1 2.0 terminates the flow of chloride ions from inside the cell to outside the cell and stops the synthesis of waxes when exposed to red light at a wavelength of 650 nm.

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