Method for detecting the transport activity of a vacuole membrane sugar transporter

Abstract

The present application belongs to the technical field of sugar transporter activity detection, and particularly relates to a method for detecting the transport activity of vacuole membrane sugar transporter. The method comprises the following steps: constructing a plant expression vector for expressing the vacuole membrane sugar transporter, transforming the expression vector into the fruit of a melon at the swelling stage to obtain transgenic melon fruit; culturing the transgenic melon fruit until the vacuole membrane sugar transporter is expressed, and then obtaining protoplasts by enzymolysis; adding the protoplasts into a sugar fluorescent analogue culture solution, incubating for a period of time, and then detecting the fluorescence signal of the vacuoles in the protoplasts, and evaluating the transport activity of the vacuole membrane sugar transporter according to the fluorescence intensity. The method of the present application is simple to operate, high in efficiency, independent of isotopes, and capable of detecting the transport activity of both endogenous and exogenous vacuole sugar transporters, while maintaining the characteristics of the plant vacuole itself, reducing the interference of the background signal in the vacuole, and improving the accuracy and sensitivity of the analysis of the transport activity of the plant vacuole membrane sugar transporter.

Description

Technical Field

[0001] The present invention relates to the technical field of sugar transporter activity detection, in particular to a method for detecting the transport activity of tonoplast sugar transporter. Background Art

[0002] Vacuolar sugar transporters are a type of protein located on the vacuole membrane that transports sugars into and out of the vacuole. They are also an important type of sugar transporter in plants. In plants, vacuolar sugar transporters are involved in multiple processes of plant growth and development and play an important role in maintaining cellular osmotic balance. In some plant organs with high sugar accumulation, vacuolar sugar transporters play an important role in sugar accumulation. There are three main types of vacuolar sugar transporters found in plants. One is H-transporter, which transports sucrose or hexose back into the vacuole. + The antiporter subfamily TMT / TST; one is the H transporter that transports sucrose out of the vacuole + There is the symporting protein SUT4, and another sugar transport protein SWEET that is independent of energy and proton gradient.

[0003] Analysis of the sugar transport activity of vacuolar sugar transporters is crucial for understanding their function. Currently, methods for analyzing the sugar transport activity of vacuolar sugar transporters primarily include yeast heterologous expression systems, Xenopus oocyte heterologous expression systems, and voltage-clamp and patch-clamp techniques. Each of these techniques has limitations. For example, the use of yeast mutants and Xenopus oocytes is a non-plant heterologous expression system. In some cases, heterologous protein expression may lead to mistargeting, and the lack of protein modifications in heterologous systems may interfere with sugar uptake analysis. Voltage-clamp and patch-clamp techniques are particularly demanding in terms of instrumentation and operator skills. Furthermore, only some transporters experience a proton gradient during sugar transport, and even when a proton gradient is present, the current is relatively weak, making it difficult to detect an effective current. This hinders the widespread application of these techniques. Therefore, there is an urgent need for new, more convenient and user-friendly methods for detecting the transport activity of vacuolar sugar transporters that retain the specific modifications endogenous to plant cells. Summary of the Invention

[0004] To address the aforementioned issues in the prior art, the present invention provides a novel method for detecting the transport activity of vacuolar sugar transporters. This method utilizes protoplasts from expanding melon fruits, which have low endogenous vacuolar sugar transport activity, as endogenous or plant-derived expression hosts for the vacuolar sugar transporter to be detected. Sugar transport activity is then detected by feeding the melon fruit protoplasts overexpressing the vacuolar sugar transporter with a fluorescent sugar analog. This method is simple to operate, highly efficient, is isotope-independent, and can detect the transport activity of both endogenous and exogenous vacuolar sugar transporters. It also maintains the inherent properties of the plant vacuole, reducing interference from background signals within the vacuole and improving the accuracy and sensitivity of plant vacuolar sugar transporter activity analysis.

