CN107058555A - A kind of screening technique of high cell density microalgae mutant - Google Patents
A kind of screening technique of high cell density microalgae mutant Download PDFInfo
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Abstract
The invention discloses a kind of screening technique of high cell density microalgae mutant, comprise the following steps：(1) resistant gene is converted into microalgae, the mutation microalgae list algae for being transferred to resistance gene sequences by the acquisition of solid plate resistance screening falls, and collects each single algae and falls to obtain microalgae mutant library；(2) fall from each single algae in microalgae mutant library and cultivated after sampling mixing in liquid medium within, and continuous passage；(3) the specific rflp analysis method detection of some alternative resistance gene sequences is often passed, terminates screening when testing result, which is shown, only remains a kind of microalgae mutant, obtains the high cell density microalgae mutant；When testing result, which is shown, also remains 2~5 kinds of microalgae mutant, carry out single algae using solid medium and fall the separation acquisition high cell density microalgae mutant.Screening technique of the present invention without high optical condition and special detection equipment, can quickly, simply, efficiently complete screening process.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of screening side of high cell density microalgae mutant
Method.
Background technology
Microalgae has the features such as growth rate is fast, growth cycle is short, fat content is high because of it, it is considered to be most potential
Grease bioenergy.Chlamydomonas reinhardtii (Chlamydomonas reinhardti) is biological as the single cell mode of research microalgae,
The potential energy microalgae for being adapted to large-scale production is turned into.But, Chlamydomonas reinhardtii compared with other business microalgaes biomass compared with
Small, relatively low stand density and biomass limits it as the possibility of extensive production of energy.
Insertion mutation is a kind of conventional mutant strain screening technique, is widely used to the special phenotype mutant strain of Chlamydomonas reinhardtii
Screening.The sequence of insertion typically all contains marker gene, such as Aph VIII (Sizova, I., Fuhrmann, M.and
Hegemann,P.(2001) A Streptomyces rimosus aphVIII gene coding for a new type
phosphotransferase provides stable antibi-otic resistance to Chlamydomonas
Reinhardtii.Genetics, 277,221-229.), Ble (Lumbreras, V., Stevens, D.R.and Purton,
S.(1998)Efficient foreign gene expression in Chlamydomonas reinhardtii
Mediated by an endogenous intron.The Plant Journal, 14,441-447.) etc., it is easy to mutation
Strain is screened.
There are some researches show：To improve biomass, pigment is reduced, the insertion mutation strain for removing feeler etc. is reachable under bloom
To purpose, but it is infeasible under low illumination.This is primarily due under the conditions of bloom, is gone the cell of feeler to float on the surface and is obtained
More illumination are obtained, more light sources can be received than wild type.But abactinal biomass is compared with wild type under low light
It is similar, because now intensity of illumination is on surface and beneath (Polle, J.E.W., Kanakagiri, the S.D.and of being more or less the same
Melis,A.(2003) tla1,a DNA insertional transformant of the green alga
Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll
antenna size.Planta,217,49-59.).Although can improve chlamydomonas biomass under bloom, strong luminous energy consumption is big, unfavorable
Used in large scale investment.Therefore, how to screen under common intensity of illumination still there is the microalgae strain compared with high-biomass to become
It is more important.
The content of the invention
The present invention is screened for lacking suitable method in the prior art still to be had compared with Gao Sheng under common intensity of illumination
The problem of microalgae strain of object amount, there is provided a kind of screening technique of high cell density microalgae mutant.
A kind of screening technique of high cell density microalgae mutant, comprises the following steps：
(1) resistant gene is converted into microalgae, the mutation for being transferred to resistance gene sequences is obtained by solid plate resistance screening
Single algae of microalgae falls, and collects each single algae and falls to obtain microalgae mutant library；
(2) fall from each single algae in microalgae mutant library and cultivated after sampling mixing in liquid medium within, and continuously
Passage；
(3) often pass on and detected several times with the specific rflp analysis method of resistance gene sequences, when testing result is shown
Terminate screening when only remaining a kind of microalgae mutant, obtain a plant height cell density microalgae mutant；When testing result is shown
When also remaining 2~5 kinds of microalgae mutant, fall the high cell density microalgae of separation acquisition using the single algae of solid medium progress and dash forward
Variant.
The higher muton of screening cell density can not be flat by solid in the mutant strain obtained from radom insertion mutation method
Screen choosing is obtained.Because the cell being coated on flat board and asynchronous growth, and regional nutrient depletion also results in algae and fallen greatly
It is small to lack correlation with stand density.Therefore, continuous passage muton mixing algae solution in present invention selection liquid medium within, by
High-cell density mutant strain can after the high muton of density can gradually occupy advantage, enrichment in continuous passage incubation
Separated after effective Secondary Culture from algae solution.
