CN106754862A - A kind of selection of the hidden dinoflagellate mutation algae strain of Seedling height ability - Google Patents
A kind of selection of the hidden dinoflagellate mutation algae strain of Seedling height ability Download PDFInfo
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- CN106754862A CN106754862A CN201611197176.6A CN201611197176A CN106754862A CN 106754862 A CN106754862 A CN 106754862A CN 201611197176 A CN201611197176 A CN 201611197176A CN 106754862 A CN106754862 A CN 106754862A
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Abstract
The invention discloses it is a kind of based on aliphatic acid synthesis key enzyme acetyl-CoA carboxylase inhibitor (including but not limited to sethoxydim) alternatively pressure hidden dinoflagellate mutation algae strain selection, including strain atmospheric pressure at room plasma (ARTP) the mutagenesis and mutation Strain selection method under the selection pressure of the inhibitor (including but not limited to sethoxydim) of acetyl-CoA carboxylase of hidden dinoflagellate algae, and the hidden dinoflagellate algae strain under the selection pressure of the inhibitor (including but not limited to sethoxydim) of acetyl-CoA carboxylase acclimation method.The invention is characterized in that the Strain selection method of atmospheric pressure at room plasma induced-mutation technique and inhibitor (including but not limited to sethoxydim) based on acetyl-CoA carboxylase is combined, and the algae strain acclimation method of the inhibitor (including but not limited to sethoxydim) based on acetyl-CoA carboxylase, relative to existing hidden dinoflagellate algae strain selection, the characteristics of with can efficiently, quickly obtain the strain of Seedling height ability algae.
Description
Technical field
It is a kind of selection of the hidden dinoflagellate mutation algae strain of Seedling height ability the invention belongs to biological technical field.
Background technology
Because polybasic unsaturated fatty acid plays an important role to the physiological function of human body, its research and production are increasingly subject to
The attention of people.Wherein, in ω -3 classes unrighted acid DHA (Docosahexaenoic acid,
DHA) it is a kind of particularly important aliphatic acid with high nutritive value, normal development, adult brain's function to infant brain
Normal performance play an important role.Additionally, DHA plays important work for human body retina and the prevention of human body angiocardiopathy
With.Human body itself can not synthesize DHA, it is necessary to be absorbed from food.
Until now, deep sea fish oil is always the main source of commercially available DHA.But this main source has
The worry of the security to fish oil that fatal problem, such as marine pollution are caused, fish oil quality can with the species of fish, fish for
Season and place it is different and different, containing eicosapentaenoic acid (Eicosapentaenoic acid, EPA), dirty easily by environment
Dye, purifying high cost, particularly in view of the key consumer of DHA is pregnant woman and young child, its is possible unfavorable
Influence is more troubling；Additionally, influence of the atrophy year by year of world fisheries resource to the sustainable supply of DHA is also worth examining
Consider；In addition, the special odor of fish oil also limit the DHA from fish oil source as a kind of application of food additives.
Using hidden dinoflagellate (Crypthecodinium cohnii) fermenting and producing DHA, compared with traditional fish oil is originated, have
Series of advantages, such as fermentation period are short, and training method is simple, and growth of microorganism is fast, it is easy to large-scale culture；Polyunsaturated fat
Acid content is high, product quality stabilization, and preferably, unrighted acid composition is single, low without EPA or EPA content for oxidation stability,
It is easily isolated purifying；Traditional DHA that obtained from fish oil is overcome simultaneously receives many limits such as raw material, weather, the place of production, production cycle
The influence of factor processed.Therefore the DHA in the alternative fish oil sources of microbial fermentation production DHA, is with a wide range of applications.
At present, the algae kind majority of actual hidden dinoflagellate fermenting and producing application is directly separated from nature and obtains, and proterties is more single
One, it is unavoidable during the fermentation that algae strain germplasm degeneration occurs.And the excellent algae kind of conventional method seed selection is utilized, the cycle is long, workload
Greatly.Therefore, the improvement of hidden dinoflagellate algae strain germplasm is particularly significant for the sustainable development of the hidden dinoflagellate production DHA industries of promotion
's.