[0005] To achieve the above objectives, the present invention is specifically implemented through the following technical solutions:

[0006] The present invention provides a method for detecting the transport activity of a tonoplast sugar transporter, comprising the following steps:

[0007] S1. Constructing a plant expression vector expressing a tonoplast sugar transporter protein and transforming the resulting melon fruit into transgenic melon fruit at the expansion stage;

[0008] S2. culturing the transgenic melon fruit until the tonoplast sugar transporter is expressed, and then enzymatically hydrolyzing the pulp cells of the transgenic melon fruit to obtain pulp protoplasts;

[0009] S3. Adding the protoplasts to a sugar fluorescent analog culture medium and incubating for a period of time, washing the protoplasts after the incubation is completed, and then detecting the fluorescent signal in the vacuole of the protoplasts, and evaluating the transport activity of the vacuole sugar transporter according to the fluorescence intensity.

[0010] Furthermore, in step S1, the tonoplast sugar transporter is a TST / TMT, SUT or SWEET family protein, and the muskmelon is Tianbao, Emerald or Yangjiaomi.

[0011] Furthermore, in step S1, the tonoplast sugar transporter is TST2, and the melon is emerald.

[0012] Furthermore, in step S1, constructing a plant expression vector of the tonoplast sugar transporter comprises the following steps:

[0013] S11, obtaining the gene fragment of the vacuolar sugar transporter;

[0014] S12. Linearize the plant expression vector using a restriction endonuclease, construct the gene fragment into the plant expression vector by recombination, and obtain a plant expression vector expressing the tonoplast sugar transporter; wherein the plant expression vector is pH7LIC5.0 or pBWA(V)HS.

[0015] Furthermore, the plant expression vector also carries a fluorescent protein encoding gene, and the fluorescent protein is GFP, mcherry or RFP.

[0016] Furthermore, in step S1, the plant expression vector is transformed into the melon fruit in the expansion stage through Agrobacterium-mediated method.

[0017] Furthermore, in step S2, enzymatic hydrolysis of the pulp cells of the transgenic melon fruit includes the following steps: slicing the transgenic melon fruit, placing it in an enzymatic hydrolysis solution, vacuuming, and culturing on a shaker for 2-3 hours to obtain the protoplasts; wherein the enzymatic hydrolysis solution formula is: 0.6M mannitol, 10mM MES, 10mM CaCl2, 0.1% bovine serum albumin, 1.5% cellulase, 0.4% macerate and 0.005% neutral red.

[0018] Furthermore, in step S3, the sugar fluorescent analogue is Esculin (aescin, a sucrose fluorescent analogue), NBD-F (4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fructose fluorescent analogue) or 2-NBDG (2-(N-7-nitro-2,1,3-benzoxadiazole-4-amino)-2-deoxy-D-glucose, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, a glucose fluorescent analogue).

[0019] Furthermore, in step S3, the sugar fluorescent analog culture solution includes 0.6M mannitol, 10mM MES, 10mM CaCl2 and 1mM sugar fluorescent analog, and the protoplasts are added to the sugar fluorescent analog culture solution and incubated for 12-24h.

[0020] Furthermore, in step S3, the fluorescence intensity is obtained by counting the fluorescence signal at the vacuole center of the protoplast using ImageJ software.

[0021] Furthermore, in step S3, evaluating the transport activity of the tonoplast sugar transporter according to the fluorescence intensity includes the following steps: evaluating the transport activity of the tonoplast sugar transporter by comparing the fluorescence intensity difference between the tonoplast sugar transporter expression group and the control group through T test.

[0022] The advantages and positive effects of the present invention are:

[0023] 1. The present invention utilizes the protoplasts of melon fruits in the expansion stage with low endogenous tonoplast sugar transport activity as the heterologous expression host of the tonoplast sugar transport protein to be detected. The exogenous gene is easily expressed transiently and efficiently in the melon fruit. The sugar transport activity is then detected by feeding the melon fruit protoplasts that overexpress the tonoplast sugar transport protein with a sugar fluorescent analog. The method has the characteristics of simple operation, high efficiency, isotope independence, and the ability to detect the transport activity of both endogenous and exogenous tonoplast sugar transport proteins. Moreover, the tonoplast sugar transport activity of the protoplasts is low, which can reduce the interference of the background signal in the vacuole and improve the accuracy and sensitivity of the analysis of the transport activity of the tonoplast sugar transport protein. In addition, by co-transferring two different genes in one protoplast, the effect of interacting proteins on sugar transport activity can be explored.

