CN109619094B - Baizilian SK3Application of dehydrin protein in reducing cell stress injury and improving ultralow temperature preservation effect - Google Patents

Abstract

The invention discloses a agapanthus SK₃The application of the dehydrin protein in reducing the cell stress injury and improving the ultralow temperature preservation effect; the protein has the capability of eliminating active oxygen and reducing cell stress injury, can effectively improve the recovery growth rate of cells after freezing, and obviously improves the ultralow temperature preservation efficiency. The specific method is to apply prokaryotic expression method to enrich and purify agapanthus SK₃Dehydrin protein (ApSK)₃) Application of Fenton reaction to prove ApSK₃The ApSK is verified by utilizing a plant cryopreservation evaluation model under the regulation and control effect on ROS metabolism₃Improving effect on plant cell cryopreservation. The method specifically comprises the following steps: adding 2 mu mol/L ApSK into ultralow-temperature vitrification solution₃The protein improves the survival rate of the plant after freezing by about 1 time. The method disclosed by the invention has obvious optimization on eliminating active oxygen, reducing cell stress damage and improving the activity and the preservation efficiency of the cells after cryopreservation and freezing.

Description

Baizilian SK3Application of dehydrin protein in reducing cell stress injury and improving ultralow temperature preservation effect

Technical Field

The invention relates to the field of preservation of plant materials, in particular to agapanthus SK₃The application of the dehydrin protein in reducing cell stress injury and improving the cryopreservation effect can reduce the plant cell stress injury, thereby optimizing the vitrification cryopreservation effect of plants.

Background

Vitrification Cryopreservation (Vitrification Cryopreservation) refers to a set of low-temperature biological technology in which Plant germplasm resources are treated by Vitrification Solution (PVS) consisting of permeable and non-permeable protective agents in a certain proportion and then rapidly put into liquid nitrogen (-196 ℃) for storage. The cell or tissue is placed in the vitrification solution composed of permeable and non-permeable protective agents in a certain proportion, so that the material and the vitrification solution thereof are solidified into an amorphous vitrification state at a sufficiently fast cooling rate, and the material and the vitrification solution thereof are stored at a low temperature in the glass state, thereby achieving the purpose of long-term preservation of germplasm. The material for ultralow temperature preservation occupies small space, does not need additional manual maintenance, can avoid the change of the chromosome number of cells and tissues due to long-term subculture and possible degeneration and pest invasion of in vitro materials in the long-term asexual propagation process, and is a preferred method for long-term preservation of excellent germplasm resources in nearly ten years. However, in the process of cryopreservation, plant tissues or cells can also face severe osmotic dehydration, ROS oxidative stress, ion toxicity, fatal ice crystal and other various stress injuries, and the death of the plant cells can be caused. Therefore, the method optimizes an ultralow-temperature preservation system through the exogenous protective substances, is an effective way for protecting plant materials and improving the preservation efficiency, has important theoretical significance and application value for in vitro preservation of isolated cells for a medium and long time, but the commonly used exogenous protective substances (such as VC, VE, GSH and the like) mainly have the functions of oxidation resistance and single protection effect, and cannot comprehensively improve the damage of ultralow-temperature composite adversity to the cell stress.

Arabidopsis seedlings are important materials for basic research of ultralow temperature preservation of plants. The protective effect of the exogenous additive on plant materials and the optimization effect of an ultralow-temperature preservation system can be quickly evaluated by counting the growth recovery rate after freezing.

Dehydrin (Dehydrin) belongs to the second family of Abundant Proteins in the Late embryonic development stage (Late embryo genesis Abundant Proteins II, LEAII) and has the characteristics of high hydrophilicity, disorder and oxidation resistance. Plants accumulate large amounts of dehydrin proteins in the late embryonic development stage and under stress to cope with abiotic stress conditions such as drought, high temperature and high salinity. Many researches prove that the phytoalexin has the functions of preventing protein denaturation, stabilizing plasma membrane structure, combining excess ions, relieving stress injury and the like, and is a protective protein closely related to severe dehydration of plant cells. The special protective function of the dehydrin has important application potential, but the development and application of cells in vitro in related technical fields are not carried out at present.

