Method for regulating and controlling synthesis of unsaturated fatty acid in synechocystis and application thereof
阅读说明：本技术 一种调控集胞藻中不饱和脂肪酸合成的方法及其应用 (Method for regulating and controlling synthesis of unsaturated fatty acid in synechocystis and application thereof ) 是由 陈高 钟怀荣 曹月蕾 范仲学 崔晓艳 于金慧 李燕乐 路晓媛 于 2020-03-09 设计创作，主要内容包括：本发明涉及一种丝氨酸/苏氨酸激酶基因在集胞藻不饱和脂肪酸合成中的应用,构建了集胞藻PCC6803中的spkD基因敲除突变株和spkG基因敲除突变株；本发明首次公开了集胞藻PCC6803中的spkD和spkG基因在改变集胞藻合成不饱和脂肪酸中的重要作用,突变株在正常培养条件下细胞中的C18:2、C18:3n6、C18:3n3、C18:4等脂肪酸的含量明显低于野生型的集胞藻；spkD和spkG两种基因在培养前期与集胞藻多不饱和脂肪酸的产量呈负相关,为下一步通过协同调控spkD和spkG基因表达进而提高蓝藻中亚油酸、α-亚麻酸和十八碳四烯酸等多不饱和脂肪酸的含量奠定基础。(The invention relates to an application of serine/threonine kinase gene in synechocystis unsaturated fatty acid synthesis, and constructs a spkD gene knockout mutant strain and a spkG gene knockout mutant strain in synechocystis PCC 6803; the invention discloses the important function of spkD and spkG genes in synechocystis PCC6803 in changing the synthesis of unsaturated fatty acid by synechocystis for the first time, and the content of fatty acid such as C18:2, C18:3n6, C18:3n3, C18:4 and the like in the mutant strain in cells under normal culture conditions is obviously lower than that of wild synechocystis; the spkD and spkG genes are negatively related to the yield of polyunsaturated fatty acid of synechocystis before culture, and a foundation is laid for further improving the content of polyunsaturated fatty acid such as linoleic acid, alpha-linolenic acid, stearidonic acid and the like in the blue algae by synergistically regulating the expression of the spkD and spkG genes.)
The application of the spkG gene in unsaturated fatty acid synthesis, wherein the nucleotide sequence of the spkG gene is shown as SEQ ID NO. 18.
2. The use according to claim 6, wherein the spkG gene is used as a gene for regulating the expression level of linoleic acid, γ -linolenic acid, α -linolenic acid and stearidonic acid.
3. A method II for regulating and controlling the synthesis of unsaturated fatty acids in synechocystis sp is characterized by comprising the following steps:
a, performing PCR amplification on synechocystis PCC6803 genome DNA by using primer pairs spkG-F and spkG-R to obtain a spkG gene;
b, connecting the amplified spkG fragment with a pClone007 simple vector, and then transforming the fragment into escherichia coli DH5 alpha for cloning to obtain a 007-spkG plasmid;
c, carrying out enzyme digestion on the 007-spkG plasmid by using BamH I endonuclease, cutting the spkG gene, taking the spkG gene segment before the enzyme digestion site of the BamH I endonuclease as an upstream arm of homologous recombination, and taking the spkG gene segment after the enzyme digestion site as a downstream arm of homologous recombination;
d, digesting the plasmid pBluescript-Kan by using BamHI endonuclease, recovering a kanamycin fragment, and connecting the kanamycin fragment with the plasmid 007-spkG prepared in the step c by using BamHI endonuclease to prepare a plasmid 007-spkG-Kan;
e, transforming the recombinant vector 007-spkG-kan prepared in the step d into synechocystis PCC6803, and screening to prepare a synechocystis mutant strain with the spkG gene knocked out.
4. The method II for regulating and controlling the synthesis of unsaturated fatty acids in Synechocystis according to claim 3, wherein in step a, the spkG gene is obtained by PCR amplification using Synechocystis PCC6803 genomic DNA as a template, and the nucleotide sequences of primers for PCR amplification are SEQ ID NO.3 and SEQ ID NO. 4.
5. The method for regulating and controlling the synthesis of unsaturated fatty acids in synechocystis according to claim 4, wherein in step a, the PCR amplification system is as follows:
2 XM 5 HiPer plus Taq HiFi PCR mix 10 uL, template DNA 1uL, primer spkG-F1 uL, primer spkG-R1 uL, ddH2O 7μL，20 mu L in total;
the PCR amplification procedure was as follows:
pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 25s, renaturation at 60 ℃ for 25s, and lmin extension at 72 ℃ for 35 cycles, final extension at 72 ℃ for 5min, and storage at 4 ℃.
6. A Synechocystis mutant knockout spkG gene prepared in accordance with claim 3 is used for the synthesis of unsaturated fatty acids.
Technical Field
The invention relates to a method for regulating and controlling synthesis of unsaturated fatty acid in synechocystis and application thereof, belonging to the technical field of biology.
Background
Cyanobacteria (Cyanobacteria) is a class of prokaryotes with photoautotrophic capabilities. The cyanobacteria cells can grow autotrophically, can survive without depending on external organic matters, can utilize simple inorganic matters to synthesize the organic matters to provide energy for life activities, and protein produced by exogenous gene expression can be fully folded and assembled, which means that the cyanobacteria hardly forms inclusion bodies, and most of the cyanobacteria and cell extracts thereof are nontoxic to human beings and animals, and are good receptors for transgenic research. Synechocystis PCC6803(Synechocystis sp. PCC6803) as a unicellular blue-green algae has the characteristics of simple structure, simple genetic background, easy culture, convenient vector construction, safe extract, low activity of intracellular protease and the like, and is a good blue-green algae genetic engineering receptor.
When the external environment changes, the blue algae rapidly senses the change by two main signal transduction systems including serine/threonine kinases (STKs), and changes related metabolites by accurately regulating gene expression, so that the blue algae can make positive and rapid response to the change of the external environment, and can survive in various adverse circumstances.
