CN106995817B

CN106995817B - Construction of high concentration CO resistance by using gene of coding chloroplast carbonic anhydrase2Application of industrial engineering microalgae capable of rapidly growing

Info

Publication number: CN106995817B
Application number: CN201610052610.5A
Authority: CN
Inventors: 魏力; 王勤涛; 辛一; 徐健
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2016-01-26
Filing date: 2016-01-26
Publication date: 2020-08-14
Anticipated expiration: 2036-01-26
Also published as: CN106995817A

Abstract

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a method for constructing high-concentration CO resistance by using a gene encoding chloroplast carbonic anhydrase₂And the industrial engineering microalgae which can grow rapidly. Construction of high concentration CO resistance gene of chloroplast carbonic anhydrase gene₂And the industrial engineering microalgae which can grow rapidly. The method disclosed by the invention is used for fixing industrial CO by using industrial microalgae₂The waste gas lays a foundation and a feasible method.

Description

Construction of high concentration CO resistance by using gene of coding chloroplast carbonic anhydrase2Application of industrial engineering microalgae capable of rapidly growing

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a method for constructing high-concentration CO resistance by using a gene encoding chloroplast carbonic anhydrase₂And the industrial engineering microalgae which can grow rapidly.

Background

With the increasing shortage of global fossil energy and the global warming caused by the development of modern agriculture and industry, the development of clean and renewable new energy is a necessary trend, and the microalgae bioenergy is a promising rescuer. According to statistics, the CO in the air in nature₂40% of the total carbon fixation is accomplished by photosynthetic organisms such as marine microalgae. This is due to the wide distribution of microalgae in the marine ecosystem, to the higher carbon fixation rates and to the faster growth rates (4-16 hours for microalgae) than terrestrial plants. At the same time, the user can select the desired position,since microalgae can utilize solar energy and CO₂To synthesize neutral lipids (triacylglycerols), polysaccharides (such as starch) and other high value-added products (such as polyunsaturated fatty acids and astaxanthin), their carbon capturing capabilities are being exploited for industrial applications in the cleaning of fuels or materials, etc.

However, exploitation of microalgal carbon capture for large-scale industrial CO₂Carbon abatement and biological energy sources still face some significant challenges. First, since the carbon dioxide concentration of industrial flue gas is 10% -30%, this captures CO by microalgae₂The precondition is that the microalgae needs to have higher photosynthetic carbon sequestration efficiency, faster growth rate and high CO₂Concentration tolerance. Second, to cope with global warming and ocean acidification becoming more severe, it is also necessary to recognize that CO should be increased₂The response of microalgae at concentration and its mechanism. Third, high CO₂Culturing microalgae under the concentration to a certain extent avoids intolerance of high-concentration CO₂The pollution of bacteria or fungi meets the requirements of biological control. Thus, for screening with high CO₂Phenotypic tolerant microalgae, using many research strategies, have met with little success, mainly due to: (1) the carbon fixing mechanism of the microalgae is lack of systematic knowledge, and at present, the knowledge is relatively clear and only comprises blue algae and chlamydomonas; (2) the screening effect achieved by traditional strategies such as chemical mutagenesis, physical mutagenesis and adaptive evolution is not obvious; (3) in most microalgae, a complete genetic manipulation tool has not been established; (4) in addition, many studies have shown that high concentrations of CO can be tolerated₂The microalgae of (A) are significantly deficient in the carbon concentration mechanism (CO)₂Concentration Mechanism, CCM), therefore, microalgae CCM activity may limit high CO₂And (4) tolerance.

In general, for most microalgae, the most suitable CO for growth₂The concentration is 1-5%, it is fortunate that the old microalgae grow on a lower CO₂Concentration environment (less than 0.04%), therefore most microalgae have evolved a CO suitable for growth in low carbon dioxide environment₂The concentration mechanism (CCM) which is mainly composed of a series of enzymes involved in carbonic anhydrase and inorganic carbon transportersPromoting reaction and assisting concentration of CO at carbon fixing site₂Concentration, thereby improving the carbon fixation efficiency. In prokaryotes, cyanobacteria and eukaryotic microalgae (such as chlamydomonas, diatoms and the like), a network model of a CCM mechanism is basically established, the regulation and control of CCM key components (carbonic anhydrase, bicarbonate transporter and the like) are clearly known, and genes of CCM related components (such as bicarbonate transporter) are over-expressed by a genetic engineering means, so that the CCM related components can be screened to be at the air level (0.04% CO)₂) The introduction of cyanobacteria Rubisco into higher plant tobacco of mutants having a faster growth phenotype at concentrations can improve photosynthesis efficiency, but still cannot solve high CO₂The concentration tolerates this bottleneck problem. However, at high CO₂CCM mechanisms are normally switched off in concentration cultures, especially when they live in very high CO in nature₂In environmental microalgae, their CCM mechanism is not present, and it can be seen that CCM activity may affect high CO₂The tolerance of (2). Therefore, the system recognizes that CCM metabolic networks and rational engineering are the only way to solve this bottleneck problem.

