CN102191241A - Engineering bacteria capable of producing fatty acid, and preparation method and application thereof - Google Patents
Engineering bacteria capable of producing fatty acid, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses engineering bacteria capable of producing fatty acid, and a preparation method and application thereof. The engineering bacteria capable of producing the fatty acid is prepared by inactivating asr1131 protein of Anabaena sp.PCC 7120, wherein the amino acid sequence of the asr1131 protein is shown as the sequence 3 in the sequence table. In the engineering bacteria prepared by the method, free calcium ion concentration in the cell is increased; and thylakoid membranes are increased and filled in the whole cell. Compared with wild bacteria, the engineering bacteria have more intima components, higher growth speed, higher total fatty acid content and higher content of various important fatty acids.
Description
Technical field
The present invention relates to a kind of product lipid acid engineering bacteria and its production and application.
Background technology
Lipid acid is a kind of important basic chemical industry raw material, derives from oil, animal oil or plant wet goods.As Nonrenewable resources, oil is limited and face the danger of transition exploitation in the margin in the whole world.In order to protect environment, save energy, renewable resources obtains paying attention to and utilizing gradually.The various lipid acid that extract from vegetable and animals oils have been widely used in food, medicine, a plurality of fields such as chemical industry.
By the biotechnology transformation, many microorganism strains also are applied to lipid acid production, comprise bacterium, yeast, mould and algae etc.Bacterium comprises Lactobacterium acidophilum, muddy rhodococcus etc.Research in the yeast mainly concentrates in some candiyeasts and the rhodotorula.In the mould representative strain belong to that spore is mould more, Mucor or aspergillus.Algae is more common in little Trentepohlia and Porphyridium cruentum belongs to.
Summary of the invention
The purpose of this invention is to provide a kind of product lipid acid engineering bacteria and its production and application.
The invention provides dna fragmentation P, comprise homology arm T from the upstream to the downstream successively
1, dna fragmentation M and homology arm T
2Described homology arm T
1With homology arm T
2Can with the proteic encoding gene generation of asr1131 homologous recombination, the described asr1131 albumen of deactivation; Described dna fragmentation M is a marker gene; The proteic aminoacid sequence of described asr1131 is shown in the sequence 3 of sequence table.
The nucleotide sequence of the proteic encoding gene of described asr1131 can be shown in the sequence 4 of sequence table.
Described marker gene fragment can be antibiotics resistance gene, as kalamycin resistance gene.
The nucleotide sequence of described dna fragmentation P specifically can be shown in the sequence 5 of sequence table.
The expression cassette, recombinant vectors, transgenic cell line or the reorganization bacterium that contain described dna fragmentation P all belong to protection scope of the present invention.
Described recombinant vectors specifically can be described dna fragmentation P is inserted the recombinant plasmid I II that the multiple clone site of pRL277 plasmid obtains.
Described reorganization bacterium specifically can be described recombinant plasmid I II, plasmid pRL443 and plasmid pRL528 is imported the reorganization bacterium that intestinal bacteria HB101 obtains.
The method of producing the lipid acid engineering bacteria for preparing provided by the invention is an asr1131 albumen among the thread cyanobacteria Anabaena of the deactivation fresh water sp.PCC 7120, obtains producing the lipid acid engineering bacteria; The proteic aminoacid sequence of described asr1131 is shown in the sequence 3 of sequence table.
Arbitrary described dna fragmentation P can be imported the thread cyanobacteria Anabaena of described fresh water sp.PCC 7120, the homologous recombination by dna fragmentation P and the thread cyanobacteria Anabaena of fresh water sp.PCC 7120 realizes described deactivation.Specifically described reorganization bacterium and the thread cyanobacteria Anabaena of fresh water sp.PCC 7120 can be cultivated the described deactivation of realization altogether.
More than the product lipid acid engineering bacteria for preparing of arbitrary described method also belong to protection scope of the present invention.
The thread cyanobacteria Anabaena of fresh water sp.PCC 7120, it is a kind of type strain that is commonly used to study cyanobacteria light and effect and nitrogen fixation, this cyanobacteria is the photoautotrophy growth, have the cell inner membrance (thylakoid membrane) of arranging by rule in the cell, these endomembrane systems are rich in film fat and phycobiliprotein.With a calcium binding protein deactivation among the thread cyanobacteria Anabaena of the fresh water sp.PCC 7120, obtained engineering bacteria Δ 1131 among the present invention.Engineering bacteria Δ 1131 intracellular free calcium ion concentrations raise, and thylakoid membrane increases and is full of whole cell, and interior membrane component is more than wild bacterium, and the speed of growth is faster than wild bacterium, and total fatty acid content is higher than wild bacterium, and a lot of important fatty acid content also are higher than wild bacterium.
The advantage of engineering bacteria Δ 1131 is as follows:
1, than the product lipid acid bacterial strain of using usually at present, culture condition is simple.Engineering bacteria Δ 1131 can carry out photoautotrophy, only needs illumination and minimal medium, does not produce poison and obnoxious flavour.The used substratum of culturing bacterium and yeast mostly is organic substratum, and the part mould can produce the harmful HUMAN HEALTH of spore, and the cultivation difficulty of little algae is higher and easily contaminated.
2, the growth comparatively quick, for the time be about 20 hours, with respect to other algae (little algae for the time 44～91 hours, Porphyridium cruentum for the time 3～5 days) have a clear superiority in.The more little algae of present research reaches low concentration and will saturatedly stop to increase when liquid culture, engineering bacteria Δ 1131 then can be kept growth always and gather in the crops more thalline.
3, it is higher that the lipid acid of Chan Shenging accounts for the ratio of biomass, is higher than 22.5%.According to bibliographical information, in 2 very high little algae algae kinds of fatty acid content, also be to have reached 22.3%, because this strain growth is quick more and cultivation is convenient, can obtain more total fatty acids in the same time.