Serine/threonine kinases (STKs) are essential for prokaryotes and are involved in activities such as growth, division and differentiation. STK, together with its homologous phosphatase, plays a central role by catalyzing reversible protein phosphorylation. Upon sensing an external stimulus, STK undergoes autophosphorylation and then transphosphorylates the substrate protein. Phosphorylation of specific amino acid residues can directly control the activity of the target protein or indirectly act by modulating protein-protein interactions.
Polyunsaturated fatty acids (PUFAs) are straight-chain fatty acids having two or more double bonds and a carbon number of 16 to 22, and mainly include linoleic acid (C18:2, LA), γ -linolenic acid (C18:3n6, GLA), α -linolenic acid (C18:3n3, ALA), stearidonic acid (C18:4, SDA), arachidonic acid (C20:4n6, AA), eicosapentaenoic acid (C20:5n3, EPA), docosahexaenoic acid (C22:6n3, DHA), and the like. Polyunsaturated fatty acids are classified into various types according to the position where the unsaturated bond starts at the end of the carbon chain methyl group of the fatty acid using the ω (omega) -numbering system. The important functions are omega-3 and omega-6, wherein LA, GLA, AA and the like belong to omega-6 polyunsaturated fatty acids, and ALA, SDA, EPA, DHA and the like belong to omega-3 polyunsaturated fatty acids.
PUFAs have an extremely important role in the health of the human body. Linoleic and linolenic acids are essential polyunsaturated fatty acids of the human body, which cannot be synthesized in the human body. Stearic acid is used in many industrial and cosmetic applications. Arachidonic acid is not synthesized in large quantities in the human body. Of the most important to the human body are eicosapentaenoic acid and docosahexaenoic acid and their analogues. PUFAs also play an irreplaceable role in animal feed and energy industries.
Currently, the sources of fatty acids such as AA are mainly meat, milk and eggs. The commercial sources of EPA and DHA are mainly deep-sea fishes. However, the production of PUFAs has been greatly limited by resource shortage caused by over-fishing of deep-sea fishes in recent years. The process for extracting PUFAs from transgenic blue-green algae is relatively simple, the quantity of blue-green algae relative to deep-sea fish resources can be inexhaustible, no special peculiar smell exists, and the cholesterol content is low. And with the change of the earth environment, the plant resources are more and more greatly influenced by region and season limitation, environmental deterioration and the like, in addition, the problems of the gradual depletion of marine fish resources, residual fishy smell and oxidation instability in the purification process of PUFAs derived from fish oil and the like are solved, and the advantages that the industrialized culture of blue algae is not influenced by the season and the region, the quantity of blue algae resources is rich, no peculiar smell exists in the production process and the like, and the unsaturated fatty acid is produced are highlighted.
In recent years, great progress has been made in obtaining a mutant strain with high yield of unsaturated fatty acid by transferring a synthetic unsaturated fatty acid regulatory enzyme gene into Synechocystis PCC 6803. For example, chinese patent document CN103014037A (application No. 201210529390.2) discloses a method for increasing the content of synechocystis PCC6803 fatty acids. The method discovers that other genes for biologically synthesizing unsaturated fatty acid regulatory enzymes can be expressed in synechocystis PCC6803, and the genes can be used for expressing corresponding regulatory enzymes in the synechocystis PCC6803 to obtain corresponding unsaturated fatty acids. Provides a research method for expanding the unsaturated fatty acid metabolic pathway in synechocystis PCC 6803.
Although the method can increase the content of unsaturated fatty acid in synechocystis, the content of unsaturated fatty acid can not be further increased. Therefore, finding new ways to further increase the content of unsaturated fatty acids becomes a new hotspot.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for regulating and controlling the synthesis of unsaturated fatty acid in synechocystis and application thereof. We use insertional inactivation to construct synechocystis 2 STKs kinase gene (spkD gene and spkG gene) knock-out mutants, and detect PUFAs (polyunsaturated fatty acids) content and fatty acid dehydrogenase gene expression in wild type and mutant. As a result, it was found that the culture temperature was 30 ℃ and the light intensity was 40. mu. mol. m-2·s-1Under the condition, the relative expression quantity of the spkD and spkG genes in the wild type synechocystis PCC6803 is obviously higher than that of spkD knockout mutant strains and spkG knock-out mutant. The fatty acid detection result firstly discovers that the 2 STKs kinase spkD genes and spkG have the function of regulating and controlling the synthesis of unsaturated fatty acid in synechocystis. The STKs kinase spkD gene and spkG play a role in regulating and controlling Synechocystis PUFAs biosynthesis.
The invention discloses that the spkD gene and the spkG gene of serine/threonine kinase have important functions in the synthesis of unsaturated fatty acid of synechocystis for the first time.
The technical scheme of the invention is as follows:
the application of the spkD gene in unsaturated fatty acid synthesis, wherein the spkD gene is used as a gene for regulating and controlling the expression level of linoleic acid, gamma-linolenic acid, alpha-linolenic acid and stearidonic acid; the nucleotide sequence of the spkD gene is shown in SEQ ID NO. 17.
A method I for regulating and controlling the synthesis of unsaturated fatty acid in synechocystis; the method comprises the following steps:
(1) carrying out PCR amplification on synechocystis PCC6803 genome DNA by using primer pairs spkD-F and spkD-R to obtain a spkD gene;
(2) connecting the amplified spkD fragment with a pClone007 simple vector, and then transforming the fragment into Escherichia coli DH5 alpha for cloning to obtain a 007-spkD plasmid;
(3) carrying out enzyme digestion on the 007-spkD plasmid by using EcoR I endonuclease, cutting the spkD gene, taking the spkD gene segment before the enzyme digestion site of the EcoR I endonuclease as an upstream arm of homologous recombination, and taking the spkD gene segment after the enzyme digestion site as a downstream arm of homologous recombination;
(4) digesting the plasmid pBluescript-Kan by using EcoR I endonuclease, recovering a kanamycin fragment, and connecting the kanamycin fragment with the plasmid 007-spkD prepared in the step (3) of likewise digesting by using the EcoR I endonuclease to prepare a plasmid 007-spkD-Kan;
(5) transforming the recombinant vector 007-spkD-kan prepared in the step (4) into synechocystis PCC6803, and screening to prepare a synechocystis mutant strain with the spkD gene knocked out;
preferably, in step (1), the spkD gene is obtained by PCR amplification using synechocystis PCC6803 genomic DNA as a template, and the nucleotide sequences of PCR amplification primers are as follows:
spkD-F：5’-ACTTACCCGTTCTGATTGA-3’ SEQ ID NO.1；
spkD-R：5’-TAACCATTGATAAGCAGAT-3’ SEQ ID NO.2。
preferably, in step (1), the PCR amplification system is as follows:
2x M5 HiPer plus Taq HiFi PCR mix 10 uL, template DNA 1uL, primer spkD-F1 uL, primer spkD-R1 uL, ddH2O7 mu L, 20 mu L in total;
the PCR amplification procedure was as follows:
pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 25s, renaturation at 60 ℃ for 25s, and lmin extension at 72 ℃ for 35 cycles, final extension at 72 ℃ for 5min, and storage at 4 ℃.