Atmospheric pressure at room plasma (ARTP) be refer to it is producing temperature between 25-40 DEG C under atmospheric pressure, have
The plasma of high activity particle (including the helium atom in excitation state, oxygen atom, nitrogen-atoms, OH free radicals etc.) concentration is penetrated
Stream.Because ARTP temperature in itself is low, active particle concentration is high and wide variety, and ARTP discharge types are various, equipment is simple,
Operation is simple, operating cost is cheap, and ARTP is environmentally safe in itself in addition and damages, so atmospheric pressure at room plasma
(ARTP) mutagenesis is more and more extensive in biological field application.The active-energy particle that ARTP is rich in is to bacterial strain/plant/cell etc.
Inhereditary material causes to damage, and induces biological cell startup SOS repair mechanisms.SOS repair processes are a kind of serious forgiveness reparation high,
Therefore the abundant mismatch site of species can be produced in repair process, and is finally stablized heredity and then is formed mutant strain.
Most of screening techniques of conventional hidden dinoflagellate mutant strain are time-consuming more long, and workload is larger, inefficient.Therefore,
The seed selection for seeking a kind of efficient screening technique for the strain of hidden dinoflagellate algae is also particularly important.
Acetyl-CoA carboxylase (Acetyl CoA carboxylase) is catalysis acetyl coenzyme A+ATP+HCO3- → third
The biotin enzyme of two acyl coenzyme A+ADP+Pi reactions.This reaction governs the speed that aliphatic acid synthesizes the first stage, therefore acetyl
CoA carboxylase enzyme is the key enzyme of aliphatic acid synthesis.And sethoxydim (sethoxydim, 2 one [1 one (ethoxy imino) fourths
Base] one 5 one [2 one (ethylmercapto group) propyl group] -3- hydroxyl -2- cyclohexene -1- ketone) be the enzyme inhibitor.Sethoxydim can suppress hidden
The aliphatic acid of dinoflagellate synthesizes and causes slow-growing or even death.
Up to the present, not using sethoxydim carries out the report that hidden dinoflagellate mutant strain is screened.
It is a discovery of the invention that by carrying out atmospheric pressure at room plasma (ARTP) mutagenesis to hidden dinoflagellate, then using aliphatic acid
The inhibitor (including but not limited to sethoxydim) of the key enzyme (such as acetyl-CoA carboxylase) in synthesis is oriented screening,
The hidden dinoflagellate mutant strain of Seedling height ability can be obtained.Meanwhile, it is a discovery of the invention that using the sethoxydim of suitable concentration to hidden dinoflagellate
Domestication is oriented, the hidden dinoflagellate domestication strain of Seedling height ability can be obtained.
In a word, inhibitor (the including but not limited to dilute standing grain based on the key enzyme acetyl-CoA carboxylase in aliphatic acid synthesis
Selection pressure calmly) carries out artificially breeding to the strain of hidden dinoflagellate algae, can efficiently and rapidly obtain the mutation algae strain of Seedling height ability.
The content of the invention
It is an object of the invention to provide a kind of method for artificially breeding of the hidden dinoflagellate mutation algae strain of Seedling height ability.
Another object of the present invention is to provide a kind of hidden dinoflagellate algae strain artificial acclimation method.
