CN1560227A - Preparation and regenerating technology for protoplasm of rainy red ball alga - Google Patents
Preparation and regenerating technology for protoplasm of rainy red ball alga Download PDFInfo
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- CN1560227A CN1560227A CNA2004100075358A CN200410007535A CN1560227A CN 1560227 A CN1560227 A CN 1560227A CN A2004100075358 A CNA2004100075358 A CN A2004100075358A CN 200410007535 A CN200410007535 A CN 200410007535A CN 1560227 A CN1560227 A CN 1560227A
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Abstract
The invention relates to a microalgae, especially Haematococcus pluvialis protoplast preparing and regenerating technique. It uses a preprocessing agent prepared by acidic buffer solution, EDTA and dithiothreitol (DTT) to process microalgae cells at 25-34 deg.C for 30-60 min; uses a compound high-permeability enzyme composed of NaCl, KCl, MgSO4, CaCl2, cellulose, snailase, pectase and acidic buffer solution to eliminate cell walls so as to make the microalgae protoplast; using a storage solution composed of BBM culture solution, glucose and sucrose to store the protoplast, diluting and separating on to a regenerative culture medium containing 0.1mol/L glucose, 0.1mol/L sucrose and 0.1-0.5ppm melissyl alcohol for regenerative culture; keeping relative humidity greater than 60% and the temperature at 25 deg.C, and keeping illumination. The prepared protoplast is transparent and has high activity and birth colors and relatively circular in shape, the regenerative cycle can be shortenedby 1/3, the regeneration ratio is increased by 70%, and it can used for protoplast fusion, gene conversion, etc.
Description
The present invention relates to little algae protoplastis preparation and regeneration techniques in the cell engineering, relate to Haematococcus pluvialis protoplast preparing and regenerating technique specifically.
Astaxanthin is a kind of high-quality natural pigment, and its resistance of oxidation is stronger than β-Hu Luobusu, has physiological actions such as the tumour of inhibition and raise immunity, and extensive use is all arranged on medicine, food, herding and aquatic products.Haematocoocus Pluvialls becomes little algae of focus development gradually as the highest biology of occurring in nature content astaxanthin.But because it is the autotrophic type biology, thus poor growth, biomass is low, the production cost height, and industrial scale and market are restricted.Protoplast Technique, particularly protoplastis merge, and can exchange the genetic recombination material between sibship parents far away, can also exchange and the reconstitution cell cytoplasmic inheritance.The heterotrophic microorganism that Haematocoocus Pluvialls is fast with growth, output is high is merged hybridization, and the seed selection superior progeny remedies himself deficiency, is the desirable approach of exploitation Haematocoocus Pluvialls.Protoplastis preparation and regeneration are precondition and the gordian techniquies that merges.Therefore, Haematococcus pluvialis protoplast preparing and regeneration techniques have theory and practical value.
Show present domestic still do not have Haematococcus pluvialis protoplast preparing and regenerated report from the pertinent data retrieval.The Agus Eko Tjahjono of external rarely seen Japan etc. are at Journal of fermentation and bioengin-eering.1993,75 (3): report among the 196-200, its preparation method is: centrifugal collection microalgae cell, add the enzymolysis solution that contains 0.06% Proteinase K, 0.2mol/L sorbyl alcohol and N.F,USP MANNITOL, handle 15min down in 35 ℃, acquisition is to the protoplastis of osmotic pressure sensitivity, and the highest preparation rate is about 70%, and regeneration rate is indeterminate.And there are several problems in this method: with Proteinase K is cytohydrolist, and major ingredient such as Mierocrystalline cellulose in the pair cell wall and pectin substance do not have effect, go wall not thorough, influence syncretizing effect; With 0.2mol/L N.F,USP MANNITOL or 0.2mol/L sorbyl alcohol simplification compound is hypertonic solution, produces because of concentration is high and poisons, and reduces cytoactive, influences protoplast regeneration; The regeneration culture medium composition is identical with the ordinary culture medium composition, and regeneration efficiency is low.
Purpose of the present invention is exactly to have lytic enzyme cost height in order to overcome existing method, and the protoplasm somatocyte wall of preparation is residual too much, and influence is merged, hypertonic solution concentration height, poison big, problem such as the protoplastis activity is low, and regenerative power is weak.
The main technical schemes that adopts of the present invention for achieving the above object:
(1) pretreating agent that is made into acid (citric acid+Trisodium Citrate) damping fluid (pH4.0), ethylenediamine tetraacetic acid (EDTA) (EDTA) and dithiothreitol (DTT) (DTT) etc. soaks cell, destroy the cell walls dense structure, quicken lytic enzyme and cell walls contact area, shorten the time that discharges protoplastis, improve activity and regenerative power.