The synechocystis mutant strain with the spkD gene knocked out is used for synthesizing unsaturated fatty acid.
The application of the spkG gene in unsaturated fatty acid synthesis, wherein the spkG gene is used as a gene for regulating the expression level of linoleic acid, gamma-linolenic acid, alpha-linolenic acid and stearidonic acid; the nucleotide sequence of the spkG gene is shown in SEQ ID NO. 18.
A method II for regulating and controlling the synthesis of unsaturated fatty acids in synechocystis; the method comprises the following steps:
a, performing PCR amplification on synechocystis PCC6803 genome DNA by using primer pairs spkG-F and spkG-R to obtain a spkG gene;
b, connecting the amplified spkG fragment with a pClone007 simple vector, and then transforming the fragment into escherichia coli DH5 alpha for cloning to obtain a 007-spkG plasmid;
c, carrying out enzyme digestion on the 007-spkG plasmid by using BamH I endonuclease, cutting the spkG gene, taking the spkG gene segment before the enzyme digestion site of the BamH I endonuclease as an upstream arm of homologous recombination, and taking the spkG gene segment after the enzyme digestion site as a downstream arm of homologous recombination;
d, digesting the plasmid pBluescript-Kan by using BamHI endonuclease, recovering a kanamycin fragment, and connecting the kanamycin fragment with the plasmid 007-spkG prepared in the step c by using BamHI endonuclease to prepare a plasmid 007-spkG-Kan;
e, transforming the recombinant vector 007-spkG-kan prepared in the step d into synechocystis PCC6803, and screening to prepare a synechocystis mutant strain with the spkG gene knocked out.
Preferably, in step a, the spkG gene is obtained by performing PCR amplification using Synechocystis PCC6803 genomic DNA as a template, and the nucleotide sequence of PCR amplification primers is as follows:
spkG-F：5’-AGACTTTCTCTATTGCCTC-3’ SEQ ID NO.3；
spkG-R：5’-GGACCCAAATCCAGAAGAC-3’ SEQ ID NO.4。
preferably, in step a, the PCR amplification system is as follows:
2 XM 5 HiPer plus Taq HiFi PCR mix 10 uL, template DNA 1uL, primer spkG-F1 uL, primer spkG-R1 uL, ddH2O7 mu L, 20 mu L in total;
the PCR amplification procedure was as follows:
pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 25s, renaturation at 60 ℃ for 25s, and lmin extension at 72 ℃ for 35 cycles, final extension at 72 ℃ for 5min, and storage at 4 ℃.
The synechocystis mutant strain with the spkG gene knocked out is used for synthesizing unsaturated fatty acid.
The spkD and spkG genes are two serine/threonine kinase genes in synechocystis.
The pClone007 simple vector is a commercial product.
Advantageous effects
1. The invention discloses the important function of the spkD gene (spkD for short) and the spkG gene (spkG for short) of serine/threonine kinase in the synthesis of unsaturated fatty acid of synechocystis for the first time, and the invention knocks out the spkD and spkG in synechocystis PCC6803 by an insertional inactivation method, and then the culture temperature is 30 ℃, and the illumination intensity is 40 mu mol.m-2·s-1Under the condition, the content of polyunsaturated fatty acid in the mutant strain is changed, and the contents of linoleic acid (C18:2), gamma-linolenic acid (C18:3n6), alpha-linolenic acid (C18:3n3) and stearidonic acid (C18:4) in the knockout mutant strain are obviously lower than that of wild synechocystis sp.
2. According to the invention, a serine/threonine kinase spkD gene and a spkG gene are knocked out in synechocystis PCC6803 for the first time, which has great significance for further improving the content of unsaturated fatty acid in synechocystis, and provides theoretical support for the research of unsaturated fatty acid in synechocystis PCC 6803.
3. In the synechocystis PCC6803, after a serine/threonine kinase spkD gene and a spkG gene are knocked out in an insertional inactivation mode, under the same culture condition, the difference of the expression of four fatty acid dehydrogenase genes in a wild type mutant strain and a mutant strain is compared in different time periods, and the fatty acid dehydrogenase related gene in the two mutant strains is found to show a descending trend, the fatty acid dehydrogenase gene expression amount of the spkD knock-out mutant strain is higher than that of the spkG knock-out mutant strain, in the research of the spkD knock-out mutant strain, the fatty acid dehydrogenase gene expression amount of the mutant strain in the initial stage of culture is found to be obviously higher than that of the wild type, the fatty acid dehydrogenase gene expression of the mutant strain is most obviously different from that of the wild type at the initial 6h of culture, but the difference is smaller and smaller as the culture time is prolonged, and finally shows that the wild type is dominant, indicating that spkD knock-out has an effect on fatty acid dehydrogenase gene expression. In the study on the spkG knockout mutant, the expression level of the fatty acid dehydrogenase gene reaches the highest level at the initial 6h of culture, which is higher than that of the wild type, but the difference is small, the content of the fatty acid dehydrogenase in the wild type after 6h shows a trend of increasing, and finally, the content is higher than that of the spkG knockout mutant, which indicates that the knockout of spkG has an influence on the expression of the fatty acid dehydrogenase gene. Thus, spkD and spkG are strongly correlated with the fatty acid dehydrogenase gene in Synechocystis. And spkG has a greater effect on the fatty acid dehydrogenase gene than spkD. At 144h of culture, the knockout mutant further inhibited the expression of the fatty acid dehydrogenase gene relative to the wild type, and thus the expression of the relevant gene in the mutant was further reduced. The above results indicate that the two genes spkD and spkG are negatively correlated with Synechocystis polyunsaturated fatty acid production at pre-culture. The method provides a new idea for research on improvement of polyunsaturated fatty acids in synechocystis PCC 6803.