Nannochloropsis is a kind of unicellular photosynthetic microalgae, widely distributed in ocean, fresh water and other environmental waters. This class of microalgae can be used as a potential model to study the mechanism of inorganic carbon absorption and utilization at different carbon dioxide concentrations, mainly due to the following considerations: (1) in Seambiotic, israel, nannochloropsis has been used for flue gas large scale outdoor cultivation; (2) it is able to tolerate a wide pH range (from 6.0 to 10.2), which means that its growth is less affected by acidification and calcification (increase of CO)₂The concentration can cause acidification and reduce CO₂Concentration can lead to calcification); (3) it is reported in the literature that nannochloropsis does not produce extracellular carbonic anhydrase, which might suggest that it has an active bicarbonate transport system or is capable of utilizing bicarbonate as the sole carbon source. Thus, it may use a unique CCM mechanism to assist in carbon sequestration, and thus, such high oil yielding microalgae are emerging as research models for oil production and photosynthetic carbon fixation. However, the current knowledge and genetic modification of the nannochloropsis CCM network regulation mechanism are very deficient.

Disclosure of Invention

The invention aims to provide a gene for coding chloroplast carbonic anhydrase in construction of high concentration CO resistance₂And the industrial engineering microalgae which can grow rapidly.

In order to achieve the purpose, the invention adopts the technical scheme that:

construction of high concentration CO resistance by using gene of coding chloroplast carbonic anhydrase₂And the industrial engineering microalgae which can grow rapidly.

The gene encoding chloroplast carbonic anhydrase has the nucleotide sequence described in any one of (1) to (3) below:

(1) has a nucleotide sequence in a sequence table SEQ ID No. 1;

(2) a polynucleotide having a protein sequence encoding a nucleotide sequence set forth in SEQ ID No.1 of the sequence Listing;

(3) has a DNA sequence which has higher sequence homology with the DNA sequence of SEQ ID No.1 in the sequence table and codes the protein with the same function.

The Nannochloropsis carbonic anhydrase named CA2 is derived from industrial microalgae (Nannochloropsis oceanica IMET1), wherein SEQ ID No.1 of the sequence Listing consists of 1218 bases and encodes a complete open reading frame encoding a protein having the amino acid residue sequence of SEQ ID No.2 of the sequence Listing.

The construction is resistant to high concentration CO₂And industrial engineering microalgae which grow rapidly: through reverse genetics RNA interference technology, the carbonic anhydrase gene of chloroplast of industrial microalgae nannochloropsis oculata is specifically knocked down, and high-concentration CO tolerance is further obtained₂And engineering microalgae capable of growing rapidly.

The method specifically comprises the following steps: constructing an RNAi expression vector, namely replacing a diatom Fcp gene promoter of a carrier framework with a nannochloropsis oculata endogenous promoter by adopting the RNAi expression vector of diatoms as the framework, cloning a reverse repeat sequence of nannochloropsis oculata chloroplast carbonic anhydrase, and connecting the reverse repeat sequence to the vector to obtain the RNAi expression vector; then, electric pulse is used to transform nannochloropsis, and the transformed nannochloropsis culture solution is cultured in high-concentration CO₂Can tolerate high concentration CO under environment₂And has a high growth rateThe algae strain can resist high concentration CO₂The engineered microalgae of (1).

Wherein the growth rate is high, namely the growth rate of the transformed nannochloropsis oculata culture solution is higher than that of a wild type, and the growth rate is improved by 20-30%.

The invention constructs an expression vector for reverse genetics research RNA interference (RNAi) technology, the vector skeleton mainly comprises a promoter sequence (nannochloropsis endogenous tublin gene promoter), a ble resistance gene sequence and an inverted repeat sequence forming a hairpin structure aiming at a target gene CA2, and the principle is that the hairpin structure is formed on the basis of the inverted repeat sequences of different genes and is recognized by an RNAi element inherent to nannochloropsis, so that the specific down regulation of the target gene is realized.

The invention introduces exogenous fragments into wild type industrial microalgae nannochloropsis oculata by an electric pulse method, and obtains mutant strains by resistance screening.

