CN106893690B

CN106893690B - Preparation method of Gracilaria chilies protoplast

Info

Publication number: CN106893690B
Application number: CN201611230340.9A
Authority: CN
Inventors: 刘涛; 金月梅
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2016-12-27
Filing date: 2016-12-27
Publication date: 2020-04-07
Anticipated expiration: 2036-12-27
Also published as: CN106893690A

Abstract

The invention relates to a preparation method of Gracilaria chilies protoplasts, belonging to the field of algae cell engineering. The preparation method comprises the steps of preparing tissue enzyme solution, carrying out Gracilaria chilgii tissue enzymolysis and the like, wherein the tissue enzyme solution preparation steps are as follows: homogenizing and crushing with 1-10 times of sea water of sea cucumber intestine volume at 0 deg.C; leaching at 4 deg.C for 4-18h, adjusting pH of the extractive solution to 7.0-8.0, freezing and centrifuging for 5min, and collecting supernatant to obtain Stichopus japonicus enzyme solution; mixing the sea cucumber enzyme solution, cellulase and seawater to obtain tissue enzyme solution.

Description

Preparation method of Gracilaria chilies protoplast

Technical Field

The invention relates to a preparation method of Gracilaria chilies protoplasts, belonging to the field of algae cell engineering.

Background

Plant protoplasts are viable cells with their cell walls removed and surrounded by a plasma membrane, and their structures include cell membranes, cytoplasms (including various organelles, cytoskeletal systems, and cellular matrices), and cell nuclei. The protoplast technology is a series of technical operations carried out on the basis of the separation culture of protoplasts, and mainly comprises the cultivation and variation of new biological technologies such as protoplast plant regeneration, protoplast fusion and cell hybridization, transgenosis and the like. The protoplast culture and plant regeneration technology has the advantages of wide application range, strong feasibility and the like, and is the basis of plant bioengineering.

The preparation of the plant protoplast comprises the selection and pretreatment of materials, the tissue decomposition and the primary dissociation of the protoplast as well as the purification and the activity detection of the protoplast. The selection of appropriate materials is the basis for successful protoplast isolation; the pretreatment of the material before enzymolysis or mechanical treatment can improve the wall breaking efficiency and reduce the damage of protoplasm; reasonable enzyme preparation selection, concentration regulation and osmotic pressure regulator selection are all the keys for improving the protoplasm yield and activity. Although culture techniques for protoplasts have been rapidly developed in recent years, there are problems such as low protoplast viability and long culture period.

Gracilaria chilblains has the characteristics of fast growth and high temperature resistance, and is one of potential cultivated varieties in the south. Currently, gracilaria is mainly propagated by means of vegetative reproduction. The breeding of the seedlings is greatly influenced by environmental factors such as temperature, seawater salinity and the like, and has instability. The protoplast has totipotency, can be rapidly propagated in large quantities under the condition of manual control, and can relieve the breeding pressure of seedlings. In addition, the cell wall of Gracilaria contains thicker colloid, so that genetic operations such as cell fusion, cell hybridization and the like cannot be directly carried out, and protoplasts obtained by utilizing a conventional enzymatic lysis method in the prior art are often low in activity and unsatisfactory in yield.

CN2016101618265 discloses a method for preparing a gracilaria salicornia protoplast, which comprises subjecting gracilaria salicornia tissues to enzymolysis with abalone digestive gland enzyme solution to prepare the protoplast. However, the invention discovers that the enzymolysis effect of the enzyme solution derived from abalone digestive tissue on Gracilaria chilgii tissue is not good.

Disclosure of Invention

The invention aims to provide a preparation method of Gracilaria chilgii protoplasts. Meanwhile, according to the totipotency of the somatic cell development of the seaweed, the gracilaria chilblains tissue is decomposed by using tool enzyme to free a somatic cell protoplast, and an ideal receptor system can be provided for genetic engineering research.