(2) lytic enzyme is prepared from by cellulase, helicase and polygalacturonase, is dissolved in compound hypertonic solution (NaCl 0.1mol/L, KCl 0.1mol/L, MgSO
40.02mol/L, CaCl
20.01mol/L) in, keep the about 0.2mol/L of solution total concn.Because every kind of salt concn is relatively low, thereby alleviated the hypertonic solution pair cell effectively and poisoned degree, and then improved protoplastis significantly and prepare rate, activity and regenerative power.
(3) add triacontanol price quote in the regeneration culture medium, more help promoting cell regeneration and division, shorten the protoplast regeneration time, improve regeneration efficiency.
Prescription of the present invention:
1, the preparation of pretreating agent: in the acidic buffer (citric acid+Trisodium Citrate) that ethylenediamine tetraacetic acid (EDTA) and the 25～45mmol/L dithiothreitol (DTT) of 30～50mmol/L is dissolved in pH4.0.
2, compound hypertonic solution preparation: be made into the phosphoric acid buffer of pH6.5 and contain NaCl 0.1mol/L, KCl0.1mol/L, MgSO
40.02mol/ and CaCl
20.01mol/L compound hypertonic solution.
3, height oozes the preparation of prozyme liquid: add multiple lytic enzyme with aforesaid compound hypertonic solution and be mixed with height and ooze prozyme liquid, wherein cellulase 1～2%, helicase 0.5～1.5%, polygalacturonase 0.3～1.0%, compound hypertonic solution 95.5～98%.
4, preserve the preparation of liquid: be made into the BBM nutrient solution of routine and contain glucose 0.1mol/L, sucrose 0.1mol/L and CaCl
20.01mol/L preservation liquid.
5, regeneration culture medium: be made into the regeneration culture medium that contains triacontanol price quote 0.1～0.5ppm with aforementioned preservation liquid.
Operating process of the present invention is:
Collect the logarithmic phase frustule of cultivating 4 days, with acidic buffer (pH4.0), 30～50mmol/L ethylenediamine tetraacetic acid (EDTA) and the formulated pretreating agent of 25～45mmol/L dithiothreitol (DTT), 25～34 ℃ of constant temperature are handled 30～60min.Change height then over to and ooze in the prozyme liquid, 25～35 ℃, the constant temperature vibration, cell walls is removed in hydrolysis.Centrifugal collection gained protoplastis behind the 3h, dilution is separated on the regeneration culture medium flat board, in 25 ℃ of illumination cultivation, falls until the regeneration algae occurring.
The present invention is easy and simple to handle, and equipment requirements is low, and prepared Haematocoocus Pluvialls protoplastis is transparent, and the vigor height has comparatively chromatic colour, and form is round, and protoplastis prepares rate and surpasses 80%; Protoplastis preparation and reproduction speed are fast, can shorten 1/3rd, and regeneration rate improves more than 70%.The protoplastis that utilizes the present invention to prepare can satisfy the requirement that protoplastis merges fully, and fusion rate is relative with regeneration rate higher.
The invention will be further described below in conjunction with example.
Embodiment 1:
Get the logarithmic phase Haematocoocus Pluvialls nutrient solution 5mL that cultivated 4 days, the centrifugal 10min of 3500r/min, after the washing of equivalent sterilized water, centrifugal collection microalgae cell, add pretreating agent (the 50mmol/L EDTA that 5mL prepares in advance, the 25mmol/L dithiothreitol (DTT), the pH4.0 acidic buffer is formulated), handle 60min for 34 ℃, centrifugal collection frustule, the compound height of adding 5mL oozes enzyme liquid and (contains 0.1mol/L NaCl, 0.1mol/LKCl, 0.02mol/L MgSO
4, 0.01mol/L CaCl
2, 2.0% cellulase, 0.5% helicase, 1.0% polygalacturonase, pH6.5 phosphoric acid buffer), 25 ℃, the 60r/min vibration, hydrolysis broken wall 3h, centrifugal collection protoplastis is with containing 0.1mol/L glucose, 0.1mol/L sucrose and 0.01mol/L CaCl
2Preservation liquid dilution, be separated on the regeneration culture medium that contains the 0.5ppm triacontanol price quote, 25 ℃ of following illumination cultivation fall until the regeneration algae occurring.
Example 2:
Get the logarithmic phase Haematocoocus Pluvialls nutrient solution 5mL that cultivated 4 days, the centrifugal 10min of 3500r/min, after the washing of equivalent sterilized water, centrifugal collection microalgae cell, add 5mL pretreating agent (40mmol/L EDTA, the 35mmol/L dithiothreitol (DTT), the pH4.0 citrate buffer solution is formulated), handle 30min for 28 ℃, centrifugal collection frustule, adding 5mL height oozes composite enzyme solution and (contains 0.1mol/L NaCl, 0.1mol/L KCl, 0.02mol/L MgSO
4, 0.01mol/L CaCl
2, 1.5% cellulase, 1.0% helicase, 0.5% polygalacturonase, pH6.5 phosphoric acid buffer), 30 ℃, the 60r/min vibration, hydrolysis broken wall 3h, centrifugal collection protoplastis is with containing 0.1mol/L glucose, 0.1mol/L sucrose and 0.01mol/L CaCl
2Preservation liquid dilution, be separated to and contain on the 0.3ppm triacontanol price quote regeneration culture medium, 25 ℃ of following illumination cultivation fall until the regeneration algae occurring.
