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A kind of moss 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 such as the shortage of raw materials for collapsing enzymes, affecting the research of Physcomitrella patens, and discontinuing the production of collapsing enzymes, so as to achieve easy purchase, Economical and efficient extraction, cheap effect

Active Publication Date: 2022-06-24
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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  • A kind of moss patens protoplast and preparation method thereof
  • A kind of moss patens protoplast and preparation method thereof
  • A kind of moss patens protoplast and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Preparation of Physcomitrella patens protoplasts

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

[0055] S1. Take the Physcomitrella patens protonema, add an equal volume of sterile water and make it into a homogenate with a homogenizer, transfer it to the BCDAT medium covered with cellophane, and suck out the water until the homogenate remains on the cellophane and does not flow but Keep it in a humid state, under the illumination temperature of 25 °C and the illumination intensity of 30 μmol m -2 s -1 Under the condition, 16h light-8h dark alternately cultivated for 5d;

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

[0057] S3. Take 2 g of the protoplas...

Embodiment 2

[0059] Example 2 Preparation of 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 make it into a homogenate with a homogenizer, transfer it to the BCDAT medium covered with cellophane, and suck out the water until the homogenate remains on the cellophane and does not flow. Keep it in a moist state, under the illumination temperature of 25 °C and the illumination intensity of 25 μmol m -2 s -1 Under the condition, 16h light-8h dark alternate culture for 4d;

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

[0063] S3. Take 2 g of the protonema obtained ...

Embodiment 3

[0065] Example 3 Preparation of Physcomitrella patens protoplasts

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

[0067] S1. Take the Physcomitrella patens protonema, add an equal volume of sterile water and make it into a homogenate with a homogenizer, transfer it to the BCDAT medium covered with cellophane, and suck out the water until the homogenate remains on the cellophane and does not flow but Keep it in a moist state, under the illumination temperature of 22 °C and the illumination intensity of 80 μmol m -2 s -1 Under the condition, 16h light-8h dark alternate culture for 7d;

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

[0069] S3. Take 2 g of the protoplasts obtained in step S...

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Abstract

The invention discloses a preparation method of Physcomitrella patens protoplasts. The method comprises the following steps: S1. Take Physcomitrella patens stems and leaf bodies or protoceles, beat them into a homogenate and put them in BCDAT medium for 16 hours of light ‑8h alternate cultivation in the dark; S2. Dissolve the mixed enzyme in 6%-10% mannitol solution, vortex and mix well, then centrifuge, take the supernatant and filter to obtain the mixed enzyme solution; S3. Take the protofilament obtained in step S1 Put the body into the mixed enzyme solution prepared in step S2 and mix evenly, enzymolyze, filter, centrifuge to remove the supernatant and resuspend with 6% to 10% mannitol solution, repeat 2 to 3 times, and the obtained suspension is Xiaoli Phytophthora protoplast; wherein, the mixed enzyme described in step S2 is any two or three of cellulase, hemicellulase or pectinase. The protoplast prepared by the method has high quality and good activity, ensures the regeneration efficiency under the premise of guaranteeing the quantity of the protoplast, and has simple preparation method and low cost, and has wide 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 the order Funariales, Funariaceae and Physcomitrium. Physcomitrella patens is extremely resistant to abiotic stresses such as drought, high temperature, and ultraviolet light. It has a high homologous recombination rate, strong regeneration ability, and a short life cycle. The haploid gametophyte stage predominates in the life history, and the genome has been sequenced. Physcomitrella patens can be cultured in liquid suspension and used for large-scale cultivation in semi-continuous photoautotrophic bioreactors for the production of vaccines, human serum albumin and other drugs. It has very high basic research and application value and is a synthetic biology ideal...

Claims

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Application Information

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Patent Type & Authority Patents(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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