In one embodiment, the preparation method comprises the steps of preparing tissue enzyme solution, enzymolyzing gracilaria chile tissue and the like, wherein the steps of preparing the tissue enzyme solution are as follows: homogenizing and crushing with 1-10 times of sea water of sea cucumber intestine volume at 0 deg.C; leaching at 4 deg.C for 4-18h, adjusting pH of the extractive solution to 7.0-8.0, freezing and centrifuging for 5min, and collecting supernatant to obtain Stichopus japonicus enzyme solution; mixing the sea cucumber enzyme solution, cellulase and seawater to obtain tissue enzyme solution.

In one embodiment, the tissue enzyme solution is prepared by the steps of: homogenizing and crushing with 2 times of sea water of sea cucumber intestine volume at 0 deg.C; extracting at 4 deg.C for 12h, adjusting pH of the extractive solution to 7.6, freezing and centrifuging for 5min, and collecting supernatant to obtain holothurian enzyme solution; mixing the sea cucumber enzyme solution, cellulase and seawater to obtain tissue enzyme solution.

In a further embodiment, the tissue enzyme solution specifically comprises 2.4ml cellulase, 0.5ml holothurian enzyme solution and 17.1ml sterilized seawater.

In another embodiment, the gracilaria chile tissue enzymolysis step is to take gracilaria chile tissue, cut algae bodies with a scalpel, add prepared tissue enzyme solution for enzymolysis, then filter with 200-mesh bolting silk, remove undigested cells, cell clusters and fragments, remove supernatant fluid for centrifugation, centrifuge at 800rpm for 10min, sink single-cell protoplast, leave cell fragments in the supernatant fluid, discard the supernatant fluid, wash and centrifuge for 2-3 times with sterilized seawater containing 1mol/L glucose, collect the precipitate and suspend to 2ml, thus obtaining the protoplast.

In one embodiment, the enzymolysis condition is that the algae body tissue added with the tissue enzyme solution is placed on a constant temperature shaking bed at 25 ℃ for dark enzymolysis for 5 hours.

Drawings

FIG. 1: fluorescence detection of protoplasts (× 20 fold) left: a protoplast; and (3) right: protoplasts after fluorescent staining showing a red color

FIG. 2: microscopic image of protoplast cultured for 12 days

Detailed Description

The invention may be further understood by reference to the following examples, which illustrate some methods of making or using. However, it is to be understood that these examples do not limit the present invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.

Example 1

Preparing a tissue enzyme solution: homogenizing and crushing with 2 times of sea water of sea cucumber intestine volume at 0 deg.C; extracting at 4 deg.C for 12h, adjusting pH of the extractive solution to 7.6, freezing and centrifuging for 5min, and collecting supernatant to obtain holothurian enzyme solution; mixing the sea cucumber enzyme solution, cellulase and seawater to obtain tissue enzyme solution. The tissue enzyme solution specifically comprises 2.4ml of cellulase, 0.5ml of sea cucumber enzyme solution and 17.1ml of sterilized seawater.

Preparation of protoplast: taking 2g of Chilean hedgerow material, sucking with absorbent paper, cutting algae with a scalpel (the more broken the algae are better), adding the prepared tissue enzyme solution, and placing on a constant temperature shaking table at 25 ℃ for dark enzymolysis. Every 30min, observation and photographing. And 5h, filtering by using a 200-mesh bolting silk, removing undigested cells, cell clusters and fragments, removing supernatant, centrifuging for 10min at 800rpm, sinking the single-cell protoplast, leaving the cell fragments in the supernatant, discarding the supernatant, washing and centrifuging for 2-3 times by using sterile seawater containing 1mol/L glucose, collecting the precipitate, and suspending to 2ml to obtain the protoplast.

Example 2

Detection of protoplasts:

measurement of cell density: cell density measurement with a hemocytometer

The specific results are as follows:

comparative example 1 is a preparation method of sea cucumber enzyme solution in tissue enzyme solution, which is to use 2 times of sea water with sea cucumber intestine volume to homogenize and crush at 0 ℃; extracting at 4 deg.C for 12h, adjusting pH to 7.0, freezing and centrifuging for 5min, and collecting supernatant to obtain holothurian enzyme solution; the preparation methods of other tissue enzyme solutions and protoplasts were the same as those in example 1 of the present invention.