Disclosure of Invention

In view of the above-mentioned technical background, it is an object of the present invention to provide SK₃The application of the dehydrin in reducing the cell stress injury and improving the ultralow temperature preservation effect; the invention obtains the agapanthus dehydratum protein ApSK₃Based on the coding gene, the method for efficiently inducing and purifying ApSK by utilizing the characteristic of thermal stability of the protein and the method of biological engineering₃A method for preparing dehydrin protein, and the purified protein is applied to the field of an ultra-low temperature preservation system by an external source adding mode, ApSK₃The dehydrin has the effects of safety and broad spectrum on the optimization of plant ultra-low temperature preservation technology, and simultaneously verifies ApSK₃Can effectively remove Reactive Oxygen Species (ROS), reduce the damage of oxidative stress and the like in the preservation process, and has obvious protection effect on plant materials in the ultralow temperature process. The invention particularly provides a protein with an antioxidant effect, optimizes the existing ultralow temperature preservation technical system, improves the ultralow temperature preservation effect of plant materials, and can be applied to the long-term stable preservation of plant germplasm resources.

In order to achieve the purpose, the invention is realized by the following technical scheme:

in a first aspect, the invention provides a agapanthus dehydratum protein ApSK₃The application of the antioxidant protection protein serving as exogenously added plant cells.

Preferably, the agapanthus dehydratus protein ApSK₃Can be used for inhibiting generation of hydroxyl radical. The agapanthus dehydratum protein ApSK₃Is an in vitro purified dehydrin protein.

The dehydrin protein ApSK₃Can be used for inhibiting generation of hydroxyl radical. In plant cells, H₂O₂Can generate hydroxyl radical (OH. cndot.) through Fenton reaction, and the ApSK is added into an in-vitro Fenton reaction system₃The protein has a remarkable inhibiting effect on the generation of hydroxyl radicals.

Hydroxyl radical (OH) is an important component of ROS, and ApSK is detected₃ApSK can be proved by the scavenging effect on hydroxyl radical (OH)₃Protecting the oxidative stress of cells in the process of ultralow temperature preservation.

In a second aspect, the invention provides agapanthus dehydratum protein ApSK₃The application of the compound in an ultralow temperature preservation system as a protective agent for oxidative stress of cell tissues.

Preferably, the agapanthus dehydratus protein ApSK₃Can be used for inhibiting generation of hydroxyl radical.

In a third aspect, the invention provides a vitrification solution for plant vitrification cryopreservation, which contains 20-40% w/v glycerol, 10-20% w/v glycol, 10-20% w/v dimethyl sulfoxide, 0.2-0.6mol/L sucrose and 0.5-2 μmol/L agapanthus dehydrated protein ApSK₃The MS culture solution of (1). In the vitrification solution, the agapanthus dehydrated protein ApSK₃When the concentration reaches 0.5-2 mu mol/L, the effect is obvious.

More preferably, the vitrification solution is ApSK containing 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v dimethyl sulfoxide, 0.4mol/L sucrose and 0.5-2 μmol/L agapanthus dehydrin protein₃The MS culture solution of (1).

Preferably, the agapanthus dehydratus protein ApSK₃Is obtained by a method comprising the following steps:

a1, for expressing dehydrin protein ApSK₃Inducing the bacterial liquid, centrifuging and cleaning the induced bacterial liquid to obtain suspended bacterial liquid;

a2, boiling and cracking the suspension liquid, cooling to room temperature, centrifuging and collecting supernatant, namely the purified ApSK₃A protein sample.

Due to the structural disorder and the thermal stability of the dehydrin protein, the invention adopts a direct boiling mode to denature other proteins in a system and extracts and purifies the recombinant dehydrin protein. The dehydrin protein has good thermal stability, and can maintain the stability of the function and structure under boiling water bathThe prokaryotic expression of the dehydrin protein ApSK is performed in a boiling cracking mode₃And carrying out rapid purification and enrichment. Compared with the extraction mode of the ultrasonic lysis bacterial liquid and the protein purified by the nickel column, the method is more efficient, quicker, easier and more convenient.

