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Physcomitrella patens protoplast and preparation method thereof

A technology of Physcomitrella patens and protoplasts, which is applied in the field of Physcomitrella patens protoplasts and its preparation, can solve problems affecting the research of Physcomitrella patens, stop production of collapsing enzymes, and shortage of raw materials for collapsing enzymes, and achieve economical and efficient extraction , easy to purchase, and sufficient production

Active Publication Date: 2020-11-17
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the shortage of raw materials for the collapse enzyme, the production of the collapse enzyme was discontinued, which affected the research of domestic and foreign researchers on Physcomitrella patens

Method used

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  • Physcomitrella patens protoplast and preparation method thereof
  • Physcomitrella patens protoplast and preparation method thereof
  • Physcomitrella patens protoplast and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The preparation of embodiment 1 Physcomitrella patens protoplast

[0054] A preparation method of Physcomitrella patens protoplasts, comprising the following steps:

[0055] S1. Take the protonema of Physcomitrella patens, add an equal volume of sterile water and beat it into a homogenate through a homogenizer, then transfer it to the BCDAT medium covered with cellophane, suck out the water until the homogenate remains on the cellophane without flowing but Keep in a wet state, at a light temperature of 25°C and a light intensity of 30μmol·m -2 the s -1 Under the conditions, 16h light-8h dark alternately cultivated for 5d;

[0056] S2. Dissolve cellulase, hemicellulase and pectinase in 8% mannitol solution, vortex at 2°C for 30 minutes, then centrifuge at 2500×g for 5 minutes, take the supernatant and filter it with a 0.22um bacterial filter membrane , to obtain a mixed enzyme solution with a final concentration of 1.2%;

[0057] S3. Take 2 g of the protocelium obtai...

Embodiment 2

[0059] The preparation of embodiment 2 Physcomitrella patens protoplasts

[0060] A preparation method of Physcomitrella patens protoplasts, comprising the following steps:

[0061] S1. Take the stem and leaf body of Physcomitrella patens, add an equal volume of sterile water and beat it into a homogenate through a homogenizer, then transfer it to the BCDAT medium covered with cellophane, suck out the water until the homogenate remains on the cellophane without flowing but Keep in a wet state, at a light temperature of 25°C and a light intensity of 25 μmol m -2 the s -1 Under the conditions, 16h light-8h dark alternately cultivated for 4d;

[0062] S2. Dissolve cellulase, hemicellulase and pectinase in 10% mannitol solution, vortex at 4°C for 32 minutes, then centrifuge at 2600×g for 6 minutes, take the supernatant and filter it with a 0.24um bacterial filter membrane , to obtain a mixed enzyme solution with a final concentration of 1.5%;

[0063] S3. Take 2 g of the proto...

Embodiment 3

[0065] The preparation of embodiment 3 Physcomitrella patens protoplasts

[0066] A preparation method of Physcomitrella patens protoplasts, comprising the following steps:

[0067] S1. Take the protonema of Physcomitrella patens, add an equal volume of sterile water and beat it into a homogenate through a homogenizer, then transfer it to the BCDAT medium covered with cellophane, suck out the water until the homogenate remains on the cellophane without flowing but Keep in a wet state, at a light temperature of 22°C and a light intensity of 80 μmol m -2 the s -1 Under the conditions, 16h light-8h dark alternately cultured for 7d;

[0068] S2. Dissolve cellulase, hemicellulase and pectinase in 6% mannitol solution, vortex at 0°C for 28 minutes and mix well, then centrifuge at 2400×g for 4 minutes, take the supernatant and filter it with a 0.2um bacterial filter membrane , to obtain a mixed enzyme solution with a final concentration of 0.8%;

[0069] S3. Take 2 g of the proto...

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Abstract

The invention discloses a preparation method of physcomitrella patens protoplast. The preparation method comprises the following steps of S1, taking physcomitrella patens cormus or protonema, performing homogenization to obtain homogenate, and putting the homogenate into a BCDAT culture medium for alternate culture of 16-hour illumination and 8-hour dark treatment; S2, dissolving a mixed enzyme ina 6-10% mannitol solution, carrying out uniform vortex mixing, performing centrifuging, taking supernatant, and performing filtering to obtain a mixed enzyme solution; and S3, putting the protonema obtained in the step S1 into the mixed enzyme solution prepared in the step S2, performing uniform mixing, carrying out enzymolysis, performing filtering, performing centrifuging to remove supernatant,performing resuspending by using the 6-10% mannitol solution, and repeating the process for 2-3 times to obtain a suspension, namely the physcomitrella patens protoplast, wherein the mixed enzyme inthe step S2 is any two or three of cellulase, hemicellulase and pectinase. The protoplast prepared by the method is high in quality and good in activity, the regeneration efficiency is guaranteed on the premise that the number of protoplasts is guaranteed, and the preparation method is simple, low in cost and wide in application prospect.

Description

technical field [0001] The invention belongs to the technical field of protoplast preparation. More specifically, it relates to a Physcomitrella patens protoplast and a preparation method thereof. Background technique [0002] Physcomitrella patens (Pp) is a pioneer plant from aquatic to terrestrial, belonging to Funariales, Funariaceae, and Physcomitrium. Physcomitrella patens is extremely resistant to abiotic stresses such as drought, high temperature, and ultraviolet rays. It has a high rate of homologous recombination, strong regeneration ability, and a short life cycle. The haploid gametophyte stage is dominant in its life history, and its genome has been sequenced. Physcomitrella patens can be cultured in liquid suspension, used for large-scale cultivation in semi-continuous photoautotrophic bioreactors, and used to produce vaccines, human serum albumin and other drugs. It has very high basic research and application value, and is a synthetic biology ideal model orga...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 赵孟楷李晓政戴俊彪胡章立
Owner SHENZHEN UNIV
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