CN106995817A - A kind of encoding chloroplast carbonic anhydrase gene is building resisting high-concentration CO2And the application in the Industrial Engineering microalgae of fast-growth - Google Patents
A kind of encoding chloroplast carbonic anhydrase gene is building resisting high-concentration CO2And the application in the Industrial Engineering microalgae of fast-growth Download PDFInfo
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Abstract
The invention belongs to technical field of microbial genetic engineering, and in particular to disclose a kind of encoding chloroplast carbonic anhydrase gene and building resisting high-concentration CO2And the application in the Industrial Engineering microalgae of fast-growth.Specific coding chloroplaset carbonic anhydrase gene is building resisting high-concentration CO2And the application in the Industrial Engineering microalgae of fast-growth.The method disclosed in the present is that industrial microalgae fixes industry CO2Waste gas is laid a good foundation and possible ways.
Description
Technical field
The invention belongs to technical field of microbial genetic engineering, and in particular to disclose a kind of encoding chloroplast carbonic anhydrase base
Because building resisting high-concentration CO2And the application in the Industrial Engineering microalgae of fast-growth.
Background technology
Global warming brought with the development of global fossil energy worsening shortages, and modern industrial or agricultural etc. is asked
Topic, development cleaning, renewable new energy turn into inexorable trend, and microalgae bioenergy is exactly the salvation for being wherein sent to great expectations
It is main.According to statistics, CO in air in nature2Carbon fixation total amount 40% be by the photosynthetic organisms such as marine microalgae complete.This
Give the credit to that microalgae is widely distributed in marine ecosystems, with the carbon sequestration rate higher than land plant and possess faster
Growth rate (microalgae for when be 4-16 hours).Simultaneously as microalgae can utilize solar energy and CO2Go to synthesize neutral fats
(triacylglycerol), polysaccharide (such as starch) and other high value added products (such as polyunsaturated fatty acid and astaxanthin), therefore it
Carbon capture ability be developed applied to the commercial Application in terms of clean fuel or material.
However, the carbon capture of exploitation microalgae is applied to large-scale industry CO2It is huge that carbon emission reduction and bioenergy still suffer from some
Big challenge.First, because the gas concentration lwevel of industrial flue gas is 10%-30%, CO is so captured by microalgae2One
Precondition is exactly that microalgae need to have high light to close carbon sequestration efficiency, very fast growth rate and high CO2Concentration tolerance.Second, be
Tackle the increasingly serious of global warming and Ocean acidification, it is also desirable to which understanding improves CO2The response of microalgae and its machine under concentration
System.3rd, high CO2Microalgae is cultivated under concentration, avoids do not tolerate high concentration CO to a certain extent2Bacterium or the pollution of fungi, it is full
The foot requirement of biological control.So, there is high CO for screening2The microalgae of tolerance phenotype, has used many research strategies, but receive
Effect is little, and main cause has：(1) the carbon fixation mechanism to microalgae lacks the understanding of system, and understanding is relatively clear at present
Only blue-green algae and chlamydomonas；(2) using the screening effect of the acquirements such as conventional measures mutagenesis, physical mutagenesis and adaptive evolution
Fruit is not obvious；(3) in most microalgaes, perfect genetic manipulation instrument is not yet set up；(4) in addition, many research displays
Tolerable high concentration CO2Microalgae substantially lack Carbon concentrating mechanism (CO2Concentration Mechanism, CCM), therefore,
Microalgae CCM activity may limit high CO2Tolerance.
Generally, for most of microalgaes, the CO of optimum growth2Concentration is 1-5%, is worth rejoice
It is, because ancient micro algae growth is in a relatively low CO2Concentration environment (is less than 0.04%), therefore most microalgaes are all entered
The CO of an adaptation low carbon dioxide ambient growth is changed2Concentrating mechanism (CCM), this mechanism mainly passes through carbonic anhydrase and nothing
A series of enzymatic reactions that machine carbon transport protein is participated in, can aid in concentrating the CO in carbon fixation site2Concentration, so as to improve solid
Carbon efficiencies.In prokaryotes cyanobacteria and eucaryon microalgae (such as chlamydomonas, diatom), the network model of CCM mechanism is built substantially
Vertical, the regulation and control to CCM key components (carbonic anhydrase and bicarbonate transport protein etc.) are provided with clear understanding, and pass through
Genetic engineering means are overexpressed CCM related components gene (such as bicarbonate transport protein), can screen in air level
(0.04%CO2) there is under concentration the mutant strain of very fast growth phenotype, cyanobacteria Rubisco energy is imported in higher plant tobacco
Photosynthetic efficiency is enough improved, but still high CO can not be solved2Concentration is resistant to this bottleneck problem.But, in high CO2Concentration culture
Middle CCM mechanism is generally all to close, and is especially perched in nature in high CO2In the microalgae of environment, their CCM mechanism
All it is non-existent, it is seen then that CCM activity may affect high CO2Tolerance.Therefore, CCM metabolism networks and reason are understood systematicly
Property engineering design be solve this bottleneck problem only way.