Preferably, in step A1, the ApSK protein expressing dehydrin is obtained₃The bacterial liquid preparation method comprises the following steps: will contain ApSK₃Inoculating Escherichia coli strain of recombinant plasmid into LB liquid culture medium, culturing Escherichia coli at 37 deg.C and 200rpm to middle logarithmic growth phase, and culturing to obtain bacterial liquid OD₆₀₀0.5-1.0; IPTG is added to the final concentration of 0.2-0.5mM, and the induction expression is carried out for 4-8 h under the condition of 20-25 ℃.

More preferably, OD of the bacterial liquid₆₀₀0.5-0.7; IPTG is added to the final concentration of 0.2-0.5mM, and the induction expression is carried out for 5-6 h at the temperature of 25 ℃.

More preferably, the ApSK-containing is obtained₃The steps of recombining the plasmids include: obtaining the coded agapanthus dehydrated protein ApSK₃The base sequence of the ORF region of (1), the construction of ApSK-containing gene through enzyme digestion reaction₃The recombinant plasmid of (1).

More preferably, the encoded agapanthus dehydrin protein ApSK is obtained₃The base sequence of the ORF region of (cloning the entire length of the target gene) of (1) is as follows: obtaining two sections of African agapanthus ApSK according to the protein function annotation result of African agapanthus transcriptome sequencing (RNA-seq)₃The gene core fragment is detailed in sequence tables SEQID NO.1 and SEQID NO. 2; obtaining 648bp ORF region agapanthus ApSK by using agapanthus cDNA as template and through RACE gene full-length cloning method₃The base sequence of the protein is shown in a sequence table SEQID NO.3 in detail.

More preferably, the recombinant plasmid is pET21a-ApSK₃A plasmid; the Escherichia coli is Transetta (DE3) prokaryotic expression Escherichia coli.

Preferably, in step a2, the boiling water bath treatment conditions are: boiling at 100 deg.C for 20-60min, and shaking the centrifuge tube every 5-10min during boiling.

Preferably, in step a2, the step of centrifugally collecting is: centrifugation was carried out at 1200rpm for 10min at 4 ℃.

In a fourth aspect, the invention provides a plant vitrification ultra-low temperature preservation method based on exogenous protein addition, which comprises the following steps:

s1, plant seedling culture: sterilizing and culturing plant seeds, and taking plant seedlings which germinate for 36-72 h;

s2, treatment of the loading solution: placing the plant seedlings in a loading liquid for soaking treatment;

s3, vitrification solution treatment: placing the young plant treated in the step S2 in the vitrification solution for dehydration treatment;

s4, preserving by liquid nitrogen: and taking out the young plant treated in the step S3, and quickly storing the young plant in liquid nitrogen.

Preferably, the plant seed is arabidopsis; taking plant seedlings which germinate for 60 hours. The vitrification method ultralow temperature preservation system can be used for preserving 60h arabidopsis seedlings, and the preservation effect can be obviously improved.

Preferably, the cultivation of said arabidopsis seedling comprises the steps of: sterilizing Arabidopsis thaliana (Col-0) seed with 70% ethanol and 2% sodium hypochlorite, sowing in MS solid culture medium, performing vernalization at 4 deg.C for 48 hr, transferring into illumination incubator with photoperiod of 8-16 hr and illumination intensity of 150 μmol · m^-2·s^-1The day and night temperature is 25 ℃ and 20 ℃ respectively, and the humidity is 60-80%. Taking the germinated seedlings for 60h as samples to carry out vitrification method ultralow temperature preservation.

Preferably, in step S2, the vitrification solution is a mixture containing 20-40% w/v glycerol, 10-20% w/v ethylene glycol, 10-20% w/v dimethyl sulfoxide, 0.2-0.6mol/L sucrose and 0.5-2 μmol/L agapanthus dehydrated protein ApSK₃The MS culture solution of (1).

Preferably, in step S2, the loading liquid is MS culture liquid containing 2mol/L of glycerol and 0.4mol/L of sucrose; the soaking treatment conditions are as follows: soaking at room temperature for 15-30 min.

