KCR1 mutant suitable for steady-state regulation and control of plant potassium ions and application of KCR1 mutant

Abstract

The invention discloses a KCR1 mutant suitable for plant potassium ion steady state regulation and application thereof, and relates to the technical field of bioengineering, and the KCR1 mutant at least has T136A and G140A mutations on the basis of light-operated potassium ion channel protein KCR1. The invention also discloses a gene for coding the KCR1 mutant. The invention also discloses application of the KCR1 mutant or gene in plant culture, cultivation of new plant varieties or preparation of plant culture products. The KCR1 mutant disclosed by the invention has a blue-shift excitation spectrum compared with a wild type, can realize accurate regulation and control of plant potassium ion flow through illumination regulation and control, and has a relatively great application value in the aspects of light-controlled plant cultivation, plant potassium ion channel research and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a KCR1 mutant applicable to the regulation of plant potassium ion homeostasis and its application. Background Art

[0002] The team of John L. Spudich at the McGovern Medical School of the University of Texas Health Science Center in Houston published an article in Nature Neuroscience, reporting potassium channel rhodopsins (KCRs) from the filamentous fungus, including KCR1 and KCR2. KCR1 is a light-gated cation channel that has a higher selectivity for K + than for Na + and opens within less than 1 millisecond after photoactivation. Under physiological ionic conditions, it can generate a series of photocurrents in response to light, and the voltage dependence of the peak current shows a slight inward rectification and a reversal potential of -85 ± 2 mV. It exhibits a more red-shifted spectrum, a larger current amplitude, and a higher selectivity for K + than KCR2.

[0003] K + ion channels play a crucial role in the life activities of plants such as growth and development, metabolism, and adaptation to the environment. (1) Plant cells control the absorption and efflux of K + by regulating the opening and closing of K + ion channels, thereby regulating the osmotic pressure inside the cell. When plants are under stress conditions such as drought, the cells will absorb K + through K + ion channels, increasing the osmotic pressure inside the cell, promoting water absorption, maintaining cell turgor pressure, and keeping the normal morphology and physiological functions of the cells. (2) K + is one of the macronutrients necessary for plant growth and has an important impact on plant growth and development. K + ion channels are involved in the processes of cell elongation and division in plants. In vigorously growing parts such as the root tip and shoot tip, the activity of K + ion channels is relatively high, providing a necessary ionic environment for cell elongation and division. (3) In photosynthesis, K + ion channels are involved in the processes of photosynthetic electron transport and photophosphorylation. The accumulation of K + ions in chloroplasts can regulate the potential and pH value of the thylakoid membrane, provide a suitable environment for photophosphorylation, promote the synthesis of ATP, and provide energy for the dark reaction of photosynthesis. (4) When plants are under stress, such as salt stress and low temperature stress, they will regulate K +The expression and activity of ion channels maintain the ion balance and osmotic pressure balance within cells, thereby enhancing the stress resistance of plants. (5) Various physiological processes within plant cells are accompanied by changes in the transfer and distribution of electric charges. K + As an important cation, potassium ions enter and exit cells through ion channels, which can regulate the charge balance within cells.

[0004] Light-sensitive proteins have been preliminarily applied in plants. Researchers have used light-controlled chloride ion channel proteins to control the opening and closing of plant pollen tubes and stomata, and light-controlled calcium ion channel proteins to control plant immune responses, etc. However, there are no reports on the application of light-sensitive proteins that can regulate potassium ions in plants. The natural KCR1 protein has a more red-shifted excitation spectrum, that is, it can be partially activated under red light. This will have a certain impact on the cultivation of future transgenic plants (cultivated under red light conditions), so it is necessary to shift the excitation spectrum of KCR1 towards the blue light region to avoid the red light excitation region. SUMMARY OF THE INVENTION

[0005] To solve the above technical problems, the object of the present invention is to provide a KCR1 mutant applicable to the regulation of plant potassium ion homeostasis and its application. The KCR1 mutant of the present invention has a blue-shifted excitation spectrum compared to the wild type, and can achieve precise regulation of plant potassium ion flux through light control, which has great application value in aspects such as light-controlled plant cultivation and plant potassium ion channel research.

