Detection method for transport activity of vacuolar saccharide transporter

Abstract

The invention belongs to the technical field of activity detection of sugar transporters, and particularly relates to a method for detecting the transport activity of a vacuolar sugar transporter. The method comprises the following steps: constructing a plant expression vector for expressing a vacuolar sugar transporter, and transforming a muskmelon fruit in a swelling period to obtain a transgenic muskmelon fruit; culturing the transgenic muskmelon fruit until the vacuolar sugar transporter protein is expressed, and then performing enzymolysis to obtain protoplast; adding the protoplast into a sugar fluorescence analogue culture solution, incubating for a period of time, then detecting a fluorescence signal of vacuoles in the protoplast, and evaluating the transport activity of the vacuole sugar transporter according to the fluorescence intensity. The method disclosed by the invention is simple and convenient to operate, high in efficiency and independent of isotope, has the characteristics that the transport activity of endogenous and exogenous vacuolar sugar transporters can be detected, also keeps the characteristics of the plant vacuoles, can reduce the interference of background signals in the vacuoles, and improves the accuracy and sensitivity of the analysis of the transport activity of the plant vacuolar sugar transporters.

Description

Technical Field

[0001] The invention relates to the technical field of sugar transporter activity detection, and 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 many processes of plant growth and development and play an important role in maintaining cell osmotic balance. In some plant organs with high sugar accumulation, vacuolar sugar transporters play an important role in sugar accumulation. There are currently three main types of vacuolar sugar transporters found in plants. One is the H2O2 that 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 transporter SWEET that is independent of energy and proton gradient.

[0003] Analyzing the sugar transport activity of vacuolar sugar transporters is of great significance for analyzing the function of the protein. At present, the methods used to analyze the sugar transport activity of vacuolar sugar transporters mainly include yeast heterologous expression system, Xenopus oocyte heterologous expression system, voltage clamp and patch clamp techniques. These techniques have different limitations. For example, the use of yeast mutants and Xenopus oocytes are both non-plant heterologous expression systems. In some cases, protein heterologous expression may produce mistargeting, and the lack of protein modification in the heterologous system may also interfere with sugar uptake analysis; voltage clamp and patch clamp have particularly high technical requirements for instruments and operators, and only some transporters have proton gradients in the sugar transport process. Even if there is a proton gradient, the current is relatively weak, and it is often difficult to detect the effective current, which makes these technologies difficult to popularize and apply. Therefore, it is urgent to develop new methods for detecting the transport activity of vacuolar sugar transporters that are more convenient, easy to operate, and can retain the endogenous specific modifications of plant cells. Summary of the invention

[0004] In view of the above problems of the prior art, the present invention provides a new method for detecting the transport activity of vacuolar sugar transporters, using the protoplasts of melon fruits in the expansion stage with low endogenous vacuolar sugar transport activity as the endogenous or plant heterologous expression host of the vacuolar sugar transporter to be detected, and feeding the melon fruit protoplasts overexpressing the vacuolar sugar transporter with a sugar fluorescent analog to detect the sugar transport activity. The method is simple to operate, highly efficient, is not dependent on isotopes, and has the characteristics of being able to detect the transport activity of both endogenous and exogenous vacuolar sugar transporters, and also maintains the characteristics of the plant vacuole itself, can reduce the interference of background signals in the vacuole, and improve the accuracy and sensitivity of the analysis of the transport activity of the plant vacuolar sugar transporter.

[0005] To achieve the above object, 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 tonoplast sugar transporter protein, transforming melon fruit at the expansion stage, and obtaining transgenic melon fruit;

[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, and then detecting the fluorescence 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 melon is a sweet melon, a green melon or a honeydew melon.

[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 a 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 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 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 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 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.

[0018] Furthermore, in step S3, the sugar fluorescent analog is Esculin (a sucrose fluorescent analog), NBD-F (4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fructose fluorescent analog) 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 analog).

[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 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 a T test.

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

[0023] 1. The present invention utilizes the protoplasts of the expanded melon fruit with low endogenous vacuolar sugar transport activity as the heterologous expression host of the vacuolar sugar transport protein to be detected. The exogenous gene is easy to achieve instantaneous and efficient expression in the melon fruit. Then, the sugar transport activity is detected by feeding the melon fruit protoplasts that overexpress the vacuolar sugar transport protein with a sugar fluorescent analog. The method has the characteristics of simple operation, high efficiency, isotope-independent, and the transport activity of both endogenous and exogenous vacuolar sugar transport proteins can be detected. Moreover, the vacuolar 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 vacuolar sugar transport protein. In addition, by co-transferring two different genes in one protoplast, the influence of interacting proteins on the 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 the 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 creative work.

