A method for preparing sweet potato protoplasts and its application in subcellular localization analysis of sweet potato proteins

Abstract

The present invention provides a method for preparing sweet potato protoplasts and their application in protein subcellular localization analysis, belonging to the field of crop molecular breeding technology. The method uses sweet potato roots as the raw material for protoplast preparation, avoiding damage to the protoplasts caused by polysaccharides and polyphenols during the extraction process. Compared with other existing sweet potato protoplast preparation techniques, this method is time-efficient and simple to operate. It can efficiently prepare large quantities of protoplasts for subcellular localization analysis, improving the accuracy of sweet potato protein localization analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of crop molecular breeding, and particularly relates to a method for preparing sweet potato protoplasts and application of the method in sweet potato protein subcellular localization analysis. Background Art

[0002] Sweet potato (Ipomoeabatatas (L.) Lam.), belonging to the Convolvulaceae family and the genus Ipomoea (Ipomoea L.), is one of the most important root crops cultivated worldwide, with an annual production of approximately 113 million tons. Due to its high yield potential and ability to adapt to diverse environmental conditions, sweet potato has become an affordable source of dietary calories, protein, fiber, minerals, vitamins, and flavonoids in developing countries. To ensure sufficient production to meet growing demand, it is essential to further identify genes that contribute to high yield, high quality, and high resistance, and to analyze their functions, thus providing a theoretical basis for sweet potato breeding. Accurately understanding the function of a protein requires not only structural analysis but also, often, clarity on its subcellular localization to determine the likely site of its function.

[0003] Sweet potato protoplasts are often prepared from callus tissue induced from stem cuts or young leaves, with callus being the most common. However, callus induction is time-consuming and complex, and bacterial contamination can lead to widespread callus death. The polysaccharide and polyphenol-rich nature of sweet potato leaves complicates protoplast extraction, severely limiting the quantity and quality of the extracted material and significantly reducing the efficiency of protoplast transformation.

[0004] In addition, the current analysis of subcellular localization of sweet potato proteins is mainly conducted in protoplasts of tobacco and Arabidopsis leaves or transiently transformed tobacco leaves and onion epidermal cells. There are no patents or articles reporting direct subcellular localization analysis using sweet potato tissue. However, the evolutionary distance between sweet potato and other plants often results in proteins not being correctly targeted to their target locations, resulting in inaccurate protein localization. For example, the IbSUT4 protein is localized to the plasma membrane, but in tobacco leaf protoplasts, the protein is not only localized to the plasma membrane but also diffuses in the cytoplasm to a small extent ( Figure 1(See Wang DD, Liu HJ, Wang HX, et al., 2020. A novel sucrase transporter gene IbSUT4 involves in plant growth and responds to abiotic stress through the ABF-dependent ABA signaling pathway in sweet potato. BMC Plant Biology). Therefore, there is an urgent need to develop a rapid, simple, and efficient method for preparing sweet potato protoplasts for the analysis of sweet potato protein subcellular localization, which is of great guiding significance for clarifying the location where proteins exercise their functions and understanding the mechanism of action of genes. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for preparing sweet potato protoplasts, which can efficiently prepare a large number of protoplasts for sweet potato protein subcellular localization analysis.

[0006] The present invention also provides the use of the sweet potato protoplasts obtained by the preparation method in the subcellular localization analysis of sweet potato proteins, which can improve the accuracy of the analysis of the localization of sweet potato proteins.

[0007] The present invention provides a method for preparing sweet potato protoplasts, comprising the following steps:

[0008] The sweet potato root system was used as the material and enzymatically hydrolyzed under the combined action of cellulase and pectinase to obtain the enzymatic hydrolysis product;

[0009] Protoplasts are separated from the enzymatic hydrolysis product and resuspended to obtain a sweet potato protoplast suspension.

[0010] Preferably, the sweet potato root system is obtained by culturing sweet potato stem segments by cutting in water and collecting the roots after the roots grow out.

[0011] Preferably, the culture temperature is 25-30°C;

[0012] The culture is carried out under light, and the light intensity of the light is 1800-2200LX.

[0013] Preferably, the enzymatic hydrolysis method comprises chopping the sweet potato roots, mixing the mixture with the enzymatic hydrolysis solution, and treating the mixture in the dark at 27-29° C. for 3.5-4.5 hours.