Drawings
FIG. 1 is a schematic diagram of the knockout of synechocystis PCC6803 spkD and spkG genes;
in the figure, (A) is a spkD gene knockout schematic diagram; (B) is a spkG gene knockout schematic diagram;
FIG. 2 is PCR amplification electrophoresis detection map of Synechocystis PCC6803 spkD and spkG gene knockout mutants;
in the figure, an electrophoretogram (A) shows that M is a Trans 5k Marker, WT is a PCR amplification result of wild synechocystis, and 1 is a PCR amplification result of synechocystis with spkD gene knockout; and (B) an electrophoretogram (B), wherein M is a Trans 5k Marker, WT is a PCR amplification result of wild synechocystis, and 1 is a PCR amplification result of the synechocystis knocked out by spkG gene.
FIG. 3 is a bar graph showing the fatty acid composition content of synechocystis PCC6803 spkD and spkG gene knockout mutants and wild type;
in the figure: c18:2, linoleic acid; c18:3n6, gamma-linolenic acid; c18:3n3, alpha-linolenic acid; c18:4, stearidonic acid; others, other fatty acids.
FIG. 4 is a graph showing the change in the expression of the fatty acid dehydrogenase gene in the synechocystis PCC6803 spkD and spkG gene knockout mutant as well as in wild type;
in the figure: (A) the culture temperature is 30 ℃, and the illumination intensity is 40 mu mol.m-2·s-1Under the condition, the expression of the delta 6 fatty acid dehydrogenase gene of the strain is changed; (B) the culture temperature is 30 ℃, and the illumination intensity is 40 mu mol.m-2·s-1Under the condition, the expression of the delta 9 fatty acid dehydrogenase gene of the strain is changed; (C) the culture temperature is 30 ℃, and the illumination intensity is 40 mu mol.m-2·s-1Under the condition, the expression of the delta 12 fatty acid dehydrogenase gene of the strain is changed; (D) the culture temperature is 30 ℃, and the illumination intensity is 40 mu mol.m-2·s-1Under the condition, the expression of the strain delta 15 fatty acid dehydrogenase gene is changed.
Detailed Description
The following examples describe the use of the serine/threonine kinase genes of the present invention in the synthesis of unsaturated fatty acids from synechocystis. The invention is further described with reference to the drawings and examples, but the scope of the invention is not limited thereto.
Sources of materials
Escherichia coli strain (Escherichia coli) DH5 α was purchased from Beijing Quanjin Biotechnology Ltd;
the pClone007 simple vector was purchased from Beijing Ongko New Biotechnology Co., Ltd;
wild type synechocystis PCC6803 was purchased from the freshwater algae seed bank of the typical culture preservation committee of the academy of sciences of china; the following wild type synechocystis are all wild type synechocystis PCC6803
The plasmid pBluescript-Kan is purchased from China plasmid vector strain cell line gene collection center;
BG-11 medium was purchased from Shanghai plain Biotech, Inc.;
the cellulose nitrate filter membrane is purchased from Shanghai' an spectral experiment science and technology company, Ltd;
neutral phenol reagent was purchased from Invitrogen;
glass beads were purchased from sigma;
trans 5k Marker was purchased from Beijing Quanjin Biotechnology, Inc.;
trizol reagent was purchased from Invitrogen corporation
M-MLV reverse transcriptase was purchased from Takara, Inc., Baoji bioengineering (Dalian);
2 × SYBR Green I PCR Master Mix purchased from TaKaRa, Dalian bioengineering (Dalian) Co., Ltd;
other enzymes, reagents, kits and the like used are all common commercial products.
Example 1
Cloning and insertion of spkD gene into a quick ligation vector and cloning and insertion of spkG gene into a quick ligation vector:
performing PCR amplification on synechocystis PCC6803 genome DNA by using a primer pair, performing electrophoresis, and verifying that the spkD gene fragment length is 1.9kb and the spkD gene sequence is SEQ ID NO.17, and cutting and recovering gel; the primer pairs are as follows:
spkD-F：5’-ACTTACCCGTTCTGATTGA-3’SEQ ID NO.1
spkD-R：5’-TAACCATTGATAA GCAGAT-3’SEQ ID NO.2；
performing PCR amplification on synechocystis PCC6803 genome DNA by using a primer pair, performing electrophoresis, wherein the spkG gene fragment length is 1.9kb, the spkG gene sequence is SEQ ID NO.18, verifying correctness, cutting gel and recovering; the primer pairs are as follows:
spkG-F：5’-AGACTTTCTCTATTGCCTC-3’SEQ ID NO.3
spkG-R：5’-GGACCCAAATCCAGAAGAC-3’SEQ ID NO.4。
the PCR amplification system is as follows:
2x M5 HiPer plus Taq HiFi PCR mix 10 uL, template DNA 1uL, primer spkD-F1 uL, primer spkD-R1 uL, ddH2O7. mu.L, totaling to 20. mu.L.
2x M5 HiPer plus Taq HiFi PCR mix 10 uL, template DNA 1uL, primer spkG-F1 uL, primer spkG-R1 uL, ddH2O7. mu.L, totaling to 20. mu.L.
The PCR amplification procedure was as follows:
pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 25s, renaturation at 60 ℃ for 25s, and lmin extension at 72 ℃ for 35 cycles, final extension at 72 ℃ for 5min, and storage at 4 ℃.
