Kcr1 mutant suitable for regulating potassium ion homeostasis of plants and application thereof

Abstract

The application discloses a KCR1 mutant suitable for plant potassium ion homeostasis regulation and application thereof, relates to the technical field of bioengineering, and the KCR1 mutant has at least T136A and G140A mutations on the basis of a light-controlled potassium ion channel protein KCR1. The application also discloses a gene encoding the KCR1 mutant. The application also discloses application of the KCR1 mutant or the gene in plant culture, breeding of new plant varieties or preparation of plant culture products. The KCR1 mutant has a blue-shifted excitation spectrum compared with a wild type, and can realize precise regulation of plant potassium ion flow through light regulation, and has great application value in light-controlled plant breeding and research of plant potassium ion channels.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a KCR1 mutant suitable for regulating potassium ion homeostasis in plants and its applications. Background Technology

[0002] A team led by John L. Spudich at the McGovern Medical Center Health Sciences at the University of Texas at Houston published an article in *Nature Neuroscience* reporting potassium channel rhodopsin (KCRs) from *Nematocystis*, including KCR1 and KCR2. KCR1 is a light-controlled cation channel that... + The selectivity is higher than that for Na + It exhibits selectivity, opening within less than 1 millisecond after photoactivation. Under physiological ion conditions, it responds to light energy to generate a series of photocurrents. The voltage dependence of the peak current shows slight inward rectification and a reversal potential of -85±2mV. It displays a more redshifted spectrum, a larger current amplitude, and greater dependence on K+ than KCR2. + A higher degree of selectivity.

[0003] K + Ion channels play a vital role in plant growth, development, metabolism, and environmental adaptation. (1) Plant cells regulate K+ + The opening and closing of ion channels controls K + The absorption and excretion of K+ regulate the osmotic pressure within the cell. When plants are under adverse conditions such as drought, the cells will regulate the osmotic pressure through the absorption and excretion of K+. + Ion channel absorption of K + This increases the intracellular osmotic pressure, thereby promoting water absorption, maintaining cell turgor pressure, and preserving the normal morphology and physiological function of the cell. (2) K + It is one of the essential macronutrients for plant growth and has a significant impact on plant growth and development. K + Ion channels participate in the elongation and division of plant cells. In vigorously growing areas such as root tips and shoot tips, K+ channels are essential for cell proliferation. + Ion channels have high activity, providing the necessary ion environment for cell elongation and division. (3) In photosynthesis, K + Ion channels participate in photosynthetic electron transport and photophosphorylation. K + The accumulation of ions in chloroplasts can regulate the potential and pH of the thylakoid membrane, providing a suitable environment for photophosphorylation, promoting ATP synthesis, and providing energy for the dark reaction of photosynthesis. (4) When plants are subjected to abiotic stress, such as salt stress and low temperature stress, they will regulate K+. +The expression and activity of ion channels maintain intracellular ion and osmotic balance, thereby enhancing plant stress resistance. (5) Various physiological processes within plant cells are accompanied by charge transfer and distribution changes, K + As an important type of cation, ions can enter and exit cells through ion channels, thus regulating the charge balance within the cell.

[0004] Photosensitive proteins have seen preliminary applications in plants. Researchers have used photocontrolled chloride channel proteins to control the opening and closing of pollen tubes and stomata, and photocontrolled calcium channel proteins to control plant immune responses, but there are no reports of applications of photosensitive proteins that can regulate potassium ions in plants. The natural KCR1 protein has a more red-shifted excitation spectrum, meaning its function can be partially activated even under red light. This will have some impact on the future cultivation of transgenic plants (cultivated under red light conditions), therefore, it is necessary to shift the excitation spectrum of KCR1 towards the blue light region, avoiding the red light excitation region. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a KCR1 mutant suitable for regulating potassium ion homeostasis in plants and its applications. The KCR1 mutant of the present invention has a blue-shifted excitation spectrum compared to the wild type, and can achieve precise regulation of potassium ion flow in plants through light control. It has significant application value in light-controlled plant cultivation and research on plant potassium ion channels.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a KCR1 mutant suitable for the regulation of potassium ion homeostasis in plants is provided, which has at least T136A and G140A mutations based on the light-controlled potassium ion channel protein KCR1.

[0007] Furthermore, the amino acid sequence of the light-controlled potassium ion channel protein KCR1 is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the KCR1 mutant is shown in SEQ ID NO.2. It is the mutant T136A / G140A.

[0009] The present invention also provides a gene encoding the above-mentioned KCR1 mutant suitable for the regulation of potassium ion homeostasis in plants.

[0010] Furthermore, the nucleotide sequence of the above gene is shown in SEQ ID NO.3. It is the encoding gene for the mutant T136A / G140A.

