Garlic stomatal opening potassium channel AsKAT1 molecule and its application

Abstract

The present application discloses a garlic stomatal opening potassium ion channel AsKAT1 molecule and its application. The nucleotide sequence and amino acid sequence of the AsKAT1 molecule are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The gene is mainly expressed in stomatal guard cells. The present application constructs an animal expression vector containing the above gene AsKAT1, proves that the gene protein has the transport activity of mediating the inward absorption of potassium ions, and finds its regulatory mechanism: the potassium absorption activity of AsKAT1 is promoted by extracellular high potassium and low pH, and then the mechanism is applied to the stomatal regulation of garlic leaves in the field, which effectively promotes the opening of stomatal pores, thereby enhancing the photosynthetic efficiency and increasing the garlic yield.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a garlic stomatal opening potassium ion channel AsKAT1 molecule and application thereof. Background Art

[0002] Potassium is a key mineral nutrient element necessary for crop growth and development. Potassium ions are also the most abundant inorganic cations in crops, accounting for 2-10% of the dry weight of plants. The physiological functions of potassium ions are mainly reflected in regulating enzyme activity, regulating cell osmotic potential, affecting protein synthesis, maintaining intracellular charge balance and cell membrane potential, enhancing plant photosynthesis and plant stress resistance (Ragel P, Raddatz N, Leidi EO, Quintero FJ, Pardo JM., 2019. Regulation of K + Nutrition in Plants. Frontiers in Plant Science 10, 281.). Potassium ion channels mediate the absorption and transport of potassium in plants, among which Shaker potassium ion channels are very important for plant potassium ion absorption and transport. According to protein sequence, gene structure and functional characteristics, Shaker potassium channels can be divided into five groups: Group I (AKT1 potassium channels), mainly found in roots; Group II (KAT1 potassium channels), mainly found in leaves, especially guard cells; Group III (weak inward rectifier potassium channels), mainly found in phloem; Group IV does not generate current when it exists alone; Group V mediates plant outward current (Pilot G, Pratelli R, Gaymard F, Meyer Y, Sentenac H., 2003. Five-Group Distribution of the Shaker-like K + Channel Family in HigherPlants.Journal of Molecular Evolution 56,418-434.).

[0003] Stomata are pore-like structures surrounded by a pair of specialized epidermal cells (guard cells). Stomata are the gateways for plants to exchange gases. Plants absorb CO2 and release O2 through stomata to perform photosynthesis. KAT1 potassium channels are mainly expressed in guard cells of leaves, and increase K in guard cells by mediating the absorption of potassium ions. +The concentration increases the osmotic pressure of guard cells, causing the stomata to open. Previous studies in rice have shown that the opening efficiency of stomatal pores is closely related to the absorption activity of stomatal opening potassium ion channels (Chinese patent ZL201810959909.8). The degree of stomatal opening affects the photosynthesis of plants. Efficiently opened stomata are beneficial to improving the photosynthetic efficiency and nutrient utilization efficiency of plants, which is beneficial to the accumulation of biomass and ultimately achieving an increase in crop production capacity. At present, there have been no reports on potassium ion channels related to the opening of garlic leaves. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide a garlic stomatal opening potassium ion absorption channel gene AsKAT1. The present application firstly discovered through electrophysiological technology that extracellular high potassium and low pH regulation can improve the potassium absorption activity of the gene protein, and then applied this mechanism to the stomatal regulation of garlic leaves in the field (spraying high potassium and low pH solution on the leaves), promoting stomatal opening, enhancing photosynthetic efficiency, and increasing garlic yield.

[0005] In order to achieve the above invention objectives, this application adopts the following technical solutions:

[0006] First, the present application provides a garlic stomatal opening potassium ion channel AsKAT1 molecule, whose nucleotide sequence is shown in SEQ IN NO.3, and whose amino acid sequence is shown in SEQ IN NO.4; the AsKAT1 molecule is localized in the guard cells of garlic leaves.

[0007] Secondly, the present application provides the use of a substance that increases the activity of the potassium ion channel AsKAT1 molecule protein shown in the nucleotide sequence of SEQ IN NO.3 in increasing the stomatal conductance, photosynthetic rate, biomass and yield of garlic. The above application refers to spraying a potassium ion solution on garlic leaves during the garlic bolting period, spraying once every 6 days, for a total of 5 times, and each spraying amount is 5 ml per garlic plant to increase the biomass and yield of garlic; in the above potassium ion solution, K + The concentration is 25-50 mM, pH 5.5; the potassium ion solution is preferably a potassium chloride solution.