4, palmitinic acid, Zoomeric acid, linolenic acid, linolenic acid all have certain content, and this is some materials to the human body beneficial, and nutmeg acid is also important, but content is lower.The nutmeg acid content of engineering bacteria Δ 1131 is more than the twice of wild bacterium, and linolenic acid, Zoomeric acid and oleic content are also than wild bacterium height, and palmitinic acid is suitable with linolenic content.Linolenic acid is a kind of important substance of needed by human body, and Zoomeric acid and oleic acid are industry and edible raw material, and nutmeg acid is spices, all has the comparison application prospects.
5, when abundant fatty acid can be produced, can also gather in the crops phycobiliprotein, because the increase of thylakoid membrane can make the content of phycobiliprotein increase simultaneously, this proteinoid this also be the high-quality natural pigment on food and the cosmetic industry and can be applied to the synthesizing new fluorescent probe.Extract the method for phycobiliprotein: after cytoclasis, add the solution washing breakdown products, 10000rpm/min high speed centrifugation 10 minutes, be applied to lipid acid extraction with throw out this moment, supernatant water solution is the phycobiliprotein aqueous solution that has bluish voilet fluorescence, the phycobiliprotein precipitation of slightly being carried by the saturated intermediate processing of ammonium sulfate, the algae that can obtain slightly to carry through lyophilize but protein powder.
6, this sudden change physical efficiency is kept phenotype for a long time.
Description of drawings
Fig. 1 is that the southern of engineering bacteria Δ 1131-A identifies figure.
Fig. 2 is the growth curve of engineering bacteria Δ 1131-A and wild bacterium.
Fig. 3 is the filament (simple microscope of engineering bacteria and wild bacterium; Scale among the figure: 10 μ m); A: wild bacterium; B: engineering bacteria Δ 1131-A.
Fig. 4 is the cell picture of existing bacterial strain (Porphyridium cruentum).
Fig. 5 is engineering bacteria Δ 1131-A and 72 hours cell (transmission electron microscope of wild bacteria growing; Scale among the figure: 2 μ m); A: wild bacterium; B: engineering bacteria Δ 1131-A.
Fig. 6 is long-time back (growing 240 hours), the cell picture (transmission electron microscope of engineering bacteria Δ 1131-A cultivated; Scale among the figure: 500nm).
Fig. 7 is the gas chromatogram after the total fatty acids esterification.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, is the quality percentage composition.
Plasmid pRL277: Peking University; Reference: Conjugal transfer of DNA tocyanobacteria.Elhai J, Wolk CP.Methods Enzymol.1988; 167:747-54.
Plasmid pRL443: Peking University; Reference: Conjugal transfer of DNA tocyanobacteria.Elhai J, Wolk CP.Methods Enzymol.1988; 167:747-54.
Plasmid pRL528: Peking University; Reference: Conjugal transfer of DNA tocyanobacteria.Elhai J, Wolk CP.Methods Enzymol.1988; 167:747-54.
The thread cyanobacteria Anabaena of fresh water sp.PCC 7120 (wild bacterium, WT): available from U.S. ATCC (USS type culture collection institute; American Type Culture Collection) strain library, numbering 27893.
The pBS carrier: available from Stratagene company, catalog number: ST212207.
Intestinal bacteria HB101: available from takara company, catalog number: D9051.
Trace element solution A5:2.86g boric acid, 1.81g Manganous chloride tetrahydrate 4H
2O, 0.222g zinc sulfate, 0.39g Sodium orthomolybdate, 0.079g copper sulfate 5H
2O, 49.4g Xiao Suangu 6H
2O, adding distil water to 1 liter.
Bgl1 liquid nutrient medium: 1.5g SODIUMNITRATE, 0.04g dipotassium hydrogen phosphate, 0.075g sal epsom 7H
2O, 0.036g calcium chloride 7H
2O, 0.02g yellow soda ash, 0.006g citric acid, 0.006g ironic citrate, 1 milliliter of trace element solution A5, adding distil water to 1 liter.
The Bgl1 solid medium: add agar in the Bgl1 liquid nutrient medium, making its final concentration is 1.2% (quality percentage composition).
The resistance screening substratum: add various microbiotic mother liquors in the Bgl1 substratum (liquid/solid) respectively, the final concentration that makes penbritin is that the final concentration of 100 μ g/mL, kantlex is that the final concentration of 50 μ g/mL, metsulfuronmethyl is that the final concentration of 50 μ g/mL, paraxin is that the final concentration of 25 μ g/mL, tsiklomitsin is 12.5 μ g/mL; The penbritin mother liquor: solvent is an aseptic double-distilled water, and solute is a penbritin, and penbritin concentration is 100mg/mL; Kanamycin mother liquid: solvent is an aseptic double-distilled water, and solute is a kantlex, and the concentration of kantlex is 50mg/mL Kan; Metsulfuronmethyl mother liquor: paraxin mother liquor: solvent is a dehydrated alcohol, and solute is a paraxin, and the concentration of paraxin is 25mg/mL; The metsulfuronmethyl mother liquor: solvent is an aseptic double-distilled water, and solute is a metsulfuronmethyl, and the concentration of metsulfuronmethyl is 50mg/mL; The tsiklomitsin mother liquor: solvent is a dehydrated alcohol, and solute is a tsiklomitsin, and the concentration of tsiklomitsin is 12.5mg/mL.
Sugarcane candy resistance screening substratum: add sucrose in the resistance screening substratum, the final concentration of sucrose is 50mg/mL; Be the substratum of the pure and mild recon of resistance screening double exchange, utilize the sugarcane candy lethal gene that contains on the plasmid pRL277 to screen, reject single cross and change recon.
The preparation of embodiment 1, engineering bacteria
One, construction of recombinant plasmid
1, the segmental preparation in the upstream of asr1131 gene
Genomic dna with the thread cyanobacteria Anabaena of fresh water sp.PCC 7120 is a template, with Auele Specific Primer first (being made up of primer 1 and primer 2) is carried out pcr amplification, obtains the upstream fragment of asr1131 gene.