The obtained spkD gene and spkG gene are respectively connected with pClone007 simple vector for 10min by TA cloning method, and transformed into Escherichia coli DH5 alpha, the specific operation is as follows: the gene plasmids and Escherichia coli DH5 alpha are respectively prepared into a mixture, the mixture is placed in an ice bath for 30min, then 42 ℃ is carried out, heat shock is carried out for 90s, then the mixture is rapidly placed in the ice bath for 3min, 750 mu L of LB liquid culture medium is respectively added, and the mixture is reactivated at 37 ℃ and 170rpm for 45 min. Spreading 200 μ L of the above reactivated bacterial solution on LB solid plate containing ampicillin (50mg/mL) and X-gal (20mg/mL), spreading with glass bar, culturing at 37 deg.C for 3h until the bacterial solution is completely absorbed, inverting the plate, and standing at 37 deg.C overnight. After the single clone grows out, a white single colony is picked from an LB solid culture medium plate by a toothpick to carry out colony PCR verification, and a positive clone is screened and sequenced. And (3) picking a single colony from the solid culture medium by using a toothpick at night for colony PCR verification, picking the corresponding single colony by using the toothpick again for verification, placing the single colony in a liquid culture medium of LB + AMP50, wherein AMP50 is used for picking the corresponding single colony at the concentration of 50mg/mL, shaking the colony overnight at 37 ℃ and 180rpm, and extracting plasmids after shaking the colony to obtain 007-spkD plasmids and 007-spkG plasmids respectively.
Example 2
Construction of spkD knock-out (insertional inactivation) and spkG knock-out (insertional inactivation) plasmids:
the 007-spkD plasmid was digested with EcoRI to give 007-spkD-EcoR I. The enzyme cutting site of EcoR I is 0.9kb of spkD gene fragment, the first 0.9kb is used as the upstream arm of homologous recombination, and the last 1.0kb (the length of spkD gene fragment is 1.9kb) is used as the downstream arm of homologous recombination. The 007-spkG plasmid was digested with BamHI to give 007-spkG-BamHI. The BamH I cleavage site is in the first 0.9kb of spkG gene. The first 0.9kb was used as the upstream arm of homologous recombination, and the second 1.0kb (the length of the spkG gene fragment was 1.9kb) was used as the downstream arm of homologous recombination.
The plasmid pBluescript-Kan (providing a kanamycin-resistant fragment) was digested with EcoRI endonuclease, and the kanamycin fragment (1.2kb) was recovered by electrophoresis and excised, and ligated to the plasmid 007-spkD-EcoR I which was likewise digested with EcoR I endonuclease, to prepare the plasmid 007-spkD-Kan.
The plasmid pBluescript-Kan (providing a kanamycin-resistant fragment) was digested with BamHI endonuclease, and the kanamycin fragment (1.2kb) was recovered by electrophoresis and ligated to the plasmid 007-spkG BamHI, which was also digested with BamHI endonuclease, to prepare a plasmid 007-spkG-Kan.
Example 3
Acquisition of spkD knock-out (insertional inactivation) mutant and spkG knock-out (insertional inactivation) mutant:
logarithmic cultivation phase (OD)7300.6) (culture conditions: BG-11 liquid culture medium, light condition 40 μmol. m at 30 deg.C-2·s-1) Centrifuging 4500g of Synechocystis PCC6803 culture solution for 8min at room temperature for 30ml, and removing the supernatant; washing with fresh BG-11 liquid culture medium, centrifuging, removing culture medium, adding fresh BG-11 liquid culture medium to final concentration OD7304.8, and is used for transformation immediately, and the transformation process is specific; the collected algal solution was dispensed into 1.5ml EP tubes (400. mu.l per tube), 5. mu.g of plasmids (plasmids 007-spkD-kan and 007-spkG-kan, respectively, prepared in example 2) were added to each tube, and the tubes were incubated under low light for 6 hours (light conditions: 10. mu. mol. m.-2·s-1Temperature 30 ℃ C.), and the mixture was gently inverted and mixed once. Then, the mixed culture of Synechocystis and plasmid was applied on a nitrocellulose filter, and cultured in a light incubator for 24 hours (culture conditions: 30 ℃ C., light conditions 40. mu. light conditions)mol·m-2·s-1) Transferring the culture medium into BG-11+50ug/mL kanamycin solid culture medium for culturing, and growing monoclonal algae strains in 10 days.
Selecting monoclonal algae strain with inoculating loop, spreading on BG-11 plate with high antibiotic concentration (kanamycin is 100ug/mL, 150ug/mL respectively), culturing, separating, purifying, inoculating into BG-11 liquid culture medium, and shake culturing on shaking bed (culture condition: 30 deg.C, 180rpm, illumination condition 40 μmol. m)-2·s-1). Obtaining the spkD gene knockout (insertional inactivation) mutant strain algae liquid and the spkG gene knockout (insertional inactivation) mutant strain algae liquid respectively.
Example 4
PCR detection of spkD knockout mutants and spkG knockout mutants:
and (3) extracting DNA from the synechocystis sp and wild synechocystis sp with the spkD gene knocked out and the spkG gene knocked out respectively by adopting a neutral phenol reagent and a glass bead oscillation method. The specific operation steps are as follows: take 50mL OD730Blue algae (spkD gene knockout mutant, spkG gene knockout mutant, and wild synechocystis) of 1.8 were centrifuged at 5000rpm for 10min at 4 ℃ to collect algal cells, 0.4mL of LTE buffer and 0.4mL of neutral phenol were added, and then a suitable amount of glass beads having a diameter of about 0.17mm were added until there was 0.5mL of suspension above the glass bead interface. Shaking by vortex oscillator at 2000rpm for 1min, centrifuging at 4 deg.C and 11900rpm for 10min, collecting supernatant, adding 0.5mL phenol/chloroform (volume ratio of 1:1) with equal volume, mixing by inversion for 15s, standing for 4min, and centrifuging at 11900rpm for 10 min. Taking the supernatant to a new centrifugal tube of 1.5m1, adding 0.5mL of isopropanol, reversing, mixing evenly, and placing in a refrigerator at-20 ℃ for sedimentation for 1.5 h. Centrifuging at 4 deg.C and 11900rpm for 10min, removing supernatant, adding 1ml of 75% ethanol (V/V), shaking by inversion for several times, centrifuging at 4 deg.C and 7500rpm for 10min, discarding supernatant, air drying at room temperature until precipitate is transparent, adding 50 μ l of TE buffer solution to dissolve precipitate, and respectively preparing total DNA of spkD gene knockout strain, spkG gene knockout strain, and wild type synechocystis.