[0008] Third, the present application also provides an expression vector containing the garlic stomatal opening potassium ion absorption channel gene AsKAT1 having a nucleotide sequence as shown in SEQ ID NO.3; such as the recombinant plasmid AsKAT1-pT7TS (based on the pT7TS vector).

[0009] In one embodiment of the present application, it was found by electrophysiological techniques that the potassium absorption capacity of AsKAT1 was promoted by high extracellular potassium and low pH, whether at a membrane potential close to the resting membrane potential of plant cells or at a relatively negative membrane potential. In another embodiment of the present application, the lower epidermis of torn garlic leaves was immersed in a high potassium (50 mM K+ ) or low potassium (5 mM K + ), high pH (pH7) or low pH (pH5.5) solution, and the results showed that the stomatal opening of garlic was significantly promoted under high potassium and low pH conditions. Based on the regulatory mechanism that the potassium absorption activity of the stomatal opening potassium ion absorption channel of the garlic leaf epidermis is strongly promoted by the external high potassium and low pH, this application first sprayed a high potassium and low pH solution (25mM K + , pH 5.5), increased the stomatal conductance of garlic leaves, promoted the efficient opening of garlic leaf stomata, enhanced the photosynthetic efficiency and thus increased the biomass and yield of garlic.

[0010] This application is the first to discover the AsKAT1 gene resource in garlic and identify its function. The garlic stomatal opening potassium ion absorption channel gene AsKAT1 provided in this application is mainly located in guard cells. Its regulatory mechanism is applied to stomatal regulation of garlic leaves in the field, effectively promoting stomatal opening, thereby enhancing photosynthetic efficiency and increasing garlic yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The results of cloning and detection of garlic AsKAT1 gene;

[0012] M1 is a 250 bp DNA ladder marker, and lane 1 is AsKAT1 (PCR result of SpeI / BglII).

[0013] Figure 2 Schematic diagram of the expression pattern and precise tissue localization results of AsKAT1.

[0014] Among them, A is the expression pattern of AsKAT1, and B is the precise tissue localization result of AsKAT1.

[0015] Figure 3 AsKAT1 gene K + Schematic diagram of absorption characteristic results;

[0016] Where A is the extracellular K + Schematic diagram of the effect of concentration on AsKAT1 potassium uptake activity, B is the extracellular K + A is a statistical diagram showing the effect of concentration on the potassium uptake activity of AsKAT1, C is a schematic diagram showing the effect of extracellular pH on the potassium uptake activity of AsKAT1, and D is a statistical diagram showing the effect of extracellular pH on the potassium uptake activity of AsKAT1.

[0017] Figure 4 For different K + Schematic diagram of garlic stomatal aperture results under concentration and pH;

[0018] Among them, A is different K + Typical microscope pictures of garlic stomatal opening under different concentrations, B is different K + A is a statistical graph of garlic stomatal opening at different concentrations, C is a typical microscope picture of garlic stomatal opening at different pH values, and D is a statistical graph of garlic stomatal opening at different pH values.

[0019] Figure 5 This is a schematic diagram of the results of improving garlic photosynthetic performance and yield by field stomatal regulation (spraying high potassium and low pH solution on leaves);

[0020] Among them, A is a schematic diagram of the results of garlic stomatal conductance under stomatal regulation, B is a schematic diagram of the results of garlic photosynthetic rate under stomatal regulation, C is a schematic diagram of the results of garlic plant biomass under stomatal regulation, and D is a schematic diagram of the results of garlic yield under stomatal regulation;

[0021] Figure 6 These are photos of garlic in the stomatal regulation group and the control group in different planting areas;

[0022] Among them, A is a photo of garlic in the stomatal regulation group (JY) and the control group (CK) in Juye County, B is a photo of garlic in the stomatal regulation group (JX) and the control group (CK) in Jinxiang County, and C is a photo of garlic in the stomatal regulation group (YT) and the control group (CK) in Yutai County.

[0023] Figure 7 Schematic diagram of stomatal regulation effect in different plots;

[0024] Among them, A is a schematic diagram of garlic yield results under stomatal regulation, B is a schematic diagram of garlic plant biomass results under stomatal regulation, C is a schematic diagram of garlic bulb fresh weight results under stomatal regulation, and D is a schematic diagram of garlic bulb horizontal diameter results under stomatal regulation. DETAILED DESCRIPTION

[0025] The garlic variety used in the following examples is Jinsuan No. 3, which was donated by Jinxiang Garlic Research Institute and its approval number is Lunongshen No. 2010063.

