CN107236679A - A kind of high yield unrighted acid recombinant strain and its construction method - Google Patents
A kind of high yield unrighted acid recombinant strain and its construction method Download PDFInfo
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- CN107236679A CN107236679A CN201710369369.3A CN201710369369A CN107236679A CN 107236679 A CN107236679 A CN 107236679A CN 201710369369 A CN201710369369 A CN 201710369369A CN 107236679 A CN107236679 A CN 107236679A
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Abstract
The present invention relates to a kind of high yield unrighted acid recombinant strain, by the restructuring of the desaturase of Δ 6 into the genome of saccharomycete；The saccharomycete is rhodotorula glutinis, and the desaturase of Δ 6 is obtained by the separation of the D6D genes of cunninghamella echinulata with amplification.Invention additionally discloses a kind of construction method of high yield unrighted acid recombinant strain.The present invention has the advantages that：Identified through double digestion, the purpose fragment direction of insertion on recombinant plasmid is correct；Realize D6D stabilization, permanent expression；Real-time fluorescence quantitative PCR shows that D6D expression improves 2 times or so；Through gas chromatography analysis, the GLA in conversion bacterial strain is improved more than 3.3 times compared with wild strain.
Description
Technical field
The present invention relates to gene engineering technology field, more particularly to a kind of high yield unrighted acid recombinant strain and
Its construction method.
Background technology
Many unrighted acids are got over because of its extensive physiologically active and its important function in terms of human health, nutrition
Paid close attention to get over by people；Polypeptide drugs again because its half-life short, easily be degraded by enzymes limit application.
The building-up process of microbial grease is almost similar to animals and plants, and topmost part is exactly the synthesis of aliphatic acid, main
There are two kinds of kytoplasm enzyme system catalysis, acetyl CoA carboxylase and fatty acid synthase complex, by carbon source of acetyl-CoA by multiple
Carbochain extends synthesizing saturated fatty acid, or again by desaturation synthesis unrighted acid.It is main during desaturation
It is responsible for catalysis by various desaturases, is the key for synthesizing long-chain unsaturated fatty acid, C=C can be introduced on fatty acid carbon chain
Double bond, improves in the degree of unsaturation of aliphatic acid, yeast and is widely present fatty acid desaturase.
And the enzyme for synthesizing unrighted acid is divided into two kinds, the fatty acid desaturase that methyl orientation and carboxyl are oriented.First
The fatty acid desaturase of base orientation introduces carbon-carbon double bond at the carbon atom that methyl end is fixed, and is referred to as ω-desaturase；Carboxylic
The fatty acid desaturase of base orientation introduces carbon-carbon double bond at the carbon atom that c-terminus is fixed, and is referred to as Δ (delta)-go to satisfy
And enzyme, it is also referred to as " front-end " desaturase.First carbon carbon double bond is introduced into saturated fatty acid by Δ 9- desaturases,
Second carbon-carbon double bond is responsible for introducing by Δ 12 and Δ 6- desaturases, and the desaturase of Δ 15 is then responsible for introducing the 3rd double bond.
After cell synthesis 18C saturated fatty acids, stearic acid obtains oleic acid through stearoyl-CoA desaturase desaturations；Oil
Acid synthesizes alpha-linolenic acid (ALA) through Δ 12- desaturases catalysis generation linoleic acid through Δ 15- desaturases；Linoleic acid is through Δ 6-
Desaturase catalysis generation gamma-Linolenic acid GLA.Wherein, Δ 6- desaturases are the key enzymes in GLA route of synthesis, for the first time
It is to separate clone from blue-green algae within 1993 to obtain to clone Δ 6- desaturases, and obtaining GLA by biotechnology for the first time is
The Δ 6- delta 8 desaturase genes that blue-green algae was expressed in potato in 1996 produce GLA.GLA further dehydrogenation extension shapes in human body
Into physiological activators such as arachidonic acid (AA), prostanoid and leukotrieneses, there is important physiological significance to human body, thus
In recent years people are higher to the research temperature of Δ 6- fatty acid dehydrogenase genes, also achieve greater advance.
Although having there is many reports to show, in yeast GLA contents can increase expression external source Δ 6- fatty acid dehydrogenases
Plus, but so far, not yet occurring can be with the yeast strain of high yield gamma-Linolenic acid (GLA) using genetic engineering structure
Report.
The content of the invention
The deficiency existed for prior art described above, the first object of the present invention is to provide a kind of high yield unsaturated lipid
Fat acid recombinant strain.