Micro- plan ball algae is a kind of unicellular photosynthetic microalgae, the environment waters such as widely distributed Yu Haiyang, fresh water.This class microalgae
Research inorganic carbon absorption and using mechanism under different gas concentration lwevels can be removed as a potential model, primarily for
Some considers below：(1) in the Seambiotic companies of Israel, micro- plan ball algae has been used for the extensive outdoor breeding of flue gas；
(2) it is resistant to wider pH value range (from 6.0-10.2), it means that its growth is seldom by the shadow of acidifying and calcification
Ring and (improve CO2Concentration can cause acidifying, reduce CO2Concentration can cause calcification)；(3) according to the literature, micro- plan ball algae will not produce
Extracellular carbonic anhydrase, this may suggest that it has active transport bicarbonate system or can be by the use of bicarbonate as only
One carbon source.So, it may use a unique CCM mechanism to assist carbon sequestration, and therefore, the microalgae of this kind of high oil-producing emerges
For oil-producing and the research model of photosynthetic carbon fixation.But, the understanding to micro- plan ball algae CCM network regulation mechanism and heredity at present
Transformation is all very deficient.
The content of the invention
Resisting high-concentration CO is being built it is an object of the invention to provide a kind of encoding chloroplast carbonic anhydrase gene2And fast fast-growing
Application in long Industrial Engineering microalgae.
To achieve the above object, the present invention use technical scheme for：
A kind of encoding chloroplast carbonic anhydrase gene is building resisting high-concentration CO2And in the Industrial Engineering microalgae of fast-growth
Application.
The encoding chloroplast carbonic anhydrase gene is with the nucleotide sequence any one of following (1) to (3)：
(1) there is the nucleotide sequence in sequence table SEQ ID No.1；
(2) polynucleotides of the protein sequence with polynucleotide SEQ ID No.1 nucleotides；
(3) there is the DNA sequence dna with SEQ ID No.1 in sequence table to have higher sequence homology, and encode identical work(
Can protein DNA sequence.
Above-mentioned micro- plan ball algae carbonic anhydrase, is named as CA2, from industrial microalgae (Nannochloropsis
Oceanica IMET1), wherein, the SEQ ID No.1 in sequence table encode a complete opening by 1218 base compositions
Reading frame, it encodes the protein of the amino acid residue sequence with sequence table SEQ ID No.2.
The structure resisting high-concentration CO2And the Industrial Engineering microalgae of fast-growth：Pass through reverse genetics RNA interference technology
The micro- ball algae chloroplaset carbonic anhydrase gene specific of intending of industrial microalgae is struck low, and then acquisition is resistant to high concentration CO2And can
The engineering microalgae of fast-growth.
Specially：RNAi expression vector is built, using diatom with RNAi expression vector as skeleton, by the silicon of carrier framework
Algae Fcp gene promoters are replaced into micro- plan ball algae endogenesis promoter, and clone the reverse weight of micro- plan ball algae chloroplaset carbonic anhydrase
Complex sequences, is connected to carrier and obtains RNAi expression vector；Then intend ball algae by the way that electroporation is micro-, micro- plan ball algae after conversion
Nutrient solution is incubated at the CO of high concentration2Under environment, high concentration CO is resistant to2And the high algae strain of growth rate is resisting high-concentration CO2
Engineering microalgae.
Wherein, it is micro- after a height of conversion of growth rate to intend the growth rate that ball algae culturing liquid growth rate is faster than wild type, life
Long speed improves 20%-30%.
The present invention constructs the expression vector that RNA interference (RNAi) technology is studied for reverse genetics, its carrier framework
Mainly include promoter sequence (micro- endogenous tublin gene promoters of plan ball algae), ble resistance gene sequences and for target base
Because of the inverted repeats of CA2 formation hairpin structures, its principle is mainly based upon the inverted repeats formation hair of different genes
Clamping structure and recognized by the RNAi elements of micro- plan ball algae inherently, and then realize the specific down-regulation of target gene.
The present invention is imported exogenous sequences in the micro- plan ball algae of the industrial microalgae of wild type by the method for electric pulse, passes through resistance
Screening obtains mutant strain.