[0014] Preferably, the enzymatic hydrolysis solution is an aqueous solution containing 10 mg / mL cellulase, 4 mg / mL pectinase, 0.4 mol / L mannitol, 20 mmol / L KCl, 10 mmol / LCaCl2, 0.035% volume percentage of β-mercaptoethanol, 1 mg / mL BSA and 0.3 mol / L MES.

[0015] Preferably, the method for isolating protoplasts comprises sieving and centrifugation;

[0016] The screening method comprises pre-rinsing the cell sieve with W5 solution, pouring the enzymatic hydrolysate into the sieve, and collecting the sieve underflow;

[0017] The centrifugation method comprises centrifuging the material under the sieve, collecting the precipitate, resuspending the precipitate in W5 solution, and centrifuging again to collect the precipitate.

[0018] Preferably, the W5 solution is an aqueous solution containing 154mmol / LNaCl, 125mmol / LCaCl2, 5mmol / LKCl, 5mmol / L glucose and 2mmol / L MES;

[0019] The centrifugal speed is 100g, and the centrifugal time is 2.5 to 3.5 minutes.

[0020] Preferably, the resuspension solution is MMg solution;

[0021] The MMg solution is an aqueous solution containing 15 mmol / LMgCl2, 0.4 mol / L mannitol and 4.7 mmol / LMES.

[0022] The invention provides application of the sweet potato protoplasts obtained by the preparation method in subcellular localization analysis of sweet potato proteins.

[0023] Preferably, the method for subcellular localization analysis of the sweet potato protein is to insert the nucleotide sequence corresponding to the sweet potato protein to be located into a vector containing a fluorescent protein to obtain a recombinant expression vector encoding the target sweet potato protein;

[0024] The expression vector is transformed into the sweet potato protoplasts, and the sweet potato protoplasts are washed, resuspended, cultured in the dark, and the fluorescence position is observed under a laser confocal microscope.

[0025] The present invention provides a method for preparing sweet potato protoplasts, specifically, using sweet potato roots as materials and performing enzymolysis under the joint action of cellulase and pectinase to obtain an enzymolysis product; separating protoplasts from the enzymolysis product, and resuspending to obtain a sweet potato protoplast suspension. In view of the characteristics of sweet potato leaves being rich in polysaccharides and polyphenols and the problem that culturing callus tissue is time-consuming and complicated, the present invention selects sweet potato roots as materials to prepare protoplasts, which can not only quickly and easily obtain a large number of protoplasts, but also the obtained high-quality protoplasts can meet the transformation requirements, and efficiently and accurately realize the subcellular localization analysis of sweet potato proteins in the sweet potato protoplasts. In the embodiment, protoplasts are separated using roots, callus tissue and young leaves as materials respectively. The results show that the number of protoplasts prepared using roots as raw materials is more than that of callus tissue and young leaves, which can provide sufficient materials for protein subcellular localization analysis.

[0026] At the same time, compared with other existing sweet potato protoplast preparation technologies, the preparation method provided by the present invention has the characteristics of short time consumption and simple operation, and can efficiently prepare a large number of protoplasts.

[0027] The present invention also provides the use of the sweet potato protoplasts obtained by the preparation method for subcellular localization analysis of sweet potato proteins. When the sweet potato protoplasts prepared by the present invention were used for subcellular localization analysis of sweet potato proteins, significant fluorescence was observed under laser confocal microscopy, indicating that the GFP fusion protein was successfully expressed in the protoplasts, and the localization results were consistent with the predicted protein localization results. Thus, the method provided by the present invention for performing protein subcellular localization analysis using sweet potato protoplasts can greatly improve the accuracy of analyzing sweet potato protein localization and provide a theoretical basis for clarifying the functions of sweet potato genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of the localization of the IbSUT4 protein in tobacco leaf protoplasts are shown in the following figure:

[0029] Figure 2 This is the technical route of the subcellular localization analysis method using sweet potato protoplasts in the present invention;

[0030] Figure 3 This is a morphological diagram of the sweet potato root system, callus tissue and young leaves;