The second object of the present invention is to provide the construction method of the high yield unrighted acid recombinant strain.
To solve above-mentioned technical problem, the present invention is adopted the following technical scheme that：A kind of high yield unrighted acid restructuring
Engineered strain, Δ 6- desaturases (D6D) are incorporated into the genome of engineering bacteria.
It is preferred that, the engineering bacteria can be saccharomycete, and the saccharomycete is saccharomyces cerevisiae.The saccharomyces cerevisiae
(Saccharomyces cerevisiae)CGMMCC 2.1445。
It is preferred that, the saccharomycete is rhodotorula glutinis.
The Δ 6- desaturases (D6D) have what the separation of the D6D genes of cunninghamella echinulata and amplification were obtained.
A kind of high yield unrighted acid recombinant strain construction method, the step of it includes is as follows：
(1) expression vector pPGK1Z-rD-ME extracting；
(2) connection PGK1 genes and D6D genes；
(3) PGK1-D6D fragments are reacted with the double digestion of expression vector carrier, are connected.
The step of method of expression vector pPGK1Z-rD-ME extracting, is as follows：
(1) bacillus coli DH 5 alpha containing pPGK1Z-rD plasmids is inoculated into equipped with 5mL LB-amp culture mediums, in 37
250rpm shaken cultivations are stayed overnight at DEG C；
(2) draw 1.5mL and stay overnight bacterium solution in centrifuge tube, 12000rpm is centrifuged 1 minute at 4 DEG C, abandons supernatant；
(3) fluid nutrient medium in centrifuge tube is blotted with filter paper, bacterial sediment is suspended in 200 μ L STET buffer solutions,
Fully mixed with turbine mixer；
(4) lysozyme soln that 4mL is newly prepared is added, mixed after standing 5 minutes at room temperature；
(5) centrifuge tube is lived with boom frame, be placed in boiling water bath, accurately clocked 45 seconds, 12000rpm centrifugations at once 5 after taking-up
Minute；
(6) discarded with the sediment in sterile toothpick picking centrifuge tube, 8mL 5% is added in the supernatant of centrifuge tube
CTAB, after being mixed with blender, 13000rpm is centrifuged 5 minutes, abandons supernatant, the liquid in centrifuge tube is blotted with filter paper；
(7) 1.2M NaCl 300mL are added, sediment is fully dissolved, adds 750mL pre-cooled ethanol, are fully mixed
Afterwards, 13000rpm is centrifuged 15 minutes, abandons supernatant；
(8) the cold ethanol of 1mL 70% is taken, slow elution centrifugation inside pipe wall, 13000rpm centrifugation 5min abandon supernatant, with filter
Paper blots the liquid on tube wall, and putting makes nucleic acid precipitation spontaneously dry 5-10min in room temperature；
(9) sediment is dissolved in 50mL TE buffer solutions, and blender is mixed, and is saved backup in -20 DEG C.
Connection PGK1 genes and the primers of D6D genes are：
PGK1-Bam HIprimer1：
5′-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3′(Bam HI)
PGK1-XhoIprimer 2：
5′-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAGATGTCGCCTATTATT-3′
(XhoI)
D6D-XhoIprimer1：
5′-TCTGCTTTCTTCGCTCCGCTCGAGATGTCAGGGCAAACTCGAG-3′(XhoI)
D6D-XhoIprimer2：
5′-CATGCCATGGATCATCTAAAACATCTTTTGAGAG-3′(NcoI)。
Engineering bacteria and the mol ratio of D6D genetic fragments will be controlled 1:3-10.
The high yield unrighted acid recombinant strain is used to be used as external source lipophilic quasi-molecule, polypeptide or compound
The carrier that medicine is introduced.
The high yield unrighted acid recombinant strain is used to be used as external source lipophilic quasi-molecule, polypeptide or compound
Functional food introduces the carrier of human body.
External source lipophilic polypeptide drugs can be α-MSH or polypeptide H22LP.H22LP is that laboratory early stage screening is obtained
Broad-spectrum chemokine receptor US28 antagonistic peptides.4 seven-transmembrane chemotactics that US28 is encoded as human cytomegalovirus (HCMV) because
One of sub- acceptor, is the CC class Chemokines acceptors of a wide spectrum, to the knot of β chemotactic factor (CF)s in the cell of HCMV infection
Close and calcium current signal induction plays an important role.We are sought by carrying out membrane spaning domain to US28 and combining the prediction of mimotope
Its N-terminal and the position of chemotactic factor (CF) excitement are found out, the polypeptide of corresponding site is synthesized, finally filtering out can block US28 corresponding
The interaction of CC classes chemotactic factor (CF), with less immunogenicity and more strongly active for being used as the small molecule guide of antagonist
Thing H22LP, premenstruum (premenstrua) experiment proves that H22LP can suppress HCMV effect by directly being reached with virion effect.