Comparative example 2 is a preparation method of sea cucumber enzyme solution in tissue enzyme solution, which is to use 2 times of sea water with sea cucumber intestine volume to homogenize and crush at 0 ℃; extracting at 4 deg.C for 12h, adjusting pH to 8.0, freezing and centrifuging for 5min, and collecting supernatant to obtain holothurian enzyme solution; the preparation methods of other tissue enzyme solutions and protoplasts were the same as those in example 1 of the present invention.

Comparative example 3 the tissue enzyme solution consisted of 2.4ml cellulase, 0.5ml abalone enzyme solution and 17.1ml sterile seawater; the preparation method of abalone enzyme solution is shown in example 1 in the specification of CN2016101618265, and the preparation methods of other tissue enzyme solution and protoplast are the same as the preparation method of example 1.

Comparative example 4 is a tissue enzyme solution specifically composed of 2.0ml cellulase, 0.9ml sea cucumber enzyme solution and 17.1ml sterilized seawater, and other tissue enzyme solutions and protoplasts were prepared in the same manner as in example 1 of the present invention.

Comparative example 5 is a tissue enzyme solution specifically composed of 2.7ml cellulase, 0.2ml sea cucumber enzyme solution and 17.1ml sterilized seawater, and other tissue enzyme solutions and protoplasts were prepared in the same manner as in example 1 of the present invention.

And (3) survival rate determination: staining with 0.5% Evans Blue gave bright yellow color for live protoplasts and dark Blue for dead cells. The survival rate of live protoplasts was calculated by randomly selecting 5 fields under the microscope, and the specific results were as follows:

example 3

The protoplasts prepared in example 1 were collected and cultured in hypertonic seawater under low light with illumination intensity of 100-.

As shown in FIG. 2, after 42 hours of culture, the protoplasts had not yet formed intact cell walls, and after 4 days of culture, most of the protoplasts had regenerated the cell walls. After 7 days of culture, the cells had already divided into a plurality of cells and arranged regularly, and after 12 days of culture, the number of cells increased and arranged in different shapes.

This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.

Claims

1. A preparation method of Gracilaria chilies protoplasts comprises the steps of preparing tissue enzyme solution, carrying out enzymolysis on Gracilaria chilies tissue and the like, wherein the tissue enzyme solution preparation steps are as follows: homogenizing and crushing with 1-10 times of sea water of sea cucumber intestine volume at 0 deg.C; leaching at 4 deg.C for 4-18h, adjusting pH of the extractive solution to 7.0-8.0, freezing and centrifuging for 5min, and collecting supernatant to obtain Stichopus japonicus enzyme solution; mixing the sea cucumber enzyme solution, cellulase and seawater to obtain tissue enzyme solution;

the tissue enzyme solution specifically comprises 2.4ml of cellulase, 0.5ml of sea cucumber enzyme solution and 17.1ml of sterilized seawater.

2. The method according to claim 1, wherein the tissue enzyme solution is prepared by: homogenizing and crushing with 2 times of sea water of sea cucumber intestine volume at 0 deg.C; extracting at 4 deg.C for 12h, adjusting pH of the extractive solution to 7.6, freezing and centrifuging for 5min, and collecting supernatant to obtain holothurian enzyme solution; mixing the sea cucumber enzyme solution, cellulase and seawater to obtain tissue enzyme solution.

3. The preparation method of claim 1, wherein the enzymolysis step of Gracilaria chilies tissue comprises cutting the Gracilaria chilies tissue with a scalpel, adding the prepared tissue enzyme solution for enzymolysis, then filtering with 200 mesh bolting silk, removing undigested cells, cell clusters and fragments, removing supernatant for centrifugation, centrifuging at 800rpm for 10min, allowing single cell protoplast to sink, leaving the cell fragments in the supernatant, discarding the supernatant, washing with sterilized seawater containing 1mol/L glucose for centrifugation for 2-3 times, collecting the precipitate, and suspending to 2ml, thus obtaining the protoplast.

4. The preparation method according to claim 3, wherein the enzymolysis condition is that the alga body tissue added with the tissue enzyme solution is put on a constant temperature shaking table at 25 ℃ for dark enzymolysis for 5 hours.