Preferably, in step S3, the conditions of the dehydration process are: soaking at 0-4 deg.C for 30-60 min. Under which is a acclimation process for low temperature dehydration of plant cells to increase stress resistance of the cells.

More preferably, in step S3, the conditions of the dehydration process are: soaking at 0 deg.C for 30-60 min.

Preferably, in step S4, the time for storage in liquid nitrogen is 1 hour or more.

Preferably, the plant vitrification cryopreservation method further comprises the steps of unfreezing, washing and re-culturing the seedlings after cryopreservation;

the unfreezing is to unfreeze in a water bath at the temperature of 30-50 ℃ for 60-120 s; when the water bath is unfrozen, shaking is adopted to accelerate the unfreezing;

the washing liquid adopted by the washing is MS culture solution containing 0.8-2.0mol/L of sucrose;

the recovery culture medium adopted by the re-culture is an MS solid culture medium containing 20-40g/L of sucrose.

In the present invention, the MS culture medium (see Murashige and Skoog, 1962) contained 1900mg/L KNO₃， 1650mg/L NH₄NO₃，170mg/L KH₂PO₄，370mg/L MgSO₄·7H₂O，440mg/L CaCl₂·2H₂O， 37.3mg/L Na₂-EDTA，27.8mg/L FeSO₄·7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H₃BO₃，22.3mg/L MnSO₄·4H₂O，8.6mg/L ZnSO₄·7H₂O，0.25mg/L Na₂MoO₄·2H₂O，0.025mg/L CuSO₄·5H₂O， 0.025mg/L CoCl₂·6H₂O and the balance of water. The pH of the MS culture solution is 5.6-6.2.

In the present invention, the MS solid medium contains 1900mg/L KNO₃，1650mg/L NH₄NO₃，170mg/L KH₂PO₄，370mg/L MgSO₄·7H₂O，440mg/L CaCl₂·2H₂O，37.3mg/L Na₂-EDTA，27.8mg/L FeSO₄·7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L saltThiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H₃BO₃，22.3mg/L MnSO₄·4H₂O，8.6mg/L ZnSO₄·7H₂O，0.25mg/L Na₂MoO₄·2H₂O，0.025mg/L CuSO₄·5H₂O，0.025mg/L CoCl₂·6H₂O, 30g/L of sucrose, 10g/L of agar powder and the balance of water. The pH value of the MS solid culture medium is 5.8-6.0.

The research shows that the dehydrin protein is one of the main stress-resistant proteins accumulated by plants under the conditions of abiotic stress such as cold, drought and the like, can stabilize and maintain the membrane system structure of cells and the conformation of other proteins, and maintain the normal physiological metabolism function of the cells. Dehydrin protein ApSK used in the invention₃Is expressed in large quantity by escherichia coli strains, and is further separated and purified to obtain target protein which is added into a vitrification solution to optimize an ultra-low temperature preservation system. And (4) treating the optimized ultralow-temperature preservation system by using a loading liquid and a vitrification solution in sequence, and finally carrying out ultralow-temperature preservation in liquid nitrogen. The other processes in the adding and matching method of the exogenous protective protein can effectively improve the preservation effect of the plant material. At the same time, the dehydrin protein ApSK₃Further proved to have the function of obviously eliminating ROS, and can effectively reduce the stress injury of the isolated cells in the ultralow temperature process.

Compared with the prior art, the invention has the following beneficial effects:

the method disclosed by the invention has the advantages that the ultralow-temperature preservation effect of the plant material is obviously optimized, and the dehydrin protein ApSK₃As an exogenous protein, when the concentration of the exogenous protein in the vitrification solution reaches 0.5-2 mu mol/L, the plant material under ultralow temperature composite stress can be effectively protected, and in addition, as an antioxidant function protein, the exogenous protein can effectively eliminate ROS and protect plant cells suffering from oxidative stress. Exogenously added dehydrin protein ApSK₃The original ultra-low temperature preservation system is optimized, the recovery growth rate of the plant germplasm resources after ultra-low temperature preservation is obviously improved, and the method has great application and popularization values for realizing the medium-long term in-vitro preservation of the excellent plant germplasm resources.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows the dehydrin protein ApSK added to the vitrification solution in example 2₃The concentration is increased, and the recovery growth rate of the wild type arabidopsis 60h seedlings is gradually increased;