For monitoring whole process in real time, the present invention is tracked test to concentration effect using rflp analysis method.RFLP
(Restriction Fragment Length Polymorphism, restriction fragment length polymorphism) refers to gene
The difference of Restriction Fragment Length between type, this species diversity be on restriction enzyme site the insertion of base, missing, reset or
Caused by point mutation.By using digestion with restriction enzyme DNA, DNA fragmentation then is separated with gel electrophoresis, then DNA
Fragment is transferred on filter membrane, shows specific DNA fragmentation (Southern hybridization) using the hybridization of radiolabeled probe, finally
Carry out interpretation of result.In the present invention, resistant maker gene insertion position is different, (general using specific restriction enzyme
It is not include in resistant maker gene sequence) after processing, in the fragment of gained, the endonuclease bamhi comprising resistant maker gene is long
Degree can be variant, and the probe reused for the resistant maker gene sequence carries out Southern hybridization checks.
It is preferred that, the microalgae is Chlamydomonas reinhardtii.Screening technique of the present invention, which is applied equally to other, can carry out heredity turn
The microalgae of change, such as chlorella (Chlorella sp.), salt algae (Dunaliella salina), volvox (Volvox
Carteri), Phaeodactylum tricornutum (Phaeodactylum tricornutum) etc..
It is preferred that, the resistant gene is aadA genes, ble genes, the genes of aph VIII or als genes.
It is further preferred that the resistant gene is ble genes.The resistant gene that screening technique of the present invention is used is on the one hand
It is to be used to carry out resistance screening to mutant strain, is on the other hand to be used for the detection of rflp analysis method, so, as long as meeting at this 2 points
Gene order or fragment be applied to screening technique of the present invention.
It is preferred that, the resistant gene conversion microalgae uses electrotransformation.Certainly, it is applied to the conversion of microalgae using other
Method can also.Such as particle bombardment, also known as microparticle bombardment method, are applicable to various types of microalgaes and carry out heredity turn
Change；Bead conversion method can be used in microalgae for Cell wall deficiency.It is preferred that, inoculum density is that OD750 is 0.1 during passage
~0.2.
It is further preferred that passage number is no less than 30 times.Passage number is combined with RFLP testing results, if passed
For it is less when, RFLP testing results show mutant strain species it is seldom when, you can separated using solid medium；And
If passage number is more than 30 times, RFLP testing results are shown when mutant strain species is still more, it is necessary to proceed passage
To screen enrichment.
It is preferred that, in step (3), often pass on 5~15 times and detected with the specific rflp analysis method of resistance gene sequences.
Often pass on once, possible RFLP testing results change is smaller, it is possible to after continuous passage several times, carry out one-time detection.
It is preferred that, during rflp analysis, digestion is first carried out with Restriction Enzyme, then using the detection of Southern methods, used during detection
The probe for resistant gene of DIG marks hybridizes.
Screening technique of the present invention is mutated using resistant gene radom insertion, is then mixed each mutant of acquisition and is carried out liquid
Fast mutant strain is grown in body culture continuous passage, succeeding generations gradually occupy advantage and be enriched with, made during continuous passage
Screening effect is monitored with RFLP analysis methods, acquisition stand density can be finally screened under ordinary light conditions and compares wild type
High microalgae mutant.Screening technique of the present invention is without high optical condition, without special detection device, can quickly, easy, height
Effect ground completes screening process.
Brief description of the drawings
Fig. 1 is the growth curve chart of Secondary Culture mutant strain in embodiment 2.
Fig. 2 is continuous passage culture schematic flow sheet.
Fig. 3 is rflp analysis result figure, and wherein A, B, C represent 3 repetitions respectively, and swimming lane M is DNA marker (similarly hereinafter),
Swimming lane C is the wild type Chlamydomonas reinhardtii CC503 (similarly hereinafter) as control, and swimming lane 0,10,20,30 represents passage number respectively.
Fig. 4 be embodiment 4 in passage screening terminate after respectively to tri- groups of experiment screenings of A, B, C to mutant strain verify
Rflp analysis result figure, wherein swimming lane i1~i6 represents single separation strains (similarly hereinafter) respectively.
Fig. 5 is the rflp analysis result figure after double digestion genomic DNA in embodiment 4.
Fig. 6 is the growth curve testing result figure of screening gained 3 plant mutants strain in embodiment 5.