[0026] Figure 1 This is a microscopic examination image of protoplasts of melon fruit after enzymatic hydrolysis and neutral red staining in 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, in Example 1 of the present invention, from left to right are protoplasts of melon fruits in the expansion stage and the maturity stage;

[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 microscopic examination of protoplasts of melon fruit in the expansion stage 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 fruit in the expansion stage overexpressing TST2 fed with the sugar fluorescent analog Esculin in Example 3 of the present invention;

[0031] Figure 6This is a statistical graph of the vacuolar fluorescence intensity 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. DETAILED DESCRIPTION

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

[0033] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes 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 defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0034] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, 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 according to the reported significant figures 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 the like are non-restrictive, i.e., other steps and other ingredients that do not affect the result 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 main organelle for nutrient storage and metabolism in plant cells, and vacuole membrane transporters play a vital role in the entry and exit of nutrients into and out of the vacuole. The detection of vacuole membrane-localized sugar transporter activity needs to overcome the following difficulties: 1. In heterologous expression systems such as yeast or African clawed frog oocytes that are distantly related to plants, it is usually difficult to express active vacuole membrane sugar transporters or the protein is not expressed, resulting in no targeting signal. Even if biologically active sugar transporters are expressed by means of codon optimization, it is impossible to detect the transport activity of vacuole membrane proteins through existing growth complementation, radioactive labeled substrates, voltage clamp, patch clamp, ion flow rate measurement and other technologies because they are located on the vacuole membrane inside the cell rather than on the cell membrane outside the cell; 2. In the expression system of model plants such as tobacco that are closely related, since the vacuole membrane itself in tobacco protoplasts has a high sugar transport activity, this will cause the detection of the transport signal of the target protein to overcome the interference of background signals, and the detection difficulty is greatly enhanced.

[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 tonoplast sugar transporter protein, transforming melon fruit at the expansion stage, and obtaining transgenic melon fruit;

[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, and then detecting the fluorescence signal in the vacuole of the protoplasts, and evaluating the transport activity of the vacuole sugar transporter according to the fluorescence intensity.

[0042] The present invention found in the process of studying the protoplasts of melon fruits that the endogenous tonoplast sucrose transport activity of the protoplasts of melon fruits in the expansion stage is low, which provides a fundamental guarantee for the identification of sugar transport protein activity using the protoplasts, greatly reducing the interference of background signals; and the melon fruits in the expansion stage are easy to express exogenous genes through Agrobacterium infection. Therefore, the present invention uses the protoplasts of melon fruits in the expansion stage as the heterologous expression host of the tonoplast sugar transport protein to be detected, firstly transforms the plant expression vector expressing the tonoplast sugar transport protein into the melon fruits through the transient transformation technology, cultivates to obtain the transgenic melon fruits successfully transformed with the target protein encoding gene, then uses conventional culture to efficiently express the tonoplast sugar transport protein, and separates to obtain the protoplasts with overexpression of the tonoplast sugar transport protein, and the plant sugar transport protein expressed in the melon protoplasts usually does not produce localization changes and modification changes, and can retain the original biological activity. Afterwards, the aforementioned protoplasts are 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, and are then transported into the vacuole via the vacuole sugar transporter and accumulate in the vacuole, generating a fluorescent signal under the excitation of 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 vacuole sugar transporter can be tracked and quantified by detecting the changes in the fluorescence intensity of the vacuole, thereby evaluating the sugar transport capacity of the vacuole sugar transporter to be tested.

[0043] The present invention detects sugar transport activity by feeding the protoplasts of melon fruits that overexpress tonoplast sugar transport proteins with sugar fluorescent analogs. Exogenous genes are easily expressed transiently and efficiently in melon fruits, and the endogenous tonoplast sugar transport activity of the protoplasts of melon fruits in the expansion period 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 analysis of the transport activity of the tonoplast sugar transport protein. Moreover, the source of melon fruit protoplasts is abundant, and the operation method of protoplast feeding technology 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 calculated by constructing a standard curve between sugar fluorescent analog concentration and fluorescence intensity. This is a conventional technique in the art and will not be described in detail herein.