FIG. 2 is a diagram showing the growth recovery of 60h seedlings of Arabidopsis; wherein, the picture (A) is a real photo of the growth recovery of the arabidopsis seedlings, and the picture (B) is a survival rate result picture of the arabidopsis seedlings;

FIG. 3 is the dehydrin protein ApSK₃A graph showing the results of the hydroxyl radical formation inhibition rate;

denotes a significant difference symbol, denotes P <0.05, denotes P < 0.01.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

The experimental procedures for which specific conditions are not noted in the following examples are generally conducted under conventional conditions or conditions recommended by the manufacturer.

The specific cultivation method of the arabidopsis seedlings used in the embodiment of the invention refers to the adversity response mechanism of the arabidopsis seedlings to vitrification cryopreservation, wily, Master academic thesis of Shanghai university of transportation, 2014.

The formula of the experimental reagent in the embodiment of the invention is as follows:

1) the MS culture solution is as follows: MS culture solution containing 1900mg/L KNO₃，1650mg/L NH₄NO₃，170mg/L KH₂PO₄，370mg/L MgSO₄·7H₂O，440mg/L CaCl₂·2H₂O，37.3mg/L Na₂-EDTA，27.8mg/L FeSO₄·7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H₃BO₃，22.3mg/L MnSO₄·4H₂O，8.6mg/L ZnSO₄·7H₂O，0.25mg/L Na₂MoO₄·2H₂O，0.025mg/L CuSO₄·5H₂O，0.025mg/L CoCl₂·6H₂O, the balance being water, and the pH of the MS culture solution being 5.8.

2) The MS solid culture medium is: MS solid culture medium containing 1900mg/L KNO₃，1650mg/L NH₄NO₃， 170mg/L KH₂PO₄，370mg/L MgSO₄·7H₂O，440mg/L CaCl₂·2H₂O，37.3mg/L Na₂-EDTA， 27.8mg/L FeSO₄·7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H₃BO₃，22.3mg/L MnSO₄·4H₂O， 8.6mg/L ZnSO₄·7H₂O，0.25mg/L Na₂MoO₄·2H₂O，0.025mg/L CuSO₄·5H₂O，0.025mg/L CoCl₂·6H₂O, 30g/L of sucrose, 10g/L of agar powder and the balance of water, wherein the pH value of the MS solid culture medium is 5.8.

3) The loading liquid comprises: MS culture solution containing 2mol/L glycerol and 0.4mol/L sucrose.

4) The vitrification solution is: contains 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v dimethyl sulfoxide, 0.4mol/L sucrose, 0.5-2 μmol/L ApSK₃MS culture solution of protein.

5) The washing liquid is: MS culture medium containing 1.2mol/L sucrose.

₃Example 1 prokaryotic expression and enrichment purification of the dehydrin protein ApSK

1. Obtaining the encoded dehydrin protein ApSK₃The base sequence of ORF region of

Using agapanthus cDNA as templateObtaining the coded agapanthus hygrophicus protein ApSK by a RACE method₃The base sequence of ORF region of (SEQ ID NO.3 of the sequence Listing).

2. Construction of prokaryotic expression vectors

1) According to ApSK₃The gene ORF sequence and the multiple cloning site in the plasmid select EcoRI and XholI restriction sites, and Primer pET-SK-S/A is designed by using Primer 5.0 software to perform PCR amplification of target fragment and introduce the restriction sites.

pET-SK-S (sequence listing SEQ ID NO.1): ATCAAGGAAAAGCTCGGC

pET-SK-A (sequence table SEQ ID NO.2): TCATCGTGGCTAGCACTCT

2) For pET21 plasmid and ApSK introduced with restriction enzyme cutting site₃The gene is subjected to double enzyme digestion, 2 Xligation mix is adopted to connect enzyme digestion products, the Ligation products are transformed into escherichia coli competence, and screening of positive monoclonal strains is carried out. And (4) selecting a monoclonal colony, and carrying out PCR detection on the bacterial liquid. And (3) sending the detected qualified bacterial liquid to Shanghai bio-organism company for sequencing, comparing a sequencing result with an ORF sequence, and extracting plasmids after confirming that the carrier is constructed without errors.