Fig. 7 is that the growth rate of screening gained 3 plant mutants strain in embodiment 5 compares figure, and wherein WT represents wild type Rhein
Chlamydomonas CC503 (similarly hereinafter).
Fig. 8 is screening gained 3 plant mutants strain maximum biomass knot in HS culture mediums and 2 times of HS culture mediums in embodiment 5
Fruit compares figure.
Fig. 9 is the chlorophyll content result figure of screening gained 3 plant mutants strain in embodiment 5.
Embodiment
The original algae strain of Chlamydomonas reinhardtii：Chlamydomonas reinhardtii CC503, purchased from chlamydomonas resource center (Chlamydomonas
Resource Center) (network address：www.chlamy.org).
Plasmid pSP124S：Purchased from chlamydomonas resource center, the plasmid includes ble gene orders.
The detection of Chlamydomonas reinhardtii OD values is detected under 750nm wavelength.
HS culture mediums：It is formulated referring to document Sueoka, N. (1960) Proc.Natl.Acad.Sci.USA 46,83-91,
Specific composition is as follows：
Mother liquor 1 (salting liquid)：
NH4Cl 100.0g
MgSO4·7H2O 4.0g
CaCl2·2H2O 2.0g
Plus H2O to 1L
Mother liquor 2 (phosphate buffer)：
K2HPO4 288.0g
KH2PO4 144.0g
Plus H2O to 1L
Mother liquor 3 (trace element)：
When HS culture mediums are used：5mL mother liquors 1,5mL mother liquors 2,1mL mother liquors 3 add water to 1L.
TAP culture mediums：Formula is referring to document Gorman, D.S., and R.P.Levine (1965)
Proc.Natl.Acad.Sci.USA 54,1665-1669, specific composition is as follows：
Mother liquor 1 (TAP salt)：
NH4Cl 15.0g
MgSO4·7H2O 4.0g
CaCl2·2H2O 2.0g
Plus H2O to 1L
Mother liquor 2 (phosphate buffer)：
K2HPO4 28.8g
KH2PO4 14.4g
Plus H2O to 100mL
Mother liquor 3 (trace element)：
When TAP culture mediums are used：2.42g Tris, 25mL mother liquor 1,0.375mL mother liquors 2, the 1mL acetic acid of 1mL mother liquors 3
(glacial acetic acid), plus H2O to 1L.
Embodiment 1
Sense primer Ble_1F：5’-TGGAAGCTTAAATGCCAGAAGGAGCGCAGCC-3’；
Anti-sense primer Ble_1160R：5 '-CCGAGCTCAGCTTCAAATACGCCCAGCCCG-3 ',
Plasmid pSP124S is expanded using above-mentioned primer, ble gene orders, nucleotide sequence such as SEQ ID No.1 institutes is obtained
Show, length is 1.1kb.
Above-mentioned ble genetic fragments are transformed into Chlamydomonas reinhardtii CC503 using electrotransformation intracellular, transformant with
Screened on the TAP flat boards of 2.5 μ g/mL bleomycins (zeocin), the Chlamydomonas reinhardtii algae grown on flat board falls as converting
The mutant strains of ble genetic fragments, the algae of all acquisitions is fallen and is saved in one by one in 96 orifice plates, builds Chlamydomonas reinhardtii mutant
Library.The present embodiment obtains more than 1000 Chlamydomonas reinhardtii mutant strain through the above method.
Electric step of converting：
(1) the algae solution 2500rpm for growth early stage of taking the logarithm is centrifuged 5 minutes and is collected frustule, and liquid (GeneArt is turned through electricity
MAX Efficiency Transformation Reagent) after cleaning centrifugation 3 times, it is resuspended to final concentration of 2 × 108~3 ×
108Individual/mL.
(2) every 250 μ L cell suspensions add 2~4 μ g and linearize incubation 5 minutes at DNA, 2~8 DEG C.
(3) the 250 μ L cell suspensions containing DNA are added in the cuvette of precooling before electricity conversion.
(4) electric Transformation Parameters are：Voltage 500V, electric capacity is 50 μ F, and resistance is 800 Ω.Generally, pulse duration time is
30ms。
(5) after electricity conversion, cell recovery 15 minutes.Cell is transferred to containing the mL of 10mL TAP-40mM sucrose solutions 50
Room temperature is placed in centrifuge tube；Then algae solution is transferred in incubator, temperature is set to 26 DEG C, and intensity of illumination is 50 μ E m-2s-1Incubate
Educate 14~16h.