Advantage for present invention：
The present invention is from micro- plan ball algae by low CO2Global express spectra change dynamic, combines simultaneously during concentration induction CCM
GC-MS identifies the change of major metabolite during this, constructs the central carbon metabolism network model such as CCM, and system understanding is micro-
Intend ball algae CCM function controls network mechanism basis, and then the present invention lowers chloroplaset carbonic acid in micro- plan ball algae by RNAi means
The expression of acid anhydride enzyme, to a certain extent, is lowered or part closing CCM mechanism ----Knockdown (Gene Knockdown), screening
High CO is arrived2Concentration is resistant to and growth rate is obtained for increased mutant strain simultaneously, and it is left that growth rate can improve 30-45%
The right side, high concentration CO2Culture is prevented effectively from or reduced the pollution of miscellaneous bacteria in incubation (bacterium and fungi etc.), with biological control
Effect, and production cost can be reduced available for industrial production.The industrial algae strain that the present invention is obtained is in bioenergy industrial applications
In terms of open the new gate of a fan.
Brief description of the drawings
The structure schematic diagram for micro- plan ball algae carbonic anhydrase RNAi expression vector that Fig. 1 provides for the present invention, it is main to include opening
The inverted repeats of mover, resistant gene and target gene；
The electrophoretogram of the PCR checkings for the transformant that Fig. 2 provides for the present invention expands resistance gene sequences using PCR, finds
There is purpose band about 400bp (the third and fourth swimming lane has band) in transformant, wherein WT is wild type (the first and second swimming
Road is without band)；
The mutant strain and the CA2 genes of wild type that Fig. 3 provides for the present invention are in high and low CO2Relative expression's abundance under concentration
The carbonic anhydrase gene C A2 of change mutant strain is substantially suppressed in rna expression level, and relative expression's abundance is reduced in 60%-
80% or so；
The mutant and wild type that Fig. 4 provides for the present invention are in height CO2The experiment of phenotype drop and liquid under condition of culture
Phenotypic characterization under culture；
The micro- plan ball algae of mutant strain and wild type that Fig. 5 provides for the present invention is in high and low CO2Growth under concentration condition of culture
Curve；
The micro- plan ball algae of mutant strain and wild type that Fig. 6 provides for the present invention is in high CO2Dry weight curve map under concentration.
Fig. 7 comes from high and low CO for what the present invention was provided2Mutant strain and the micro- plan ball frustule of wild type under concentration condition of culture
Hydrogen photoproduction；
Fig. 8 comes from high CO for what the present invention was provided2Mutant strain and wild type is micro- intends ball frustule under concentration condition of culture
Fat content.
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, but the scope of the present invention is not limited to
The content.
The present invention by reverse genetic strategy RNA interference (RNAi) technology to have struck low industrial microalgae it is micro- intend in ball algae one
Individual encoding chloroplast carbonic anhydrase gene and then obtain and be resistant to high concentration CO2And it is capable of the mutant strain of fast-growth, dash forward
The growth rate of mutant is in high concentration CO2(5-10%) is significantly improved (40%) compared with wild type, and in air concentration CO2Lower life
Long speed difference is not clear aobvious, high concentration CO2Lower culture efficiently avoid the dirt such as bacterium, protozoan during large-scale culture
Dye problem, in addition, fat content is accordingly improved, increase rate is in 20%-30% or so.The method disclosed in the present is
Industrial microalgae fixes industry CO2Waste gas is laid a good foundation and possible ways.
Method used is normal experiment method unless otherwise instructed in the embodiment of the present invention.
The micro- acquisition for intending ball algae (Nannochloropsis oceanica IMET1) carbonic anhydrase complete sequence of embodiment 1
By to micro- plan ball algae genome sequencing, tentatively obtaining the complete of the chloroplaset carbonic anhydrase that Matrix attachment region is encoded
Sequence, as shown in SEQ ID No.1 and SEQ ID No.2；And SEQ ID No.2 homologous sequence SEQ ID No.3.
The entire reading frame (ORF) for obtaining its chloroplaset carbonic anhydrase is further verified by polymerase chain reaction PCR,
PCR primer sequence is：F1 is 5 ' ATGTGGCGGCGTGTGCTCGCA3 '；R1 is 5 ' CTAGACGCTTTTATTAACC3 '.PCR is anti-
System is answered to include 5XPCR reaction buffers (5X DNA buffer) 5ul, deoxyribonucleoside triphosphate (dNTP) 4ul, sulfuric acid
Magnesium (MgSO4) 2ul, forward primer (Forward primer) 2ul, reverse primer (Reverse primer) 2ul, DNA3ul,
Archaeal dna polymerase (KOD DNA poleymase) 0.5ul, ddH2O is 32.5ul, and overall reaction system is 50ul；
PCR response procedures are 95 DEG C of 5min of the first step, 95 DEG C of reaction 1min of second step, 58 DEG C of 30s, 72 DEG C of reaction 30s
(30 circulations), the 3rd 72 DEG C of step extension 7min, the 4th 4 DEG C of step reaction 5min.