Primer 1 (upstream primer): 5 '-GAACCTCGAGTACAACAACGGGAAACCGC-3 ';
Primer 2 (downstream primer): 5 '-GCTGGTCGACAGATTAAGAATACTAAAGG-3 ';
2, the segmental preparation in the downstream of asr1131 gene
Genomic dna with the thread cyanobacteria Anabaena of fresh water sp.PCC 7120 is a template, with Auele Specific Primer second (being made up of primer 3 and primer 4) is carried out pcr amplification, obtains the downstream fragment of asr1131 gene.
Primer 3 (upstream primer): 5 '-ATCGGTCGACGTGCAACCACAAGCGTCG-3 ';
Primer 4 (downstream primer): 5 '-CTGCGGATCCTCATAATTAAACACCTCTGG-3 ';
Primer 3 is introduced the SalI restriction enzyme site, and primer 4 is introduced the BamHI restriction enzyme site.
3, the upstream fragment of the asr1131 gene of inserting step 1 preparation between the XhoI of pBS carrier and SalI restriction enzyme site, the downstream fragment of the asr1131 gene of inserting step 2 preparations obtains recombinant plasmid I between SalI and the BamHI restriction enzyme site.
4, between the SalI of recombinant plasmid I restriction enzyme site, insert kalamycin resistance gene (seeing the sequence 1 of sequence table), obtain recombinant plasmid I I.
5, cut recombinant plasmid I I with XhoI and BamHI enzyme, reclaim the dna fragmentation P of about 7300bp.The nucleotide sequence of dna fragmentation P is shown in the sequence 5 of sequence table, from the 1st to 2871 upstream fragment that Nucleotide is the asr1131 gene of 5 ' end, the the 2872nd to 4204 Nucleotide is kalamycin resistance gene, and the 4205th to 7140 Nucleotide is the downstream fragment of asr1131 gene.
6, the dna fragmentation that step 5 is obtained inserts between the XhoI and BglII restriction enzyme site (BglII and BamHI are isocaudarner) of shuttle vectors pRL277, obtains recombinant plasmid I II.
Two, the preparation of engineering bacteria Δ 1131-A
1, will be transformed into intestinal bacteria HB101 in conjunction with plasmid pRL443 and helper plasmid pRL528, obtain containing the reorganization fungus beetle of resistant gene Amp (penbritin), Sm (metsulfuronmethyl), Cm (paraxin) and Tc (tsiklomitsin).
2, recombinant plasmid I II is changed in the reorganization fungus beetle, the bacterium second that obtains recombinating is with LB substratum washing 3 times.
3, with the thread cyanobacteria Anabaena of fresh water sp.PCC 7120 usefulness Bgl1 liquid nutrient mediums washing 3 times.
4, with the centrifugal suspension culture base (LB substratum and Bgl1 substratum equal-volume are mixed the substratum that obtains) that is placed on of the thread cyanobacteria of fresh water of the reorganization bacterium second of step 2 and step 3, thorough mixing left standstill 4 hours according under the culture condition in the room temperature low light.
5, cover aseptic nitrocellulose filter on the Bgl1 solid medium, then the thalline mixture is coated with on the aseptic nitrocellulose filter, illumination cultivation is until growing newborn cyanobacteria bacterium colony.
6, colony lift to sugarcane candy resistance screening substratum, screening microbiotic positive strain.
7, the microbiotic positive strain is carried out southern and identify that (probe is shown in the sequence 2 of sequence table; What probe mated is about 800bp fragment in asr1131 gene downstream, size by expection deactivation gene and that gene of card that will replace distinguishes whether replace expection deactivation gene by that gene of card fully) qualification result shows, (promptly this mutant isozygotys to have 16 bacterial strains to can't see the specific band of wild bacterium fully, do not contain the asr1131 gene), obtain 16 recons that isozygoty, wherein a strain called after engineering bacteria Δ 1131-A.The southern evaluation figure of engineering bacteria Δ 1131-A sees Fig. 1.
Three, the preparation of engineering bacteria Δ 1131-B
The same step 2 of preparation method.
Obtain 16 recons that isozygoty, wherein a strain called after engineering bacteria Δ 1131-B.
Four, the preparation of engineering bacteria Δ 1131-C
The same step 2 of preparation method.
Obtain 16 recons that isozygoty, wherein a strain called after engineering bacteria Δ 1131-C.
Five, the preparation of contrast bacterium
Replace recombinant plasmid I II, the same step 2 of preparation method with pRL277.
The growth of embodiment 2, engineering bacteria and phenotype are observed
Culture condition: in the Bgl1 substratum that contains SODIUMNITRATE (1.5g/L), cultivate; Room temperature (20-25 ℃); Intensity of illumination is about 35 μ E/m
2/ S; Feed 1%CO during aerated culture
2Adopt above-mentioned culture condition culturing engineering bacterium Δ 1131-A, engineering bacteria Δ 1131-B, engineering bacteria Δ 1131-C and wild bacterium respectively.
Make respectively three engineering bacterias and wild bacterium growth curve and according to growth curve calculate for the time (the optical density(OD) numerical value of measuring according to growth curve is got the Lg value time is mapped, present the collinear zone in the Lg value and promptly think to reach logarithmic phase, the inverse of this growth period slope of a curve count for the time) (getting the mean value of three repeated experiments).The Dai Shijun of engineering bacteria Δ 1131-A, engineering bacteria Δ 1131-B and engineering bacteria Δ 1131-C is about 19.92 hours, wild bacterium for the time be about 20 hours.The growth curve of engineering bacteria Δ 1131-A and wild bacterium is seen Fig. 2.
Observe the filament of three engineering bacterias and wild bacterium by simple microscope.Find three engineering bacteria plesiomorphisms, the cell of engineering bacteria is all obviously greater than wild bacterium.The filament picture of engineering bacteria Δ 1131-A and wild bacterium is seen Fig. 3.