(6) 2500rpm is centrifuged 5 minutes and is collected frustule, abandons supernatant, algae solution is resuspended in 200 μ L TAP culture mediums.
(7) screening mutant, many plates are carried out on the TAP culture medium flat plates containing 2.5 μ g/mL bleomycins (zeocin)
Screening collects obtains more than 1000 Chlamydomonas reinhardtii mutant strain altogether.These mutant strains are incubated at the fluid nutrient medium of 96 orifice plates
Saved backup in (the μ L of fluid nutrient medium volume 200).
Embodiment 2
More than 1000 mutant strain will be obtained in embodiment 1 and respectively takes 10 μ L algae solutions from 96 orifice plates, is centrifuged after mixing, with fresh
The HS nutrient solutions containing 2.5 μ g/mL zeocin prepared suspend, and are inoculated in the HS trainings containing 100mL (containing 2.5 μ g/mL zeocin)
Cultivated in the shaking flask of nutrient solution, culture illumination condition is continuous 50 μm of ol m-2s-1.In this case, the mutation of initial incubation
Strain (about 0.15OD) is 1, can respectively reach about 0.4OD, about 0.95OD, about 1.35OD, about 1.45 OD (Fig. 1) within 2,3,4 days.Carefully
Born of the same parents initially entered plateau at the 3rd day, chose the starting point progress continuous passage culture (Fig. 2) that this point is used as inoculation.Treat just
When the mixing algae solution that begins grows to OD750=1.0, mutant strain is seeded to continuation in 3 shaking flasks with the initial concentration for being about 0.15OD
Culture, is divided into A, B, tri- experimental groups of C.Continuous passage is carried out in platform early stage (3 days, about 1.4OD), takes fresh culture
(containing 2.5 μ g/mL zeocin) renewed vaccination (0 day, about 0.15OD), proceeds the passage of logarithmic phase and plateau.This is continuous
Passage number was 30 generations.
The RFLP of embodiment 3 is detected
For monitoring whole process in real time, the present invention is tracked test to concentration effect using RFLP.Due to RFLP collection of illustrative plates
In each unique size of insertion mutation strain at least one DNA fragmentation, in the initial incubation containing more than 1000 insertion mutations strains
In can see complexity RFLP collection of illustrative plates.With the continuation of passage, the complexity of RFLP collection of illustrative plates declines, and shows to train in continuous passage
HCD mutant strains (High-Cell-Density's writes a Chinese character in simplified form, and represents the high-cell density mutant strain screened) is gradually rich in supporting
Collection.After general 30 passages circulation, several bands are left behind in RFLP, the mutant strain containing these fragments is considered as HCD time
Select mutant strain.
For algae strain situation of change in detection succeeding generations, the rflp analysis of Ble sequence-specifics has been carried out to mutant strain.Tool
Gymnastics is made as follows：Just culture cell is collected, was passed on for the 10th generation, 20 generations, the cell sample in 30 generations, is cracked using CTAB buffer solutions,
Phenol: chloroform method extracts genomic DNA.With restriction enzyme NcoI digestion DNA, RFLP collection of illustrative plates situations are detected.Detection probe
PCR DIG probes synthetic agent box (Roche, PCR DIG Probe Synthesis are utilized from template plasmid pSP124S
Kit, article No.：11636090910) synthesize, synthetic primer is as follows：
Ble_E2_F：5'-TGGCCAAGCTGACCAGCGCCGTTCC-3'；
Ble_E3_R：5'-CCTCCGACCACTCGGCGTACAGC-3',
Last gained probe sequence is as shown in SEQ ID No.2, the probe conjugate anti digoxin antibody of alkaline phosphatase.
Southern hybridization is carried out according to DIG label films Crossing system.Comprise the following steps that, the genome after separation electrophoresis
DNA, DNA fragmentation is transferred on nylon membrane, is hybrid with it and is detected using the DIG of the above-mentioned synthesis probes marked.Detection is anti-
Should in α-DIG-AP by 1: 15000 dilution, be optimizing detection step, eluent composition is 0.1M maleic acids, 3M NaCl,
0.3%Tween20, pH 8.0.
In the passage assays of 3 repetitions in example 2, find there is 1,3 and 3 advantage respectively in experiment A, B, C
Fragment (Fig. 3).One advantage fragment STN_A1 present in experiment A exists in experimental group B and C.Exist in experiment B
Fragment STN_B3 there is also (STN_C2) in C.Therefore, carry out after continuous passage, enrichment obtains the possible HCD of at least four
Mutant strain.