The present invention has the advantages that：Foreign gene D6D expression vector pPGK1Z-rD-D6D are successfully constructed, it is real
Existing high efficient expression of the external source D6D genes in rhodotorula glutinis GM4 bacterial strains, is identified, the purpose fragment on recombinant plasmid through double digestion
Direction of insertion is correct；Recombinant plasmid is imported in rhodotorula glutinis, inverted bacterial strain PCR identifications, recombinant plasmid successfully imports viscous red
In yeast and it is inserted into the genome of rhodotorula glutinis, realizes D6D stabilization, permanent expression；Real-time fluorescence quantitative PCR shows
D6D expression improves 2 times or so；Through gas chromatography analysis, the GLA in conversion bacterial strain is improved compared with wild strain
More than 3.3 times.
Brief description of the drawings
Fig. 1 is the Technology Roadmap of rhodotorula glutinis integrating expression vector pPGK1Z-rD-D6D structure；
Fig. 2 is colonial morphology figure observation photo；
Fig. 3 is the PCR amplified band figures of PGK1 (A) and D6D (B) gene；
Fig. 4 be rhodotorula glutinis conversion bacterial strain GM4-D6D Insert Fragment D6D PCR detections figure (wherein 1,2,3 roads attach most importance to
Group bacterial strain, 4,5,6 roads are empty carrier bacterial strain)；
Fig. 5 is that recombinant plasmid pPGK1Z-rD-D6D double digestions qualification figure (is compareed, wherein 1 road is with pPGK1Z-rD
PPGK1Z-rD-D6D, 2 roads are pPGK1Z-rD)；
Fig. 6 is rhodotorula glutinis D6D gene expression dose figures.
Specific embodiment
In order to preferably illustrate present invention, illustrated below with some preferably specific embodiments.But these have
Body embodiment is simply to illustrate that present disclosure, rather than present invention is limited.
2.1 experiment materials and instrument explanation
2.1.1 strain
Rhodotorula glutinis (Rhodotorula glutinis) GM4 is to screen and preserve through conventional meanses；
Saccharomyces cerevisiae (Saccharomyces cerevisiae) 2.1445 is purchased from China General Microbiological culture presevation pipe
Reason center (CGMMCC)；
Cunninghamella echinulata (Cunninghamella echinulata) MIAN6；
Escherichia coli (Escherichia coli) DH5 α.
2.1.2 plasmid
Expression vector pPICZ-rD, builds and preserves.
2.1.3 culture medium and main agents
(1) YPD culture mediums (1L)：
Yeast extract (Yeast extract) 10g
Peptone (Peptone) 20g
Glucose (Dextrose) 20g
Distilled water 1000mL
1 × 10 after dissolving5Pa sterilizing 20min, use another addition agar 20g (Agar) during solid medium.
(2) YPD-zeocin culture mediums (1L)：
YPD culture mediums add the zeocin that concentration is 50 μ g/mL in addition.
(3) Luria-Bertani (LB) culture medium (1L)：
Old (Typtone) 10g of tryptose
Yeast extract (Yeast extract) 5g
NaCl 10g
By above-mentioned material plus distilled water 950mL, PH is adjusted to 7.0-7.2 with NaOH solution, 1L, 1 × 10 is added water to5Pa goes out
Bacterium 20min, using separately adding agar (Agar) 20g during solid medium.
(4) LB-ampicillin culture mediums (g/L)：
LB culture mediums add the ampicillin that concentration is 100 μ g/mL in addition.
(5) less salt LB-zeocin culture mediums：
Old (Typtone) 10g of tryptose
Yeast extract (Yeast extract) 5g
NaCl 5g
By above-mentioned material plus distilled water 950mL, PH is adjusted to 7.0-7.2 with NaOH solution, 1L, 1 × 10 is added water to5Pa goes out
Bacterium 20min, adds the zeocin that concentration is 50 μ g/mL, using separately adding agar (Agar) 20g during solid medium in addition.