[0026] Unless otherwise specified, the raw materials and reagents involved in the following examples were purchased from commercial channels.

[0027] The culture medium involved in the embodiment:

[0028] Improved garlic nutrient solution: 1.25mM NH4NO3, 0.3mM KH2PO4, 0.35mM K2SO4, 1mM CaCl2·2H2O, 1mM MgSO4·7H2O, 0.5mM Na2SiO3, 20μM NaFeEDTA, 20μM H3BO3, 9μM MnCl2·4H2O, 0.32μM CuSO4·5H2O, 0.77μM ZnSO4·7H2O, 0.39μM Na2MoO4·2H2O, pH 6.5.

[0029] Example 1 Cloning and bioinformatics analysis of garlic AsKAT1 gene

[0030] 1. Cloning of the Garlic AsKAT1 Gene

[0031] In this example, a polysaccharide and polyphenol plant total RNA extraction kit (Beijing Tiangen) was used to extract the total RNA of garlic Jinsuan No. 3 grown in hydroponics for 10 days and used it as a template. 1 μg of RNA sample was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novizan) to obtain the reverse transcription product cDNA. According to the results of the laboratory's previous sequencing of the garlic transcriptome, the AsKAT1 gene with a complete coding reading frame was selected in the garlic transcriptome database, and the upstream primer F1 (SEQ ID NO.1) and the downstream primer R1 (SEQ ID NO.2) were designed; high-fidelity enzymes were used for PCR amplification, the PCR reaction system was 50 μL, 2×Phanta Max Master Mix was 25 μL, cDNA was 1 μL, and the upstream and downstream primers (both at a concentration of 10 μmol / L) were 1 μL each, and ddH2O was added to make up to 50 μL.

[0032] PCR reaction program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 2 min, 30 cycles; extension at 72°C for 10 min; storage at 4°C.

[0033] 1% agarose gel electrophoresis detection, a single product of about 1.8 kb was obtained, such as Figure 1The target fragment was recovered by using FastPureGel DNA Extraction Mini Kit (Nanjing Novozyme), and connected to the pT7TS vector to obtain the AsKAT1-pT7TS plasmid. Nanjing Qingke Biotechnology Co., Ltd. was commissioned to sequence and obtain the cDNA sequence of garlic AsKAT1. Its cDNA nucleotide sequence (1869 bp in size, the last three TAAs in the sequence list are terminators) and its encoded amino acid sequence (622 aa in size) are shown in SEQ IN NO.3 and SEQ IN NO.4, respectively.

[0034] Example 2 Expression pattern and precise tissue localization of AsKAT1

[0035] 1. Expression pattern of garlic AsKAT1 gene

[0036] After the garlic seedlings germinated in clear water were transferred to the improved garlic nutrient solution for 7 days, the garlic roots, pseudostems, upper epidermis of leaves, and lower epidermis of leaves were sampled, and the garlic RNA from each part was extracted, and the reverse transcription was performed using the HiScript III RTSuperMix for qPCR (+gDNA wiper) kit (Nanjing Novozyme) to obtain the reverse transcription product cDNA. Using this cDNA as a template, real-time quantitative RT-qPCR analysis was performed with specific primers F2 (SEQ ID NO.5) and R2 (SEQ ID NO.6). EF-1α was used as an internal reference gene, and the primers were F3 (SEQ ID NO.7) and R3 (SEQ ID NO.8).

[0037] The real-time quantitative PCR reaction system was 20 μL: 2×ChamQ Universal SYBR qPCR Master Mix was 10 μL, upstream and downstream primers (both at a concentration of 10 μmol / L) were 0.4 μL each, and cDNA was 1 μL, and ddH2O was added to make up to 20 μL.

[0038] Real-time quantitative PCR reaction program: 95°C pre-denaturation for 30 sec; 95°C denaturation for 15 sec, 60°C annealing for 30 sec, 72°C extension for 30 sec, 40 cycles; 95°C treatment for 15 sec, 60°C treatment for 60 sec, 95°C treatment for 15 sec to obtain the melting curve.

[0039] The results are as follows Figure 2 As shown in Figure A, the AsKAT1 gene is mainly expressed in the lower epidermis of garlic leaves and is almost undetectable in the roots, indicating that the AsKAT1 gene has obvious tissue specificity in garlic plants.