Compare by the cell of three engineering bacterias of transmission electron microscope observing and wild bacterium and with the existing bacterial strain of a strain.Existing bacterial strain be a kind of be the eucaryon algae through being commonly used to produce the red algae bacterial strain (Porphyridium cruentum) of lipid acid, the cell picture is seen Fig. 4 (quote from " http://www.bio.mtu.edu/the_wall/phycodisc/RHODOPHYTA/ ").Find three engineering bacteria plesiomorphisms, obvious difference is arranged with the cell inner membrance of wild bacterium.The engineering bacteria cell interior is full of by inner membrance, and cell obviously becomes big, and long-time structure of cultivating the back cell interior is fine and close more, the intimal thickening random arrangement that becomes.There is tangible cavity wild mycetocyte inside.The inner membrance of existing strain cell inside is a lot, the similar existing bacterial strain of the richness of the cell inner membrance of engineering bacteria (mutants which had is the protokaryon algae).The cell picture of engineering bacteria Δ 1131-A and wild bacterium is seen Fig. 5.After long-time the cultivation, the cell picture of engineering bacteria Δ 1131-A is seen Fig. 6.
Embodiment 3, product lipid acid ability are identified
Four repeated experiments are set, results averaged among the embodiment.
One, cultivates
Respectively engineering bacteria Δ 1131-A, engineering bacteria Δ 1131-B, engineering bacteria Δ 1131-C, wild bacterium and contrast bacterium are inoculated in 500 milliliters of Bgl1 liquid nutrient mediums that contain SODIUMNITRATE (1.5g/L), inoculation back OD value is 0.5; Culture condition: room temperature (20-25 ℃); Intensity of illumination is about 35 μ E/m
2/ S; Feed 1%CO during aerated culture
2Cultivate results after 7 days.
Two, weigh
Thalline weigh (with dry weight basis) with results.Engineering bacteria Δ 1131-A is about 0.52g, and engineering bacteria Δ 1131-B is about 0.54g, and engineering bacteria Δ 1131-C is about 0.5g, and wild bacterium is about 0.4g, and the contrast bacterium is about 0.38g.Slightly faster than wild bacterium and contrast bacterium, the thalline output of engineering bacteria is about 1.3 times of wild bacterium thalline output to the growth velocity of three engineering bacterias.
Three, total fatty acid content is measured
Extract the lipid acid of engineering bacteria Δ 1131-A, engineering bacteria Δ 1131-B, engineering bacteria Δ 1131-C, wild bacterium and contrast bacterium respectively, extracting method is as follows:
1, changes in the centrifuge tube that weighs up in advance after the thalline that reclaims is ground in liquid nitrogen, calculate thalline actual mass (0.4g);
2, every 1g thalline adds 10ml extraction damping fluid (water: ethanol: ether: pyridine: ammoniacal liquor=15: 15: 5: 1: 0.018; Volume ratio);
3, placed 15 minutes for 60 ℃, take out the back for a moment, centrifugal 5 minutes of 5000rpm/min at equilibrium at room temperature;
4, supernatant is changed in the clean centrifuge tube.
5, continue to add extraction damping fluid glass stick mixing in the precipitation, repeating step 3 and 4 twice.
6, all supernatants are rotated evaporates to dryness at 50 ℃, be total fatty acids, take by weighing the quality of total fatty acids.
The total fatty acids quality of engineering bacteria Δ 1131-A is 90mg, and the total fatty acids quality of engineering bacteria Δ 1131-B is 75mg, and the total fatty acids quality of engineering bacteria Δ 1131-C is 90mg, and wild bacterium is about 57mg, and the total fatty acids quality of contrast bacterium is about 58mg.For etc. the thalline of quality, the 1.3-1.6 that the total fatty acids quality of engineering bacteria is about wild bacterium is doubly.
Four, the mensuration of each fatty acid content
The total fatty acids of each bacterium that step 3 is obtained is the laggard promoting the circulation of qi analysis of hplc of esterification respectively, and the step of esterification is as follows:
1, in the total fatty acids that step 3 obtains, adds 4mL 2% methyl alcohol sulfuric acid (be made up of methyl alcohol and sulfuric acid, the quality percentage composition of methyl alcohol is 2%), placed 5 hours, and then this reaction system was equilibrated to room temperature for 85 ℃;
2, the 0.15M sodium chloride aqueous solution that in reaction system, adds 1/2 volume, the vibration mixing;
3, add isopyknic normal hexane, behind the thermal agitation mixing, left standstill 5 minutes;
4,3000rpm/min is centrifugal 3 minutes;
5, with the careful sucking-off of upper strata normal hexane layer, change in the new centrifuge tube;
6, repeating step is 3 to 5 twice;
7, the normal hexane with 3 collections merges, and is dissolved in again in the normal hexane after nitrogen dries up, and uses gas chromatographic analysis.
Engineering bacteria Δ 1131-A and the correlated gas chromatogram of wild bacterium are seen Fig. 7.
The position of each characteristic peak of three engineering bacterias, wild bacterium and contrast bacterium overlaps, and engineering bacteria be described, to contrast bacterium consistent with various lipid acid kinds in the wild bacterium.In three engineering bacterias, wild bacterium and contrast bacterium: palmitinic acid, Zoomeric acid, linolenic acid all have certain content, and nutmeg acid also has certain content, but content is lower; The nutmeg acid content of engineering bacteria is more than the twice of wild-type, and linolenic acid, Zoomeric acid and oleic content are also than wild bacterium height, and palmitinic acid and linolenic content are suitable with wild bacterium.
The measurement result of each fatty acid content sees Table 1.