The hidden dinoflagellate algae strain method for artificially breeding of the present invention, including strain atmospheric pressure at room plasma (ARTP) mutagenesis of hidden dinoflagellate algae
Algae strain is screened with the selection pressure of the inhibitor of aliphatic acid synthesis key enzyme acetyl-CoA carboxylase, specific behaviour
Work includes：
1) mutagenesis is carried out to the strain of hidden dinoflagellate algae using the mutagenesis of atmospheric pressure at room plasma：Lured using room temperature atmospheric plasma
Become instrument carries out mutagenesis to hidden dinoflagellate algae solution；
2) by mutagenic treatment after hidden dinoflagellate algae solution coat on solid C9N2 culture mediums, carried out under regular culture conditions
Culture, while using the algae solution of non-mutagenic treatment as control；
3) after algae falls and grows, every piece of effective flat board picking is preserved to new C9N2 culture medium solids again compared with macrocolony
On flat board；
4) cultivated in single bacterium colony being chosen into liquid C9N2 culture mediums；
5) hidden dinoflagellate mutagenic fungi algae solution is coated on the solid C9N2 culture mediums containing sethoxydim, under regular culture conditions
Cultivated, while using the algae solution of non-mutagenic treatment as control.Fallen artificial situation long according to the algae on flat board, select the algae of survival
Fall, you can obtain the hidden dinoflagellate mutant strain of high-biomass；
6) the growth characteristics analysis of hidden dinoflagellate mutant strain：By the hidden dinoflagellate mutant strain of gained in the fluid nutrient medium of C9N2
Culture, whether identification mutant strain maximum biomass is higher than original algae strain of setting out.
The algae kind belongs to for hidden dinoflagellate；The inhibitor is sethoxydim；The hidden dinoflagellate cell concentration of the mutagenic treatment is
106~1085%~20% glycerine of addition in individual/mL, algae solution, distance is 2mm between plasma emission source and sample, processes work(
Rate is 100W, and throughput is 10SLM (10L/min), and the mutagenic treatment time is 0~100s, diluting cells in mutagenic processes, wash-out
Algae strain uses liquid C9N2 culture mediums on slide glass.
Step (2) the solid C9N2 culture mediums be by glucose 9g, yeast extract 2g, sea salt 25g, agar 15g,
Add water to 1L to form, pH is 6.5；
Step (4) the liquid C9N2 culture mediums are, by glucose 9g, yeast extract 2g, sea salt 25g, to add water to 1L
Form, pH is 6,5.
The concentration of the step (5) and step (6) sethoxydim is for about 5 μm of ol/L~500 μm ol/L；Step (4) and
When being cultivated in step (6) fluid nutrient medium, shaking table parameter is set to 20~200rpm of rotating speed, 4~40 DEG C of temperature.
A kind of hidden dinoflagellate algae strain artificial acclimation method, it is characterised in that (such as dilute in the inhibitor of acetyl-CoA carboxylase
Standing grain is determined, but is not limited to sethoxydim) selection pressure under the hidden dinoflagellate algae method tamed of strain, concrete operations are included：
1st, hidden dinoflagellate algae solution is coated on the solid C9N2 culture mediums containing various concentrations sethoxydim, is entered under normal condition
Row culture, at the same using not plus sethoxydim solid plate as control.Fallen artificial situation long according to the algae on flat board, determine sethoxydim
Initial suitable inhibition concentration；
2nd, hidden dinoflagellate is inoculated into the liquid C9N2 culture mediums containing suitable concentration sethoxydim, is cultivated under normal condition；
3rd, when hidden dinoflagellate cell quantity reaches 107~108During individual/mL, passed on；
4th, whne hidden dinoflagellate can the normal growth in containing a certain concentration sethoxydim culture medium when, properly increase sethoxydim concentration
Continue to cultivate；Until by repeatedly passage algae strain analyzed by table, its grow highest biomass apparently higher than it is original go out
Hair algae strain.
Described algae kind belongs to for hidden dinoflagellate；Step (1) the solid C9N2 culture mediums are by glucose 9g, yeast extract
2g, sea salt 25g, agar 15g, add water to 1L and form, and pH is 6.5.
Step (2) the liquid C9N2 culture mediums are, by glucose 9g, yeast extract 2g, sea salt 25g, to add water to 1L
Form, pH is 6.5.
The concentration of the step (1) and step (4) sethoxydim is 5 μm of ol/L~500 μm ol/L.
When being cultivated in the step (2) and step (4) fluid nutrient medium, shaking table parameter is set to 20~200rpm of rotating speed,
4~40 DEG C of temperature.