(6) Potato dextrose agar (PDA) culture medium (g/L)：
Potato leaching liquor 200g, glucose 20g, agar (Agar) 20g, 1 × 105Pa sterilizes 20min after dissolving, uses liquid
Agar need not be added during body culture medium.
(7) STET buffer solutions：
Tris-HCl：
12.11gTris is dissolved in 60mL sterilized waters, and concentrated hydrochloric acid adjusts pH value to be 8.0,
EDTA：
Na2EDTA 18.61g, are dissolved in 60mL sterilized waters, and PH is adjusted to 8.0,
Take Tris-HCl solution 12.5mL, EDTA solution 25ml, Trion X-100 20g, 8% sucrose solution 150mL
It is sufficiently mixed and constant volume is produced to 250mL.
(8) TE buffer solutions：
10.0mM Tris-HCl, 1.0mM EDTA, pH are adjusted to 7.5.
(9) 10%SDS (lauryl sodium sulfate)：
10g SDS are dissolved in 90mL deionized waters, and PH is adjusted to 7.2.
(10) plasmid extraction liquid
Solution I：50mmol/L glucose (Dextrose), 10mmol/L EDTA, 25mmol/L Tris-HCl (pH
8.0), autoclaving, 4 DEG C of preservations；
Solution II：0.2mol/L NaOH (being diluted from 10mol/L mother liquor), 1%SDS, matching while using；
Solution III：KAc 29.4g, acetic acid (Acetic Acid) 11.5mL, ddH2O 28.5mL, 4 DEG C of preservations；
(11) 10xDNA buffer link buffer
The buffer preserving prepared is in -20 DEG C.
(12) Tricine SDS-PAGE electrophoresis reagents
A. cathode buffer liquid (10x)：
242.28g Tris are weighed, 750mL is dissolved in and goes in aseptic deionized water, then are adjusted PH with 1.0mol/L hydrochloric acid
To 8.9,1L is added water to.
B. negative pole cathode buffer liquid (10x)：
121.14g Tris are weighed, 179.16g Tricine, 10g SDS are dissolved in 650mL and gone in aseptic deionized water,
Addition is gone in aseptic deionized water to final volume 1L.
C. gel buffer liquid (3x)：
363.0g Tris, 3.0g SDS are weighed, is dissolved in 600mL, sterile ionized water, then with 1.0mol/L hydrochloric acid
PH is adjusted to 8.5, adds water and is diluted to 1000mL.
D. fixer：
500mL methanol is measured with graduated cylinder, 100mL glacial acetic acid measures 0.1moL ammonium acetates, plus aseptic deionized water constant volume is arrived
1000mL。
E. dyeing liquor：
Mas bright blue G-250200mg is weighed, is dissolved in 100mL95% ethanol, 200mL85% phosphoric acid is added, plus
Water constant volume is to 1000mL, cotton filtering.
F. destainer：
Acetic acid 100mL is measured, ethanol 50mL, plus aseptic deionized water constant volume shake up to 1000mL.
G. sample buffer：
0.5mol/L Tris-HCl (pH6.8) 2mL, mercaptoethanol 1mL, anhydrous glycerol 2mL is measured, SDS 0.4g are weighed,
Bromophenol blue 0.02g, pH value is adjusted to 7, plus aseptic deionized water is made into 20mL.
H.AB-3 storing liquids (49.5%T, 3%C)：
19.2g acrylamides are weighed, 0.6g methylene diacrylamides, dissolving constant volume terminates in 40mL aseptic deionized waters, mistake
Filter.
I.AB-6 storing liquids (49.5%T, 3%C)：
18.6g acrylamides are weighed, 1.2g methylene diacrylamides, dissolving constant volume terminates in 40mL aseptic deionized waters, mistake
Filter.
J.16% separation gel
K.10% squeegee
L.4% glue is concentrated
Other reagents：
2.1.4 key instrument
2.2.1 the separation and amplification of target gene
2.2.1.1 the separation and amplification of saccharomyces cerevisiae PGK1 genes
1st, separate：
(1) saccharomyces cerevisiae is inoculated in 50mL/250mL YPD fluid nutrient mediums, 30 DEG C, and 180rpm is cultivated to OD660Reach
(12000r/min, 5min, 4 DEG C) is centrifuged when 3, supernatant is abandoned, thalline is collected；
(2) twice, thalline is resuspended with cold sterilized water after centrifugation in aseptic water washing thalline, and bacteria suspension is transferred to containing liquid nitrogen
Somatic cells are crushed with being ground in the mortar of 1g alumina；
(3) broken somatic cells are transferred in 5mL DNA Extraction buffers (50mM TRIS, 10mM MgCl2,
50mM NaCl, 1% (wt/vol) SDS, pH 7.4) centrifuge tube in；
(4) by phenol/chloroform extracting repeatedly and methanol precipitation step, saccharomyces cerevisiae DNA is separated.