PCR amplification is carried out by taking the Synechocystis knockout spkD gene, the Synechocystis knockout spkG gene and wild Synechocystis DNA as templates and respectively taking spkD-F SEQ ID NO.1, spkD-R SEQ ID NO.2, spkG-F SEQ ID NO.3 and spkG-R SEQ ID NO.4 as primer pairs.
The PCR amplification system is as follows:
10×PCR buffer 2uL，dNTP(2.5mM each)2.5uL，Mg2+2uL, Taq enzyme (5U/uL)0.5uL, primer F (10uM)1uL, primer R (10uM)1uL, ddH2O 20uL。
The specific amplification procedure was as follows:
pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 25s, renaturation at 60 ℃ for 25s, and lmin extension at 72 ℃ for 35 cycles, final extension at 72 ℃ for 5min, and storage at 4 ℃.
The detection of PCR products by electrophoresis is shown in FIG. 2. In the figure, in an electrophoretogram (A), M is a Trans 5k Marker, WT is a wild synechocystis PCR amplification result, and 1 is a spoD gene knockout synechocystis PCR amplification result; and (B) an electrophoretogram (B), wherein M is a Trans 5k Marker, WT is a PCR amplification result of wild synechocystis, and 1 is a PCR amplification result of the synechocystis knocked out by spkG gene.
Example 5
And (3) detecting the synechocystis PCC6803 spkD gene knockout algal strain and the spkG gene knockout algal strain by gas chromatography-mass spectrometry:
wild type Synechocystis PCC6803, spkD knock-out algal strain, spkG knock-out algal strain were inoculated into 50ml BG-11 liquid medium (culture conditions: 30 ℃, 180rpm, illumination conditions 40. mu. mol. m.)-2·s-1) OD in logarithmic growth phase730Algal cells were collected by centrifugation at 5.0 and dried under vacuum.
Accurately weighing 0.2g of crushed dry algae powder, putting the crushed dry algae powder into a mortar, adding liquid nitrogen, and repeatedly grinding the powder;
extracting with 7.0ml methanol-chloroform (2: 1, V: V), mixing with ultrasound for 10min, centrifuging for 15min and 6000rpm, collecting the organic phase extractive solution, adding 1.5ml methanol-chloroform (2: 1, V: V) into the residue, centrifuging, collecting the organic phase extractive solution, adding 1.5ml methanol-chloroform (2: 1, V: V) into the residue, and centrifuging; the organic phase extract was retained.
And (3) combining the organic phase extracting solutions, transferring the organic phase extracting solutions into a separating funnel, adding 2.5ml of chloroform and 3.0ml of 1% sodium chloride solution, standing for layering, recovering a lower layer, placing the lower layer into a fat extracting bottle, volatilizing the solvent to constant weight by nitrogen at 50 ℃, and weighing on a balance to obtain the total weight of the total algae fat.
The algae total lipids are further methyl-esterified and subjected to phase chromatography-mass spectrometry detection, and the analysis data of the fatty acid content is shown in fig. 3.
As shown in FIG. 3, the contents of linoleic acid (C18:2), gamma-linolenic acid (C18:3n6), alpha-linolenic acid (C18:3n3) and stearidonic acid (C18:4) in the wild type were significantly higher than those of the spkD and spkG gene knock-out mutants.
Example 6
RNA isolation and cDNA synthesis:
in the exponential growth phase (culture conditions: BG-11 liquid medium, 30 ℃, 180rpm, illumination conditions 40. mu. mol. m-2·s-1) Wild-type synechocystis PCC6803, spkD knock-out algal strains and spkG knock-out algal strains at the same cell concentration were cultured and harvested, and total RNA was isolated using Trizol reagent, first strand cDNA was synthesized using M-MLV reverse transcriptase, and cDNA was synthesized by modifying oligonucleotide (dT) according to the instructions of the M-MLV reverse transcriptase manufacturer.
Detecting the expression change of the fatty acid dehydrogenase gene of synechocystis PCC6803 spkD and spkG gene knockout algal strains:
carrying out quantitative real-time PCR detection of serine/threonine kinase expression by using Bio-Rad iQ5, and carrying out PCR amplification by using the following gene specific primers to obtain a cDNA sequence; the reactions were prepared according to the instructions of the 2 XSSYBR Green I PCR Master Mix (TaKaRa) manufacturer and qRT-PCR was performed using Bio-Rad iQ 5.
The gene-specific primers were as follows:
primer pair for amplifying the rnpB gene:
rnpB-F：5’-GTGAGGACAGTGCCACAGAA-3’SEQ ID NO.5
rnpB-R：5’-GGCAGGAAAAAGACCAACCT-3’SEQ ID NO.6
primer pair for amplifying 16SrRNA gene:
16S rRNA-F：5’-AGCGTCCGTAGGTGGTTATG-3’SEQ ID NO.7
16S rRNA-R：5’-CTACGCATTTCACCGCTACA-3’SEQ ID NO.8
primer pair for amplifying Δ 6 fatty acid dehydrogenase gene:
D6-RT-F：5’-GCCATTGATGACGAGTG-3’SEQ ID NO.9
D6-RT-R：5’-TAGCCAGCGATAGTTAGAG-3’SEQ ID NO.10
primer pair for amplifying Δ 9 fatty acid dehydrogenase gene:
D9-RT-F：5’-GGCATTGGCATTACTTT-3’SEQ ID NO.11
D9-RT-R：5’-CCTTATTAGAATCGTGGG-3’SEQ ID NO.12
primer pair for amplification of Δ 12 fatty acid dehydrogenase gene:
D12-RT-F：5’-TGGACAGGGACAGCCTTAAC-3’SEQ ID NO.13
D12-RT-R：5’-TTTTGTTGGTGTGGAGGTGA-3’SEQ ID NO.14
primer pair for amplification of Δ 15 fatty acid dehydrogenase gene:
D15-RT-F：5’-TCGCCTCAAACAAAGC-3’SEQ ID NO.15
D15-RT-R：5’-AATCGGATAGAAGAACCAG-3’SEQ ID NO.16
the reaction system is as follows:
each PCR was performed in a total volume of 20. mu.L containing 2 XSSYBR Green I PCR Master Mix (TaKaRa), 100nM concentration of each primer and 1. mu.L (1: 20 dilution by volume) of template cDNA in 3 replicates per sample.