One aspect of the present invention, the present invention is lured hidden dinoflagellate by using atmospheric pressure at room plasma (ARTP) technology
Become, the inhibitor (including but not limited to sethoxydim) of the key enzyme in then being synthesized using aliphatic acid is screened, and can be obtained
The hidden dinoflagellate mutant strain of Seedling height ability.On the other hand, the present invention by using acetyl-CoA carboxylase inhibitor (for example
Sethoxydim, but be not limited to sethoxydim) selection pressure domestication is oriented to the strain of hidden dinoflagellate algae, be obtained in that Seedling height ability
Hidden dinoflagellate tames strain.
On the other hand, the inhibitor (including but not limited to sethoxydim) of utilization acetyl-CoA carboxylase of the present invention
Alternatively pressure is oriented domestication to the strain of hidden dinoflagellate algae, i.e., cultivated in the fluid nutrient medium containing suitable concentration sethoxydim
The mutant strain of hidden dinoflagellate, screening and enrichment tolerance acetyl-CoA carboxylase inhibitor.First, hidden dinoflagellate algae solution is coated and is contained
Have on the solid C9N2 culture mediums of various concentrations sethoxydim, cultivated under normal condition, while the solid not add sethoxydim
Flat board is used as control.Fallen artificial situation long according to the algae on flat board, determine the initial suitable inhibition concentration of sethoxydim；Secondly, will be hidden
Dinoflagellate is inoculated into the liquid C9N2 culture mediums containing suitable concentration sethoxydim, is cultivated under normal condition；When hidden dinoflagellate cell number
Amount reaches 107~108During individual/mL, passed on；Finally, treat that hidden dinoflagellate can be containing normal in a certain concentration sethoxydim culture medium
During growth, properly increase sethoxydim concentration and continue to cultivate.Tamed by the orientation of certain algebraically, you can obtain growth ability raising
Hidden dinoflagellate domestication strain.
The growth characteristics analysis of the hidden dinoflagellate mutant strain of the present invention, the hidden dinoflagellate mutant strain of gained is in C9N2 fluid nutrient mediums
(glucose 9g, yeast extract 2g, sea salt 25g, add water to 1L, and pH is 6.5) to cultivate, shaking table arrange parameter be rotating speed 100~
200rpm, 24~28 DEG C of temperature.Step is：Taken during inoculation during fresh cells 5mL that OD490nm is 0.8 adds 50mL culture mediums,
Every group is done 3 Duplicate Samples, is the light absorption value under 490nm with UV-1750 spectrophotometric determination wavelength, and per 12h, measurement once, is painted
Block diagram processed.
Atmospheric pressure at room plasma (ARTP) technical equipment used in the present invention is simple, operation is simple, operating cost is low
It is honest and clean, environmentally safe and infringement, while Mutagenic Effect is obvious；Using the key enzyme acetyl coenzyme A in fatty acid synthesis process
The inhibitor (including but not limited to sethoxydim) of carboxylase is oriented screening, greatly reduces workload, improves screening effect
Rate.Selected good strains in the field for seed accordingly, with respect to traditional hidden dinoflagellate algae and educated, be it is a kind of efficiently, efficiently new method.
The inhibitor (including but not limited to sethoxydim) of acetyl-CoA carboxylase used herein alternatively pressure pair
The method that hidden dinoflagellate algae strain is oriented domestication, orientation domestication effect is a kind of the efficient quick of Seedling height ability algae kind substantially
Selection.
Brief description of the drawings
Fig. 1 hidden first round of dinoflagellate ATCC 30556 mutagenic obtained mutant strain is solid containing 10 μm of C9N2 of ol/L sethoxydims
Growth figure on body flat board.
The hidden dinoflagellate ATCC 30,556 second of Fig. 2 take turns mutagenic obtained mutant strain solid containing 20 μm of C9N2 of ol/L sethoxydims
Growth figure on body flat board.
Original algae strain strain and growth comparison diagram (wherein, the culture medium of mutant strain of setting out of hidden dinoflagellate under Fig. 3 C9N2 culture mediums
It is C9N2：Glucose 9g, yeast extract 2g, sea salt 25g, add water to 1L, and pH is 6.5.Culture to 48h, it is original set out algae strain and
The growth of Mutant Cells reaches maximum, wherein a mutant strain is than original algae strain balanced growth 10.28%, two of setting out
15.55%) secondary mutant strain sets out algae strain balanced growth than original.