2nd, PCR is expanded：
According to PGK1 gene orders, primer is designed.
- the CGC of forward primer 5 'GGATCCTATTTAGATTCCTGACTTCAACTC-3′(Bam HI)
- the TATCCG of reverse primer 5 'CTCGAGTGTTTTATATTTGTTGAAAAAGTAG-3′(XhoI)
PCR reaction systems are：
PCR reaction conditions：
5 μ L pcr amplification products are taken to treat to analyze through 2% agarose gel electrophoresis.
2.2.1.2 the separation and amplification of cunninghamella echinulata D6D genes
1st, separate：
The extraction operating process of cunninghamella echinulata total serum IgE refers to Wan and Zhang et al. method [47].
(1) cunninghamella echinulata is in 28 DEG C in PDA liquid medium, and 240rpm/min grows 3 days, and mycelia passes through mistake
Filter is collected, and is rinsed with phosphate buffer；
(2) rapid after liquid nitrogen grinding be transferred to added with being extracted in appropriate TRIzol (Invitrogen) centrifuge tube is added
Total serum IgE；
(3) mRNA is extracted from total serum IgE with Oligotex mRNA Mini Kit (Qiagen).
2nd, PCR is expanded：
According to cunninghamella echinulata D6D gene orders (the GenBank accession number announced：
DQ177498), primer is designed.
D6D primers：
- the CCG of forward primer 5 'CTCGAGATGTCAGGGCAAACTCGAG-3′(XhoI)
- the CATG of reverse primer 5 'CCATGGATCATCTAAAACATCTTTTGAGAG-3′(NcoI)
3rd, reverse transcription (RT) is reacted：
(1) every time in RT reactions, according to the form below adds each component：
(2) 70 DEG C are placed 5 minutes, are then immediately placed in 5 minutes on ice；
(3) it is added sequentially following component：
(4) 37 DEG C are reacted 1 hour.
15 minutes terminating reactions of (5) 75 DEG C of incubations, are stored in -20 DEG C or are directly used in downstream experiment.
4th, PCR reacts：
PCR reaction systems are：
PCR reaction conditions：
5 μ L pcr amplification products are taken to treat to analyze through 2% agarose gel electrophoresis.
2.2.1.3 gel electrophoresis is reclaimed with PCR primer
1st, gel electrophoresis：
(1) 2% Ago-Gel is prepared：2g agaroses are weighed, 100mL 1 × TAE electrophoretic buffers are added, micro-wave oven adds
Heat, until thawing completely, adds 1.5 μ l 10mg/ml EB, shake up；
(2) Ago-Gel liquid is cooled to 70 DEG C or so, poured into the glue groove put well, cooling until into gel,
Gel and glue groove are taken out, are positioned in electrophoresis tank, 1xTAE electrophoretic buffers are added until submergence gel；
(3) sample-loading buffer (bromophenol blue) is mixed into DNA sample, sample is added to the sulculus of gel slab with liquid-transfering gun
It is interior；
(4) after being loaded, it is powered carries out electrophoresis immediately, voltage 120V, when the near gel top of bromophenol blue, stops electrophoresis；
(5) gel is taken out, 30min is dyed with the EB/1xTAE solution containing 0.5 μ g/ml, then cleaned with distilled water
15min；
(6) observe, it was observed that after band, taken pictures preservation using gel imaging system under uviol lamp.
2nd, PCR primer is reclaimed：
Go out target DNA band using ultraviolet photodevelopment, cut corresponding agar band with slicer, insert in centrifuge tube；
(1) Binding Buffer II (7~4 μ l/1mg agar gels) are added, are shaken up in 60 DEG C of water-baths, until completely
Dissolving；
(2) put the agar gel of dissolving into UNIQ-10 pillars 5min, filtrate, 8000rpm normal temperature are collected with collecting pipe
Centrifuge 15min
(3) waste liquid of collection is discarded, UNIQ-10 posts are removed, enters 600 μ l Wash Solution, 8000rpm centrifugations
2min；
(4) repeat step (3)；
(5) UNIQ-10 posts are taken out to be put in new centrifuge tube, 40 μ l Elution Buffer are added in pillar film center,
5min is placed in 50 DEG C；
(6) at room temperature 12000rpm centrifuge 1min, liquid be reclaim DNA, -20 DEG C freeze it is standby.