[0011] This invention also provides the application of the above-mentioned KCR1 mutant or gene in plant culture.

[0012] This invention also provides the application of the above-mentioned KCR1 mutant or gene in the breeding of new plant varieties.

[0013] This invention also provides the application of the above-mentioned KCR1 mutant or gene in the preparation of plant culture products.

[0014] The present invention has the following beneficial effects:

[0015] 1. The excitation spectrum of the KCR1 mutant in this invention shifts towards the blue light region compared to the wild type, thus avoiding excitation by red light. After stable expression in plants, cultivation under red light will not activate the protein's function and will not affect the plant. The KCR1 protein can be activated under green light irradiation to regulate plant growth and development, facilitating the study of KCR1 protein. + The impact on plant growth and development.

[0016] 2. This invention applies the KCR1 mutant to plants, enabling millisecond-level plant KCR1 reduction through light exposure. + The regulation of channel opening and closing has significant practical application and scientific research value.

[0017] 3. The KCR1 mutant of this invention can achieve single-cell / tissue-specific KCR1 synthesis that is impossible to achieve using traditional methods. + Channel regulation (e.g., root epidermal cells vs. guard cells), and K + Precise flow control.

[0018] 4. The KCR1 mutant of this invention can achieve K+ regulation in plants under adverse conditions (drought, salt stress) through light regulation. + Steady-state regulation, in analyzing K + The dynamic functionality of the channel under adversity, and the development of light-controlled K + It has significant application value in the efficient utilization of new crop varieties. Attached Figure Description

[0019] Figure 1 The image shows the results of the detection of the electrophysiological characteristics of KCR1 (TAGA).

[0020] Figure 2 Figure showing the growth of different transgenic Arabidopsis thaliana lines under red light conditions;

[0021] Figure 3 Figure showing the growth of different transgenic Arabidopsis thaliana lines under green light conditions;

[0022] Figure 4 The graph shows the changes in cell membrane potential of KCR1(GATA) transgenic Arabidopsis thaliana under green light irradiation;

[0023] Figure 5This is a map showing the expression localization of KCR1(GATA) and eYFP in transgenic Arabidopsis thaliana.

[0024] Figure 6 Figure showing root growth of different transgenic Arabidopsis thaliana lines under white light irradiation;

[0025] Figure 7 Figure showing root growth of different transgenic Arabidopsis thaliana lines under red light irradiation;

[0026] Figure 8 A graph showing the statistical results of taproot length in transgenic Arabidopsis thaliana under different irradiation conditions;

[0027] Figure 9 The image shows the results of fluorescence observation and cell membrane potential detection in KCR1(TAGA) transgenic tobacco.

[0028] Figure 10 This diagram shows the phenotypic changes of different transgenic tobacco plants under white light cultivation. Detailed Implementation

[0029] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0030] Example 1

[0031] A KCR1 mutant (denoted as KCR1(TAGA)) suitable for regulating potassium ion homeostasis in plants has T136A and G140A mutations based on the light-controlled potassium ion channel protein KCR1 (SEQ ID NO.1). The amino acid sequence of this mutant is shown in SEQ ID NO.2.

[0032] The gene encoding this KCR1(TAGA) mutant has the nucleotide sequence shown in SEQ ID NO.3.

[0033] Example 1: Construction and physiological property testing of the KCR1 mutant

[0034] (1) The KCR1(TAGA) mutant was constructed by double enzyme digestion.

[0035] Primers with mutation sites T136A and G140A were designed to amplify the KCR1 fragment by PCR using wild-type KCR1 (wt), thus completing the site-directed mutagenesis of KCR1. The KCR1 (TAGA) gene was then ligated into the expression vector pGEM by double digestion with BamHI and HindIII, with yellow fluorescent protein YFP used as an indicator tag for expression localization.

[0036] (2) Expression of KCR1 (TAGA) in Xenopus laevis eggs

[0037] Normal and healthy oocytes were selected, rinsed with culture medium, and cultured in an incubator for later use. The gene expression vector of the light-controlled ion channel protein was linearized, and the corresponding mRNA was synthesized using an in vitro transcription kit. The synthesized mRNA was injected into toad oocytes using microinjection technology. After injection, the oocytes were incubated in an incubator for 3 days to allow the light-controlled ion channel protein to be expressed in the oocytes.