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

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

[0008] Further, the amino acid sequence of the KCR1 mutant is as 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 applicable to the regulation of plant potassium ion homeostasis.

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

[0011] The present invention also provides the application of the above KCR1 mutant or gene in plant cultivation.

[0012] The present invention also provides the use of the above-mentioned KCR1 mutant or gene in cultivating new plant varieties.

[0013] The present invention also provides the use of the above-mentioned KCR1 mutant or gene in preparing plant culture products.

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

[0015] 1. The excitation spectrum of the KCR1 mutant of the present invention is shifted towards the blue light region compared to the wild type, avoiding the excitation of red light. After stable expression in plants, the culture under red light will not activate the function of this protein and will not affect the plants. The KCR1 protein can be activated under green light irradiation to regulate the growth and development of plants, so as to study the + influence on plant growth and development.

[0016] 2. Applying the KCR1 mutant of the present invention to plants can achieve the regulation of the opening and closing of plant K + channels at the millisecond level through light irradiation, which has important practical application and scientific research value.

[0017] 3. Using the KCR1 mutant of the present invention can achieve single-cell / tissue-specific K + channel regulation (such as root epidermal cells vs. guard cells) that cannot be achieved by traditional methods, as well as precise manipulation of K + flux.

[0018] 4. Using the KCR1 mutant of the present invention can achieve the regulation of plant K + homeostasis under stress (drought, salt stress) environments through light regulation, and has great application value in analyzing the dynamic function of K + channels under stress and developing new crop varieties with efficient light-controlled K + utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a detection result diagram of the electrophysiological characteristics of KCR1 (TAGA);

[0020] Figure 2 It is a growth situation diagram of different transgenic Arabidopsis thaliana lines under red light conditions;

[0021] Figure 3 It is a growth situation diagram of different transgenic Arabidopsis thaliana lines under green light conditions;

[0022] Figure 4 It is a diagram of the change in the cell membrane potential of KCR1 (GATA) transgenic Arabidopsis thaliana under green light irradiation;

[0023] Figure 5Expression localization map of KCR1(GATA) and eYFP in transgenic Arabidopsis

[0024] Figure 6 Root growth of different transgenic Arabidopsis lines under white light illumination

[0025] Figure 7 Root growth of different transgenic Arabidopsis lines under red light illumination

[0026] Figure 8 Statistical results of the primary root length of transgenic Arabidopsis under different irradiations

[0027] Figure 9 Fluorescence observation and cell membrane potential detection results of KCR1(TAGA) transgenic tobacco

[0028] Figure 10 Plant phenotype changes of different transgenic tobaccos under white light culture Detailed implementation mode

[0029] The principles and characteristics of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0030] Example 1

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

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

[0033] Experimental example 1 Construction and physiological property detection of KCR1 mutant

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

[0035] Design primers with mutation sites of T136A and G140A, amplify the KCR1 fragment from wild-type KCR1 (wt) by PCR to complete the site-directed mutagenesis of KCR1; then, through double digestion with BamH I and Hind III, ligate the KCR1 (TAGA) gene to the expression vector pGEM, using yellow fluorescent protein YFP as the indication tag for expression localization.

[0036] (2) Express KCR1 (TAGA) in Xenopus laevis oocytes

[0037] Select morphologically normal and healthy oocytes, rinse them with culture medium, and place them in an incubator for standby; linearize the gene expression vector of the light-gated ion channel protein, and then synthesize the corresponding mRNA using an in vitro transcription kit; use microinjection technology to inject the synthesized mRNA into the Xenopus oocytes; after injection, incubate the oocytes in an incubator for 3 days to express the light-gated ion channel protein in the oocytes.