Embodiment 2
1) structure of RNAi expression vector
The structure of RNAi expression vector is using phir-PtGUS as carrier framework, first by pcr amplification reaction with micro- plan ball
Algae genome is template, and amplification obtains 229bp fragment (CA2 gene order 1395bp to 1623bp positions) and 404bp fragments
(CA2 gene order 1395bp to 1798bp positions) two PCR fragments；
Wherein, the primer of amplification 229bp fragment is：
CA2_Fw(5’CGGAATTCGGGCATAGAGTGCGAATTGA3’；Include EcoRI sites)/CA2_Rv1 (5 '
GCTCTAGACGCTTTTATTAACCCCATCC3’；Include XbaI sites)；
The primer of fragment for expanding 404bp is：
CA2_Fw(5’CGGAATTCGGGCATAGAGTGCGAATTGA3’；Include EcoRI sites)/CA2_Rv2 (5 '
GCTCTAGAATCCTGGTCGTCAAAGAACG3’；Include XbaI sites).
It is above-mentioned enter performing PCR amplification reaction system be 50ul：Including 5XPCR reaction buffers (5X DNA buffer)
5ul, deoxyribonucleoside triphosphate (dNTP) 4ul, magnesium sulfate (MgSO4) 2ul, forward primer (Forward primer) 2ul,
Reverse primer (Reverse primer) 2ul, DNA 3ul, recombinant DNA polymerase (KOD DNA poleymase) 0.5ul,
ddH2O is 32.5ul, and overall reaction system is 50ul；
PCR response procedures are 95 DEG C of 5min of the first step, 95 DEG C of reaction 1min of second step, 60 DEG C of 30s, 72 DEG C of reaction 30s
(30 circulations), the 3rd 72 DEG C of step extension 7min, the 4th 4 DEG C of step reaction 5min.
Contain a common fragment by above-mentioned two fragments of acquisition for 229bp, purpose formation reverse complementary sequence (neck ring
Hairpin structure).Then limit endonuclease digestion two above PCR fragment with EcoRI and XbaI and use PCR primer purification kit
Purified, while digesting phir-PtGUS carriers with EcoRI and being carried out with calf intestinal alkaline phosphatase at fragment dephosphorylation
Purified after reason.Again by EcoRI and XbaI two PCR fragments digested and linearized vector (after phir-PtGUS EcoRI digestion
Linear fragment) it is attached with T4 ligases and obtains plasmid phir-PtCA2.Micro- startup for intending ball algae Beta-tubulin genes
Son uses primer Notub_fw (5 ' GCGAGCTCGCCAGCTGCCTCAT3 '；Include SacI sites) and Notub_rv (5 '
GGCCATGG3TGTGTCCGCCGCC’；Include NcoI sites) it is amplified, then with SacI and NcoI digestion with restriction enzyme,
Carrier phir-PtCA2 is also with SacI and NcoI digestion with restriction enzyme and purifying simultaneously, then again by Beta-tubulin
The promoter fragment of gene is attached with linearized vector (promoter of Beta-tubulin genes) with T4DNA ligases
Obtain carrier phir-NgCA2, its carrier comprising the promoter regions of Beta-tubulin genes, resistant gene ble, CA2 it is positive and negative
To repetitive sequence and the terminator of fcp genes, composition expression cassette (as shown in Figure 1).
2) the PCR amplifications of RNAi expression cassettes
With PCR primer (F1：5’CCCAGTCACGACGTTGTAAAACG3’；R1：5’GGAAACAGCTATGACCATG 3’)
The expression vector of amplification above example 2 obtains including tub gene promoters, resistant gene ble, can form hairpin structure
CA2 sequences and transcription terminator sequences, PCR reaction systems are 5XPCR reaction buffers (5X DNA buffer) 5ul, deoxidation
Ribonucleotide triphosphate (dNTP) 4ul, magnesium sulfate (MgSO4) 2ul, forward primer (primer F1) 2ul, reverse primer
(primer R1) 2ul, template DNA (DNA) 3ul, archaeal dna polymerase (KOD DNA poleymase) 0.5ul, ddH2O
32.5ul, overall reaction system is 50ul；PCR response procedures are 95 DEG C of 5min of the first step, 95 DEG C of second step, 56 DEG C of 1min 30s 72
DEG C 45s (30 circulations), the extension of the 3rd 72 DEG C of step 7min, the 4th 4 DEG C of step 5min.After the completion for the treatment of pcr amplification reaction, by what is obtained
PCR primer electrophoresis detection and the purifying of further PCR primer, make its concentration reach 3-5ug/ul, are put in -20 DEG C of preservations, treat next
Walk standby during electroporation.