The invention has the advantages that:

the invention uses nannochloropsis oculata with low CO₂The global expression profile changes dynamically in the concentration induction CCM process, changes of main metabolites in the process are identified by combining GC-MS, a CCM and other central carbon metabolism network model is constructed, the basis of a CCM function control network mechanism of nannochloropsis minitans is systematically understood, and further the invention down-regulates the expression of chloroplast carbonic anhydrase in nannochloropsis minitans by an RNAi means, down-regulates or partially closes the CCM mechanism to a certain extent-Gene knock-down (Gene knock-down), and screens high CO₂The concentration tolerance and the growth rate of the mutant strain are increased at the same time, the growth rate can be improved by about 30 to 45 percent, and the high-concentration CO is₂The culture effectively avoids or reduces the pollution of mixed bacteria (bacteria, fungi and the like) in the culture process, has biological control effect, can reduce the production cost, and can be used for industrial production. The industrial algae strain obtained by the invention opens up a new gate in the aspects of biological energy industrial application and the like.

Drawings

FIG. 1 is a schematic diagram of the construction of a Nannochloropsis oculata carbonic anhydrase RNAi expression vector provided by the present invention, mainly comprising a promoter, a resistance gene and an inverted repeat sequence of a target gene;

FIG. 2 is a PCR-verified electropherogram of the transformant according to the present invention, which shows that the transformant has a band of interest of about 400bp (with bands in the third and fourth lanes) by PCR amplification of the resistance gene sequence, wherein WT is a wild type (with no band in the first and second lanes);

FIG. 3 shows the high and low CO contents of the mutant and wild type CA2 genes provided by the present invention₂The carbonic anhydrase gene CA2 of the mutant strain with the relative expression abundance change under the concentration is obviously inhibited at the RNA expression level, and the relative expression abundance is reduced by about 60-80%;

FIG. 4 shows the high-low CO of the mutant and wild type provided by the present invention₂Performing phenotypic drop experiments under the culture conditions and phenotypic characterization under liquid culture;

FIG. 5 shows the high and low CO content of the mutant and wild type nannochloropsis oculata provided by the present invention₂Growth curve under concentration culture condition;

FIG. 6 shows the high CO content of the mutant and wild type nannochloropsis₂Dry weight graph of concentration.

FIG. 7 shows the results of high and low CO according to the present invention₂Photosynthetic oxygen release rate of mutant strain and wild type nannochloropsis cell under concentration culture condition;

FIG. 8 shows the high CO content provided by the present invention₂Oil content of mutant strain and wild type nannochloropsis cell under concentration culture condition.

Detailed Description

The invention is described in more detail below with reference to the figures and examples, without limiting the scope of the invention.

The invention uses reverse genetics strategy RNA interference (RNAi) technology to knock down a coding chloroplast carbonic anhydrase gene in industrial microalgae nannochloropsis oculata so as to obtain a gene capable of tolerating high-concentration CO₂And can grow rapidly at a high CO concentration₂(5-10%) is obviously improved (40%) compared with wild type, and CO concentration in air is obviously increased₂Low growth rate difference, high concentration of CO₂The lower culture effectively avoids the bacteria in the large-scale culture processAnd protozoa, and the like, and in addition, the oil content is correspondingly improved by about 20 to 30 percent. The method disclosed by the invention is used for fixing industrial CO by using industrial microalgae₂The waste gas lays a foundation and a feasible method.

The methods used in the examples of the present invention are all routine experimental methods unless otherwise specified.

EXAMPLE 1 acquisition of the complete sequence of Carbonic anhydrase from Nannochloropsis oceanica IMET1

Sequencing the nannochloropsis oculata genome to obtain the complete sequence of chloroplast carbonic anhydrase encoded by nuclear genome, as shown in SEQ ID No.1 and SEQ ID No. 2; and the homologous sequence of SEQ ID No.2 SEQ ID No. 3.

Further obtaining a complete reading frame (ORF) of chloroplast carbonic anhydrase through PCR verification of polymerase chain reaction, wherein the PCR primer sequence is as follows: f1 is 5 'ATGTGGCGGCGTGTGCTCGCA 3'; r1 is 5 'CTAGACGCTTTTATTAACC 3'. The PCR reaction system comprises 5XPCR reaction buffer solution (5 XDNA buffer)5ul, deoxyribonucleoside triphosphate (dNTP)4ul, magnesium sulfate (MgSO)₄)2ul, Forward primer (Forward primer)2ul, Reverse primer (Reverse primer)2ul, DNA3ul, DNA polymerase (KOD DNA poleunase) 0.5ul, ddH₂O is 32.5ul, and the total reaction system is 50 ul;

the PCR reaction procedures are respectively the first step of reaction at 95 ℃ for 5min, the second step of reaction at 95 ℃ for 1min, the second step of reaction at 58 ℃ for 30s and the third step of reaction at 72 ℃ for 30s (30 cycles), the third step of extension at 72 ℃ for 7min and the fourth step of reaction at 4 ℃ for 5 min.