3) The successfully constructed pET21a-ApSK₃The plasmid is transferred into Transetta (DE3) prokaryotic expression escherichia coli, inoculated into 100mL LB liquid medium and cultured under the conditions of 37 ℃ and 200rpm shaking table until OD is in the middle logarithmic growth phase₆₀₀After culturing for about 0.5 to 0.7 hours, 1mL of the suspension was taken out of the EP tube and used as a pre-induction bacterial suspension sample. Expression of the protein ApSK₃The optimal induction conditions for E.coli of (1) are: adding 0.2-0.5mM IPTG, and inducing at 25 deg.C for 5-6 h.

4) And (3) taking 200mL of induced bacteria liquid, centrifugally collecting thalli, washing twice with sterile water, suspending, placing in a boiling water bath, boiling for 20-60min, cracking the thalli and removing non-heat stable protein, and shaking a centrifugal tube every 5min during the boiling water bath. Cooling to room temperature, centrifuging at 12000rpm for 10min at 4 deg.C, collecting the supernatant after boiling and cracking, i.e. purified ApSK₃And (5) detecting the purity of the protein by SDS-PAGE.

5) The protein concentration was determined by Coomassie Brilliant blue method and stored in a refrigerator at-80 ℃ for further use.

₃Example 2 addition of dehydrin protein ApSK to optimize vitrification cryopreservation System

1. And (3) culturing arabidopsis seedlings: sterilizing Arabidopsis seeds for 15s by using 70 percent ethanol, washing the Arabidopsis seeds with sterile water for 4 times, then sterilizing the Arabidopsis seeds with an aqueous solution containing 20 percent by weight of NaClO and 0.01 percent by weight of Tween 20 for 10min, and washing the Arabidopsis seeds with the sterile water for 6 times; then vernalizing the disinfected seeds for 48h at 4 ℃, and culturing on MS solid culture medium under the conditions of illumination culture for 8h every day and light intensity of 150 mu mol/m²The culture temperature is 25 ℃; and then culturing for 16h in the dark at the culture temperature of 20 ℃, and culturing the seeds for 60h to obtain the arabidopsis thaliana seedlings with the seedling age of 60 h.

2. Transferring the seedlings of arabidopsis thaliana which sprout for 60 hours to a loading solution, and soaking for 20min at room temperature;

3. removing the loading solution by suction, adding vitrification solution, and dehydrating at 0 deg.C for 50 min;

4. and putting the freezing pipe into liquid nitrogen for ultralow temperature preservation for more than 1 h.

And (3) after the step 3 is finished, directly placing the arabidopsis seedlings soaked in the vitrification solution in liquid nitrogen for ultralow temperature preservation without removing the vitrification solution.

The seedlings germinated in 60h from Arabidopsis were divided into experimental and control groups according to the above procedure, each group was set with 3 parallel experiments.

The vitrification solution of the experimental group 1 contains 1 mu mol/L ApSK₃A protein.

The vitrification solution of the experimental group 2 contains 2 mu mol/L ApSK₃A protein.

The control group differed in that no ApSK was included in the vitrification solution₃Proteins, others were the same as experimental groups.

5. Preserving in liquid nitrogen for more than 1h, taking out, quickly placing into 40 deg.C water bath, thawing for 90s, and gently shaking; removing the vitrification solution by suction, adding a washing solution, treating at room temperature for 40min, and replacing the washing solution every 10 min; and (4) after the washed arabidopsis seedlings are transferred to an MS solid medium for recovery culture for 10 days, calculating and comparing the recovery growth rates of arabidopsis in the experimental group and the control group.