Hidden dinoflagellate is original under Fig. 4 C27N6 culture mediums sets out that (wherein, culture medium is with the growth comparison diagram of mutant strain for algae strain
C9N2：Glucose 27g, yeast extract 6g, sea salt 25g, add water to 1L, and pH is 6.5.Culture to 96h, the original algae strain and prominent of setting out
The growth of mutant cell reaches maximum, wherein a mutant strain is than original algae strain balanced growth 17.97% of setting out, it is secondary
20.31%) mutant strain sets out algae strain balanced growth than original.
The original algae strain of setting out of hidden dinoflagellate (wherein, is cultivated with the growth comparison diagram of mutant strain under Fig. 5 C33N7.33 culture mediums
Base is C9N2：Glucose 33g, yeast extract 7.33g, sea salt 25g, add water to 1L, and pH is 6.5.Culture is original to set out to 120h
Algae strain and the growth of Mutant Cells reach maximum, wherein a mutant strain is than original algae strain balanced growth of setting out
14.10%, 15.29%) secondary mutant strain sets out algae strain balanced growth than original.
Dry cell weight comparison diagram (its of strain is tamed in the original algae strain of setting out of hidden dinoflagellate with sethoxydim under Fig. 6 C9N2 culture mediums
In, culture medium is C9N2：Glucose 9g, yeast extract 2g, sea salt 25g, add water to 1L, and pH is 6.5.Culture to 48h, it is original go out
The strain of hair algae and the growth of domestication strain cell reach maximum, wherein setting out algae strain after taming for 60 generations, sethoxydim are tolerated
Concentration is improved to 20 μm of ol from 5 μm of ol, tames dry cell weight balanced growth of the strain than original algae strain of setting out 22.97%).
Specific embodiment
The following examples are in order that those skilled in the art more fully understands the present invention but is not limited to this
Invention.
The present invention is described in further detail below by embodiment and with reference to accompanying drawing.
It is of the present invention that mutagenesis is carried out to the strain of hidden dinoflagellate algae using atmospheric pressure at room plasma, that is, use atmospheric pressure at room etc.
Gas ions mutagenesis instrument carries out mutagenesis.Wherein, the hidden dinoflagellate cell concentration of mutagenic treatment is 106~108Addition in individual/mL, algae solution
5%~20% glycerine, distance is 2mm between plasma emission source and sample, and processing power is 100W, and throughput is 10SLM,
The mutagenic treatment time is 0~100s, and diluting cells in mutagenic processes elute algae strain on slide glass and use liquid C9N2 culture mediums.
After mutagenesis operation is completed, by mutagenic treatment after hidden dinoflagellate algae solution coat solid C9N2 culture mediums (Portugal
Grape sugar 9g, yeast extract 2g, sea salt 25g, agar 15g, add water to 1L, pH be 6.5) on, trained under regular culture conditions
Support, while using the algae solution of non-mutagenic treatment as control.
After algae falls and grows, every piece of effective flat board picking preserves flat to new C9N2 culture mediums solid again compared with macrocolony
On plate.
Single bacterium colony chosen into liquid C9N2 culture mediums (glucose 9g, yeast extract 2g, sea salt 25g, add water to 1L, and pH is
6.5) culture in.Hidden dinoflagellate mutagenic fungi algae solution is coated on the solid C9N2 culture mediums containing sethoxydim, regular culture conditions
Under cultivated, while using the algae solution of non-mutagenic treatment as control.
Fallen artificial situation long according to the algae on flat board, the algae for selecting survival falls, you can obtain the hidden dinoflagellate of growth ability improvement
Mutant strain.
The hidden dinoflagellate mutant strain that the first time mutagenesis can also be obtained carries out second mutagenesis, repeats the above steps.
Screening obtains the more outstanding secondary mutant strain of hidden dinoflagellate of growth ability proterties.
