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Agapanthus sk 3 Application of dehydrin in reducing cell stress damage and improving cryopreservation effect

A dehydrin protein, vitrification ultra-low temperature technology, applied in the fields of application, plant preservation, botany equipment and methods, etc., can solve the problems of in vitro development and application without cells, achieve significant effect of ultra-low temperature preservation, remove ROS, and improve growth recovery rate effect

Active Publication Date: 2021-05-28
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The special protective function of dehydrin has important application potential, but it has not been developed and applied in vitro in related technical fields.

Method used

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  • Agapanthus sk <sub>3</sub> Application of dehydrin in reducing cell stress damage and improving cryopreservation effect
  • Agapanthus sk <sub>3</sub> Application of dehydrin in reducing cell stress damage and improving cryopreservation effect
  • Agapanthus sk <sub>3</sub> Application of dehydrin in reducing cell stress damage and improving cryopreservation effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Dehydrin ApSK 3 Prokaryotic expression and enrichment purification

[0062] 1. Obtain the coding dehydrin protein ApSK 3 The base sequence of the ORF region

[0063] Using Agapanthus japonicus cDNA as a template, the encoding protein ApSK of Agapanthus abaciens was obtained by RACE method 3 The base sequence of the ORF region (sequence listing SEQ ID NO.3).

[0064] 2. Construction of prokaryotic expression vector

[0065] 1) According to ApSK 3 For the ORF sequence of the gene and the multiple cloning site in the plasmid, EcoRI and XholⅠ restriction sites were selected, and the primer pET-SK-S / A was designed using Primer 5.0 software, and the target fragment was amplified by PCR and the restriction site was introduced.

[0066] pET-SK-S (SEQID NO.1 of the sequence listing): ATCAAGGAAAAGCTCGGC

[0067] pET-SK-A (SEQID NO.2 of the sequence listing): TCATCGTGGCTAGCACTCT

[0068] 2) For the pET21 plasmid and the ApSK after introducing the restriction sit...

Embodiment 2

[0072] Embodiment 2 adds dehydrin protein ApSK 3 , optimize the vitrification cryopreservation system

[0073] 1. Cultivation of Arabidopsis seedlings: Disinfect Arabidopsis seeds with 70% ethanol for 15 seconds, rinse them with sterile water for 4 times, then disinfect them with an aqueous solution containing 20 wt% NaClO and 0.01 wt% Tween 20 for 10 min, rinse them with sterile water 6 times; then the sterilized seeds were vernalized at 4°C for 48 hours and cultured on MS solid medium. 2 , at a culture temperature of 25° C.; then cultivated in the dark for 16 h at a culture temperature of 20° C., and cultured the seeds for a total of 60 h to obtain Arabidopsis seedlings with a seedling age of 60 h.

[0074] 2. Transfer Arabidopsis thaliana seedlings germinated for 60 hours to the loading solution for soaking at room temperature for 20 minutes;

[0075] 3. Absorb the loading solution, add vitrification solution, and dehydrate at 0°C for 50 minutes;

[0076] 4. Put the fr...

Embodiment 3

[0085] Example 3 Validation of Dehydrin ApSK 3 Protects against oxidative stress

[0086] 1. Add 0.2mL 0.2mM FeSO 4 Mix with 0.2mL 1mM bromopyrogallol, then add 0.2mL 0.5% H 2 o 2 .

[0087] 2. Add BSA protein solution and ApSK to the experimental group respectively 3 Protein solution, the protein concentration was set to 0.02, 0.05 and 0.1mg / mL in turn; the negative control group only added 0.2mL 0.5% H 2 o 2 , no protein solution was added; the blank group did not add H 2 o 2 and protein solution.

[0088] 3. Measure the absorbance value of the sample at a wavelength of 550nm. The absorbance value of the experimental group was As, and the absorbance value of the negative control group was A C , the absorbance value of the blank group is A 0 .

[0089] 4. Calculate the BSA and ApSK of the experimental group 3 Relative inhibition rate of protein solution to hydroxyl radical (OH·) generation.

[0090]

[0091] The result is as image 3 As shown, ApSK 3 It c...

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Abstract

The invention discloses a kind of Agapanthus SK 3 The application of dehydrin in reducing cell stress damage and improving the effect of cryopreservation; this protein has the ability to remove active oxygen and reduce cell stress damage, can effectively improve the growth rate of cells after freezing, and significantly improve the efficiency of cryopreservation. The specific method is to apply the method of prokaryotic expression to enrich and purify Agapanthus SK 3 type dehydrin protein (ApSK 3 ), using the Fenton reaction to prove that ApSK 3 The regulatory effect on ROS metabolism, using the plant cryopreservation evaluation model to verify ApSK 3 Improved effect on cryopreservation of plant cells. Specifically include: adding 2 μmol / L ApSK to the ultra-low temperature vitrification solution 3 protein, which increases the survival rate of plants after freezing by about 1 times. The method disclosed in the present invention is significant for removing active oxygen, reducing cell stress damage, improving cell activity after cryopreservation and optimizing storage efficiency.

Description

technical field [0001] The invention relates to the field of preservation of plant materials, in particular to a kind of Agapanthus SK 3 The application of dehydrin in reducing cell stress damage and improving cryopreservation effect can reduce plant cell stress damage, thereby optimizing plant vitrification cryopreservation effect. Background technique [0002] Vitrification cryopreservation (Vitrification Cryopreservation) refers to the treatment of plant germplasm resources with a vitrification solution (Plant Vitrification Solution, PVS) composed of a certain proportion of permeable and non-permeable protective agents, and then quickly put into liquid nitrogen (-196 ° C). ) a set of cryobiological techniques for preservation. Cells or tissues are placed in a vitrification solution composed of a certain proportion of permeable and non-permeable protective agents, so that the material and its vitrified solution solidify into a non-crystalline vitrified state at a sufficie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N3/00
CPCA01N3/00
Inventor 张荻吕珊吕可杨舟
Owner SHANGHAI JIAOTONG UNIV
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