Example 3:
Get the logarithmic phase Haematocoocus Pluvialls nutrient solution 5mL that cultivated 4 days, the centrifugal 10min of 3500r/min, after the washing of equivalent sterilized water, centrifugal collection microalgae cell, add 5mL pretreating agent (30mmol/L EDTA, the 45mmol/L dithiothreitol (DTT), the pH4.0 citrate buffer solution is formulated), handle 40min for 25 ℃, centrifugal collection frustule, adding 5mL height oozes composite enzyme solution and (contains 0.1mol/L NaCl, 0.1mol/L KCl, 0.02mol/L MgSO
4, 0.01mol/L CaCl
2, 1.0% cellulase, 1.5% helicase, 0.8% polygalacturonase, pH6.5 phosphoric acid buffer), 35 ℃, the 60r/min vibration, hydrolysis broken wall 3h, centrifugal collection protoplastis is with containing 0.1mol/L glucose, 0.1mol/L sucrose and 0.01mol/L CaCl
2Preservation liquid dilution, be separated to and contain on the 0.1ppm triacontanol price quote regeneration culture medium, 25 ℃ of following illumination cultivation fall until the regeneration algae occurring.
Claims (5)
1, a kind of composite hydrolytic enzyme that utilizes is the broken wall agent, with BBM is the Haematococcus pluvialis protoplast preparing and the regeneration techniques of substratum, it is characterized in that frustule 25～34 ℃ of constant temperature processing of acidic buffer pretreating agent, the 30～60min that contains ethylenediamine tetraacetic acid (EDTA), dithiothreitol (DTT), after change the compound height that contains cellulase, helicase, polygalacturonase over to and ooze degradation of cell wall in the enzyme solution, be separated on the regeneration culture medium that contains triacontanol price quote and regenerate.
2, preparation according to claim 1 and regeneration techniques is characterized in that the pretreating agent prescription is:
Ethylenediamine tetraacetic acid (EDTA) 30～50m mol/L
Dithiothreitol (DTT) 25～45m mol/L
Acidic buffer (citric acid+Trisodium Citrate, pH4.0)
3, preparation according to claim 1 and regeneration techniques is characterized in that compound hypertonic solution prescription (volumetric molar concentration) is:
NaCl 0.1mol/L
KCl 0.1mol/L
MgSO
4 0.02mol/L
CaCl
2 0.01mol/L
Phosphoric acid buffer (citric acid+Trisodium Citrate, pH6.5)
4, preparation according to claim 1 and regeneration techniques is characterized in that using height to ooze consist of (weight percent) of prozyme liquid:
Cellulase 1～2%
Helicase 0.5～1.5%
Polygalacturonase 0.3～1.0%
Compound hypertonic solution 95.5～98%
5, preparation according to claim 1 and regeneration techniques is characterized in that having added in the regeneration culture medium triacontanol price quote, and concentration is 0.1～0.5ppm.
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| CNA2004100075358A CN1560227A (en) | 2004-02-25 | 2004-02-25 | Preparation and regenerating technology for protoplasm of rainy red ball alga |
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| CN102283125A (en) * | 2010-12-13 | 2011-12-21 | 湖北省烟草科研所 | High-efficiency tobacco pollen in-vitro liquid culture method |
| CN102293154A (en) * | 2010-12-13 | 2011-12-28 | 湖北省烟草科研所 | High-efficiency mutagenesis method of nicotiana tabacum L. pollen |
| CN102283125B (en) * | 2010-12-13 | 2013-03-27 | 湖北省烟草科研所 | High-efficiency tobacco pollen in-vitro liquid culture method |
| CN102160524A (en) * | 2011-01-10 | 2011-08-24 | 中国海洋大学 | Separation culture and plant regeneration method for kappaphycus seaweed protoplast |
| CN102144544B (en) * | 2011-01-10 | 2013-03-06 | 中国海洋大学 | Method for separating Eucheuma seaweed protoplast and culturing regenerated plant |
| CN102144544A (en) * | 2011-01-10 | 2011-08-10 | 中国海洋大学 | Method for separating Eucheuma seaweed protoplast and culturing regenerated plant |
| CN102257955A (en) * | 2011-05-06 | 2011-11-30 | 中国科学院新疆生态与地理研究所 | Method for establishing syntrichia caninervis mitt. individual plant cloning system through asexual propagation |
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