The growth characteristics analysis of hidden dinoflagellate mutant strain, the hidden dinoflagellate mutant strain of the gained (grape in C9N2 fluid nutrient mediums
Sugared 9g, yeast extract 2g, sea salt 25g, add water to 1L, and pH is 6.5) to cultivate, and shaking table arrange parameter is 100~200rpm of rotating speed,
24~28 DEG C of temperature.Step is：Taken during inoculation in the fresh cells 5mL addition 50mL culture mediums that OD490nm is 0.8, every group is done 3
Individual Duplicate Samples, are the light absorption value under 490nm with UV-1750 spectrophotometric determination wavelength, and per 12h, measurement once, draws column
Figure.
Embodiment 1
Atmospheric pressure at room plasma (ARTP) the mutagenesis instrument developed using Wuxi source clear sky wood bio tech ltd, it is right
Hidden dinoflagellate (Crypthecodinium cohnii) ATCC 30556 carries out mutagenesis.
1. hidden dinoflagellate algae solution cell concentration is adjusted to 106~108Individual/mL, uses liquid C9N2 culture mediums during dilution.It is dilute
Release after finishing, the glycerine of addition final concentration of 5~20%.
2., in metal slide glass is placed in into the appropriate calcination of alcolhol burner flame envelope in super-clean bench, batch cultur ware is put into after cooling
In, take 10~20 μ L algae solutions and be uniformly applied on metal slide glass.
3. the culture dish that will be equipped with specimen slides is put into ARTP mutagenesis instrument operating rooms, and it is right to be placed in slide glass with aseptic nipper
Answer hole position, and shutoff operation room door.
4. distance is 2mm between plasma emission source and sample, and processing power is 100W, and throughput is 10SLM, mutagenesis
Process time is 0~100s.The setting processing time, sample is processed successively.
5. after sample treatment is finished, slide glass is put into the EP pipes equipped with 1mL liquid C9N2 culture mediums with aseptic nipper.
6. EP pipes are shaken into 1~3min on the oscillator, make to be attached to the algae solution on slide glass and be eluted in liquid, form new
Algae solution.
7. pair new algae solution is suitably diluted, and is taken 100 μ L and is coated on solid medium.Simultaneously with non-mutagenic treatment algae
Liquid is used as control.
8. the solid medium flat board of coating is positioned in constant incubator, 25 DEG C of temperature, is cultivated under dark condition.
9. after algae falls and grows after algae falls and grows, from every piece of effective flat board picking algae fall be relatively large in diameter algae strain, then
It is secondary to preserve to new C9N2 culture medium solid plates.
10. cultivated in single bacterium colony being chosen into liquid C9N2 culture mediums, use 100mL triangular flasks, liquid amount 20mL.Liquid
C9N2 culture mediums are, by glucose 9g, yeast extract 2g, sea salt 25g, to add water to 1L and form, and pH is 6.5.Shaking table parameter is set to
100~200rpm of rotating speed, 24~28 DEG C of temperature.
11. hidden dinoflagellate mutagenic fungi algae solution is coated the solid C9N2 cultures containing 5 μm of ol/L~200 μm ol/L of sethoxydim
On base, it is positioned in constant incubator, 24~28 DEG C of temperature, is cultivated under dark condition.Solid C9N2 culture mediums are by glucose
9g, yeast extract 2g, sea salt 25g, agar 15g, add water to 1L and form, and pH is 6.5.
The growth characteristics analysis of 12. hidden dinoflagellate mutant strains, the hidden dinoflagellate mutant strain of gained is trained in C9N2 fluid nutrient mediums
Support.Liquid C9N2 culture mediums are, by glucose 9g, yeast extract 2g, sea salt 25g, to add water to 1L and form, and pH is 6.5.Shaking table sets
Parameter is put for 100~200rpm of rotating speed, 24~28 DEG C of temperature.Step is：The fresh cells 5mL that OD490nm is 0.8 is taken during inoculation
Add in 50mL culture mediums, every group is done 3 Duplicate Samples, is the extinction under 490nm with UV-1750 spectrophotometric determination wavelength
Value, per 12h, measurement once, draws block diagram.It is determined that the hidden dinoflagellate mutant strain biomass for obtaining is higher than original algae strain of setting out.