ApSK added to vitrification solution₃The concentration is increased, the recovery growth rate of 60h seedlings of arabidopsis thaliana of 2 experimental groups is gradually improved, and when the concentration reaches 2 mu mol/L, ApSK₃The protective effect on the arabidopsis seedlings is obviously improved, and the recovery growth rate is improved from 23.7% to 46.6% (see figure 1). The specific effect is shown in fig. 2, wherein CK in fig. 2 represents the control group: 60h arabidopsis seedlings in ultralow temperature vitrification solution without adding ApSK₃A protein; . FIG. 2(A) shows the experimental group in which 2. mu. mol/L ApSK was added to the vitrification solution₃After an ultralow-temperature preservation system is optimized, the recovery growth of the arabidopsis seedlings is obviously better; FIG. 2(B) is a bar graph showing the protein ApSK when dehydrated in a vitrification solution₃When the concentration reaches 2 mu mol/L, the recovery growth rate of the plant material after being preserved at ultralow temperature can be greatly improved.

₃Example 3 verification that the dehydrin protein ApSK has protective effect on oxidation stress resistance

1. 0.2mL of 0.2mM FeSO₄Mixing with 0.2mL of 1mM bromopyrogallol, and adding 0.2mL of 0.5% H₂O₂。

2. Respectively adding BSA protein solution and ApSK into experimental groups₃The protein concentration of the protein solution is set to be 0.02, 0.05 and 0.1mg/mL in sequence; negative control group added 0.2mL of 0.5% H alone₂O₂No protein solution is added; blank group without addition of H₂O₂And a protein solution.

3. The absorbance of the sample was measured at a wavelength of 550 nm. The absorbance values of the experimental group and the negative control group are respectively As and A_CAbsorbance value of blank group is A₀。

4. Calculating the experimental groups BSA and ApSK₃The protein solution has a relative inhibition rate on the generation of hydroxyl radicals (OH & ltcndot & gt).

The results are shown in FIG. 3, ApSK₃Can obviously inhibit Fenton reaction and generation of hydroxyl radical, and the influence on the reaction is gradually enhanced along with the increase of protein concentration. ApSK at a concentration of 0.05mg/mL₃The formation of hydroxyl free radicals can be prevented by about 60 percent, and the inhibition rate is obviously higher than 9 percent of that of BSA. These results show that ApSK₃Under stress conditions, Fenton reaction can be inhibited, generation of hydroxyl free radicals is reduced, and the active effect is played in the ROS removing process. Further proving that ApSK₃In the process of ultralow temperature preservation, the damage of oxidative stress on plant materials can be reduced, the germplasm resource ultralow temperature preservation system is further optimized, and the survival rate of the plant materials subjected to recovery culture is improved. Has great application and popularization value.

The invention discloses an application of dehydrin protein in reducing stress injury and improving the effect of ultralow temperature preservation; the protein has the capability of eliminating active oxygen and reducing cell stress injury, can effectively improve the recovery growth rate of cells after freezing, and obviously improves the ultralow temperature preservation efficiency. The specific method is to apply prokaryotic expression method to enrich and purify agapanthus SK₃Dehydrin protein (ApSK)₃) Application of Fenton reaction to prove ApSK₃The ApSK is verified by utilizing a plant cryopreservation evaluation model under the regulation and control effect on ROS metabolism₃Improving effect on plant cell cryopreservation. The method specifically comprises the following steps: adding 2 mu mol/L ApSK into ultralow-temperature vitrification solution₃The protein improves the survival rate of the plant after freezing by about 1 time. The method disclosed by the invention has obvious optimization on eliminating active oxygen, reducing cell stress damage and improving the activity and the preservation efficiency of the cells after cryopreservation and freezing.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

SEQUENCE LISTING

<110> Shanghai university of transportation

Application of agapanthus SK3 dehydrin protein in reducing cell stress injury and improving ultralow temperature preservation effect