[0040] 2. Precise tissue localization of the garlic AsKAT1 gene

[0041] In situ PCR (refer to the method disclosed in Athman A., Tanz SK, Conn VM, Jordans C., Mayo G.M., Ng WW, et al. 2014. Protocol: a fast and simple in situ PCR method for localizing gene expression in plant tissue. Plant Methods 10, 29.) was used to determine the precise tissue localization characteristics of AsKAT1 in the lower epidermis of garlic leaves, and specific primers F4 (SEQ ID NO.9) and R4 (SEQ ID NO.10) were designed. Under the same primer conditions, no reverse transcription was performed as a negative control.

[0042] The results are as follows Figure 2 As shown in B, AsKAT1 is mainly localized in guard cells, suggesting that it may play a role in regulating stomatal opening.

[0043] Example 3 AsKAT1 gene K + Absorption characteristics analysis

[0044] In this example, the CDS of the AsKAT1 gene was cloned into the oocyte expression vector pT7TS vector (Invitrogen). The cRNA was synthesized using the mMESSAGE mMACHINE T7 transcription kit (Thermo Fisher Scientific), and 30 ng of cRNA was injected into the mature oocytes of Xenopus laevis (Shanghai Stem Cell Institute). The oocytes injected with cRNA were incubated at 16°C for 24-48 h in ND96 solution (mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES-NaOH, pH 7.4), and then the current was measured using a two-electrode voltage clamp. The clamping voltage was -40 mV, the step voltage was 10 mV, the applied voltage range was from -160 mV to +20 mV, and the stimulation interval was 3 s.

[0045] In this embodiment, the method for introducing cRNA into frog eggs is a conventional technique in the art, such as the literature "Véry AA, Bosseux C, Gaymard F, Sentenac H, Thibaud J B., 1994. Level of expression in Xenopus oocytes affects some characteristics of a plant inward-rectifying voltage-gated K+ channel. Pfliigers Arch 428, 422-424".

[0046] 1. AsKAT1 potassium uptake activity is regulated by extracellular K + Concentration Control

[0047] The oocytes were stabilized by clamping and treated with solutions of different potassium concentrations (X = 5, 10, 50, 100 mM K + ) for perfusion, the perfusion solution composition: X mM KCl, (100-X) mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4.

[0048] The experimental results are as follows Figure 3 As shown in Figure A, whether under -90 mV (close to the resting membrane potential of plant cells) or -140 mV (hyperpolarized negative membrane potential), the potassium uptake ability of AsKAT1 is promoted by extracellular potassium ions. Figure 3 (B) also shows that with the increase of external potassium ion concentration, the transport activity of AsKAT1 increases, and its K + Absorption capacity is significantly increased.

[0049] 2. AsKAT1 potassium uptake activity is regulated by extracellular pH

[0050] The clamped and stabilized oocytes were perfused with solutions of different pH (pH=5.4, 6.4, 7.4). The components of the perfusion solution were: 50 mM KCl, 50 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.0 mM HEPES (pH 7.4) or 5.0 mM MES (pH 5.4 or 6.4).

[0051] The experimental results are as follows Figure 3 As shown in Figure C, at different membrane potentials (-90 mV and -140 mV), the potassium uptake capacity of AsKAT1 was promoted by extracellular acidification. Figure 3 (D) also shows that with the decrease of extracellular pH, the transport activity of AsKAT1 increases, and its K + Absorption capacity is significantly increased.

[0052] Example 4 Effect of different potassium concentrations and pH on garlic stomatal aperture

[0053] After culturing garlic with uniform germination and growth in the modified garlic nutrient solution for 14 days, the lower epidermis was torn off with tweezers and divided into two groups. One group was immersed in high pH (5mM K + , pH 7) or low pH (5 mM K +, pH 5.5) solution; one group was immersed in low potassium (5mM K + , pH 7) or high potassium (50 mM K + The above solutions were prepared from KCl, 10 mM MES was added, and the pH was adjusted to 7 or 5.5 using NaOH. The osmotic pressure of each solution was maintained at 200 mosmol kg by adding sorbitol. -1 The stomata were opened after 1 h of light induction. The photos were taken with a Nikon 80i microscope, and the length and width of the stomata were measured with image J software to calculate the stomatal opening (stomatal opening = stomatal width / stomatal length).

[0054] The experimental results are as follows Figure 4 As shown, where A is different K + Microscope photos of garlic stomatal opening under different concentrations, B is different K + The statistical graph of garlic stomatal opening under different concentrations shows that, under the same pH condition, the increase of K+ concentration from 5mM to 50mM significantly increased the garlic stomatal opening, with an average increase of 69%, indicating that the opening of garlic stomata depends on the concentration of potassium ions in guard cells. C is a microscope photo of garlic stomatal opening under different pH conditions, and D is the statistical result of garlic stomatal opening under different pH conditions. It can be seen that under the physiological K + At the concentration (5mM), the decrease in pH (from 7 to 5.5) significantly increased the garlic stomatal aperture, with an average increase of 38%, indicating that acidification is conducive to the opening of garlic stomata. These changes are consistent with the electrophysiological results of the AsKAT1 gene, indicating that the increased AsKAT1 transport activity under high potassium and low pH is conducive to the entry of potassium ions into guard cells, thereby promoting the opening of garlic plants' stomata.