Embodiment 4
Embodiment 2 is passed on to the mutant strain progress solid plate list algae obtained after 30 times and falls screening, it is single per plate picking 5-6
Algae falls to carry out RFLP checking analyses again.As a result show, 5 mutant strains in experiment A all show the same STN_A1 of a bar segment
Fragment is similar.Test in B, the 2 of separation, 4,6 is similar with STN_B1 fragments, and the 3 of separation are similar with STN_B3 with 5 fragments, separation
It is 1 similar with STN_B2 fragments.Test in C, the fragment of all separation is all similar with STN_C1, do not find to include with STN_C2 and
The separation strains (Fig. 4) of STN_C3 clip sizes.
To verify the fragment observed in separation strains really with consistent, the base of each separation strains being enriched with after 30 passages
Because group has carried out rflp analysis after NcoI and KpnI double digestion.Clip size after double digestion is with enrichment after 30 passages
Consistent (Fig. 5) of algae strain.As a result show, separation strains are the sociales in all insertion mutation strains.Therefore, including STN_B1
(separation strains STN_A1_i1-i5；STN_B1_i2, i4 and i6；STN_C1_i1 and i3-i6), STN_B2 (separation strains STN_B2_
) and the mutant strain of STN_B3 (separation strains STN_B3_i3 and i5) fragment is respectively designated as HCD1, HCD2 and HCD3 mutant strains i1.
Therefore, the present invention is enriched with by the way of continuous passage obtains the 3 plant height cell density mutant strains with more than, continuous to pass
The mutant strain of fast-growth or dense growth can be separated for method.
Embodiment 5
To analyze the growth rate and maximum cell density of HCD mutant strains, mutant strain is 50 μm of ol in continuous illumination intensity
m-2s-1HS culture mediums in grow, determine its growth curve (Fig. 6), growth rate and biomass.As a result show, HCD1 and
HCD2 growth rate is above wild type (p-value<0.001) (Fig. 7).HCD1, HCD2 for being grown in HS culture mediums and
HCD3 maximum cells density (maximum cell dry weight, CDW) 22%-40% higher than wild type；When culture medium is in 2 times of HS culture mediums
During growth, the high 50%-100% of maximum cell density ratio wild type (Fig. 8) of HCD mutant strains.Meanwhile, chlorophyll content (Fig. 9)
Shown with cellular morphology, HCD mutant strains have no significant difference compared with wild type.To sum up, HCD mutant strains have had been provided with being better than
The stand density and biomass of wild type Chlamydomonas reinhardtii.
Claims (9)
1. a kind of screening technique of high cell density microalgae mutant, it is characterised in that comprise the following steps：
(1) resistant gene is converted into microalgae, the mutation microalgae for being transferred to resistance gene sequences is obtained by solid plate resistance screening
Single algae fall, collect each single algae and fall to obtain microalgae mutant library；
(2) fall sampling from each single algae in microalgae mutant library, cultivated after mixing in liquid medium within, and continuously pass
Generation；
(3) the specific rflp analysis method detection of some alternative resistance gene sequences is often passed, one is only remained when testing result is shown
Terminate screening when planting microalgae mutant, obtain the high cell density microalgae mutant；When testing result show also remain 2~
During 5 kinds of microalgae mutant, carry out single algae using solid medium and fall the separation acquisition high cell density microalgae mutant.
2. screening technique as claimed in claim 1, it is characterised in that the microalgae is Chlamydomonas reinhardtii.
3. screening technique as claimed in claim 1, it is characterised in that the resistant gene is aadA genes, ble genes, aph
VIII gene or als genes.
4. screening technique as claimed in claim 3, it is characterised in that the resistant gene is ble genes.
5. screening technique as claimed in claim 1, it is characterised in that the resistant gene conversion microalgae uses electrotransformation.
6. screening technique as claimed in claim 1, it is characterised in that inoculum density is that OD750 is 0.1~0.2 during passage.
7. screening technique as claimed in claim 6, it is characterised in that passage number is no less than 30 times.
8. screening technique as claimed in claim 1, it is characterised in that in step (3), often passes on 5~15 times and uses resistant gene
The rflp analysis method detection of sequence-specific.
9. screening technique as claimed in claim 1, it is characterised in that during rflp analysis, first carries out digestion with Restriction Enzyme, then
Detected using Southern methods, the probe for resistant gene marked during detection with DIG hybridizes.
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| CN108993425A (en) * | 2018-07-24 | 2018-12-14 | 浙江海洋大学 | A kind of compound biological adsorption agent and its application |
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