[0038] (3) Detection of electrophysiological characteristics of KCR1 (TAGA)

[0039] Oocytes expressing KCR1(TAGA) were transferred to recording wells and perfused with extracellular fluid. The extracellular fluid composition was designed to mimic the physiological environment, containing appropriate ion concentrations and pH values. Electrophysiological recordings of membrane current changes in the oocytes were performed using voltage-clamp techniques. First, light stimulation at different wavelengths was applied, and the properties of KCR1(TAGA) under different wavelength light conditions were detected. The results are as follows: Figure 1 As shown in the figure, Figure A is the excitation spectrum, Figure B is the detection result of the reversal potential, and Figure C is the result under different illumination conditions (green light 532nm, 60μW / mm). 2 Red light 635nm, 720μW / mm 2 Blue light 450nm, 67μW / mm 2 The change in photocurrent (with an illumination time of 5 s).

[0040] Depend on Figure 1 As shown in Figure A, the excitation spectrum of the KCR1(TAGA) mutant exhibits a significant blue shift compared to the wild type; Figure B, based on the detection of the inversion potential, indicates that the ion selectivity of the mutant has not changed significantly; Figure C similarly shows that the excitation spectrum of KCR1(TAGA) has shifted towards the blue light region.

[0041] Experimental Example 2: Cultivation and Identification of Transgenic Arabidopsis thaliana

[0042] (1) Obtaining the target gene and constructing the vector

[0043] The KCR1(TAGA) gene was ligated into the pCAMBIA3300 vector using enzyme digestion and ligation. Positive clones were screened and recombinant plasmids were extracted by transforming E. coli competent cells.

[0044] (2) Agrobacterium-mediated transformation

[0045] The recombinant plasmid was introduced into Agrobacterium competent cells, and the transformed Agrobacterium was screened for positive clones on a solid culture medium containing the corresponding antibiotics.

[0046] (3) Cultivation and training of Arabidopsis thaliana

[0047] After sowing, Arabidopsis thaliana seeds were placed in a light incubator, maintaining a temperature of approximately 22℃ and a light intensity of 100-120 μmol·m⁻¹. -2 ·s -1 The light duration was 16 hours / day and the dark duration was 8 hours / day. Subsequently, single colonies of Agrobacterium containing the recombinant plasmid were picked and inoculated into LB broth containing the corresponding antibiotic, and cultured at 28°C with shaking until the logarithmic growth phase. The bacterial cells were collected by centrifugation and resuspended in osmotic medium containing 5% sucrose and 0.02% Silwet L-77, and the OD of the bacterial culture was adjusted. 600 The value was reduced to 0.4. The Arabidopsis plant was inverted so that the inflorescence was immersed in Agrobacterium tumefaciens solution for 5-10 minutes, and gently shaken. Then the plant was placed upright and covered with plastic wrap to keep it moist for 24 hours.

[0048] (4) Screening and identification of transgenic Arabidopsis thaliana

[0049] The transformed Arabidopsis plants were cultured until the seeds matured, and the seeds were harvested and sown in the soil. Positive plants were screened by Basta spraying, and the selected positive plants were identified by fluorescence. Finally, transgenic seedlings with resistance were selected.

[0050] (5) Identification of KCR1 (TAGA) function

[0051] Healthy leaves expressing KCR1 (TAGA) were selected, and the changes in Arabidopsis cell membrane voltage under light stimulation were detected by current clamp technique to determine the function of the protein.

[0052] (6) Phenotypic changes in transgenic Arabidopsis thaliana under different light conditions

[0053] Arabidopsis thaliana plants were placed under different light conditions to observe changes in plant growth.

[0054] Different transgenic Arabidopsis strains under red light (650nm, 23μW / mm) 2 The growth situation after 23 days under the following conditions is as follows: Figure 2As shown in the figure, Figure A shows the phenotypes of different transgenic Arabidopsis thaliana lines, Figure B shows the fresh weight comparison of different transgenic Arabidopsis thaliana lines, and Figure C shows the dry weight comparison of different transgenic Arabidopsis thaliana lines. The results show that the KCR1(TAGA) transgenic Arabidopsis thaliana exhibited the same growth as the control group expressing eYFP and the wild-type Col-0, and there was no significant difference in the fresh weight and dry weight of the plants. This indicates that red light did not activate the function of KCR1, and the transgenic plants could grow normally under red light.

[0055] Different transgenic Arabidopsis thaliana lines under green light (530nm, 30μW / mm) 2 Growth under the following conditions: Figure 3 As shown in the figure. The results showed that under green light growth conditions, the transgenic Arabidopsis thaliana KCR1(TAGA) was significantly affected. Compared with the control group, after 4 days of growth under green light conditions, the transgenic Arabidopsis thaliana KCR1(TAGA) showed yellowing of leaves.

[0056] Furthermore, in green light (532nm, 150μw / mm) 2 The changes in cell membrane potential of KCR1(GATA) transgenic Arabidopsis thaliana were tested under 5s irradiation conditions, and the results are as follows: Figure 4 As shown, light stimulation activates KCR1, mediating potassium ion efflux, which leads to inhibited plant growth and development, indicating that the functional expression of KCR1 (TAGA) affects normal plant growth.