The measurement result of each fatty acid content of table 1
Sequence table
＜110〉Peking University
＜120〉produce lipid acid engineering bacteria and its production and application
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ctactgggct?atctggacaa?gggaaaacgc?aagcgcaaag?agaaagcagg?tagcttgcag 180
tgggcttaca?tggcgatagc?tagactgggc?ggttttatgg?acagcaagcg?aaccggaatt 240
gccagctggg?gcgccctctg?gtaaggttgg?gaagccctgc?aaagtaaact?ggatggcttt 300
cttgccgcca?aggatctgat?ggcgcagggg?atcaagatct?gatcaagaga?caggatgagg 360
atcgtttcgc?atgattgaac?aagatggatt?gcacgcaggt?tctccggccg?cttgggtgga 420
gaggctattc?ggctatgact?gggcacaaca?gacaatcggc?tgctctgatg?ccgccgtgtt 480
ccggctgtca?gcgcaggggc?gcccggttct?ttttgtcaag?accgacctgt?ccggtgccct 540
gaatgaactg?caggacgagg?cagcgcggct?atcgtggctg?gccacgacgg?gcgttccttg 600
cgcagctgtg?ctcgacgttg?tcactgaagc?gggaagggac?tggctgctat?tgggcgaagt 660
gccggggcag?gatctcctgt?catctcacct?tgctcctgcc?gagaaagtat?ccatcatggc 720
tgatgcaatg?cggcggctgc?atacgcttga?tccggctacc?tgcccattcg?accaccaagc 780
gaaacatcgc?atcgagcgag?cacgtactcg?gatggaagcc?ggtcttgtcg?atcaggatga 840
tctggacgaa?gagcatcagg?ggctcgcgcc?agccgaactg?ttcgccaggc?tcaaggcgcg 900
catgcccgac?ggcgaggatc?tcgtcgtgac?ccatggcgat?gcctgcttgc?cgaatatcat 960
ggtggaaaat?ggccgctttt?ctggattcat?cgactgtggc?cggctgggtg?tggcggaccg 1020
ctatcaggac?atagcgttgg?ctacccgtga?tattgctgaa?gagcttggcg?gcgaatgggc 1080
tgaccgcttc?ctcgtgcttt?acggtatcgc?cgctcccgat?tcgcagcgca?tcgccttcta 1140
tcgccttctt?gacgagttct?tctgagcggg?actctggggt?tcgaaatgac?cgaccaagcg 1200
acgcccaacc?tgccatcacg?agatttcgat?tccaccgccg?ccttctatga?aaggttgggc 1260
ttcggaatcg?ttttccggga?cgccggctgg?atgatcctcc?agcgcgggga?tctcatgctg 1320
gagttcttcg?cccaccccgg?tcgac 1345
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cgctgcgatt?accaaactag?ctgcttctcc?atactggatt?ccaccccatt?gctaaaagcc 60
gtagttttac?tgatatttat?tttggcataa?gccaaactta?gcagtgtgga?caggaagatt 120
taaagtggta?cagagctaca?ataccgagtg?tgtagcgtta?cgatgtggct?gtaaggtaag 180
ctgatgtgca?tttaaactgg?gatttttgaa?agctgtactc?cttgctgtgt?cgagattgct 240
cgctcgattt?taagttgggt?tatggtgggc?aatgcccacc?ctacagatac?ttagacagca 300
attatctgcc?tactaattta?tcgcgcaatt?gcttgatgcg?atcgcgcaat?tttgccgcct 360
cttcaaattc?cagttttttc?gatgcttctt?tcatctgtgc?ttctaatttg?tcaatcaagt 420
taggaatctc?ctctaacggc?aattcatcaa?tatgttcttc?aacaactttt?aagtctgttg 480
cattcaaccg?tcgagacact?tccaagaaag?acaaaattgc?attactcgat?tttttgacaa 540
tcggttgtgg?tgtaattcca?tgcagcttat?tatatgcaac?ctgaataccg?cgccgtctgt 600
ctgtttcttc?aatagcttta?atcatgctat?ctgtcatatt?atcagcatat?aaaattgctt 660
gtcctctgat?gtgtcgtgct?gctctcccaa?tggtttgaat?taaagaacgt?tccgcgcgta 720
agaaaccttc?tttatcggca?tccataatcg?ctactaggga?aacttcaggt?aaatctaaac 780
cttcccgtag?taggttcaca?cccaccaaca?catcaaagct?accttggcgt?aaatcctg 838
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＜213〉the thread cyanobacteria Anabaena of fresh water sp.
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Met?Ala?Thr?Glu?Gln?Glu?Leu?Gln?Set?Leu?Phe?Asn?Thr?Leu?Asp?Arg
1 5 10 15
Asp?Gln?Asp?Gly?Lys?Ile?Ser?Ile?Asn?Glu?Leu?Phe?Leu?Ser?Pro?Gly
20 25 30
Leu?Ser?Ala?Val?Ile?Ser?Ser?Glu?Thr?Asn?Thr?Asn?Ser?Pro?Gln?Glu
35 40 45
Leu?Leu?Val?Gln?Tyr?Asp?Ser?Asp?Gln?Asp?Gly?Ser?Ile?Thr?Phe?Glu
50 55 60
Glu?Leu?Lys?Lys?Ala?Val?Lys?Lys?Ala?Ser?Asn?Leu?Thr
65 70 75
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＜213〉the thread cyanobacteria Anabaena of fresh water sp.