2.2.2 external source D6D integration vectors pPGK1Z-rD-D6D structure is expressed
2.2.2.1 integration vector pPGK1Z-rD-D6D structure route map
Using homologous recombination principle design D6D gene integration expression vectors, constructing technology route is shown in Fig. 1：
2.2.2.2 expression vector pPGK1Z-rD-ME extracting
PPGK1Z-rD plasmids are what this laboratory built and preserved, and plasmid is extracted according to following steps：
(1) bacillus coli DH 5 alpha containing pPGK1Z-rD plasmids is inoculated into equipped with 5mLLB-amp culture mediums, in 37
250rpm shaken cultivations are stayed overnight at DEG C；
(2) draw 1.5mL and stay overnight bacterium solution in centrifuge tube, 12000rpm is centrifuged 1 minute at 4 DEG C, abandons supernatant；
(3) fluid nutrient medium in centrifuge tube is blotted with filter paper, bacterial sediment is suspended in 200 μ L STET buffer solutions,
Fully mixed with turbine mixer；
(4) lysozyme soln that 4mL is newly prepared is added, mixed after standing 5 minutes at room temperature；
(5) centrifuge tube is lived with boom frame, be placed in boiling water bath, accurately clocked 45 seconds, 12000rpm centrifugations at once 5 after taking-up
Minute；
(6) discarded with the sediment in sterile toothpick picking centrifuge tube, 8mL 5% is added in the supernatant of centrifuge tube
CTAB, after being mixed with blender, 13000rpm is centrifuged 5 minutes, abandons supernatant, the liquid in centrifuge tube is blotted with filter paper；
(7) 1.2M NaCl 300mL are added, sediment is fully dissolved, adds 750mL pre-cooled ethanol, are fully mixed
Afterwards, 13000rpm is centrifuged 15 minutes, abandons supernatant；
(8) the cold ethanol of 1mL 70% is taken, slow elution centrifugation inside pipe wall, 13000rpm centrifugation 5min abandon supernatant, with filter
Paper blots the liquid on tube wall, and putting makes nucleic acid precipitation spontaneously dry 5-10min in room temperature；
(9) sediment is dissolved in 50mL TE buffer solutions, and blender is mixed, and is saved backup in -20 DEG C.
2.2.2.3 over-lap PCR connects PGK1 and D6D
In order to realize foreign gene D6D high efficient expressions under strong promoter PGK1 drive, we will with Overlap PCR
Strong promoter PGK1 genes and the connection of D6D genetic fragments, to ensure between two genetic fragments without other sequences so that avoid because
The introducing of other genes is rough to cause destination gene expression abnormal.
Overlap PCR primers for PGK1 and D6D are as follows：
PGK1-Bam HIprimer1：
5′-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3′(Bam HI)
PGK1-XhoIprimer 2：
5′-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAGATGTCGCCTATTATT-3′(XhoI)
D6D-XhoIprimer1：
5′-TCTGCTTTCTTCGCTCCGCTCGAGATGTCAGGGCAAACTCGAG-3′(XhoI)D6D-
XhoIprimer2：
5′-CATGCCATGGATCATCTAAAACATCTTTTGAGAG-3′(NcoI)
Respectively with PGK1-Bam HI primer1, PGK1-XhoIprimer 2 and D6D-XhoIprimer1, D6D-
XhoIprimer2 expands PGK1 and D6D respectively, PGK1 and D6D fragments is reclaimed after 5 circulations, with PGK1 the and D6D fragments of recovery
For co-template, PGK1-Bam HI primer1 and D6D-XhoIprimer2 are that upstream and downstream primer carries out Overlap PCR,
Obtain PGK1-D6D fragments.
Overlap PCR reaction systems are：
PCR reaction conditions：
2.2.2.4 double digestion reaction, the connection of PGK1-D6D fragments and carrier
The 1st, Overlap PCR primers and pPGK1Z-rD are carried out to NcoI and Bam HI double digestions respectively
Plasmid pPGK1Z-rD NcoI/Bam HI double digestion reaction systems：
Fragment PGK1-D6D NcoI/Bam HI double digestion reaction systems：
Endonuclease reaction was in 37 DEG C of water-baths 3 hours.