[0024] 2. The melon fruit protoplasts of the present invention are rich in sources, and the protoplast feeding technology is simple to operate, which is conducive to repeated experiments, ensures the authenticity and reliability of the results, and reduces experimental costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is a microscopic image of protoplasts stained with neutral red after enzymatic hydrolysis of muskmelon fruit according to Example 1 of the present invention;

[0027] Figure 2 This is a fluorescence microscopic examination of protoplasts of melon fruits fed with Esculin, a fluorescent sugar analogue, according to Example 1 of the present invention. From left to right, protoplasts of melon fruits at the expansion stage and the maturity stage are shown.

[0028] Figure 3 This is a microscopic examination of the expression of green fluorescent protein in melon fruit after Agrobacterium-mediated transient transformation in Example 2 of the present invention;

[0029] Figure 4 This is a fluorescence microscopy image of protoplasts of melon fruits overexpressing green fluorescent protein fed with the sugar fluorescent analog Esculin in Example 2 of the present invention;

[0030] Figure 5 This is a fluorescence microscopic examination of protoplasts of melon fruits in the expansion stage that were fed with the sugar fluorescent analog Esculin in Example 3 of the present invention and overexpressed TST2;

[0031] Figure 6This is a statistical graph of the vacuolar fluorescence intensity of protoplasts of melon fruits overexpressing TST2 during the expansion stage fed with the sugar fluorescent analog Esculin in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. The embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Given the information contained herein, it will be readily apparent to those skilled in the art that various modifications may be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the processes, properties, or components defined herein, as these embodiments and other descriptions are intended only to illustrate specific aspects of the present invention. Indeed, various modifications to the embodiments of the present invention that are apparent to those skilled in the art or related fields are intended to be within the scope of the appended claims.

[0034] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention to express amounts, percentages, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant digits and by conventional rounding methods.

[0035] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art. The meanings of the terms "comprise," "include," "contain," "have," and similar words are non-restrictive, and other steps and other ingredients that do not affect the results can be added. The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below.

[0037] The vacuole is the primary organelle for nutrient storage and metabolism in plant cells, and vacuolar membrane transporters play a crucial role in nutrient transport in and out of the vacuole. Detecting the activity of vacuolar-localized sugar transporters faces the following challenges: 1. In heterologous expression systems distantly related to plants, such as yeast or Xenopus oocytes, it is often difficult to express active vacuolar membrane sugar transporters, or the proteins are not expressed at all, resulting in a lack of targeting signals. Even if biologically active sugar transporters are expressed through codon optimization, their localization to the vacuolar membrane within the cell rather than on the outer cell membrane makes it difficult to detect their transport activity using existing techniques such as growth complementation, radiolabeled substrates, voltage clamp, patch clamp, and ion flux measurements. 2. In closely related model plant expression systems, such as tobacco, the vacuolar membrane itself has high sugar transport activity, which means that detecting the target protein's transport signal requires overcoming background signal interference, making detection significantly more difficult.

[0038] Based on this, an embodiment of the present invention provides a method for detecting the transport activity of a tonoplast sugar transporter, comprising the following steps:

[0039] S1. Constructing a plant expression vector expressing a tonoplast sugar transporter protein and transforming the resulting melon fruit into transgenic melon fruit at the expansion stage;

[0040] S2. culturing the transgenic melon fruit until the tonoplast sugar transporter is expressed, and then enzymatically hydrolyzing the pulp cells of the transgenic melon fruit to obtain pulp protoplasts;

[0041] S3. Adding the protoplasts to a sugar fluorescent analog culture medium and incubating for a period of time, washing the protoplasts after the incubation is completed, and then detecting the fluorescent signal in the vacuole of the protoplasts, and evaluating the transport activity of the vacuole sugar transporter according to the fluorescence intensity.