3) micro- acquisition for intending ball algae mutant strain
The above-mentioned RNAi expression cassettes fragment that obtains is imported by the micro- plan ball algae of wild type using electroporation method.Specific implementation
Method is：The micro- plan ball algae of wild type is cultivated to concentration in f/2 culture mediums and reaches 3 × 107(condition of culture is 25 to cells/ml
DEG C, continuous illumination culture, light intensity is 50umol/m-2S-1, throughput is 100ml/min), microalgae cell is collected by centrifugation and outwells
Clear liquid, frustule three times is then washed with the 375mM of 4 DEG C of precoolings sorbitol solution and frustule is resuspended with it, makes frustule
Concentration is 8 × 108cells/ml；Frustule solution after concentration is divided into 200ul a, then added in every part on 3ug
Acquisition linearized vector and 1ul salmon sperm dnas (15ug/ml) are stated, 10min is placed after mixing on ice；Mixture is transferred to again
2mm electric shock cups, with 2200V (HV), 600 ohm, 50 microfarads (uf) are shocked by electricity, immediately that frond is fresh with 5ml after electric shock
F/2 media transfers enter sterile hoary hair's glass tube, cultivate (being recovered with dim light) in illumination shaking table after shading, rotating speed is set
It is set to 100rpm, 25 DEG C of recovery 48h；It is centrifuged into 5min in 5000g after recovery, most supernatant is abandoned, then with the corns of 1ml 20%
Starch is resuspended after frond, on the flat board for being poured on the f/2 of agar content 0.8%, is uniformly spread out and is blown flat board in superclean bench
Sealed after dry, be put in 25 DEG C of illumination cultivations, after selecting transformant after 3-4 weeks.
20% cornstarch is alternately washed 3 times, then use 10ml to take 2g cornstarch with absolute ethyl alcohol and ultra-pure water
The resuspension of 70% ethanol, washed 4 times with f/2 culture mediums, then be resuspended with f/2 nutrient solutions of the 10ml containing 0.8%PEG8000.
It is previously prepared agar plate on the f/2 of the agar content 0.8% flat board, then adds 3-5ug/ml's
Zeocin antibiotic)
The PCR checkings of the transformant of embodiment 3
Transformant is selected with toothpick in fresh f/2 culture mediums (the Zeocin antibiotic for including 3-5ug/ml), light is put in
According to incubator culture, its growth 2-3 weeks is treated, frustule is collected by centrifugation, with genome DNA extracting reagent kit (OMEGA HP DNA
Extration kit) genomic DNA of transformant is extracted, while the micro- genomic DNA for intending ball algae of wild type is also extracted, as
The control of subsequent experimental.After extracting genome and being quantified with Nannodrop, with forward primer F1 (5 '
TTATCAACGGCATACCGGCACTG3 ') and reverse primer R1 (5 ' CTGATGAACAGGGTCACGTCGT3 ') enter performing PCR amplification
Reaction, reaction system is 50ul：5XPCR reaction buffers (5X DNA buffer) 5ul, deoxyribonucleoside triphosphate
(dNTP) 4ul, magnesium sulfate (MgSO4) 2ul, forward primer (primer F1) 2ul, reverse primer (primer R1) 2ul, masterplate
DNA (DNA) 3ul, archaeal dna polymerase (KOD plus DNA poleymase) is 0.5ul, ddH2O 32.5ul, overall reaction system
For 50ul.Response procedures are：95 DEG C of 5min of the first step, 95 DEG C of second step, 72 DEG C of 1min56 DEG C of 30s 45s (30 circulations), the 3rd
Step 72 DEG C of extensions 7min, the 4th 4 DEG C of step 5min.After the completion of pcr amplification reaction, agarose gel electrophoresis detection sees whether use
Transformant DNA is that template has 400bp bands, and wild type does not have band, as shown in Figure 2, it was demonstrated that exogenous sequences have been integrated
To micro- plan ball algae genome, electroporation success obtains mutant strain, mutant strain can be respectively labeled as M2, M4.
Embodiment 4 is micro- to intend ball algae mutant strain CA2 genes relative expression levels measurement
By above-mentioned acquisition mutant strain and wild type respectively in different CO2Concentration (different CO2Concentration is that high concentration is 5%CO2,
Low concentration is 0.04%CO2) under cultivate, intensity control is unanimously in 50umol/m2/ s, Ventilation Rate is in 100ml/min.Treat microalgae
Collect microalgae when culture is to exponential phase, then 5000g centrifugation 5min use liquid nitrogen quick freeze, be stored in -80 DEG C it is standby.