2nd, coupled reaction
After endonuclease reaction, PGK1-D6D fragments and carrier pPGK1Z-rD use QIAquick Gel Extraction Kit recovery purifying, Ran Houyong respectively
Carrier pPGK1Z-rD and PGK1-D6D fragment after T4DNA ligases connection double digestion, construction recombination plasmid pPGK1Z-rD-
D6D。
Coupled reaction reaction system is as follows:
The mol ratio of 16 DEG C of water-bath reaction overnights, carrier and exogenous dna fragment will be controlled 1:3-10.
2.2.3 recombinant plasmid pPGK1Z-rD-D6D electricity converts rhodotorula glutinis competent cell
2.2.3.1 the preparation of rhodotorula glutinis competence
1st, picking yeast single bacterium colony is seeded in 5mL YPD fluid nutrient mediums, and in 30 DEG C, 250rpm shaken cultivations are stayed overnight；
2nd, 100mL YPD fluid nutrient mediums are forwarded to 1% inoculum concentration, in 30 DEG C, 250rpm shaken cultivations to cell
OD600≈1.4；
3rd, bacterium solution is in 4 DEG C, and 3000g centrifugation 5min sedimentation cells are abandoned supernatant, are resuspended with 100mL sterilized waters；
4th, repeat step three；
5th, 4 DEG C, 3000g centrifugation 2min sedimentation cells abandon supernatant, cell are resuspended with the 1M sorbierites of 20mL ice precoolings；
6th, 4 DEG C, 3000g centrifugation 2min sedimentation cells abandon supernatant, cell are resuspended with 200 μ L 1M sorbierites, for converting；
2.2.3.2 plasmid linearization
About 10 μ g recombinant plasmids pPGK1Z/rD/D6D are subjected to single endonuclease digestion with Sac I, the temperature when consumption of enzyme, digestion,
With reference to shop instruction, the time used in complete degestion will be paid special attention to, can neither be partially digested, also can not be by plasmid digest
Fall, this has highly important effect to the efficiency that electricity turns.
Digestion system is as follows:
2.2.3.3 electricity conversion
By the recombinant plasmid linearized electricity conversion to rhodotorula glutinis GM4.
1st, the DNA to be transformed that the competent cell and 20 μ L (about 10 μ g) that oneself prepares by 80 μ L have been linearized is mixed
Close, be added in the electricity conversion cup of 0.2cm precoolings；
2nd, it will be equipped with the electricity conversion cup ice bath 5min of mixed liquor；
3rd, the parameter of electroporation is adjusted:Voltage 1.5kV, electric capacity 25uF, the Ω of resistance 200, the electric shock duration is about 5ms
Left and right；
4th, after electric shock terminates, the 1M sorbitol solutions of 1mL precoolings are added into conversion cup immediately, is slightly inhaled with rifle and beats mixed
It is even, then gone in sterilized centrifuge tube, 30 DEG C of standing lh, then YPD culture mediums fresh addition 1mL, 30 DEG C,
200rpm shakes 1 hour；
5th, 3000rpm at room temperature, centrifuges 4min, and thalline is resuspended with 200 μ L ddH2O；
6th, the liquid that will disappear is coated on YPD flat boards (containing 50 μ L Zeocin), is placed in 30 DEG C of incubators and cultivates 2-3d, Zhi Daochang
Untill going out single bacterium colony.
2.2.3.4 recombinate the PCR detections of rhodotorula glutinis
1st, the preferable single bacterium colony of growing way is transferred in YPD fluid nutrient mediums in picking YPD resistant panels, 30 DEG C, 250rpm
Shaken cultivation is to OD600More than 2；
2nd, take 1mL bacterium solutions 12000rpm to centrifuge 2min, abandon supernatant, add 100 μ L TE buffer solutions and thalline is resuspended, water-bath is boiled
5-10min is boiled, -20 DEG C of refrigerators is immediately placed on and freezes 15min, stand dissolves bacteria suspension at room temperature, 12000rpm centrifugation 5min,
The μ L of supernatant 5 are taken to enter performing PCR detection for template.
Reaction system is as follows：
PCR reaction conditions
2.2.3.5 the digestion verification of plasmid
After bacterium solution PCR preliminary identifications, the plasmid in correct positive colony is extracted, counterweight is distinguished with Nco I and Bam HII
Group plasmid carries out double digestion identification, and reaction temperature is 37 DEG C.