[0042] During research on melon fruit protoplasts, the present invention discovered that the endogenous tonoplast sucrose transport activity of melon fruit protoplasts in the expansion phase is low, which provides a fundamental guarantee for using these protoplasts to identify sugar transporter activity and greatly reduces background signal interference. Furthermore, melon fruit in the expansion phase is easily accessible for exogenous gene expression via Agrobacterium infection. Therefore, the present invention utilizes melon fruit protoplasts in the expansion phase as heterologous expression hosts for the tonoplast sugar transporter to be detected. First, a plant expression vector expressing the tonoplast sugar transporter is transformed into the melon fruit using transient transformation technology. Transgenic melon fruit successfully transformed with the target protein encoding gene is then cultured to obtain transgenic melon fruit. Subsequently, conventional culture is performed to efficiently express the tonoplast sugar transporter, and protoplasts overexpressing the tonoplast sugar transporter are isolated. The plant sugar transporter expressed in the melon protoplasts generally does not undergo localization or modification changes, retaining its original biological activity. The aforementioned protoplasts are then used as transporters for sugar uptake and incubated with sugar fluorescent analogs for a period of time. The sugar fluorescent analogs pass through the cell membrane into the cytoplasm, are then transported into the vacuole via the tonoplast sugar transporter and accumulate in the vacuole, generating a fluorescent signal under excitation by excitation light. Since the transport amount of the fluorescent substance can be calculated by measuring the fluorescence intensity, the transport process and sugar uptake activity of the tonoplast sugar transporter can be tracked and quantified by detecting changes in the fluorescence intensity of the vacuole, thereby evaluating the sugar transport capacity of the tonoplast sugar transporter to be tested.

[0043] The present invention detects sugar transport activity by feeding a sugar fluorescent analog to a melon fruit protoplast that overexpresses a tonoplast sugar transport protein. Exogenous genes are easily expressed transiently and efficiently in melon fruits, and the endogenous tonoplast sugar transport activity of the melon fruit protoplasts in the expansion stage is low, which can ensure that the fluorescent signal detected in the vacuole is specifically produced by the overexpressed tonoplast sugar transport protein, thereby improving the accuracy and sensitivity of the tonoplast sugar transport activity analysis. Moreover, the source of melon fruit protoplasts is abundant, and the protoplast feeding technology operation method is simple, which is conducive to repeated experiments, ensures the authenticity and reliability of the results, and reduces the experimental cost. In addition, the detection system of the present invention can realize the co-transfection of two different genes in one protoplast, which can be used to analyze the influence of interacting proteins on sugar transport activity.

[0044] In the present invention, sugar transport activity can be determined qualitatively and quantitatively. Qualitative determination is achieved by detecting fluorescence intensity with an instrument, while quantitative determination is achieved by calculating a standard curve between the concentration of sugar fluorescent analogs and fluorescence intensity. This is a conventional technique in the art and will not be described in detail here.

[0045] Instruments for detecting fluorescence signals include, but are not limited to, laser confocal microscopes.

[0046] Optionally, the tonoplast sugar transporter is a Tonoplast Sugar Transporter (TST, also called TMT), sucrose transporter (SUT) or sugars will eventually be exported transporter (SWEET) family protein.

[0047] In a specific embodiment, the tonoplast sugar transporter may be CmTST2, and its nucleotide coding sequence is shown in SEQ ID NO.1.

[0048] Sugar fluorescent analogs are selected according to the type of tonoplast sugar transporter to be detected. For example, when detecting the glucose transport activity of a sugar transporter, the glucose fluorescent analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) can be selected; when detecting fructose transport activity, the fructose fluorescent analog 4-fluoro-7-nitro-2,1,3-benzoxadiazole (4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F) can be selected; and when detecting sucrose transport activity, the sucrose fluorescent analog esculin can be selected.