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aaaatctcca?ttaatgagct?ttttttaagc?cctggtttaa?gtgcagtcat?ctcatcagaa 120
acgaatacca?atagccccca?agagttgcta?gtacagtatg?attcagacca?agacggtagt 180
attacctttg?aagagttaaa?gaaagcagtt?aagaaagcaa?gtaatttaac?ctag 234
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<223>
<400>5
ctcgagtaca?acaacgggaa?accgccttta?gggaagccga?acaacgctta?aaaaatctag 60
aaaatcaaca?acagcgttta?caagaacgca?tccaagaagc?ccaacagcgc?atcaccgaat 120
atcaaaccca?acaaaccact?tgtacagaag?cgattaatcg?cgtctctcaa?caaaccacaa 180
ccataaacgc?acaaatcacc?caaacccgcg?ccaaactcag?cgaacttgaa?caacacctcg 240
gcgcagaaaa?acaaaaacgc?gacacaattg?aacaagaagt?gcgatcgcac?ctcctgcgtc 300
agcaacaatt?agaatgggaa?attcaaaaac?tggaagaaac?ccaacttaaa?cgccgggaag 360
acttaaccgc?cttacaaagc?cagttacagg?aactagtccc?agaactaccc?aaccctttgc 420
cagaagtccc?agacaaggta?gacttagaag?aattgcagaa?agaattgcga?tcgctcgcca 480
aacgcctcca?agcaatggaa?cctgtgaata?tgctggcgtt?ggaagaatac?gaacgtaccc 540
aaaagcgcct?agaagaacta?tcccaaaaat?tacagacctt?agaaggagaa?cgcaccgaac 600
tactcctacg?cattgaaaac?ttcaccacct?tgcgccaaat?cgcctttaaa?gaagccttcg 660
acgctgtaaa?tgaaaatttc?caatcaatct?tcgccaccct?ttccgacggc?gacggctacc 720
tgcaattaga?taacccagaa?gatcccttta?acagtggctt?gaacttagtc?gcccacccca 780
aaggtaaacc?agtccaaaga?ctcgcctcca?tgtccggtgg?tgaaaaatcc?ctcaccgcct 840
taagctttat?cttcgccttg?caacgctacc?gcccctcgcc?attttacgcc?tttgatgaag 900
tagatatgtt?cctcgatggg?gcaaacgtag?agcgattatc?aagaatgatt?aaacaacaag 960
cccagcaggc?acaattcata?gttgtgagtt?tgcgccgtcc?gatgatagaa?tcagccgaac 1020
gcacaatcgg?cgttactcaa?gcacgaggag?cttatactca?agttttggga?attaagttat 1080
catcctcgaa?tacatctgct?tgagtttttg?ttaataatag?tgtatagata?aaccggattc 1140
gagatcagga?ctccgtatag?aatgacctct?gaacaaataa?ttaggcgttc?cgacatatta 1200
aatacgcagg?taattacccg?cgacaacggc?aaacggttag?gaatcgtcag?tcaagtttgg 1260
gtggatattg?atcaaagaga?ggttgtggct?cttggtttgc?gagacagcct?gatctctatc 1320
tctggtctgc?cccgctatat?gtaccttagc?agcatccacc?aatatggcga?tgtcatctta 1380
gttgataacg?aagatgtcat?cgaagatatt?gaagttgaag?ccctcagtaa?cttgattaac 1440
tgggaagtaa?tcaccgaaac?aggcgaagtt?ttaggtagag?tgcggggttt?ccgatttgaa 1500
gccgaaacag?gtaaactcaa?caccatagtc?atcgcctcct?tgggagttcc?ccaaattccc 1560
gaccagtttt?taagcaccta?cgaaatatcc?atagaagaag?tagtcagcac?cggccccaac 1620
aggttgattg?tctttgaagg?agccgaagaa?cgggtaaatc?agttaacagt?cggtgttctc 1680
gaacgcctgg?gtattggtaa?agcaccttgg?gaacgggata?cagaagaaga?atatggttat 1740
tccgcacccc?gcgctgtcgc?accatcaaat?cagctaccta?gcggcgtacc?cttacagcca 1800
cccaaaccca?aagtccgcac?ccccgaaccc?gtagccgagg?aagaatggac?tgaagattac 1860
atcgaagaag?aaagaccaca?gcgccaagtc?atgaaggcgc?gacaatacga?gtcaattcaa 1920
tacgaagaag?acgaagaaga?caactggagt?gaagccacgg?gtagagacag?atatcagccc 1980
ccgtctcaac?cctacagcaa?accatacacc?gaagattacg?acgattatga?tgacgatgta 2040
gaaagtgatg?cgtgggaaga?cgtaccaccc?caaccagtaa?atattcccaa?gaaagtcaaa 2100
gaaagacaac?cagaatacga?agaagaaggc?ggatattaat?tttttggtga?ttcatgatta 2160
aacctctcct?atttgtttgt?aggagaggtt?taattttttg?cttaaggatt?aatagcagaa 2220
ttagccattt?tgatgagaga?attgcgatcg?cctgccttca?acccaaaact?atactgcacc 2280
ccttgctgtc?gccacgtcaa?agtcgcatca?gaacaattag?ccccacaacg?aaaatccaca 2340
aaatagccag?caataccctt?agctaaagcc?acccttctcc?ccttgaggcg?cggcgttgaa 2400
cgcgtgacag?cctccgccga?aaccacacct?aaacgacaag?ccgtcccacc?agtacaatca 2460
ggactgaaac?ccaacagaat?ctcatatttc?ttcggtgtag?ccgtttctat?aatggcgtaa 2520
attggatttg?tcccatccga?ctcaggaatg?tatttaggca?ataaaatctt?aatttgactc 2580
ttttgcttta?acttcggcaa?aatcggttga?aaaactttgt?gtggctgaga?attagctgtt 2640
ctagctggtc?gttgtgctga?tagctgcggt?tgattatttg?tattagctat?agccgacgga 2700
taaaaattgt?tgctgccgac?taatatcagt?aatattgata?aattagtgag?actttttaaa 2760
ccaaaaatca?taaagtaaaa?cctatttcaa?cgggtgtatt?taacttatgt?ttcccatttt 2820
ttagactaat?cggtgcttca?tactcacctt?tagtattctt?aatctgtcga?cggtatcgat 2880
aagcttcacg?ctgccgcaag?cactcagggc?gcaagggctg?ctaaaggaag?cggaacacgt 2940
agaaagccag?tccgcagaaa?cggtgctgac?cccggatgaa?tgtcactact?gggctatctg 3000
gacaagggaa?aacgcaagcg?caaagagaaa?gcaggtagct?tgcagtgggc?ttacatggcg 3060
atagctagac?tgggcggttt?tatggacagc?aagcgaaccg?gaattgccag?ctggggcgcc 3120
ctctggtaag?gttgggaagc?cctgcaaagt?aaactggatg?gctttcttgc?cgccaaggat 3180
ctgatggcgc?aggggatcaa?gatctgatca?agagacagga?tgaggatcgt?ttcgcatgat 3240
tgaacaagat?ggattgcacg?caggttctcc?ggccgcttgg?gtggagaggc?tattcggcta 3300
tgactgggca?caacagacaa?tcggctgctc?tgatgccgcc?gtgttccggc?tgtcagcgca 3360
ggggcgcccg?gttctttttg?tcaagaccga?cctgtccggt?gccctgaatg?aactgcagga 3420