The reaction procedure was as follows:
pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 10s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 42 cycles; one additional cycle: denaturation at 95 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 5min and extension at 95 ℃ for 10 s.
Obtaining a fatty acid dehydrogenase gene expression change diagram in the spkD gene knockout mutant strain, the spkG gene knockout mutant strain and the wild type, as shown in FIG. 4, comparing the expression difference of four fatty acid dehydrogenase genes in the wild type and the mutant strain respectively in different periods of time under the same culture conditions, finding that the fatty acid dehydrogenase related genes in the two mutant strains show a descending trend, the fatty acid dehydrogenase gene expression amount of the spkD knockout mutant strain is higher than that of the spkG knockout mutant strain, in the research on the spkD knockout mutant strain, finding that the fatty acid dehydrogenase gene expression amount of the mutant strain is obviously higher than that of the wild type in the initial stage of culture, and the fatty acid dehydrogenase gene expression of the mutant strain is most obviously different from that of the wild type in the initial 6h of culture, but the difference is smaller and smaller as the culture time is prolonged, and finally shows that the wild type is dominant, indicating that spkD knock-out has an effect on fatty acid dehydrogenase gene expression. In the study on the spkG knockout mutant, the expression level of the fatty acid dehydrogenase gene reaches the highest level at the initial 6h of culture, which is higher than that of the wild type, but the difference is small, the content of the fatty acid dehydrogenase in the wild type after 6h shows a trend of increasing, and finally, the content is higher than that of the spkG knockout mutant, which indicates that the knockout of spkG has an influence on the expression of the fatty acid dehydrogenase gene. Thus, spkD and spkG are strongly correlated with the fatty acid dehydrogenase gene in Synechocystis. And spkG has a greater effect on the fatty acid dehydrogenase gene than spkD. At 144h of culture, the knockout mutant further inhibited the expression of the fatty acid dehydrogenase gene relative to the wild type, and thus the expression of the relevant gene in the mutant was further reduced. The above results indicate that the two genes spkD and spkG are negatively correlated with Synechocystis polyunsaturated fatty acid production at pre-culture. The method provides a new idea for research on improvement of polyunsaturated fatty acids in synechocystis PCC 6803.
The above results indicate that the culture temperature was 30 ℃ and the light intensity was 40. mu. mol. m-2·s-1Under the condition, the relative expression quantity of the spkD and spkG genes in the wild type synechocystis PCC6803 is obviously higher than that of the spkD knockout mutant strain and the spkG gene knockout mutant strain. Meanwhile, fatty acid detection results show that spkD and spkG have the function of regulating and controlling the synthesis of unsaturated fatty acid in synechocystis. The levels of linoleic acid (C18:2, LA), gamma-linolenic acid (C18:3n6, GLA), alpha-linolenic acid (C18:3n3, ALA) and stearidonic acid (C18:4, SDA) in the wild type were significantly higher than in the spkD and spkG knock-out mutants. The STKs kinase spkD gene and spkG gene play a role in regulating and controlling Synechocystis PUFAs biosynthesis.
Meanwhile, a foundation is laid for improving the contents of polyunsaturated fatty acids such as LA, ALA and SDA in the cyanobacteria cells by synergistically regulating the spkD and spkG gene expression in the next step, and theoretical basis and experimental scheme are provided for industrially producing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by utilizing microalgae.
SEQUENCE LISTING
<110> center for researching biotechnology of academy of agricultural sciences of Shandong province
<120> method for regulating and controlling synthesis of unsaturated fatty acid in synechocystis and application thereof
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<400> 1
acttacccgt tctgattga 19
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
taaccattga taagcagat 19
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<400> 3
agactttctc tattgcctc 19
<210> 4
<211> 19
<212> DNA
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<400> 4
ggacccaaat ccagaagac 19
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
gtgaggacag tgccacagaa 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
ggcaggaaaa agaccaacct 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
agcgtccgta ggtggttatg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
ctacgcattt caccgctaca 20
<210> 9
<211> 17
<212> DNA
<213> Artificial sequence
<400> 9
gccattgatg acgagtg 17
<210> 10
<211> 19
<212> DNA
<213> Artificial sequence
<400> 10
tagccagcga tagttagag 19