The experiment measurement of mutant strain gene expression abundance is divided into following three step：
First step RNA extraction, carries out RNA extractions, first by the way of liquid nitrogen grinding by the frustule of freezen protective
Then broken frustule, general liquid nitrogen grinding 5-10min adds the rapid fully mixings of 1ml Trizol (Invitrogen companies)
And add 200ul chloroforms 10min is centrifuged under 12000rpm, Aspirate supernatant is transferred in new eppendorf pipes and added
Volume of chloroform, fully vibration are mixed, and stand after 2-3min to centrifuge 10min under 12000rpm, supernatant is transferred to new centrifugation
Manage and add isometric isopropanol precipitation at room temperature 15min, centrifuge after 15min, then washed with 75% ethanol after precipitation, drying is added
The ddH of 50ul DEPC processing2After O, measurement RNA concentration, -80 are stored in standby；
Second step, cDNA preparation, RNA samples are according to Takara reagentsRT reagent Kit
With gDNA Eraser kit process description carries out cDNA synthesis using random primer, and remarks genomic DNA removes anti-
It is 20ul, total serum IgE, 5X reaction buffer and gDNA Eraser enzymes including 2ug to answer system；
3rd step, quantitative fluorescent PCR reaction, passes through480II is (general using the quickly startup of Roche
Green fluorescence) fluorescent quantitation qRT-PCR quantified.PCR reaction system is the 1ul cDNA (reverse transcriptions from previous step
Prepare), forward and reverse primer each 1ul, 10ul 2X Roche FastStart SYBR Green Master (ROX), ddH2O adds
Plus 7ul complements to 20ul, wherein primer sequence is forward primer：5’ATCCAGGAACAATTATTTCGCT3’；Reverse primer：5’
GAGTCGGAACAAGCAATAA3’.Reaction condition is performed with reference to Roche kits explanation.Real-time fluorescence qPCR result datas
Analysis uses 2-ΔΔCtMethod, wherein Δ Δ Ct values are (CtCA2 genes-CtReference gene)Mutant strain-(CtCA2 genes-CtReference gene)Wild type。
The gene expression abundance of versus wild type of the CA2 genes in mutant strain is calculated by above method, is as a result confirmed
The CA2 gene expression abundances of mutant strain (M2 or M4) are either in low CO2Or in high CO2It is substantially less than wild under condition of culture
Type CA2 expression, it was demonstrated that CA2 genes are suppressed in transcriptional level, about lower 60%-80%, as shown in figure 3, further card
Real CA2 genes are silenced in mutant strain M2, M4.
Embodiment 5 is micro- to intend the resistance to CO of ball algae2The growth phenotype measurement of mutant strain
The micro- plan ball algae of mutant strain and wild type is fallen to be placed in f/2 culture mediums in 50ml triangles with toothpick picking list algae respectively
Cultivated 2-3 weeks in bottle, then measure OD750Under cell density, then renewed vaccination makes inoculation dense to fresh f/2 culture mediums
Degree is consistent, i.e. identical OD750Value=0.5-1.0, then continuous passage culture 2-3 times, is seeded to 200ml's by microalgae
Column reactor culture (in reactor culture medium be f/2 culture mediums), after exponential phase is grown into, by mutant strain and wild
The micro- plan ball algae of type is diluted to identical concentration (said determination OD respectively750Value is identical), absorption 10ul dilutions drip to pre- respectively
On the f/2 solid agar flat boards first prepared (agar concentration is 0.8% and without antibiotic), each algae sample is dripped to respectively
(in two CO on two pieces of flat boards2Cultivated under concentration, be 0.04% and 5% CO2 concentration respectively), dried up on superclean bench
Afterwards, placed 2-3 days under room temperature, unglazed or low luminous environment, flat board is then positioned over simple incubator (number of patent application：
201410339234.9), and one of incubator is passed through air concentration CO2, another is passed through high concentration CO2(5%) or
It is passed through low concentration CO2(0.04%), light intensity 50umol/m2/ s, relative close type culture screening can be carried out with this incubator, led to
The CO of incubator internal stability can be controlled by overregulating incubator air inlet and gas outlet valve2Environment, irregularly observes microalgae
The growing state (Fig. 4) of (mutant strain and wild type).Meanwhile, microalgae will be also inoculated with from 200ml column reactors to fresh liquid
The physical signs such as growth curve and dry weight is measured in body culture medium.Measurement result such as Fig. 5, shown in 6, has found mutant strain in high CO2
Growth is faster than wild type under concentration, and dry weight increases by 30% or so (Fig. 6) after being cultivated through 10 days.Therefore, the side of Knockdown is passed through
Method obtains the high CO of tolerance2Concentration cuts the mutant strain for being capable of fast-growth, is laid to cultivate industrial microalgae under industrialization flue gas
Solid conditions for sports industry.