[0031] Figure 4 The following are the results of microscopic examination of protoplasts from sweet potato roots, callus tissue and young leaves (the arrows point to the protoplasts);

[0032] Figure 5 This is the GFP vector map;

[0033] Figure 6 This is the result of subcellular localization analysis of the positive control GFP protein;

[0034] Figure 7 The results of subcellular localization analysis of EDL3 and PP2CA proteins. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing sweet potato protoplasts, comprising the following steps:

[0036] The sweet potato root system was used as the material and enzymatically hydrolyzed under the combined action of cellulase and pectinase to obtain the enzymatic hydrolysis product;

[0037] Protoplasts are separated from the enzymatic hydrolysis product and resuspended to obtain a sweet potato protoplast suspension.

[0038] In the present invention, the sweet potato root system is preferably obtained by culturing sweet potato stem cuttings in water, and then collecting the roots after they grow. The culturing temperature is preferably 25-30°C, more preferably 28°C. The light intensity is preferably 1800-2200 lux, more preferably 2000 lux. When the roots grow to 2-3 cm, the roots are cut and used as material for enzymatic hydrolysis.

[0039] In the present invention, the enzymatic hydrolysis method is preferably to chop the sweet potato roots and mix them with an enzymatic hydrolysis solution, and then dark-treat them at 27-29°C for 3.5-4.5 hours. The temperature of the dark treatment is preferably 28°C. The time of the dark treatment is preferably 4 hours. The enzymatic hydrolysis solution is preferably an aqueous solution containing 10 mg / mL cellulase, 4 mg / mL pectinase, 0.4 mol / L mannitol, 20 mmol / L KCl, 10 mmol / LCaCl2, 0.035% β-mercaptoethanol by volume, 1 mg / mL BSA, and 0.3 mol / LMES. 1.5-2.5 g of the sweet potato roots are added to every 15 mL of the enzymatic hydrolysis solution. During the mixing, the sweet potato roots are preferably chopped into pieces of 2-3 mm.

[0040] In the present invention, in addition to using the sweet potato hydroponic root system as the material for enzymatic hydrolysis, the sweet potato callus and the young leaves of the sweet potato were also used as the materials for enzymatic hydrolysis in the same way. The results showed that the number of protoplasts prepared from the root system was relatively large, reaching 1×10 7 / g, while the number of protoplasts prepared from sweet potato callus was 4×10 4 The number of protoplasts prepared from young leaves is the least, only 15 per gram. It can be seen that the present invention is conducive to obtaining more protoplasts by using root system as material for preparing protoplasts.

[0041] After obtaining the enzymatic hydrolysis product, the present invention separates protoplasts from the enzymatic hydrolysis product and resuspends the product to obtain a sweet potato protoplast suspension.

[0042] In the present invention, the method for isolating protoplasts preferably includes sieving and centrifugation. The sieving method is preferably to pre-rinse the cell sieve with W5 solution, pour the enzymatic hydrolyzate into the sieve, and collect the sieve material. The centrifugation method is preferably to centrifuge the sieve material, collect the precipitate, resuspend it with W5 solution, and centrifuge again to collect the precipitate. The W5 solution is preferably an aqueous solution containing 154mmol / LNaCl, 125mmol / LCaCl, 5mmol / LKCl, 5mmol / Lglucose and 2mmol / LMES. The centrifugal speed is preferably 100g, and the centrifugal time is preferably 2.5 to 3.5min, more preferably 3min.

[0043] In the present invention, the resuspension solution is preferably an MMg solution. The MMg solution is preferably an aqueous solution containing 15 mmol / LMgCl2, 0.4 mol / L mannitol and 4.7 mmol / L MES.

[0044] The invention provides application of the sweet potato protoplasts obtained by the preparation method in subcellular localization analysis of sweet potato proteins.

[0045] In the present invention, the method for subcellular localization analysis of the sweet potato protein is preferably as follows: inserting a nucleotide sequence corresponding to the sweet potato protein to be located into a vector containing a fluorescent protein to obtain a recombinant expression vector containing the target sweet potato protein; transforming the recombinant expression vector into the sweet potato protoplasts, washing and resuspending the sweet potato protoplasts, culturing in the dark, and observing the fluorescence position under a laser confocal microscope.