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

[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 vacuole sugar transporter to be detected. For example, when detecting the glucose transport activity of the 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; 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 to detect the fructose transport activity; the sucrose fluorescent analog Esculin can be selected to detect the sucrose transport activity.

[0049] Preferably, in step S1, constructing a plant expression vector of a 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 endonuclease, construct the gene fragment into the plant expression vector by recombination method, and obtain the plant expression vector expressing the tonoplast sugar transporter.

[0052] In step S11, the gene fragment can be obtained by whole gene synthesis or by 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 from 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. Therefore, by detecting the fluorescent signal of the fluorescent protein, it is possible to quickly determine 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] The plant expression vector can be integrated into the melon fruit genome or retained in the form of a plasmid. Methods of gene transformation include gene gun method, electric shock method, microinjection method or Agrobacterium-mediated method. This is a well-known technology 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 method. The specific operation includes: injecting the Agrobacterium bacterial solution containing the plant expression vector into the melon fruit by syringe, the bacterial solution injection volume is 0.5mL, the needle insertion depth is 0.5cm, and the bacterial solution concentration OD 600 =0.4.

[0059] The Agrobacterium strain described 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, enzymatic hydrolysis of 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) macrozyme (Macerozyme R10) and 0.005% (wt) neutral red.

[0062] Optionally, in step S3, the sugar fluorescent analog culture solution includes 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 hours.

[0063] In step S3, the fluorescence intensity is obtained by counting the fluorescence signal at the vacuole center of the protoplast by ImageJ software. The evaluation of sugar transport activity is performed by comparing the fluorescence intensity difference between the vacuole sugar transporter expression group and the control group by T test. The control group is the same as 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 can transport the added specific substrate and the sugar transport activity is high.

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

[0065] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the 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 development stage of melon fruit includes three cycles, namely, fruit setting period, fruit expansion period, and fruit maturity period. Among them, the fruit expansion period is from the "hair removal" of young fruits to the basic determination of fruit size ("fixed size"), and the main growth characteristics during this period are rapid expansion and development of fruit volume, and the growth center of the whole plant is stable on the fruit; the maturity period is from the determination of fruit size to the maturity of fruit, and the main growth characteristics during this period are biochemical changes inside the fruit, a large amount of sugar conversion and accumulation, the flesh becomes crisp or softened, and emits different aromas, and the maturity characteristics are obvious. In this embodiment, the emerald melon fruits in the maturity and expansion periods are taken as raw materials for obtaining protoplasts.

[0072] The sampled fruit is sliced ​​to 0.5-1.0 mm to obtain a fruit slice to be separated; the fruit slice is put into an enzymatic solution, vacuumed for 15 minutes, and shaken on a shaker for 2-3 hours, during which the enzymatic state is examined under a microscope; the enzymatic 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) macrozyme R10 and 0.005% (wt) neutral red. After the enzymatic solution is completed, it is filtered using a 100 μm filter, centrifuged at 50×g, and washed 2-3 times using a washing buffer (including 0.6M mannitol, 10mM MES and 10mM CaCl2), and the protoplast activity is confirmed under a microscope.

[0073] Figure 1 The microscopic examination of protoplasts of melon fruit after enzymatic hydrolysis and neutral red staining is shown, in which 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] Take another protoplast that has been washed and resuspended with washing buffer, add 100mM Esculin stock solution, adjust the final concentration of Esculin to 1mM, and culture in a 23℃ incubator for 12h. Then use washing buffer to wash twice to remove the feeding solution, and after the protoplasts sink to the bottom, aspirate them and place them in a glass-bottomed dish, and observe and photograph them under a laser confocal microscope. The excitation light of Esculin is 405nm, the absorption light is set to 410-450nm, and the microscope is focused on the cell equatorial plane.

[0075] Figure 2 The figure shows the fluorescence distribution of protoplasts of melon fruits in the swelling and ripening stages 12 hours after being fed with Esculin. 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 sucrose can be transported to the pulp cytoplasm but not to the vacuole during the swelling stage of melon fruits, indicating that the protoplasts of melon fruits in the swelling stage have higher cell membrane sucrose transport activity but lower vacuole sucrose transport activity, and are good materials for the determination of vacuole sucrose transport activity, and are suitable for identifying the transport activity of vacuole sugar transport proteins.