<130> DAG37812

<160> 3

<170> PatentIn version 3.5

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ccgccgtggt aaccgaacag gacgtggtcg tcgcggccgc ggccgccgaa gcggaagata 480

cgaccgtcgt cgtggagaag atcgaggaga ccgtcgtcgt ggagaagatc gaggaggaag 540

agaagaaagg gttcctcgat aagatcaaac agaagctccc cggccacaac aagaaggagg 600

ctgccgccgc cgccgaagtc gaggcgccgg cggcgaagga gagtgctagc cacgatgaag 660

gaggggagaa gaagggcatt ttcggaaaga tcatggacaa gataccaggg tatcacaagg 720

aggacaagga gaccgagaag gctcattaga ggaggttatt aattatgtgt cgctgtttat 780

tttatgtgtg tatgttttga atattaaatg tttgtgttga tcgagtgagt gctttggtta 840

ctgttttgtt tttgatttgt tagggttgtt tctttagtaa gttgagcatg caggatgtgt 900

atggagcttg cttttttgtg cgtggcaaca atcattttgt gttttaaaat gatgagagat 960

ggagtttgga gctaggttat gaatgaatgg ctggtgttga tgtatttttc catcgtggat 1020

aaatgtttta caaaaaaaaa aaaaaaaaaa aaaaaaa 1057

Claims (7)

1. The vitrification solution for plant vitrification cryopreservation is characterized by comprising 20-40% w/v of glycerol, 10-20% w/v of glycol, 10-20% w/v of dimethyl sulfoxide, 0.2-0.6mol/L of sucrose and 0.5-2 mu mol/L of agapanthus dehydrated protein ApSK₃The MS culture solution of (1), the agapanthus hygrophicus protein ApSK₃Is obtained by a method comprising the following steps:

a1, for expressing dehydrin protein ApSK₃Inducing the bacterial liquid, centrifuging and cleaning the induced bacterial liquid to obtain suspended bacterial liquid;

a2, boiling and cracking the suspended bacteria liquidAfter being cooled to room temperature, the mixture is centrifuged to collect supernatant fluid, namely the purified ApSK₃A protein sample.

2. The vitrification solution for plant vitrification cryopreservation of claim 1 where the ApSK expressing dehydrin protein is obtained in step a1₃The bacterial liquid preparation method comprises the following steps: will contain ApSK₃Of recombinant plasmidsTransettaInoculating the strain in LB liquid culture medium, culturing Escherichia coli at 37 deg.C and 200rpm to middle logarithmic growth phase, at which point the OD of the strain solution₆₀₀= 0.5-1.0; IPTG is added to the final concentration of 0.2-0.5mM, and the induction expression is carried out for 4-8 h under the condition of 20-25 ℃.

3. The vitrification solution for plant vitrification cryopreservation according to claim 1 wherein the conditions of the boiling water bath treatment in step a2 are: boiling at 100 deg.C for 20-60min, and shaking the centrifuge tube every 5-10min during boiling.

4. A plant vitrification ultra-low temperature preservation method based on exogenous protein addition is characterized by comprising the following steps:

s1, plant seedling culture: sterilizing and culturing plant seeds, and taking plant seedlings which germinate for 36-72 h;

s2, treatment of the loading solution: placing the plant seedlings in a loading liquid for soaking treatment;

s3, vitrification solution treatment: placing the young plant seedlings treated in the step S2 in the vitrification solution of claim 1 for dehydration treatment;

s4, preserving by liquid nitrogen: and taking out the young plant treated in the step S3, and quickly storing the young plant in liquid nitrogen.

5. The plant vitrification cryopreservation method based on the exogenously added protein as claimed in claim 4, wherein in step S2, the loading liquid is MS culture solution containing 2mol/L of glycerol and 0.4mol/L of sucrose; the soaking treatment conditions are as follows: soaking at room temperature for 15-30 min.

6. The vitrification cryopreservation method for plants based on the exogenously added protein as claimed in claim 4, wherein in step S3, the conditions of dehydration treatment are: soaking at 0-4 deg.C for 30-60 min;

in step S4, the time for storing in liquid nitrogen is 1 hour or more.

7. The vitrification cryopreservation method for plants based on the exogenously added protein as claimed in claim 4, further comprising the steps of thawing, washing and re-culturing the seedlings after cryopreservation;

the thawing is carried out in a water bath at the temperature of 30-50 ℃ for 60-120 s;

the washing liquid adopted by the washing is MS culture solution containing 0.8-2.0mol/L of sucrose;

the recovery culture medium adopted by the re-culture is an MS solid culture medium containing 20-40g/L of sucrose.

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