[0049] Preferably, in step S1, constructing a plant expression vector of the tonoplast sugar transporter comprises the following steps:

[0050] S11, obtaining the gene fragment of the vacuolar sugar transporter;

[0051] S12. Linearize the plant expression vector using restriction endonucleases, construct the gene fragment into the plant expression vector by recombination, and obtain a plant expression vector expressing the tonoplast sugar transporter.

[0052] In step S11, the gene fragment can be obtained by whole gene synthesis or PCR amplification.

[0053] In step S12, the plant expression vector can be selected from pH7LIC5.0 or pBWA(V)HS.

[0054] In a specific embodiment, when the tonoplast sugar transporter is CmTST2 and the plant expression vector is pH7LIC5.0, the nucleotide sequences of the upstream and downstream primers for amplifying CmTST2 are shown in SEQ ID NO. 2-3.

[0055] In order to facilitate the screening of transgenic melon fruits, a fluorescent protein encoding gene is also inserted into the plant expression vector. The fluorescent protein is GFP, mcherry or RFP, which is connected to the C-terminus or N-terminus of the tonoplast sugar transporter and expressed together with the tonoplast sugar transporter. In this way, by detecting the fluorescent signal of the fluorescent protein, it can be quickly determined whether the tonoplast sugar transporter is efficiently expressed.

[0056] For example, the expression vector containing the fluorescent protein encoding gene GFP can be pH7LIC5.0-N-GFP or pBWA(V)HS-GFP, and the GFP protein is connected to the N-terminus of the inserted gene.

[0057] Plant expression vectors can be integrated into the melon fruit genome or retained as plasmids. Genetic transformation methods include biolistic methods, electroporation, microinjection, or Agrobacterium-mediated methods. These are well-known techniques in the art and will not be described in detail here.

[0058] In a preferred embodiment, the plant expression vector is transformed into the melon fruit at the expansion stage by Agrobacterium-mediated transformation. The specific operation includes: injecting the Agrobacterium solution containing the plant expression vector into the melon fruit by syringe, the injection volume of the solution is 0.5 mL, the needle insertion depth is 0.5 cm, and the concentration of the solution is OD 600 =0.4.

[0059] The Agrobacterium strain mentioned above is Agrobacterium EHA105 or GV3101.

[0060] After the plant expression vector is transformed into the melon fruit at the expansion stage, the tonoplast sugar transporter protein is expressed for 3-5 days.

[0061] Optionally, in step S2, enzymatically hydrolyzing the transgenic melon fruit to obtain protoplasts comprises the following steps: slicing the transgenic melon fruit, placing it in an enzymatic hydrolysis solution, evacuating the solution, and culturing it on a shaker for 2-3 hours to obtain the protoplasts; wherein the enzymatic hydrolysis solution comprises: 0.6M mannitol, 10mM 4-morpholineethanesulfonic acid (MES), 10mM CaCl2, 0.1% (wt) bovine serum albumin (BSA), 1.5% (wt) cellulase (Cellulose RS), 0.4% (wt) macerase (Macerozyme R10) and 0.005% (wt) neutral red.

[0062] Optionally, in step S3, the sugar fluorescent analog culture solution comprises 0.6 M mannitol, 10 mM MES, 10 mM CaCl2 and 1 mM sugar fluorescent analog, and the protoplasts are added to the sugar fluorescent analog culture solution and cultured for 12-24 h.

[0063] In step S3, the fluorescence intensity is obtained by counting the fluorescence signal at the vacuole center of the protoplast using ImageJ software. The sugar transport activity is assessed by comparing the difference in fluorescence intensity between the vacuole sugar transporter expression group and the control group using a T test. The control group is identical to the expression group except that the vacuole sugar transporter is not expressed. Fluorescence intensity is positively correlated with the transport activity of the vacuole sugar transporter. An increase in fluorescence intensity indicates that the vacuole sugar transporter is able to transport the added specific substrate and has high sugar transport activity.