[0057] (7) Effects of functional expression of KCR1(TAGA) on plant root growth

[0058] The expression localization of KCR1(GATA) and eYFP in transgenic Arabidopsis thaliana is as follows: Figure 5 As shown in the figure. The results indicate that KCR1(TAGA) can be expressed on the root cell membrane in the root system.

[0059] Different transgenic Arabidopsis strains under white and red light (650nm, 20μW / mm) 2 Root growth under irradiation conditions are as follows: Figure 6 and Figure 7 As shown, the statistical results of the taproot length of Arabidopsis thaliana are as follows: Figure 8 As shown in the figure (n=5, p<0.0001). The results showed that, under white light culture conditions, the root growth of transgenic Arabidopsis thaliana stably expressing KCR1(TAGA) was significantly inhibited compared with the control group; however, under red light conditions, the root growth of transgenic Arabidopsis thaliana with KCR1(TAGA) was not affected; in summary, the functional expression of KCR1(TAGA) inhibited the root growth of plants.

[0060] Experiment 3: Cultivation and identification of growth phenotype of KCR1(TAGA) transgenic tobacco

[0061] (1) Preparation of transgenic tobacco by leaf disc method

[0062] Select vigorous and healthy tobacco plants and take their tender leaves. After rinsing thoroughly with tap water, immerse the leaves in 75% alcohol for 60 seconds to sterilize the surface. Next, rinse three times with sterile water, then immerse the leaves in a 0.2% mercuric chloride solution for 10 minutes to kill surface bacteria and fungi. Finally, rinse five times with sterile water until no mercuric chloride residue remains in the rinsing solution. Place the sterilized leaves in sterile petri dishes and use a sterile punch to create leaf discs approximately 8 mm in diameter. Select the upper and middle parts of the leaves, avoiding tissue near the veins, to ensure cell viability and regeneration capacity of the leaf discs. Then, immerse the prepared leaf discs in a bacterial solution expressing KCR1 (TAGA) Agrobacterium for 5 minutes to ensure full contact with the Agrobacterium. Then, excess bacterial culture was blotted from the surface of the leaf discs with sterile filter paper. The leaf discs were then inoculated into culture dishes containing solid MS medium supplemented with 2 mg / L 6-benzylaminopurine (6-BA) and 0.2 mg / L naphthaleneacetic acid (NAA) to promote leaf cell division and differentiation. The culture dishes were sealed and incubated for 2 days at 25°C, with a light intensity of 1000-1500 lx and a photoperiod of 16 h / day, allowing Agrobacterium to transfer the target gene into tobacco leaf cells.

[0063] (2) Rooting culture

[0064] When the adventitious buds grow to 2-3 cm in height, they are cut off and transferred to a rooting medium containing 1.0 mg / L indolebutyric acid (IBA) for rooting culture. After culturing on the rooting medium for 1-2 weeks, white adventitious roots will gradually grow from the base of the adventitious buds, forming complete transgenic tobacco plants.

[0065] (3) Detection and identification of transgenic plants

[0066] After the transgenic tobacco plants grew to a certain size, positive plants were identified by fluorescence observation and detection of plant surface membrane potential. The results are as follows: Figure 9 As shown, Figure A illustrates the expression localization of KCR1(TAGA) in tobacco leaf epidermal cells, and Figure B shows the expression localization under green light (532 nm, 180 μW / mm²). 2 Changes in surface membrane potential of KCR1(TAGA) transgenic tobacco under stimulation.

[0067] (4) Phenotypic changes in transgenic tobacco stably expressing KCR1(TAGA)

[0068] Transgenic tobacco expressing YFP was used as a control. Transgenic tobacco stably expressing KCR1 (TAGA) was cultured under white light for 72 hours, and phenotypic changes in the plants were observed. The results are as follows: Figure 10 As shown in the figure. The results showed that, compared with the control group, transgenic tobacco plants stably expressing KCR1(TAGA) exhibited a phenotypic change of yellowing leaves, indicating that the functional expression of KCR1(TAGA) can also affect the growth of tobacco plants.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A KCR1 mutant suitable for regulating potassium ion homeostasis in plants, characterized in that, It contains T136A and G140A mutations based on the light-controlled potassium ion channel protein KCR1; The amino acid sequence of the light-controlled potassium ion channel protein KCR1 is shown in SEQ ID NO.1; The amino acid sequence of the KCR1 mutant is shown in SEQ ID NO.

2.

2. The gene encoding the KCR1 mutant of claim 1, applicable to the regulation of potassium ion homeostasis in plants.

3. The gene as described in claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.3.

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