<210> 11
<211> 17
<212> DNA
<213> Artificial sequence
<400> 11
ggcattggca ttacttt 17
<210> 12
<211> 18
<212> DNA
<213> Artificial sequence
<400> 12
ccttattaga atcgtggg 18
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence
<400> 13
tggacaggga cagccttaac 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
ttttgttggt gtggaggtga 20
<210> 15
<211> 16
<212> DNA
<213> Artificial sequence
<400> 15
tcgcctcaaa caaagc 16
<210> 16
<211> 19
<212> DNA
<213> Artificial sequence
<400> 16
aatcggatag aagaaccag 19
<210> 17
<211> 1816
<212> DNA
<213> Artificial sequence
<400> 17
acttacccgt tctgattgat tttggtgctg ttaaggaaac tatgggggct gttactttag 60
gctcaggttc tactgttagc tctgtagtta ttggcactag gggatttatg gctcctgaac 120
aaagttcggg acgttctgtt ttcagcaccg atctttatgc tttaggactg actattattt 180
acactcttac caagaagttg ccagttgaat tttctagcga ccagcaaaca ggccaattag 240
actggcaaag tcatgtatct aaaatcgatt ctgtcttggc taaagtaatc aataaagcca 300
tagaaatgga gcctagtcgt cgttattcca gtgccgaagc aatgtatcag gctctacact 360
ccttaattag ttctggggct gagccagcac ttccaatgga aactgttcga gttgcaccta 420
gtaatgaatt tttagtcacg agaagttcca ccaaaacggc tgaaactgtt gttaagcccg 480
tcggtaactc tcataataac tactcgaaca acaatggaaa atcaaaaatt gcaacgctgt 540
taactgttct tattgggatt attgtggtca ctgctggttt aggaggtggg tttataatta 600
ctcaacaaat taaagaagct gaagctaggg cggctcaagc tgaaaaagaa aaacaggaag 660
cagagcaaaa acgaatagaa gcagagcaaa aaattgccga gaatgaaaaa cgtcaaaggg 720
aattagaaca aaaacgagta gaagaagaac gtcaaagact agctgctgag gccgaacgag 780
ctaagcaaga aaggcagcgt ttagccgcag aaagacagag agttcaagtt ttagcaaatc 840
aggcaaaggc tatggccagc ggtgctagcg caactattgg aggaattcct ggatctaaaa 900
atattcgctc tggtccggga acagattatg gcgttattac tcaaggctat acaggtgagg 960
gtttagacat cttagatagt agtactgatt ccagtggcca tgtttggtat aaagtttatc 1020
actatggttc aggatcaacg gggtggattg ccagtcaatt agtaaacttt tagacttgat 1080
tctaaggaaa atgctaaaat ttttaaaaaa taactttcaa ttggtaaact tttttgttgc 1140
cgctctgtta gtgggaatgt tgtatggttg gtggtactta cctaatgcta aacaattatc 1200
tattaacgaa actagtggca ttcataatca agaaaaaatt ccatctcccc ccccccttga 1260
agatagagag ctttcctctc ttccggttcc cactccgact ccaactaatc tcgaaccatt 1320
aataactgaa gtccccgaaa tccagactag ccccctattg ccaggacttc caccagatta 1380
tgaacaatta tccccgctag cagaacagca aggatcaaga gaagttccca acaatattgc 1440
ggttaaaact aatttaggtc attattcttt ccctgaaaat agccagcaaa gattagtaaa 1500
agtaggtgaa tattacggcc gcagtgaatc tttagatcaa gaagcagcca ctgcttttaa 1560
aaagatgcaa gctgatgctc aagttcaagg agtaagatta actattattt ctggatttcg 1620
ttctattgcc tctcaggatg ctttatttca aaatcaaatt aaaagaaaag gtggtaaaga 1680
agcagcggca aggtttagtg cccctcccgg acacagtgaa caccacactg gctatgcttt 1740
agatatagga gatggtgcta atccagcaaa tgatttaaaa ataaattttg aaaatacatc 1800
tgcttatcaa tggtta 1816
<210> 18
<211> 1915
<212> DNA
<213> Artificial sequence
<400> 18
agactttctc tattgcctcc cagacaacaa caacagtaat aatcgcctag gacaaaggta 60
agatggaatc actccccgtc ctaggcgtat tttttatggt aaagactgcc aatgtggcgg 120
ttatgccgct gtttccccgc tcccaatata gaatcattgg acagatcgga cagggacagt 180
tcgggcgggt ttactgtgct attcaccgga ctacgggcaa aatgtatgcc ctgaaggatt 240
tagaacatcg ggtatttccc actaataaat ttctgcggga actgtcctat ctgttgaccc 300
tgcgccatcc caacattgtg gcttgccatg gtttggagta ccatcctggg ggccgctatt 360
tggtgatgga ttactgtgag gggggaactc tgcgggacat tattgatggg gacggagatt 420
tatgtttggc gggaaaaata gatttattaa gacaaatatt gctgggtttg gcccaagccc 480
atcagcatga cattgtccac tgtgacctta aaccggaaaa tatcctgtta ataccccggg 540
cagagggatg gcaggtaaaa gtatcagatt ttggcattgc tcgcctaaca gctaaaacgg 600
gcaatcctaa tttcagcaag ggttacactg gttctcccgc ctatatggcc ccggaacggt 660
tctacggcaa gttttctgta gcttccgaca tttatgctgt gggtattttg ctgtacgagc 720
tcatcgtcgg cgatcgcccg ttttcaggat ttcctaaagc gttgcaggca gcccatctca 780
atgtccgctt aaccttaccg ccggaatttc ctcccttact tgcccccatt gtccaacggg 840
ctttggagaa attaccccaa agacgatttc ccaatgccac agccatggcc agtgatttgg 900
ccacggttca aaaacaaata ttggaacagg atccccccag gggtaacggc tatctttacc 960
atcacttggc tccagctccg ctagccttta cggccacagt caaacacagt actcccttgt 1020
tatttcccat tagtcaccta actggggcag gactttggtt atacctaggc aatggggccg 1080
aactgtacct atgggagtat ggcgatggaa atgtggagca ccatcctttg ccccgctggg 1140
cgttgggttt accgggaacc atagctaatc tggaagtaaa tcaagaccaa attagcctct 1200
taatccaggg aggtgaacca ggagaatggc aattttacca gtggcgagaa accctacttg 1260
gcgctctctc tctccccaaa cctcgcatta atttgcgggc agagcgtctg ttggccaatc 1320
taagccccgg tggtaaaact ttggcagtgg tagtaggcga tcgggacagt gaaaaaggcc 1380
attttcaatt atggcgcact gaccacagct tacctgtggc tgcagccgta gtaattcgat 1440
ggccggatca actgctcacc ctagaccaaa accatggcct gttggtgcag tctcaatcaa 1500
cggcaagtta ttgtcatact atttttttcc tgtttaatcg gcgggggtcc ctgtttccag 1560
cctttcgcct ttcttttttg gtatttcaat tggtagttaa tcgctactcc cgcaaccatc 1620
tgtttggctt ggccgataat gctccctaca ctggtatttt gattcgtctg caacccctga 1680
aagtcaatcg cattgcccta actatccagc cccaattcat tgagcctttt ccctggggtt 1740
atctactagc cgatcgccat ggggaagtag ctttgttgga ctatgaaggt tttttatttg 1800
gcaatttttc cctcggggaa accatcacgg cgatcgcccc catgggtcgc tatctctgtt 1860
tatttgctac ctggcagggc aatgggggaa ctttacgtct tctggatttg ggtcc 1915