cgaggcagcg?cggctatcgt?ggctggccac?gacgggcgtt?ccttgcgcag?ctgtgctcga 3480
cgttgtcact?gaagcgggaa?gggactggct?gctattgggc?gaagtgccgg?ggcaggatct 3540
cctgtcatct?caccttgctc?ctgccgagaa?agtatccatc?atggctgatg?caatgcggcg 3600
gctgcatacg?cttgatccgg?ctacctgccc?attcgaccac?caagcgaaac?atcgcatcga 3660
gcgagcacgt?actcggatgg?aagccggtct?tgtcgatcag?gatgatctgg?acgaagagca 3720
tcaggggctc?gcgccagccg?aactgttcgc?caggctcaag?gcgcgcatgc?ccgacggcga 3780
ggatctcgtc?gtgacccatg?gcgatgcctg?cttgccgaat?atcatggtgg?aaaatggccg 3840
cttttctgga?ttcatcgact?gtggccggct?gggtgtggcg?gaccgctatc?aggacatagc 3900
gttggctacc?cgtgatattg?ctgaagagct?tggcggcgaa?tgggctgacc?gcttcctcgt 3960
gctttacggt?atcgccgctc?ccgattcgca?gcgcatcgcc?ttctatcgcc?ttcttgacga 4020
gttcttctga?gcgggactct?ggggttcgaa?atgaccgacc?aagcgacgcc?caacctgcca 4080
tcacgagatt?tcgattccac?cgccgccttc?tatgaaaggt?tgggcttcgg?aatcgttttc 4140
cgggacgccg?gctggatgat?cctccagcgc?ggggatctca?tgctggagtt?cttcgcccac 4200
cccggtcgac?gtgcaaccac?aagcgtcgtc?agtttagaaa?aaacgcggcg?actttagcgg 4260
aaaacatcaa?atttgagctt?ctgcgctggg?atgtttttgt?ttgaactaaa?actttgctgt 4320
tctaagtgag?aacgttcttg?ttttaaacga?gaacgttttc?gttttagttg?ctaacttttg 4380
ccaaatcaca?tctggtaatt?ctattttgcc?ttcagtggtg?acgtttgcta?gaaattcgta 4440
cgctttcata?ttatgtttat?tgttcaccag?ttctaatact?attttgacgc?tgcgattacc 4500
aaactagctg?cttctccata?ctggattcca?ccccattgct?aaaagccgta?gttttactga 4560
tatttatttt?ggcataagcc?aaacttagca?gtgtggacag?gaagatttaa?agtggtacag 4620
agctacaata?ccgagtgtgt?agcgttacga?tgtggctgta?aggtaagctg?atgtgcattt 4680
aaactgggat?ttttgaaagc?tgtactcctt?gctgtgtcga?gattgctcgc?tcgattttaa 4740
gttgggttat?ggtgggcaat?gcccacccta?cagatactta?gacagcaatt?atctgcctac 4800
taatttatcg?cgcaattgct?tgatgcgatc?gcgcaatttt?gccgcctctt?caaattccag 4860
ttttttcgat?gcttctttca?tctgtgcttc?taatttgtca?atcaagttag?gaatctcctc 4920
taacggcaat?tcatcaatat?gttcttcaac?aacttttaag?tctgttgcat?tcaaccgtcg 4980
agacacttcc?aagaaagaca?aaattgcatt?actcgatttt?ttgacaatcg?gttgtggtgt 5040
aattccatgc?agcttattat?atgcaacctg?aataccgcgc?cgtctgtctg?tttcttcaat 5100
agctttaatc?atgctatctg?tcatattatc?agcatataaa?attgcttgtc?ctctgatgtg 5160
tcgtgctgct?ctcccaatgg?tttgaattaa?agaacgttcc?gcgcgtaaga?aaccttcttt 5220
atcggcatcc?ataatcgcta?ctagggaaac?ttcaggtaaa?tctaaacctt?cccgtagtag 5280
gttcacaccc?accaacacat?caaagctacc?ttggcgtaaa?tcctgcaata?tttcaatccg 5340
ttgaatagaa?ttaatttctg?aatgtaaata?ccgcactttc?acgccgtgtt?cttgcagata 5400
ttctgttaaa?tcttccgcca?tccgcttagt?taaagtggta?atcaataccc?tttcatggag 5460
gtcgattcta?tctttaattt?ctcctaacaa?atcatcaatt?tgtccttctg?tgggacgaac 5520
ggaaatttct?ggatcaatta?cgcctgtagg?gcgaatgact?tgctcaacta?tgcgattttc 5580
ggaaatctct?aattcccaat?ttcctggggt?agcagaaaca?aaaatacact?ggttaacttt 5640
ttgccaaaat?tcctctgctt?ttaaaggacg?gttatcagca?gcactaggaa?gcctaaatcc 5700
atgctcaatt?aatactttct?tcctagcttg?gtcgccgtta?tacattccgc?gaatttgcgg 5760
gactgtaacg?tgagattcgt?caatgactaa?caaccaatct?ttcgggaaat?aatcaattaa 5820
actttctggt?ggttcccccg?cttgtctacc?tgctaaatgg?cgagaatagt?tttctacgcc 5880
gttgcaataa?cccacttccc?gcagcatttc?taaatcgtaa?cgggtgcgtt?ggtcgatgcg 5940
ttgcgcttct?accaatttgc?cagtttcttc?taattctgct?ttgcgctgtt?ttaattcata 6000
ggcgatatct?tcgcaagcta?cctctaaacg?ttcctctggg?gtgacaaagt?gacgcgcagg 6060
atagatattt?actgcttgta?aactgttgat?aatttcccct?gtcacggggt?caatgtagcg 6120
aattgcgtca?atttcatcgc?caaaaaattc?tacccgaata?atcctgtctt?cgtaagcggg 6180
gccgatttct?aatacatcac?cccggacgcg?gaaacgtccc?cttcccattt?ctacatcgtt 6240
gcggctgtat?tgtacagagg?ctaaatcccg?caaaatctcc?cgttgattta?cttccatccc 6300
aatttgcaga?ggaatagcgg?ctttaaggta?ttcggcgggg?attcccaaac?cgtaaataca 6360
gctaatagaa?gccacaacaa?tcacatcacg?acgctcgaat?agcgatcgcg?tggctgaatg 6420
tcgtaacata?tcaatctcat?cgttaatcgc?cgccgttttt?tcaatatacg?tatcggtaac 6480
gggaatatag?gcttctggtt?gatagtaatc?gtaataactg?acgaaatact?caacagcgtt 6540
gttgggaaag?aactcgcgca?attcattaca?tagttgcgcg?gccaaggttt?tattatgcgc 6600
cagaactagc?gttggtctcc?caattttctc?aatcactgcg?gcgatggaaa?atgtcttacc 6660
tgttcccgta?gcacctaata?aggtttggta?acggttacct?ccttcgatgc?tagcaactaa 6720
ctgggcgatc?gcgcttggtt?gatcacctgt?cggactaaag?ggagcttgca?gaccaaattc 6780
tgtcatacat?ctgtactaaa?gataccctat?ctcatgttag?cgataaatta?atcaggctag 6840
tgctaagtac?tgagttccta?ctcaaaagct?aacagtcagc?agtcagccgt?caacattcaa 6900
cggtcaacag?tcaacagccc?atagtaattg?ttcttacctt?tgctccccca?ccttttcaat 6960
tagcaatttt?ctttgacaaa?ttttcagaat?ttaagtttac?ttatatgtca?cttttgaaga 7020
tacttatata?tatgcaaggt?gctttaaggg?taaactcact?tatataggaa?ataatttgat 7080
ttttccttaa?gtattgtaaa?gttaagttaa?taacccagag?gtgtttaatt?atgaggatcc 7140