[0064] The melon variety that can be used in the present invention is preferably thin-skinned melon, and can be specifically selected from Tianbao, Lubaoshi or Yangjiaomi.

[0065] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0066] The gene and primer sequences used in the following examples are shown in Table 1, where primer sequence F represents an upstream primer and R represents a downstream primer.

[0067] Table 1 Gene and primer sequences used in the examples of the present invention

[0068]

[0069]

[0070] Example 1 Melon fruit protoplast hydrolysis and sugar fluorescent analog feeding experiment

[0071] The developmental stages of melon fruit include three cycles: fruit setting, fruit expansion, and fruit ripening. The fruit expansion period, from the time the young fruit "sheds" until the fruit is basically set in size ("set"), is characterized by rapid fruit volume expansion and growth, with the entire plant's growth center stabilizing on the fruit. The ripening period, from the time the fruit sets in size until the fruit matures, is characterized by biochemical changes within the fruit, the conversion and accumulation of large amounts of sugar, the crispness or softening of the flesh, the emission of different aromas, and distinct ripening characteristics. In this example, Emerald Melon fruits in the ripening and expansion stages were used as raw materials for obtaining protoplasts.

[0072] The sampled fruit was sliced ​​into 0.5-1.0 mm slices to obtain fruit slices to be separated. The fruit slices were placed in an enzymatic hydrolysis solution, vacuumed for 15 minutes, and shaken on a shaker for 2-3 hours, during which the enzymatic hydrolysis process was observed under a microscope. The enzymatic hydrolysis solution contained: 0.6 M mannitol, 10 mM 4-morpholineethanesulfonic acid (MES), 10 mM CaCl2, 0.1% (wt) bovine serum albumin (BSA), 1.5% (wt) cellulase (Cellulose RS), 0.4% (wt) macerase (Macerozyme R10), and 0.005% (wt) neutral red. After enzymatic hydrolysis, the solution was filtered through a 100 μm filter, centrifuged at 50 × g, and washed 2-3 times with washing buffer (comprising 0.6 M mannitol, 10 mM MES, and 10 mM CaCl2). Protoplast activity was confirmed under a microscope.

[0073] Figure 1 The figure shows a microscopic examination of protoplasts of melon fruit after enzymatic hydrolysis and neutral red staining. A large number of protoplasts are visible, and the protoplasts are stained red, indicating that a large number of active melon fruit protoplasts can be obtained in this example.

[0074] Wash and resuspend the protoplasts in washing buffer and add 100mM esculin stock solution to a final esculin concentration of 1mM. Incubate in a 23°C incubator for 12 hours. Wash the protoplasts twice with washing buffer to remove the feed solution. After the protoplasts settle to the bottom, aspirate them and place them in a glass-bottomed dish. Observe and photograph them under a laser confocal microscope. Excitation for esculin is 405nm, and absorption is set to 410-450nm. Focus the microscope on the cell equator.

[0075] Figure 2 The fluorescence distribution of protoplasts of melon fruits in the swelling and ripening stages 12 hours after being fed with Esculin is shown. It can be seen that Esculin fluorescence can be observed in the cytoplasm of fruits in both stages; there is basically no Esculin fluorescence signal in the vacuole of protoplasts of melon fruits in the swelling stage, while there is obvious Esculin fluorescence in the vacuole of protoplasts of melon fruits in the ripening stage, indicating that during the swelling stage of melon fruits, sucrose can be transported to the pulp cytoplasm but not to the vacuole, indicating that the protoplasts of melon fruits in the swelling stage have higher cell membrane sucrose transport activity but lower tonoplast sucrose transport activity, which is a good material for the determination of tonoplast sucrose transport activity and suitable for the identification of the transport activity of tonoplast sugar transport proteins.