[0046]

[0047] The present invention has no particular limitation on the type of fluorescent protein, and any fluorescent protein commonly used in the art for subcellular localization can be used, such as GFP protein. The vector containing the fluorescent protein is preferably CaMV35S-sGFP (pCaMV35S-sGFP), which can be purchased through conventional commercial channels.

[0048] After constructing the recombinant expression vector, verification is preferably performed. The verification method is preferably to transform the ligation product obtained above into Escherichia coli, culture it, extract the plasmid, and sequence it. A positive plasmid is considered if the sequencing result is consistent with the expected result and is used for subsequent transformation.

[0049] The present invention does not particularly limit the transformation method; transformation methods well known in the art may be employed, such as PEG-mediated transformation. The PEG-mediated transformation method preferably comprises mixing the recombinant expression vector and protoplasts in the presence of PEG, incubating at room temperature for 8 minutes, adding W5 solution, gently mixing, allowing to stand for 2 minutes, and centrifuging to remove the PEG. The protoplasts are then washed with W5 solution, and the pellet is resuspended in WI solution. After incubation in the dark for 16-24 hours, fluorescence is observed under a laser confocal microscope. The WI solution is preferably an aqueous solution of 0.5 mmol / L mannitol, 20 mmol / L KCl, and 0.3 M MES. IbEDL3 and IbPP2CA proteins are localized to the nucleus, consistent with previous predictions from Plant-mPLoc (http: / / www.csbio.sjtu.edu.cn / bioinf / plant-multi / ) that IbEDL3 and IbPP2CA are localized to the nucleus.

[0050] The sweet potato protein subcellular localization analysis method provided by the present invention is carried out based on the high-quality sweet potato protoplasts obtained by the above-mentioned preparation method. The method can effectively improve the accuracy of analyzing the localization of the sweet potato protein.

[0051] The following describes in detail a method for preparing sweet potato protoplasts provided by the present invention and its application in subcellular localization analysis of sweet potato proteins in conjunction with the examples. However, they should not be construed as limiting the scope of protection of the present invention.

[0052] The reagent formulas and primers used in the present invention are shown in Tables 1 to 6:

[0053] 1. Enzyme hydrolysate (prepared and used immediately)

[0054]

[0055] Table 2W5 solution (pH = 5.8121 ° C sterilization 20min)

[0056]

[0057]

[0058] Table 3 MMg solution (pH = 5.61, sterilized at 21°C for 20 min)

[0059] Reagents 100mL <![CDATA[15mmol / LMgCl2]]> 0.1425g 4.7mmol / LMES 1.7mL (0.3MMES) 0.4 mol / L mannitol 7.287g

[0060] Table 4 WI solution (pH = 5.71, sterilized at 21°C for 20 min)

[0061] Reagents 100mL 0.5mmol / LMannitol 9.109g 4mmol / LMES 1.33mL (0.3MMES) 20mmol / LKCl 0.149g

[0062] Table 5 40% PEG (prepared and used)

[0063] Reagents 5mL PEG4000 2g 0.8 mol / L mannitol 0.73g <![CDATA[100mmol / LCaCl2]]> 0.055g

[0064] Table 6 Primer information

[0065]

[0066] Example 1

[0067] A method for preparing sweet potato protoplasts

[0068] 1. Material culture (see Figure 3 )

[0069] 1) Root culture: Sweet potato stem cuttings were placed in water, placed in a 28°C culture room, and cultured under light for 4 days. When the roots grew to 2-3 cm, the roots were cut for protoplast isolation.

[0070] Callus Culture: Cut sweet potato stem segments approximately 1 cm long from the tissue culture flask and place them horizontally on MSD medium to induce callus. Once callus has grown, propagate to a sufficient number for protoplast isolation.

[0071] Young leaves: Take the sweet potato seedlings grown in tissue culture bottles and cut the young leaves for protoplast isolation.

[0072] 2. Preparation of Protoplasts

[0073] ⑴ Use a sharp blade to cut the above materials into 2mm pieces and immediately immerse them in a culture dish containing 15mL of enzymatic hydrolysis solution, place it in a 28℃ incubator, and culture it in the dark for 4h. During this period, gently shake the culture dish every 1h to ensure sufficient enzymatic hydrolysis.