Example 2

1) Construction of RNAi expression vectors

Constructing an RNAi expression vector by taking phir-PtGUS as a vector skeleton, and amplifying to obtain two PCR fragments of 229bp (position 1395bp to 1623bp of CA2 gene sequence) and 404bp (position 1395bp to 1798bp of CA2 gene sequence) by taking a nannochloropsis oculata genome as a template through PCR amplification reaction;

wherein, the primers for amplifying the 229bp fragment are as follows:

CA2_ Fw (5 'CGGAATTCGGGCATAGAGTGCGAATTGA 3'; contains the EcoRI site)/CA 2_ Rv1(5 'GCTCTAGACGCTTTTATTAACCCCATCC 3'; contains the XbaI site);

the primers for amplifying the 404bp fragment are as follows:

CA2_ Fw (5 'CGGAATTCGGGCATAGAGTGCGAATTGA 3'; containing the EcoRI site)/CA 2_ Rv2(5 'GCTCTAGAATCCTGGTCGTCAAAGAACG 3'; containing the XbaI site).

The reaction systems for PCR amplification were all 50 ul: comprises 5XPCR reaction buffer (5 XDNA buffer)5ul, deoxyribonucleoside triphosphate (dNTP)4ul, magnesium sulfate (MgSO)₄)2ul, Forward primer (Forward primer)2ul, Reverse primer (Reverse primer)2ul, DNA3ul, recombinant DNA polymerase (KOD DNA poleunase) 0.5ul, ddH₂O is 32.5ul, and the total reaction system is 50 ul;

the PCR reaction procedures are respectively the first step of reaction at 95 ℃ for 5min, the second step of reaction at 95 ℃ for 1min, the second step of reaction at 60 ℃ for 30s and the third step of reaction at 72 ℃ for 30s (30 cycles), the third step of extension at 72 ℃ for 7min and the fourth step of reaction at 4 ℃ for 5 min.

The two fragments obtained above contained a common fragment of 229bp, which was targeted to form the reverse complement (neck-loop hairpin). The two PCR fragments were then digested with EcoRI and XbaI restriction endonucleases and purified using the PCR product purification kit, while the phir-PtGUS vector was digested with EcoRI and purified after fragment dephosphorylation with bovine small intestine alkaline phosphatase. The EcoRI and XbaI digested two PCR fragments were then ligated with a linearized vector (the EcoRI digested linear fragment of phir-PtGUS) using T4 ligase to obtain plasmid phir-PtCA 2. The promoters of the Torulopsis minor Beta-tubulin gene were amplified using primers Notub _ fw (5 'GCGAGCTCGCCAGCTGCCTCAT 3'; containing SacI site) and Notub _ rv (5 'GGCCATGG 3 TGTGTCCGCCGCC'; containing NcoI site), then digested with SacI and NcoI restriction enzymes, and the vector phir-PtCA2 was also digested with SacI and NcoI restriction enzymes and purified, and then the promoter fragment of the Beta-tubulin gene was ligated with the linearized vector (the promoter of the Beta-tubulin gene) using T4DNA ligase to obtain vector phir-NgCA2, which comprises the promoter region of the Beta-tubulin gene, the resistant ble gene, the forward and reverse repeat sequence of CA2, and the terminator of the fcp gene, constituting an expression cassette (see FIG. 1).

2) PCR amplification of RNAi expression cassettes

The expression vector of example 2 above was amplified with PCR primers (F1: 5 'CCCAGTCACGACGTTGTAAAACG 3'; R1: 5 'GGAAACAGCTATGACCATG 3') to obtain a DNA comprising the tub gene promoter, the resistant gene ble, the CA2 sequence capable of forming a hairpin structure, and the transcription terminator sequence, 5. mu.l of 5XPCR reaction buffer (5 XDR DNA buffer), 4. mu.l of deoxyribonucleoside triphosphates (dNTPs), and magnesium sulfate (MgSO 4)₄)2ul, forward primer (primer F1)2ul, reverse primer (primer R1)2ul, template DNA (DNA)3ul, DNA polymerase (KOD DNA polymease) 0.5ul, ddH2O32.5ul, and total reaction system is 50 ul; the PCR reaction program is that the first step is 95 ℃ for 5min, the second step is 95 ℃ for 1min, the 56 ℃ for 30s, 72 ℃ for 45s (30 cycles), the third step is 72 ℃ for extension for 7min, and the fourth step is 4 ℃ for 5 min. After the PCR amplification reaction is finished, carrying out electrophoresis detection on the obtained PCR product and further purifying the PCR product to ensure that the concentration of the PCR product reaches 3-5ug/ul, storing at-20 ℃, and standing for later use when electric pulse transformation is carried out in the next step.