Embodiment 2
The present embodiment is substantially the same manner as Example 1, and the initial algae strain for simply using is for the hidden dinoflagellate obtained by embodiment 1 is dashed forward
Mutant, carries out the second wheel mutagenesis with screening.
Embodiment 3
The growth characteristics analysis of hidden dinoflagellate mutant strain, the hidden dinoflagellate mutant strain of gained is trained in the liquid of different concentration of glucose
Support culture in base.Shaking table arrange parameter is 100~200rpm of rotating speed, 24~28 DEG C of temperature.Step is：OD490nm is taken during inoculation
In for 0.8 fresh cells 5mL addition 50mL culture mediums, every group is done 3 Duplicate Samples, with UV-1750 spectrophotometric determination ripples
Light absorption value under a length of 490nm, per 24h, measurement once, draws block diagram.
The formula of the hidden dinoflagellate culture medium of the different concentration of glucose of table 1
| Culture medium title | Glucose (g/L) | Yeast extract (g/L) | Sea salt (g/L) |
| C9N2 | 9 | 2 | 25 |
| C27N6 | 27 | 6 | 25 |
| C33N7.33 | 33 | 7.33 | 25 |
Embodiment 4
1. hidden dinoflagellate algae solution is coated on the solid C9N2 culture mediums containing various concentrations sethoxydim, entered under normal condition
Row culture, at the same using not plus sethoxydim solid plate as control.Fallen artificial situation long according to the algae on flat board, determine sethoxydim
Initial suitable inhibition concentration.
2. hidden dinoflagellate is inoculated into the liquid C9N2 culture mediums containing suitable concentration sethoxydim, is cultivated under normal condition.
3. when hidden dinoflagellate cell quantity reaches 107~108During individual/mL, passed on.
4. whne hidden dinoflagellate can the normal growth in containing a certain concentration sethoxydim culture medium when, properly increase sethoxydim concentration
Continue to cultivate.
5. the growth characteristics analysis of hidden dinoflagellate domestication strain, the hidden dinoflagellate domestication strain of gained is trained in C9N2 fluid nutrient mediums
Support.Liquid C9N2 culture mediums are, by glucose 9g, yeast extract 2g, sea salt 25g, to add water to 1L and form, and pH is 6.5.Shaking table sets
Parameter is put for 100~200rpm of rotating speed, 24~28 DEG C of temperature.Step is：The fresh cells 5mL that OD490nm is 0.8 is taken during inoculation
Add in 50mL culture mediums, every group is done 3 Duplicate Samples, is the extinction under 490nm with UV-1750 spectrophotometric determination wavelength
Value, per 12h, measurement once, draws block diagram.It is determined that the hidden dinoflagellate mutant strain biomass for obtaining is higher than original algae strain of setting out.
Claims (10)
1. a kind of hidden dinoflagellate algae strain method for artificially breeding, it is characterised in that：Including hidden dinoflagellate algae strain atmospheric pressure at room plasma
(ARTP) algae strain is sieved under the selection pressure of mutagenesis and the inhibitor in aliphatic acid synthesis key enzyme acetyl-CoA carboxylase
Choosing, concrete operations include：
(1) mutagenesis is carried out to the strain of hidden dinoflagellate algae using the mutagenesis of atmospheric pressure at room plasma：Use room temperature atmospheric plasma mutagenesis
Instrument carries out mutagenesis to hidden dinoflagellate algae solution；
(2) by mutagenic treatment after hidden dinoflagellate algae solution coat on solid C9N2 culture mediums, trained under regular culture conditions
Support, while using the algae solution of non-mutagenic treatment as control；
(3) after algae falls and grows, every piece of effective flat board picking is preserved to new C9N2 culture medium solid plates again compared with macrocolony
On；
(4) cultivated in single bacterium colony being chosen into liquid C9N2 culture mediums；
(5) hidden dinoflagellate mutagenic fungi algae solution is coated on the solid C9N2 culture mediums containing sethoxydim, is entered under regular culture conditions
Row culture, while using the algae solution of non-mutagenic treatment as control.Fallen artificial situation long according to the algae on flat board, select the algae of survival
Fall, you can obtain the hidden dinoflagellate mutant strain of high-biomass；
(6) the growth characteristics analysis of hidden dinoflagellate mutant strain：The hidden dinoflagellate mutant strain of gained is trained in the fluid nutrient medium of C9N2
Support, whether identification mutant strain maximum biomass is higher than original algae strain of setting out.