[0074] ⑵ Rinse the 40μm cell strainer and 50mL conical centrifuge tube with appropriate amount of W5 solution.

[0075] ⑶ Pour the enzymatic solution into the cell strainer and add an equal volume of W5 solution to rinse the culture dish and the strainer. Centrifuge at 100g for 3 minutes at room temperature (set the centrifuge speed setting to 1 for both the acceleration and deceleration).

[0076] (4) Use a cut pipette tip to slowly remove the supernatant, add 5 mL of W5 solution to resuspend the protoplasts, and centrifuge at 100 g for 2 minutes at room temperature.

[0077] ⑸Use the cut tip of the pipette to slowly remove the supernatant, add 1mL of W5 solution to resuspend the protoplasts, and take 100μL for microscopic examination ( Figure 4 ), the residue is placed on ice for 30min.Select that quantity in microscope visual field is large and protoplast quality is good (edge ​​is complete, rounded, and impurity is less in solution) and proceed to next step experiment.By microscopy and utilizing the quantity of hemocytometer counting plate statistics protoplast, calculate the output of protoplast according to formula 1.

[0078] Protoplast yield (pieces / g) = (number of protoplasts in 20 squares × 10 4 ) / enzymatically hydrolyzed sample mass formula I.

[0079] The results are shown in Table 7. The results showed that the protoplasts prepared from the hydroponic root system were large in quantity and had intact edges.

[0080] Table 7 Blood cell counting plate statistical protoplast yield

[0081]

[0082]

[0083] Example 2

[0084] Analysis of subcellular localization of IbEDL3 and IbPP2CA proteins using sweet potato protoplasts

[0085] A. Cultivating sweet potato roots

[0086] The sweet potato stem segments were inserted into water and placed in a culture room at 28°C. They were cultured under light for 4 days. When the roots grew to 2-3 cm, the roots were cut for protoplast isolation.

[0087] B. Preparation of Protoplasts

[0088] ⑴ Use a sharp blade to cut the roots into 2mm pieces and immediately immerse them in a culture dish containing 15mL of enzymatic hydrolysis solution. Place them in a 28℃ incubator and culture them in the dark for 4 hours. During this period, gently shake the culture dish every 1 hour to ensure sufficient enzymatic hydrolysis of the roots.

[0089] ⑵ Rinse the 40μm cell strainer and 50mL conical centrifuge tube with appropriate amount of W5 solution.

[0090] ⑶ Pour the enzymatic solution into the cell strainer and add an equal volume of W5 solution to rinse the culture dish and the strainer. Centrifuge at 100g for 3 minutes at room temperature (set the centrifuge speed setting to 1 for both the acceleration and deceleration).

[0091] (4) Use a cut pipette tip to slowly remove the supernatant, add 5 mL of W5 solution to resuspend the protoplasts, and centrifuge at 100 g for 2 minutes at room temperature.

[0092] ⑸ Use a cut pipette tip to slowly remove the supernatant, add 1 mL of W5 solution to resuspend the protoplasts, take 100 μL for microscopic examination, and place the rest on ice for 30 minutes.

[0093] ⑹Use the cut tip of the pipette to slowly remove the supernatant, add a certain volume of MMg solution to make the protoplast concentration 1~2×10 5 / mL for the next plasmid transformation.

[0094] C. Construction of recombinant expression vector

[0095] Primers containing restriction enzyme sites (SalI and SpeI) were designed using PrimerPrimer 5.0 (see Table 6). PCR amplification was performed to obtain the ORF regions of the IbEDL3 and IbPP2CA genes, containing restriction enzyme sites but without a stop codon. The PCR amplification reaction system consisted of 25 μL of PrimerSTARMax, 1 μL of primer F, 1 μL of primer R, 1 μL of template cDNA, and 22 μL of ddH2O, for a total of 50 μL. The reaction procedure was as follows: initial denaturation at 98°C for 5 min; 32 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 15 s; and extension at 72°C for 2 min.

[0096] The recombinant expression vector CaMV35S:IbEDL3 / IbPP2CA-GFP was constructed by ligating the coding sequence of the target gene with restriction sites obtained by the amplification described above with the linearized CaMV35S-GFP plasmid after restriction digestion under the action of T4 enzyme. The ligation product was transformed into Escherichia coli and cultured. After screening and culture, the plasmid that was successfully sequenced was extracted using a plasmid extraction kit (TIANGEN, high-purity plasmid extraction kit) (see Figure 5 ).