3) Obtaining of Nannochloropsis mutant strains

The RNAi expression cassette fragment is introduced into wild nannochloropsis oculata by electric pulse transformation, and the specific implementation method is that the wild nannochloropsis oculata is cultured in f/2 culture medium until the concentration reaches 3 × 10⁷cells/ml (culturing at 25 deg.C, continuous light culturing, light intensity of 50 umol/m)^-2S^-1Aeration amount is 100ml/min), centrifuging to collect microalgae cells, pouring out supernatant, washing the microalgae cells with 375mM sorbitol solution pre-cooled at 4 ℃ for three times, and re-suspending the microalgae cells to make the concentration of the microalgae cells be 8 × 10⁸cells/ml; dividing the concentrated algae cell solution into 200ul parts, adding 3ug of the above linearized vector and 1ul of salmon sperm DNA (15ug/ml) into each part, mixing, and standing on ice for 10 min; transferring the mixture into a 2mm electric shock cup, performing electric shock by 2200V (HV), 600 ohm and 50 microfarads (uf), immediately transferring the algae into a sterile white-head glass tube by 5ml of fresh f/2 culture medium after electric shock, culturing in a light shaking table (recovering by using weak light) after shading, setting the rotating speed at 100rpm, and recovering for 48h at 25 ℃; centrifuging at 5000g for 5min after recovery, discarding supernatant, re-suspending the algae with 1ml 20% corn starch, and pouring into agar with content of 08% f/2 plate, spreading out evenly, drying the plate on a clean bench, sealing, placing in 25 ℃ for illumination culture, and selecting transformants after 3-4 weeks.

The 20% corn starch is prepared by taking 2g of corn starch, alternately washing the corn starch with absolute ethyl alcohol and ultrapure water for 3 times, then re-suspending the corn starch with 10ml of 70% ethyl alcohol, washing the corn starch with f/2 culture medium for 4 times, and then re-suspending the corn starch with 10ml of f/2 culture medium containing 0.8% PEG 8000.

On the f/2 plate with 0.8% agar content, preparing agar plate in advance, and adding 3-5ug/ml Zeocin antibiotic

Example 3 PCR validation of transformants

The transformants were picked with toothpicks from fresh f/2 medium (containing 3-5ug/ml Zeocin antibiotic), cultured in a light incubator until they grew for 2-3 weeks, centrifuged to collect algal cells, and the genomic DNA of the transformants was extracted with a genomic DNA extraction kit (OMEGA HP DNAextraction kit) and the genomic DNA of wild type nannochloropsis was also extracted as a control for the subsequent experiments. After genome extraction and quantification with Nannodrop, PCR amplification reaction was performed with forward primer F1 (5 'TTATCAACGGCATACCGGCACTG 3') and reverse primer R1 (5 'CTGATGAACAGGGTCACGTCGT 3'), in a 50 ul: 5XPCR reaction buffer (5 XDNA buffer)5ul, deoxyribonucleoside triphosphate (dNTP)4ul, magnesium sulfate (MgSO)₄)2ul, forward primer (primer F1)2ul, reverse primer (primer R1)2ul, template DNA (DNA)3ul, DNA polymerase (KOD plus DNA polymease) 0.5ul, ddH2O 32.5.5 ul, and total reaction system 50 ul. The reaction procedure is as follows: the first step is at 95 deg.C for 5min, the second step is at 95 deg.C for 1min and at 56 deg.C for 30s and at 72 deg.C for 45s (30 cycles), the third step is at 72 deg.C for 7min, and the fourth step is at 4 deg.C for 5 min. After the PCR amplification reaction is completed, agarose gel electrophoresis is carried out for detection, whether a 400bp band exists by using transformant DNA as a template and a wild type does not have the band exists is judged, as shown in figure 2, the fact that the exogenous fragment is integrated into the nannochloropsis, electric pulse transformation is successful, mutant strains are obtained, and the mutant strains can be respectively marked as M2 and M4.

Example 4 measurement of relative expression level of the Nannochloropsis mutant CA2 Gene

Respectively subjecting the mutant strain and wild type strain to different CO₂Concentration (different CO)₂High concentration of 5% CO₂Low concentration of 0.04% CO₂) Culturing under the condition that the light intensity is controlled to be consistent to 50umol/m²The aeration rate was 100 ml/min. Collecting microalgae when the microalgae is cultured to logarithmic phase, centrifuging for 5min at 5000g, rapidly freezing with liquid nitrogen, and storing at-80 deg.C.