Embodiment 6 is micro- to intend ball algae resisting high-concentration CO2The physiological phenotype measurement of mutant strain
By above-mentioned resistance to high CO2Micro- plan ball algae mutant strain of concentration and the hydrogen photoproduction of wild-type cell, which are determined, to be used
Clark-II types oxygen electrode (Hansatech companies of Britain) is carried out, and the temperature of reactive tank is 4 DEG C (water-bath adjustment), light during measure
300 μm of ol m are adjusted to anti-dazzling screen by force-2s-1.By micro- plan ball algae mutant strain and wild-type cell culture to exponential phase,
Frustule and 15ml centrifuge tubes, each two parts of sample collection are collected with 5000g centrifugations 5min, portion is used to determine photosynthetic oxygen evolution speed
Rate, it is a in addition to be used for the measurement of chlorophyll content.The microalgae cell of collection first (is passed through N in advance with carbon-free water2Exclude solution
In CO2) washing, then it is suspended in again in the buffer solution of different pH value, is followed successively by 20mmol L-1 MES buffer solutions,
(pH6.0,7.8 or 9.0), buffer solution in the carbon-free water of 20mmol L-1Tris buffer solutions and 20mmol L-1 APES buffer solutions
Configuration seawater then determine hydrogen photoproduction under condition of different pH of mutant strain and wild-type cell respectively.It is simultaneously every
Individual sample leaves and takes a content for being used for measuring chlorophyll a, and the measurement of chlorophyll a uses the method that methanol is extracted, using following
This formula calculates the content of chlorophyll a (chla)：
Chla content (ug/ml)=16.5169XA665-8.0962XA652
Hydrogen photoproduction is converted with the content of chlorophyll a.Measurement result shows, the photosynthetic oxygen evolution of Mutant Cells
Speed is under the conditions of pH6.0, and the hydrogen photoproduction of mutant strain is significantly increased, and under the conditions of pH9.0, mutant strain it is photosynthetic
Put oxygen speed is significantly reduced again, therefore mutant strain can be resisted due to CO2PH reductions caused by concentration rise, to a certain extent
Show that the microenvironment inside acid resistance, possible chloroplaset is improved, i.e., pH gets a promotion, so fast-growth is shown,
As shown in Figure 7.
Then, the measure of fat content is carried out to above-mentioned acquisition mutant strain, grease is extracted to be taken out using methanol/chloroform method
Carry, crude oil fat is weighed after being dried through nitrogen evaporator, as a result show that mutant strain fat content increase by 20% under regular culture conditions is left
The right side, as shown in figure 8, fat content increases to 0.25mg/mg dry cell weights by original 0.18mg/mg dry weights.
SEQ ID No.1
ATGTGGCGGCGTGTGCTCGCATATTCCTCGAGATTCGATGTAACACATCTCAATAGCGAGGTAACGCTATCCACGTA
TCTCACCATTGTCTCCACCCTCTCTATTAACCCCATCTTTCTATGCAGTTCGCAAAAATCTCCGATACGCGGCCCTG
CGGTTAATCCAACAAGTCAAGACCTTGAAACGTCGGAAATGAGCAACCCGCCGTCTCCTGTCGTGCCGTCTCCCAGC
CTTACACAATCAAATCATGACATGCTCACCAGCATCCGCAGCCACCTAGACGACCAAGAGGTCAAGATCCAGGAACA
ATTATTTCGCTTCCAGGGCATTAAGAGGGCCATTGACCGCATTTGCAAGAATGACGAGGCCTACATGGACTCCCCCG
ATATTGTCAAGGCACAGACCACGCTCGAAGATCGCCCGGAGCTGGAAAGGAAAGGAGAGACTGCTGCTGAAGCCTTG
CGCCTGCTCA AAGTGGGCAACGCTGCCTTTTTGCGCGATGAGGTTGTGCCTGTCCACCGCTCTGACAGACATATTG
GCCTACATTTTGCCCAGAAACCCCACGCCATAATTATTGCTTGTTCCGACTCTCGGGTCCCGCCGGAGATGATCTTT
AATTGCGCGTTTGGTGAACTCTTCGTCATCCGCCTTGCCGGCAACACCGTCGATGCACTCGCTCGCGCCAGCCTCCT
TTACGCGGTGCAACACCTCCAATCTCCCCTGGTCGTTGTTCTGGGCCACGAAAAGTGCGGCGCCGTCACAGCCGCCT
TGCAGCCGGAAGAGGAGCTGGCGGATGCTCCCGGGGACATTAAGACCTTGGTGCGAAACATCAAAAGGGGCATAGAG
TGCGAATTGATTGACCCGAGCGCCCACATTTTCGGCGATGAAAGACTGTTGTGCGCGATTGTCTGCAACGTGCATTA
CGTGGCCCGCACCCTGTCGGAACACCCCGATATCCGCCCATTTATTGACCGAAGGAAACTGTCCGTCGTGGGCGCCT
ACTACGCTTTTAGTGGGGTGGTGTCGTTCTTTGACGACCAGGATGAGGGAGGAAGGATGGAGAGAGGGCGGGTGTTT