[0076] Example 2 Reliability Verification of Transient Expression in Muskmelon Fruit Protoplasts and Sugar Fluorescent Analog Feeding Technology

[0077] The plant binary vector pH7LIC5.0-N-GFP with GFP tag was transformed into Agrobacterium, and the melon fruit at the swelling stage was infected with Agrobacterium. The above vector was transformed into Agrobacterium (strain GV3101 or EH105) by freeze-thaw method, and single clones were selected for colony PCR verification and shaken to OD 600 After centrifugation at an OD of 0.5-0.8, the supernatant was removed and the bacteria were resuspended in infection solution (including 10 mM MES, 10 mM MgCl2, 200 μM acetosyringone (AS)) to make the concentration of Agrobacterium tumefaciens to be OD 600 = 0.4. After 1-2 hours of standing, the injection was carried out on Emerald melon fruits in the expansion stage. After 3 days of standing at room temperature, the fruits were cut open and samples were taken for microscopic examination to determine the fluorescence intensity.

[0078] Figure 3 The microscopic images of green fluorescent protein (GFP) expression after transient transformation of melon fruit in the expansion stage by Agrobacterium are shown. It can be found that a large amount of transformed fruit pulp with GFP fluorescence can be clearly observed for protoplast hydrolysis 3 days after overexpression, and the reproducibility is good.

[0079] Enzymatic feeding: The fluorescent pulp was enzymatically hydrolyzed and washed according to the method of Example 1; Esculin was prepared into a 100mM mother solution and added to the culture medium with protoplasts at a volume ratio of 1:100 (final concentration is 1mM), and the culture medium was gently shaken to mix the Esculin evenly. The protoplasts were placed in a 23°C incubator and cultured for 12 hours. After that, the supernatant of the protoplast solution was aspirated and washed 3-4 times with washing buffer. Finally, the protoplasts were aspirated and placed on a glass-bottomed dish and photographed using a laser confocal microscope. The microscope was focused on the equatorial plane of the cell, and the location of the gene could be clearly seen. At least 30 protoplasts were photographed for each treatment.

[0080] Figure 4 The fluorescence distribution of GFP-overexpressing melon fruit protoplasts fed with Esculin for 12 hours after expansion is shown. It can be seen that there is no obvious Esculin fluorescence signal in the vacuoles of the fruit protoplasts after transient GFP transfection, indicating that Esculin has cytoplasmic membrane sugar transport activity but lacks tonoplast sugar transport activity.

[0081] Example 3 Identification of sucrose transport activity of CmTST2 using melon fruit protoplasts

[0082] Construction of a fluorescently tagged CmTST2 overexpression vector: Using melon (Cucumis melo) fruit cDNA as a template, RT-PCR amplification was performed using CmTST2-F / R primers, and the target product was recovered. The CmTST2 coding sequence is shown in SEQ ID NO. 1. The plant expression vector pH7LIC5.0-N-GFP was linearized and recovered using the restriction endonuclease StuI. The target gene amplification product and the linearized vector were mixed and ligated using a seamless cloning kit (purchased from Abclonal, Cat. No. RK21020). The product was then transformed into Escherichia coli HD5α, and sequencing confirmed the successful construction of the vector.

[0083] Agrobacterium transformation: The CmTST2 overexpression vector was transformed into Agrobacterium, and Agrobacterium was used to infect Emerald Melon fruits at the expansion stage (15 days after pollination). The Agrobacterium infection procedure was the same as in Example 2. A GFP empty vector was used as a control group. During fruit infection, the experimental group (CmTST2-GFP group) and the control group (GFP group) were injected at opposite equatorial locations on the same fruit.

[0084] Protoplast preparation: After the candidate sugar transporter gene CmTST2-GFP is expressed, the melon fruit is enzymatically hydrolyzed to prepare protoplasts. The enzymatic hydrolysis procedure of the fruit protoplasts is the same as that in Example 1.

[0085] Sugar Fluorescent Analog Feeding: Protoplasts from melon fruits overexpressing CmTST2 were cultured in a medium containing a sugar fluorescent analog (0.6 M mannitol, 10 mM MES, 10 mM CaCl2, and 1 mM sucrose fluorescent analog, Esculin) and incubated in a 23°C incubator for 12 h. After washing away the medium, the protoplasts were aspirated and placed on a glass-bottomed dish. The protoplasts were then photographed under a laser confocal microscope, and the vacuolar fluorescence intensity was compared between the protoplasts and the control group. At least 30 protoplasts were imaged for each treatment (N > 30). Fluorescence intensity was calculated using ImageJ software, and the results were tested using SPSS t-tests to determine if there were any differences compared to the control group.