The experimental measurement of the expression abundance of the mutant strain comprises the following three steps:

firstly, extracting RNA, namely extracting RNA from cryopreserved algae cells, firstly crushing the algae cells by adopting a liquid nitrogen grinding mode, grinding the crushed algae cells for 5-10min by using general liquid nitrogen, then adding 1ml of Trizol (Invitrogen company), quickly and fully mixing the crushed algae cells uniformly, adding 200ul of chloroform, centrifuging the mixture for 10min at 12000rpm, sucking supernate, transferring the supernate into a new eppendorf tube, adding isometric chloroform, fully oscillating and mixing the mixture uniformly, standing the mixture for 2-3min, centrifuging the mixture for 10min at 12000rpm, transferring the supernate into a new centrifuge tube, adding isometric isopropanol, precipitating the supernate for 15min at room temperature, centrifuging the mixture for 15min, washing the precipitate by using 75% ethanol, drying the precipitate by blowing, and adding 50ul of DDH treated by DEPC₂O, measuring the concentration of RNA, and storing at-80 for later use;

second, cDNA preparation, RNA samples according to Takara reagent

The procedure of RT reagent kit with gDNA Eraser kit shows that random primers are used for cDNA synthesis, and the remark genome DNA removal reaction system is 20ul, which comprises 2ug of total RNA, 5X reaction buffer and gDNA Eraser enzyme;

in the third step, a fluorescent quantitative PCR reaction is carried out

480II (Roche Rapid Start Universal Green fluorescence) quantitative qRT-PCR. The reaction system for PCR was 1ul of cDNA (reverse transcription preparation from the previous step), 1ul and 10ul of each of forward and reverse primers 2X Roche FastStart SYBR Green Master (ROX), ddH₂O adds 7ul to complement to 20ul, wherein the primer sequence is a forward primer: 5 'ATCCAGGAACAATTATTTCGCT 3'; reverse primer:5 'GAGTCGGAACAAGCAATAA 3'. Reaction conditions were performed according to the Roche kit instructions. Analysis of real-time fluorescence qPCR results data use 2^-ΔΔCtMethod wherein the Δ Δ Ct value is (Ct)_{CA2 gene}-Ct_{Internal reference gene})_{Mutant strain}-(Ct_{CA2 gene}-Ct_{Internal reference gene})_{Wild type}。

The gene expression abundance of the CA2 gene in the mutant relative to the wild type was calculated by the above method, and as a result, it was confirmed that the CA2 gene expression abundance of the mutant (M2 or M4) was found to be either at low CO₂Or in high CO₂Significantly lower expression under culture conditions than wild-type CA2, confirmed that CA2 gene was repressed at transcriptional level, down-regulated by approximately 60% -80%, as shown in fig. 3, further confirmed that CA2 gene was silenced in mutants M2, M4.

Example 5 Nannochloropsis CO-resistance₂Measurement of growth phenotype of mutant strains

Picking single algae colony from mutant strain and wild type nannochloropsis oculata with toothpick, culturing in f/2 culture medium in 50ml triangular flask for 2-3 weeks, and measuring OD₇₅₀Cell density of the cells, and then re-inoculating to fresh f/2 medium to maintain the same inoculation concentration, i.e., the same OD₇₅₀Setting the value at 0.5-1.0, continuously subculturing for 2-3 times, inoculating microalgae into 200ml column reactor, culturing (culture medium in reactor is f/2 culture medium), and diluting mutant strain and wild type nannochloropsis oculata to the same concentration (OD determined as above) after logarithmic growth phase₇₅₀Same values), 10ul of each dilution was pipetted onto a pre-prepared f/2 solid agar plate (agar concentration 0.8% and no antibiotics added) and each algal sample was pipetted onto two plates (on two CO plates)₂Culturing at concentration of 0.04% and 5% CO2, respectively), blow-drying on a clean bench, standing at room temperature in a dark or low-light environment for 2-3 days, and placing the plate in a simple incubator (patent application No.: 201410339234.9) and introducing one of the incubators with CO at an air concentration₂The other is filled with high-concentration CO₂(5%) or by introducing low-concentration CO₂(0.04%) light intensity 50umol/m²S, with which the phase can be performedFor closed culture screening, stable CO in the incubator can be controlled by adjusting the air inlet and air outlet valves of the incubator₂Environment, growth of microalgae (mutant and wild type) was observed at irregular intervals (fig. 4). Meanwhile, the physiological indexes such as growth curve, dry weight and the like are measured by inoculating microalgae into a fresh liquid culture medium from a 200ml column reactor. The results of measurement are shown in FIGS. 5 and 6, and it was found that the mutant strain was in high CO₂Growth at concentration was faster than wild type, with a 30% increase in dry weight after 10 days of culture (figure 6). Thus, tolerance to high CO was obtained by the Gene knockdown method₂The mutant strain which can grow rapidly is cut in concentration, and a solid industrialization foundation is laid for cultivating industrial microalgae under the gas of an industrial flue.