GGGGGTGTGGAGGGAAACGGATCCTTGTGCTTAAGTTGTTCCGGAACTTTGTCCGGAAACTCGAGTCCGATGGCGGG
GGGAGGGAGCAAGGGATCCAGTCCGCCTCCGAGCGTGCAGGATGGGGTTAATAAAAGCGTCTAG
SEQ ID No.2
MWRRVLAYSSRFDVTHLNSEVTLSTYLTIVSTLSINPIFLCSSQKSPIRGPAVNPTSQDLETSEMSNPPSPVVPSPS
LTQSNHDMLTSIRSHLDDQEVKIQEQLFRFQGIKRAIDRICKNDEAYMDSPDIVKAQTTLEDRPELERKGETAAEAL
RLLKVGNAAFLRDEVVPVHRSDRHIGLHFAQKPHAIIIACSDSRVPPEMIFNCAFGELFVIRLAGNTVDALARASLL
YAVQHLQSPLVVVLGHEKCGAVTAALQPEEELADAPGDIKTLVRNIKRGIECELIDPSAHIFGDERLLCAIVCNVHY
VARTLSEHPDIRPFIDRRKLSVVGAYYAFSGVVSFFDDQDEGGRMERGRVFGGVEGNGSLCLSCSGTLSGNSSPMAG
GGSKGSSPPPSVQDGVNKSV
SEQ ID No.3
MTGSVGTSVKPSISQQNHEVLERIRHHLDDQEHKIHEQLNRFQLIKKAVERICKNDDAYMQSPDIVKAQTTLEDSPL
ERKGNTAAEALELLKAGNAAFLRDEVPAKQCSDRHIGLHFAQKPHAIIIACSDSRVPPEMIFNCAFGELFVIRLAGN
TIDALARASLLYAVQHLHSPLVVVLGHEKCGAVTAALQPPEALAEAPRDIKTLVKKIQSGIECALLEPGLEDVDDER
LLCAIVCNVHSVARTLSEHPDIRPFIEQKALSVVGAYYAFNGVVSFFDDGEDGNATGKEVGSTVNGQASLCMSCSGT
IHRSSSPTADPSQKVNSRLSVSEGLSKNT
Claims (4)
1. a kind of encoding chloroplast carbonic anhydrase gene is building resisting high-concentration CO2And in the Industrial Engineering microalgae of fast-growth
Using.
2. the encoding chloroplast carbonic anhydrase gene as described in claim 1 is building resisting high-concentration CO2And the industry of fast-growth
Application in engineering microalgae, it is characterised in that：The encoding chloroplast carbonic anhydrase gene is to appoint with following (1) into (3)
Nucleotide sequence described in one：
(1) there is the nucleotide sequence in sequence table SEQ ID No.1；
(2) polynucleotides of the protein sequence with polynucleotide SEQ ID No.1 nucleotides；
(3) there is the DNA sequence dna with SEQ ID No.1 in sequence table to have higher sequence homology, and coding identical function egg
The DNA sequence dna of white matter.
3. the encoding chloroplast carbonic anhydrase gene as described in claim 1 is building resisting high-concentration CO2And the industry of fast-growth
Application in engineering microalgae, it is characterised in that：By reverse genetics RNA interference technology by industrial microalgae it is micro- intend ball algae chloroplaset
Carbonic anhydrase gene specific strikes low, and then acquisition is resistant to high concentration CO2And it is capable of the engineering microalgae of fast-growth.
4. the encoding chloroplast carbonic anhydrase gene as described in claim 3 is building resisting high-concentration CO2And the industry of fast-growth
Application in engineering microalgae, it is characterised in that：RNAi expression vector is built, using diatom with RNAi expression vector as skeleton,
The diatom Fcp gene promoters of carrier framework are replaced into micro- plan ball algae endogenesis promoter, and clone micro- plan ball algae chloroplaset carbon
The inverted repeats of acid anhydrides enzyme, is connected to carrier and obtains RNAi expression vector；Then intend ball algae by the way that electroporation is micro-,
It is micro- after conversion to intend the CO that ball algae culturing liquid is incubated at high concentration2Under environment, high concentration CO is resistant to2And the high algae of growth rate
Strain is resisting high-concentration CO2Engineering microalgae.
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| CN107841495B (en) * | 2017-11-27 | 2020-11-06 | 浙江大学 | Method for rapidly screening nuclear mutagenesis carbon fixing algae strain by utilizing photosynthetic oxygen evolution electronic separation |
| CN110066817A (en) * | 2019-05-21 | 2019-07-30 | 上海海洋大学 | Kelp α type carbonic anhydrase gene Sj α-CA2 and its coding albumen and application |
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| KR20220020766A (en) * | 2020-08-12 | 2022-02-21 | 고려대학교 산학협력단 | Carbonic anhydrase complex and method for enhancing carbon dioxide biofixation and lipid production through the complex |
| KR102582851B1 (en) | 2020-08-12 | 2023-09-26 | 고려대학교 산학협력단 | Carbonic anhydrase complex and method for enhancing carbon dioxide biofixation and lipid production through the complex |
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