[0086] Figure 5 The fluorescence distribution of the sugar fluorescent analogue Esculin in melon fruit protoplasts overexpressing TST2 at the expansion stage after 12 hours of feeding, among which green is GFP protein and blue is Esculin fluorescence. The four small figures are four parallel experiments. It can be seen that after TST2 is expressed in melon protoplasts, the fluorescence brightness of Esculin in the vacuole is significantly enhanced compared with the control group. After counting the fluorescence intensity in the vacuole, it was found that the Esculin intensity of TST2-GFP was significantly higher than that of the control group (see Figure 6 ), demonstrating that TST2 protein has sucrose transport activity.

[0087] 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 and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the transport activity of tonoplast sugar transporter, characterized in that: The following steps are involved: S1. Constructing a plant expression vector expressing a tonoplast sugar transporter protein, transforming the fruit of muskmelon at the expansion stage to obtain transgenic muskmelon fruit, wherein the tonoplast sugar transporter protein is a TST / TMT family protein, and the muskmelon is an emerald; S2. culturing the transgenic melon fruit until the tonoplast sugar transporter is expressed, and then enzymatically hydrolyzing the pulp cells of the transgenic melon fruit to obtain pulp protoplasts; S3. Adding the protoplasts to a sugar fluorescent analog culture medium and incubating for a period of time, washing the protoplasts after the incubation is completed, and then detecting the fluorescent signal in the vacuole of the protoplasts, and evaluating the transport activity of the vacuole sugar transporter according to the fluorescence intensity.

2. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, wherein In step S1, constructing a plant expression vector for the tonoplast sugar transporter comprises the following steps: S11, obtaining the gene fragment of the tonoplast sugar transporter; S12. Linearize the plant expression vector using a restriction endonuclease, construct the gene fragment into the plant expression vector by recombination, and obtain a plant expression vector expressing the tonoplast sugar transporter; wherein the plant expression vector is pH7LIC5.0 or pBWA(V)HS.

3. The method for detecting the transport activity of tonoplast sugar transporter according to any one of claims 1 to 2, wherein: The plant expression vector also carries a fluorescent protein encoding gene, and the fluorescent protein is GFP, mcherry or RFP.

4. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, wherein In step S1, the plant expression vector is transformed into muskmelon fruit in the expansion stage through Agrobacterium-mediated method.

5. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, wherein In step S2, enzymatic hydrolysis of the pulp cells of the transgenic melon fruit comprises the following steps: The transgenic melon fruit is sliced, placed in an enzymatic hydrolysis solution, vacuumed, and cultured on a shaking table for 2-3 hours to obtain the protoplasts; wherein the enzymatic hydrolysis solution has a formula of: 0.6M mannitol, 10mM MES, 10mM CaCl2, 0.1% bovine serum albumin, 1.5% cellulase, 0.4% macerate, and 0.005% neutral red.

6. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, wherein In step S3, the sugar fluorescent analog is Esculin, NBD-F or 2-NBDG.

7. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, wherein In step S3, the sugar fluorescent analog culture solution includes 0.6M mannitol, 10mM MES, 10mM CaCl2 and 1mM sugar fluorescent analog, and the protoplasts are added to the sugar fluorescent analog culture solution and incubated for 12-24h.

8. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, wherein In step S3, the fluorescence intensity is obtained by counting the fluorescence signal at the vacuole center of the protoplast using ImageJ software; Evaluating the transport activity of the tonoplast sugar transporter according to fluorescence intensity comprises the following steps: evaluating the transport activity of the tonoplast sugar transporter by comparing the fluorescence intensity difference between the tonoplast sugar transporter expression group and the control group through T test.

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