Example 6 Nannochloropsis resists high concentrations of CO₂Measurement of physiological phenotype of mutant strains

Subjecting the above to high CO resistance₂The photosynthetic oxygen evolution rate of nannochloropsis oculata mutant strain and wild type cell was measured by Clark-II type oxygen electrode (Hansatech, UK), the temperature of the reaction tank was 4 deg.C (water bath adjustment), and the light intensity was adjusted to 300. mu. mol m with a light-shielding film^-2s^-1. Culturing nannochloropsis oculata mutant strains and wild type cells to logarithmic growth phase, centrifuging at 5000g for 5min, collecting algae cells and 15ml centrifuge tubes, collecting two samples, one for measuring photosynthetic oxygen release rate, and the other for measuring chlorophyll content. The collected microalgae cells are first treated with carbohydrate-free water (N is introduced in advance)₂Removing CO from the solution₂) Washed and then suspended in buffers with different pH values, namely 20mmol of L-1 MES buffer, 20mmol of L-1Tris buffer and 20mmol of L-1 APES buffer in sequence in carbon-free water (pH6.0, 7.8 or 9.0), and the buffers are prepared by using seawater and then respectively measuring the photosynthetic oxygen release rate of the mutant strain and the wild type cell under different pH conditions. Meanwhile, a part of chlorophyll a is reserved for measuring the content of chlorophyll a, the content of chlorophyll a (chla) is calculated by adopting a methanol extraction method according to the following formula:

the content (ug/ml) of Chla is 16.5169XA665-8.0962XA652

Chlorophyll a content for photosynthetic oxygen release rateThe amount is converted. The measurement result shows that the photosynthetic oxygen release rate of the mutant strain cell is obviously improved under the condition of pH6.0, and the photosynthetic oxygen release rate of the mutant strain cell is obviously reduced under the condition of pH9.0, so that the mutant strain can resist CO₂The decrease in pH caused by the increase in concentration shows acid resistance to some extent, and it is likely that the microenvironment inside the chloroplast is improved, that is, the pH is raised, so that rapid growth is exhibited, as shown in fig. 7.

And then, measuring the oil content of the obtained mutant strain, extracting the oil by adopting a methanol/chloroform method, drying the crude oil by a nitrogen blowing instrument, and weighing, wherein the result shows that the oil content of the mutant strain is increased by about 20% under the normal culture condition, and as shown in figure 8, the oil content is increased from the original 0.18mg/mg dry weight to 0.25mg/mg cell dry weight.

SEQ ID No.1

ATGTGGCGGCGTGTGCTCGCATATTCCTCGAGATTCGATGTAACACATCTCAATAGCGA GGTAACGCTATCCACGTATCTCACCATTGTCTCCACCCTCTCTATTAACCCCATCTTTC TATGCAGTTCGCAAAAATCTCCGATACGCGGCCCTGCGGTTAATCCAACAAGTCAAGAC CTTGAAACGTCGGAAATGAGCAACCCGCCGTCTCCTGTCGTGCCGTCTCCCAGCCTTAC ACAATCAAATCATGACATGCTCACCAGCATCCGCAGCCACCTAGACGACCAAGAGGTCA AGATCCAGGAACAATTATTTCGCTTCCAGGGCATTAAGAGGGCCATTGACCGCATTTGC AAGAATGACGAGGCCTACATGGACTCCCCCGATATTGTCAAGGCACAGACCACGCTCGA AGATCGCCCGGAGCTGGAAAGGAAAGGAGAGACTGCTGCTGAAGCCTTGCGCCTGCTCA AAGTGGGCAACGCTGCCTTTTTGCGCGATGAGGTTGTGCCTGTCCACCGCTCTGACAGA CATATTGGCCTACATTTTGCCCAGAAACCCCACGCCATAATTATTGCTTGTTCCGACTCTCGGGTCCCGCCGGAGATGATCTTTAATTGCGCGTTTGGTGAACTCTTCGTCATCCGCC TTGCCGGCAACACCGTCGATGCACTCGCTCGCGCCAGCCTCCTTTACGCGGTGCAACAC CTCCAATCTCCCCTGGTCGTTGTTCTGGGCCACGAAAAGTGCGGCGCCGTCACAGCCGC CTTGCAGCCGGAAGAGGAGCTGGCGGATGCTCCCGGGGACATTAAGACCTTGGTGCGAA ACATCAAAAGGGGCATAGAGTGCGAATTGATTGACCCGAGCGCCCACATTTTCGGCGAT GAAAGACTGTTGTGCGCGATTGTCTGCAACGTGCATTACGTGGCCCGCACCCTGTCGGA ACACCCCGATATCCGCCCATTTATTGACCGAAGGAAACTGTCCGTCGTGGGCGCCTACT ACGCTTTTAGTGGGGTGGTGTCGTTCTTTGACGACCAGGATGAGGGAGGAAGGATGGAG AGAGGGCGGGTGTTTGGGGGTGTGGAGGGAAACGGATCCTTGTGCTTAAGTTGTTCCGG AACTTTGTCCGGAAACTCGAGTCCGATGGCGGGGGGAGGGAGCAAGGGATCCAGTCCGC CTCCGAGCGTGCAGGATGGGGTTAATAAAAGCGTCTAG