Claims (10)
1.DNA fragment P comprises homology arm T successively to the downstream from the upstream
1, dna fragmentation M and homology arm T
2Described homology arm T
1With homology arm T
2Can with the proteic encoding gene generation of asr1131 homologous recombination, the described asr1131 albumen of deactivation; Described dna fragmentation M is the marker gene fragment; The proteic aminoacid sequence of described asr1131 is shown in the sequence 3 of sequence table.
2. dna fragmentation P as claimed in claim 1 is characterized in that: the nucleotide sequence of the proteic encoding gene of described asr1131 is shown in the sequence 4 of sequence table; Described marker gene fragment is the antibiotics resistance gene fragment.
3. dna fragmentation P as claimed in claim 2 is characterized in that: the nucleotide sequence of described dna fragmentation P is shown in the sequence 5 of sequence table.
4. the expression cassette, recombinant vectors, transgenic cell line or the reorganization bacterium that contain arbitrary described dna fragmentation P among the claim 1-3.
5. recombinant vectors as claimed in claim 4 is characterized in that: the recombinant plasmid I II that described recombinant vectors obtains for the multiple clone site of described dna fragmentation P being inserted the pRL277 plasmid.
6. reorganization bacterium as claimed in claim 4 is characterized in that: described reorganization bacterium is for importing the reorganization bacterium that intestinal bacteria HB101 obtains with recombinant plasmid I II, plasmid pRL443 and plasmid pRL528; The recombinant plasmid that described recombinant plasmid I II obtains for the multiple clone site of described dna fragmentation P being inserted the pRL277 plasmid.
7. preparing the method for producing the lipid acid engineering bacteria, is the asr1131 albumen among the thread cyanobacteria Anabaena of the deactivation fresh water sp.PCC 7120, obtains producing the lipid acid engineering bacteria; The proteic aminoacid sequence of described asr1131 is shown in the sequence 3 of sequence table.
8. method as claimed in claim 7, it is characterized in that: described deactivation is that arbitrary described dna fragmentation P in the claim 1 to 3 is imported the thread cyanobacteria Anabaena of fresh water sp.PCC 7120, and the homologous recombination by dna fragmentation P and the thread cyanobacteria Anabaena of fresh water sp.PCC 7120 realizes.
9. method as claimed in claim 8 is characterized in that: described homologous recombination is by cultivating realization altogether with claim 6 described reorganization bacterium and the thread cyanobacteria Anabaena of fresh water sp.PCC 7120.
10. the product lipid acid engineering bacteria that arbitrary described method prepares in the claim 7 to 9.
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| CN112825817A (en) * | 2019-11-25 | 2021-05-25 | 北京大学 | Method for indoor feeding and breeding Kallima inachus under full-artificial condition |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7314974B2 (en) * | 2002-02-21 | 2008-01-01 | Monsanto Technology, Llc | Expression of microbial proteins in plants for production of plants with improved properties |
| CN101473038A (en) * | 2006-03-15 | 2009-07-01 | 马泰克生物科学公司 | Plant seed oils containing polyunsaturated fatty acids |
| CN101765661A (en) * | 2007-06-01 | 2010-06-30 | 索拉兹米公司 | In microorganism, produce oil |
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2010
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7314974B2 (en) * | 2002-02-21 | 2008-01-01 | Monsanto Technology, Llc | Expression of microbial proteins in plants for production of plants with improved properties |
| CN101473038A (en) * | 2006-03-15 | 2009-07-01 | 马泰克生物科学公司 | Plant seed oils containing polyunsaturated fatty acids |
| CN101765661A (en) * | 2007-06-01 | 2010-06-30 | 索拉兹米公司 | In microorganism, produce oil |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112825817A (en) * | 2019-11-25 | 2021-05-25 | 北京大学 | Method for indoor feeding and breeding Kallima inachus under full-artificial condition |
| CN112825817B (en) * | 2019-11-25 | 2022-03-08 | 北京大学 | Method for indoor feeding and breeding Kallima inachus under full-artificial condition |
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