[0097] D. PEG method for plasmid transformation

[0098] All steps of PEG-mediated plasmid transformation of sweet potato protoplasts were carried out at 25-28°C.

[0099] ⑴ Take a 2 mL round-bottom centrifuge tube rinsed with calf serum (BSA), add 10 μL of plasmid DNA first, then add 100 μL of protoplasts, mix gently, then add 110 μL of 40% PEG, and mix by pipetting.

[0100] ⑵Leave at room temperature for 8 minutes.

[0101] ⑶ Slowly add 440 μL of W5 solution, mix gently, and let it stand for 2 minutes.

[0102] (4) Centrifuge at 100g for 1 min to remove PEG, and slowly add 1 mL of W5 solution to resuspend the precipitate.

[0103] (5) Centrifuge at 100g for 1 min, remove the supernatant, slowly add 1mL of WI solution to resuspend the precipitate, incubate in the dark for 16 to 24 hours, and observe the fluorescence under a laser confocal microscope.

[0104] The results are as follows Figure 6 and Figure 7 As shown, compared with the positive control ( Figure 6 ), IbEDL3 and IbPP2CA proteins are localized in the nucleus ( Figure 7 This is consistent with the previous results of Plant-mPLoc (http: / / www.csbio.sjtu.edu.cn / bioinf / plant-multi / ) predicting that IbEDL3 and IbPP2CA are localized in the cell nucleus.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing sweet potato protoplasts, characterized in that: The following steps are involved: The sweet potato root system is used as a material and enzymatically hydrolyzed under the combined action of cellulase and pectinase to obtain an enzymatic hydrolysis product; the length of the sweet potato root system is 2 to 3 cm; Separating protoplasts from the enzymatic hydrolysis product and resuspending them to obtain a sweet potato protoplast suspension; The enzymatic hydrolysis method comprises chopping the sweet potato root system and mixing it with an enzymatic hydrolysis solution; the enzymatic hydrolysis solution is an aqueous solution composed of 10 mg / mL cellulase, 4 mg / mL pectinase, 0.4 mol / L mannitol, 20 mmol / L KCl, 10 mmol / L CaCl2, 0.035% by volume beta-mercaptoethanol, 1 mg / mL BSA and 0.3 mol / L MES.

2. The method for preparing sweet potato protoplasts according to claim 1, wherein The sweet potato root system is obtained by cutting sweet potato stem segments and culturing them in water, and collecting the roots after they grow out.

3. The preparation method according to claim 2, characterized in that The culture temperature is 25-30°C; The culture is carried out under light, and the light intensity of the light is 1800-2200 LX.

4. The method for preparing sweet potato protoplasts according to claim 1, wherein The enzymatic hydrolysis method comprises the following steps: chopping the sweet potato root system, mixing the mixture with the enzymatic hydrolysis solution, and then treating the mixture in the dark at 27-29° C. for 3.5-4.5 hours.

5. The method for preparing sweet potato protoplasts according to claim 1, wherein The method for separating protoplasts includes sieving and centrifuging; The screening method comprises pre-rinsing the cell sieve with W5 solution, pouring the enzymatic hydrolysate into the sieve, and collecting the sieve underflow; The centrifugation method comprises centrifuging the material under the sieve, collecting the precipitate, resuspending the precipitate in W5 solution, and centrifuging again to collect the precipitate.

6. The method for preparing sweet potato protoplasts according to claim 5, wherein The W5 solution is an aqueous solution containing 154 mmol / L NaCl, 125 mmol / L CaCl2, 5 mmol / L KCl, 5 mmol / L glucose and 2 mmol / L MES; The centrifugal speed is 100 g, and the centrifugal time is 2.5-3.5 min.

7. The method for preparing sweet potato protoplasts according to any one of claims 1 to 6, wherein The resuspension solution is MMg solution; The MMg solution is an aqueous solution containing 15 mmol / L MgCl2, 0.4 mol / L mannitol and 4.7 mmol / L MES.

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