(a) Sequence characteristics:

● length: 1218

● type: base sequence

● chain type: single strand

● topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: nannochloropsis oceanica IMET1

SEQ ID No.2

MWRRVLAYSSRFDVTHLNSEVTLSTYLTIVSTLSINPIFLCSSQKSPIRGPAVNPTSQD LETSEMSNPPSPVVPSPSLTQSNHDMLTSIRSHLDDQEVKIQEQLFRFQGIKRAIDRIC KNDEAYMDSPDIVKAQTTLEDRPELERKGETAAEALRLLKVGNAAFLRDEVVPVHRSDR HIGLHFAQKPHAIIIACSDSRVPPEMIFNCAFGELFVIRLAGNTVDALARASLLYAVQH LQSPLVVVLGHEKCGAVTAALQPEEELADAPGDIKTLVRNIKRGIECELIDPSAHIFGD ERLLCAIVCNVHYVARTLSEHPDIRPFIDRRKLSVVGAYYAFSGVVSFFDDQDEGGRME RGRVFGGVEGNGSLCLSCSGTLSGNSSPMAGGGSKGSSPPPSVQDGVNKSV

(a) Sequence characteristics:

● length: 405

● type: amino acid sequence

● chain type: single strand

● topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: nannochloropsis oceanica IMET1

SEQ ID No.3

MTGSVGTSVKPSISQQNHEVLERIRHHLDDQEHKIHEQLNRFQLIKKAVERICKNDDAY MQSPDIVKAQTTLEDSPLERKGNTAAEALELLKAGNAAFLRDEVPAKQCSDRHIGLHFA QKPHAIIIACSDSRVPPEMIFNCAFGELFVIRLAGNTIDALARASLLYAVQHLHSPLVV VLGHEKCGAVTAALQPPEALAEAPRDIKTLVKKIQSGIECALLEPGLEDVDDERLLCAI VCNVHSVARTLSEHPDIRPFIEQKALSVVGAYYAFNGVVSFFDDGEDGNATGKEVGSTV NGQASLCMSCSGTIHRSSSPTADPSQKVNSRLSVSEGLSKNT

(a) Sequence characteristics:

● length: 337

● type: amino acid sequence

● chain type: single strand

● topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: nannochloropsis oceanica IMET1

Claims

1. Construction of high concentration CO resistance by using gene of coding chloroplast carbonic anhydrase₂And the application in the industrial engineering nannochloropsis which grows rapidly;

the sequence of the gene encoding chloroplast carbonic anhydrase is shown in SEQ ID No. 1.

2. The use of the gene encoding chloroplast carbonic anhydrase of claim 1 in the construction of high concentration CO tolerance₂The application of the industrial engineering nannochloropsis which grows rapidly is characterized in that: through reverse genetics RNA interference technology, the carbonic anhydrase gene of chloroplast of industrial microalgae nannochloropsis oculata is specifically knocked down, and high-concentration CO tolerance is further obtained₂And can quickly grow the engineering nannochloropsis.

3. The use of the gene encoding chloroplast carbonic anhydrase of claim 2 in the construction of high concentration CO tolerance₂The application of the industrial engineering nannochloropsis which grows rapidly is characterized in that: constructing an RNAi expression vector, namely replacing a diatom Fcp gene promoter of a carrier framework with a nannochloropsis oculata endogenous promoter by adopting the RNAi expression vector of diatoms as the framework, cloning a reverse repeat sequence of nannochloropsis oculata chloroplast carbonic anhydrase, and connecting the reverse repeat sequence to the vector to obtain the RNAi expression vector; then, electric pulse is used to transform nannochloropsis, and the transformed nannochloropsis culture solution is cultured in high-concentration CO₂Can tolerate high concentration CO under environment₂The algae strain with high growth rate can resist high concentration CO₂The engineered nannochloropsis.