[0076] Example 2 Reliability verification of melon fruit protoplast transient expression and sugar fluorescent analog feeding technology

[0077] The plant binary vector pH7LIC5.0-N-GFP with GFP tag was transferred into Agrobacterium, and the melon fruit in the expansion stage was infected with Agrobacterium. The above vector was transferred into Agrobacterium (strain GV3101 or EH105) by freeze-thaw method, and single clones were selected for colony PCR verification and shaken until OD 600 After centrifugation at OD 0.5-0.8, remove the supernatant, and resuspend the bacteria in infection solution (including 10 mM MES, 10 mM MgCl2, 200 μM acetosyringone (AS)) to make the concentration of Agrobacterium tumefaciens reach OD 600 =0.4. After 1-2 hours of placement, the fruit was injected into the swelled melon, the melon variety was Emerald. After 3 days of placement at room temperature, the fruit was cut open and the sample was taken for examination under a microscope to observe the fluorescence intensity.

[0078] Figure 3 The microscopic images of green fluorescent protein (GFP) expression after Agrobacterium transiently transformed melon fruit in the expansion stage 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 repeatability is good.

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

[0080] Figure 4 The fluorescence distribution of melon fruit protoplasts overexpressing GFP at the expansion stage after feeding Esculin for 12 hours is shown. It can be seen that there is no obvious Esculin fluorescence signal in the vacuole of the fruit protoplasts after transient GFP transfection, which 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 CmTST2 overexpression vector with fluorescent label: Using Cucumis melo fruit cDNA as 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 restriction endonuclease StuI, the target gene amplification product was mixed with the linearized vector, connected using a seamless cloning kit (purchased from Abclonal, catalog number RK21020), transformed into Escherichia coli HD5α and sequenced to verify the successful construction of the vector.

[0083] Agrobacterium transformation: The CmTST2 overexpression vector was transferred into Agrobacterium, and the Agrobacterium was used to infect the Emerald Melon fruit at the expansion stage (15 days after pollination). The Agrobacterium infection operation was the same as in Example 2. The GFP empty vector was used as the control group. When the fruit was infected, the experimental group (CmTST2-GFP group) and the control group (GFP group) were injected at opposite equatorial planes of 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 fruit protoplast enzymatic hydrolysis operation is the same as in Example 1.

[0085] Sugar fluorescent analogue feeding: The melon fruit protoplasts overexpressing CmTST2 were cultured with a culture medium containing sugar fluorescent analogues, including 0.6M mannitol, 10mM MES, 10mM CaCl2 and 1mM sucrose fluorescent analog Esculin, and placed in a 23° incubator for 12h. After washing away the culture medium, the protoplasts were aspirated and placed on a glass-bottomed dish, placed under a laser confocal microscope and photographed, and the difference in vacuolar fluorescence intensity between them and the control group was compared. At least 30 protoplasts were photographed for each treatment (N>30). The fluorescence intensity was calculated using ImageJ software, and the calculated results were subjected to a T test using SPSS to detect whether there was a difference with the control.

[0086] Figure 5 The fluorescence distribution of the sugar fluorescent analogue Esculin in the protoplasts of the expanding melon fruit overexpressing TST2 for 12 hours is shown. The green is GFP protein, the blue is Esculin fluorescence, and 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, and after counting the fluorescence intensity in the vacuole, it is found that the Esculin intensity of TST2-GFP is significantly higher than that of the control group (see Figure 6 ), proving 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 protection scope 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 tonoplast sugar transporter protein, transforming melon fruit at the expansion stage, and obtaining transgenic melon fruit; 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, and then detecting the fluorescence 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, characterized in that: In step S1, the tonoplast sugar transporter is a TST / TMT, SUT or SWEET family protein, and the melon is a sweet melon, a green melon or a melon.

3. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, characterized in that: In step S1, constructing a plant expression vector of a tonoplast sugar transporter comprises the following steps: S11, obtaining the gene fragment of the vacuolar sugar transporter; S12. Linearize the plant expression vector using 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.

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

5. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, characterized in that: In step S1, the plant expression vector is transformed into melon fruit at the expansion stage through Agrobacterium-mediated method.

6. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, characterized in that: 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 hydrolysate, vacuumed, and cultured on a shaking table for 2-3 hours to obtain the protoplasts; wherein the enzymatic hydrolysate 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.

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

8. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, characterized in that: 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.

9. The method for detecting the transport activity of tonoplast sugar transporter according to claim 1, characterized in that: 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 the 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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