2. method according to claim 1, it is characterised in that the algae kind is hidden dinoflagellate category；The inhibitor is sethoxydim；Institute
The hidden dinoflagellate cell concentration for stating mutagenic treatment is 106~1085%~20% glycerine of addition, plasma emission in individual/mL, algae solution
Distance is 2mm between source and sample, and processing power is 100W, and throughput is 10SLM (10L/min), the mutagenic treatment time is 0~
100s, diluting cells in mutagenic processes, algae strain uses liquid C9N2 culture mediums on wash-out slide glass.
3. method according to claim 1, it is characterised in that step (2) the solid C9N2 culture mediums are by glucose 9g, ferment
Mother leaching powder 2g, sea salt 25g, agar 15g, add water to 1L and form, and pH is 6.5.
4. method according to claim 1, it is characterised in that step (4) the liquid C9N2 culture mediums are by glucose 9g, ferment
Mother leaching powder 2g, sea salt 25g, add water to 1L and form, and pH is 6,5.
5. method according to claim 1, it is characterised in that the concentration of step (5) and step (6) sethoxydim is for about 5 μ
Mol/L~500 μm ol/L；When being cultivated in step (4) and step (6) fluid nutrient medium, shaking table parameter be set to rotating speed 20~
200rpm, 4~40 DEG C of temperature.
6. a kind of hidden dinoflagellate algae strain artificial acclimation method, it is characterised in that in inhibitor (such as dilute standing grain of acetyl-CoA carboxylase
It is fixed, but be not limited to sethoxydim) selection pressure under the method tamed to the strain of hidden dinoflagellate algae,
Concrete operations include：
(1) hidden dinoflagellate algae solution is coated on the solid C9N2 culture mediums containing various concentrations sethoxydim, is carried out under normal condition
Culture, at the same using not plus sethoxydim solid plate as control.Fallen artificial situation long according to the algae on flat board, determine sethoxydim
Initial suitable inhibition concentration；
(2) hidden dinoflagellate is inoculated into the liquid C9N2 culture mediums containing suitable concentration sethoxydim, is cultivated under normal condition；
(3) when hidden dinoflagellate cell quantity reaches 107~108During individual/mL, passed on；
(4) whne hidden dinoflagellate can the normal growth in containing a certain concentration sethoxydim culture medium when, properly increase sethoxydim concentration after
Continuous culture；Until the algae strain by repeatedly passage is analyzed by table, the highest biomass of its growth sets out apparently higher than original
Algae strain.
7. method according to claim 6, it is characterised in that described algae kind is hidden dinoflagellate category；Step (1) the solid C9N2
Culture medium is, by glucose 9g, yeast extract 2g, sea salt 25g, agar 15g, to add water to 1L and form, and pH is 6.5.
8. method according to claim 6, it is characterised in that step (2) the liquid C9N2 culture mediums are by glucose 9g, ferment
Mother leaching powder 2g, sea salt 25g, add water to 1L and form, and pH is 6.5.
9. method according to claim 6, it is characterised in that the concentration of step (1) and step (4) sethoxydim is 5 μm of ol/
L~500 μm ol/L.
10. method according to claim 6, it is characterised in that when being cultivated in step (2) and step (4) fluid nutrient medium, shaking table
Parameter is set to 20~200rpm of rotating speed, 4~40 DEG C of temperature.
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| CN109355192B (en) * | 2018-11-02 | 2022-06-21 | 厦门大学 | Domestication method for improving oil production capacity of dunaliella salina |
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| CN110628758A (en) * | 2019-10-08 | 2019-12-31 | 昆明藻能生物科技有限公司 | Method for improving yield and yield of crypthecodinium cohnii DHA |
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