[0038] (3) Detection of the electrophysiological properties of KCR1 (TAGA)

[0039] Transfer the oocytes expressing KCR1 (TAGA) to the recording chamber and perfuse them with extracellular fluid, the composition of which needs to simulate the physiological environment, including appropriate ion concentrations and pH values. Use the voltage clamp technique to electrophysiologically record the changes in the membrane current of the oocytes. First, apply light stimuli of different wavelengths to detect the properties of KCR1 (TAGA) under different wavelength illumination conditions, and the results are as Figure 1 shown, where Figure A is the excitation spectrum, Figure B is the detection result of the reversal potential, and Figure C is the photocurrent change under different illumination conditions (green light 532 nm, 60 μW / mm 2 ; red light 635 nm, 720 μW / mm 2 ; blue light 450 nm, 67 μW / mm 2 ; illumination time is 5 s).

[0040] As Figure 1 can be seen, Figure A shows that compared with the wild type, the excitation spectrum of the KCR1 (TAGA) mutant has a significant blue shift; the detection of the reversal potential in Figure B shows that the ion selectivity of the mutant has not changed significantly; Figure C also 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

[0042] (1) Acquisition of the target gene and vector construction

[0043] The KCR1 (TAGA) gene was ligated to the pCAMBIA3300 vector by enzymatic digestion and ligation methods. After transforming Escherichia coli competent cells, positive clones were screened and recombinant plasmids were extracted.

[0044] (2) Agrobacterium transformation

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

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

[0047] The sown Arabidopsis thaliana was placed in a light incubator, maintaining a temperature of about 22 °C, a light intensity of 100 - 120 μmol·m -2 ·s -1 , with a light duration of 16 h / day and a dark duration of 8 h / day. Subsequently, single colonies of Agrobacterium containing the recombinant plasmid were picked and inoculated into LB liquid medium containing the corresponding antibiotics, and cultured with shaking at 28 °C until the logarithmic growth phase. The bacterial solution was centrifuged to collect the bacteria, and resuspended with an osmotic medium containing 5% sucrose and 0.02% Silwet L-77, and the OD 600 value of the bacterial solution was adjusted to 0.4. The Arabidopsis thaliana plants were inverted so that the inflorescences were immersed in the Agrobacterium bacterial solution for 5 - 10 min, gently shaken, and then the plants were placed upright and covered with plastic wrap to maintain moisture for 24 h.

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

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

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

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

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

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

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

[0055] The growth conditions of different transgenic Arabidopsis thaliana lines after being cultured under green light (530 nm, 30 μW / mm 2 ) are as follows Figure 3 shown. The results show that under the green light growth conditions, the transgenic Arabidopsis thaliana expressing KCR1(TAGA) was significantly affected. Compared with the control group, after growing under green light for 4 days, the transgenic Arabidopsis thaliana expressing KCR1(TAGA) showed yellowing of the leaves.

[0056] Furthermore, the change in the cell membrane potential of the transgenic Arabidopsis thaliana expressing KCR1(GATA) was tested under the condition of green light (532 nm, 150 μw / mm 2 ) irradiation for 5 s, and the results are as follows Figure 4 shown. It can be seen that light stimulation activates KCR1-mediated potassium ion efflux, resulting in the inhibition of plant growth and development, indicating that the functional expression of KCR1(TAGA) affects the normal growth of plants.

[0057] (7) The effect of the 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 shown. The results show that in the roots, KCR1(TAGA) can be localized and expressed on the root cell membrane.

[0059] The root growth conditions of different transgenic Arabidopsis thaliana lines under white light and red light (650 nm, 20 μW / mm 2 ) irradiation are respectively as follows Figure 6 and Figure 7 shown, and the statistical results of the main root length of Arabidopsis thaliana are as follows Figure 8 shown (n = 5, p < 0.0001). The results show that under the white light culture conditions, compared with the control group, the root growth of the transgenic Arabidopsis thaliana stably expressing KCR1(TAGA) was also significantly inhibited; but under red light conditions, the root growth of the transgenic Arabidopsis thaliana expressing KCR1(TAGA) was not affected; generally speaking, the functional expression of KCR1(TAGA) inhibited plant root growth.