[0055] Example 5 Photosynthetic performance and agronomic indicators of garlic under stomatal regulation (spraying high potassium and low pH solution on leaves)

[0056] This example was conducted in a garlic field in Kangqiao Village, Jinxiang County, Shandong Province in 2021. The experimental group sprayed 25mM potassium ion solution (pH 5.5) on garlic leaves from the bolting stage, once every 6 days, for a total of 5 times, with each spraying amount of about 5 ml per garlic plant. The same spraying amount was used as a control. The potassium ion solution used for spraying was prepared with potassium chloride to 25mM K + The solution was adjusted to a pH of about 5.5 using vinegar.

[0057] The sowing, water and fertilizer management of garlic were carried out in accordance with the local commonly used technical regulations for garlic planting and management (T / JXDSZZ002-2020) (Hong Huayang, Wei Tianqi, Zhou Hongmei, et al. 2024. Organic matter compensation scheme and preliminary mechanism for repairing the decline of soil productivity in continuous garlic cropping. Acta Pedologica Sinica, 1-13.).

[0058] During the bulb expansion period of garlic, the stomatal conductance and net photosynthetic rate (Pn) of the third leaf of garlic were measured using a portable photosynthetic meter (LI-6400XT) from 9:00 to 11:00 a.m. The working conditions of the photosynthetic meter were: light intensity 1000mmol·m -2 s -1 , CO2 concentration 400mmol·mol –1 , flow rate 500 μmol s -1 .

[0059] The results showed that compared with the control sprayed with water, the stomatal conductance (e.g. Figure 5 A in the middle) and photosynthetic rate (as shown in Figure 5 (B) significantly increased by 41% and 22% respectively.

[0060] The results of field yield measurement showed that compared with the water spraying control, the aboveground biomass of garlic plants sprayed with high potassium and low pH solution (such as Figure 5 C) and garlic yield (as shown in Figure 5 (D) increased significantly, by 19% and 16% respectively.

[0061] Example 6 Effects of stomatal regulation measures in different plots of land

[0062] In order to verify the yield-increasing effect of stomatal regulation measures in different locations, this embodiment implemented stomatal regulation measures in typical fields in emerging garlic planting areas (Xuzhou City and Juye County of Heze City) and traditional garlic planting areas (Jinxiang County, Yutai County of Jining City and Lanling County of Linyi City) in 2024. The experimental scheme is the same as that of Example 5.

[0063] The experimental results are as follows Figure 6 , Figure 7 As shown, BYJ represents the garlic planting area of ​​Bayi Town, Xuzhou City, JY represents the garlic planting area of ​​Juye County, Heze City, SN represents the garlic planting area of ​​Suining County, Xuzhou City, JX represents the garlic planting area of ​​Jinxiang County, Jining City, LL represents the garlic planting area of ​​Lanling County, Linyi City, and YT represents the garlic planting area of ​​Yutai County, Jining City. The garlic of the stomatal regulation group and the control group harvested in the embodiment are as shown in FIG. Figure 6 shown.

[0064] After statistics, it was found that compared with the control group (CK), the stomatal regulation group sprayed with 25mM KCl (pH5.5) had a significant yield increase effect in different plots, with the yield increase rate ranging from 11 to 29% (such as Figure 7 At the same time, garlic biomass also increased significantly by 13-37% (as shown in Figure 7Further determination of the fresh weight and transverse diameter of a single garlic bulb revealed that the fresh weight of garlic bulbs regulated by stomata (as shown in Figure 2B) Figure 7 C) and transverse diameter (as shown in Figure 7 D) showed different degrees of increase compared with the control, with increases of 13-25% and 7-12% respectively.

Claims

1. A garlic stomatal opening potassium ion channel AsKAT1 gene, the nucleotide sequence of which is encoded by the gene is shown in SEQ INNO.

3.

2. The garlic stomatal opening potassium ion channel AsKAT1 gene as claimed in claim 1, whose amino acid sequence is shown in SEQ INNO.

4.

3. An expression vector containing the garlic stomatal opening potassium ion channel AsKAT1 gene as claimed in claim 1.

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