[0060] Experiment Example 3: Cultivation of KCR1(TAGA) Transgenic Tobacco and Identification of Growth Phenotype

[0061] (1) Preparation of Transgenic Tobacco by Leaf Disk Method

[0062] Select vigorous and healthy tobacco plants and take their young leaves. After rinsing them clean with tap water, soak the leaves in 75% alcohol for 60 s for surface sterilization. Then, rinse them 3 times with sterile water and soak the leaves in 0.2% mercuric chloride solution for 10 min to kill the surface bacteria and fungi. Finally, rinse them 5 times with sterile water until there is no residual mercuric chloride in the rinse solution. Place the disinfected leaves in a sterile petri dish and use a sterile punch to punch the leaves into leaf disks with a diameter of about 8 mm. Try to select the upper-middle part of the leaves and avoid using the tissues near the leaf veins to ensure the cell activity and regeneration ability of the leaf disks. Subsequently, place the prepared leaf disks into the Agrobacterium liquid expressing KCR1(TAGA), soak for 5 min to allow the leaf disks to fully contact the Agrobacterium. Then, use sterile filter paper to blot dry the excess bacterial liquid on the surface of the leaf disks, and inoculate the leaf disks into a petri dish containing solid MS medium supplemented with 2 mg / L 6-benzylaminopurine (6-BA) and 0.2 mg / L naphthaleneacetic acid (NAA) to promote the division and differentiation of leaf cells. Seal the petri dish and place it in an incubator at 25 °C, with a light intensity of 1000 - 1500 lx and a light time of 16 h / day for 2 days to allow the Agrobacterium to transfer the target gene into the tobacco leaf cells.

[0063] (2) Rooting Culture

[0064] When the adventitious buds grow to 2 - 3 cm in height, cut them off and transfer them 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 at the base of the adventitious buds to form complete transgenic tobacco plants.

[0065] (3) Detection and Identification of Transgenic Plants

[0066] After the transgenic tobacco plants grow to a certain size, determine the positive plants through fluorescence observation and detection of the plant surface membrane potential. The results are as Figure 9 shown, where Figure A shows the expression localization of KCR1(TAGA) on the tobacco leaf epidermal cells, and Figure B shows the change of the surface membrane potential of KCR1(TAGA) transgenic tobacco under the stimulation of green light (532 nm, 180 μW / mm 2 ).

[0067] (4) Phenotypic Changes of Transgenic Tobacco with Stable Expression of KCR1(TAGA)

[0068] The transgenic tobacco expressing YFP was used as a control. The transgenic tobacco stably expressing KCR1(TAGA) was cultured under white light conditions for 72 h, and the phenotypic changes of the plants were observed. The results are as Figure 10 shown. The results showed that, compared with the control group, the transgenic tobacco stably expressing KCR1(TAGA) showed phenotypic changes such as yellowing of the leaves, indicating that the functional expression of KCR1(TAGA) could also affect the growth of tobacco plants.

[0069] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A KCR1 mutant applicable to the regulation of plant potassium ion homeostasis, characterized in that, Based on the light-gated potassium ion channel protein KCR1, it has at least the T136A and G140A mutations.

2. The KCR1 mutant applicable to the regulation of plant potassium ion homeostasis as described in claim 1, characterized in that, The amino acid sequence of the light-gated potassium ion channel protein KCR1 is shown in SEQ ID NO.

1.

3. The KCR1 mutant applicable to the regulation of plant potassium ion homeostasis according to claim 1, characterized in that, The amino acid sequence of the KCR1 mutant is shown in SEQ ID NO.

2.

4. A gene encoding the KCR1 mutant suitable for regulating potassium ion homeostasis in plants as claimed in claim 1.

5. The gene according to claim 4, wherein The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

6. Use of the KCR1 mutant as claimed in claim 1 or the gene as claimed in claim 4 in plant cultivation.

7. Use of the KCR1 mutant as claimed in claim 1 or the gene as claimed in claim 4 in cultivating new plant varieties.

8. Use of the KCR1 mutant as claimed in claim 1 or the gene as claimed in claim 4 in preparing plant cultivation products.

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