Endonuclease reaction system is as follows：
2.2.3.6 transformant Detection of Stability
Rhodotorula glutinis transformant is inoculated in 40mL YPD fluid nutrient mediums, 30 DEG C, 250rpm shaken cultivations 24h-
36h.1mL bacterium solutions are taken to be diluted to 10 respectively with sterilized water-2,10-3,10-4, respectively take the dilution of the different dilution factors of 200 μ L to apply respectively
Common YPD flat boards and YPD-Zeocin resistant panels are distributed in, clump count is calculated, total bacteria count is designated as respectively and with integrated plasmid
Clump count.YPD fluid nutrient mediums are forwarded to 1% inoculum concentration, 30 DEG C, 250rpm shaken cultivations, are trained using 24h as 10 from generation to generation altogether
Support 50 generations.A plate count is carried out from generation to generation every 10.
It is calculated as follows plasmid stability：
Stability (%)=clump count ÷ total bacteria count × 100% with integrated plasmid
2.2.4 transformant D6D expressions are determined
Expressions of the D6D in conversion bacterial strain is detected with real-time fluorescence quantitative PCR.Wild strain GM4 and transformed bacteria
Strain GM4-D6D is collected in YPD culture mediums after fermentation 24h, 48h, 72h respectively, and extraction wild strain is total with conversion bacterial strain
RNA, method is with reference to above 2.2.1.3；Then cDNA is obtained by reverse transcription, method is with reference to 2.2.1.3.By the cDNA after synthesis
30 times of dilution, for real-time fluorescence quantitative PCR.
D6D real-time fluorescence quantitative PCR primer is：
F:5′-ATGTCAGGGCAAACTCGAG-3′
R:5′-ATCATCTAAAACATCTTTTGAGAG-3′
Using the good cDNA of above-mentioned dilution as masterplate, according to fluorescence quantitative kit SYBRPrimeScript TM RT-PCR
Kit instructs to carry out.
The reaction system of fluorescent quantitative PCR is：
PCR amplification conditions are：
SYBR melting curve analysis is carried out to Real-time PCR primers simultaneously, each sample is parallel to do 4 secondary responses, weight
Retrial is tested 2 times, and carries out data analysis with reference to Pfaffl (2001) method.
2.2.5 bacterial strain GM4-D6D fatty acid compositional analysis are converted
1st, the extraction of bacterial strain GM4-D6D total fatty acids
(1) the GM4-D6D bacterial strains in YPD fluid nutrient mediums culture to stationary phase are collected, two are cleaned with distilled water after centrifugation
Time, 5000g centrifuges 5min in 4 DEG C and collected dries to constant weight after thalline, is put in grind into powder in mortar after weight, uses filter paper bag
Good bacterium powder is dried to constant weight again；
(2) bacterium powder that filter paper is wrapped up is put in extracting barrel, injection absolute ether submergence sample, in 70 degree or so of constant temperature
Backflow extracting 8h or so in water-bath, oil-free terminates for extracting in ether, and the grease removed after solvent is stand-by；
(3) 1mL 2% (wt/vol) sulfuric acid-methanol, 60 DEG C of esterification 2h are added to the grease of extraction；
(4) 1mL hexanes, vortex 10min extraction fatty acid methyl esters are added after cooling down；
(5) the above-mentioned μ L of hexane 800 for having extracted fatty acid methyl ester are taken to be added to progress GC analyses in vial.
2nd, GC is analyzed
Match somebody with somebody hydrogen flameionization (FID) detector and capillary chromatographic column HP-INNOWAX with Bruker 450-GC instruments
(30m×0.25mm).Column temperature heating schedule is：150 DEG C (1min), 10min rises to 230 DEG C, and keeps 2min at 230 DEG C, point
Stream is than being 10:1, the retention time of fatty acid methyl ester standard items is compareed, qualitative and quantitative analysis is carried out to different aliphatic acid.
As a result prove
2.3.1 the colonial morphology of rhodotorula glutinis
After rhodotorula glutinis GM4 grows 3 days on Bangladesh's flat board, its form is as shown in Figure 2.Bacterium colony average diameter 3~
5mm, color is red or Chinese red, gloss, and surface is smooth and neat in edge, rounded projection.
2.3.1 PCR augmentation detection purpose fragments
Enter performing PCR amplification to know the real situation gene PGK1 and D6D by designing specific primer, pass through agarose gel electrophoresis point
Analysis, (A), which is shown in 800bp or so and amplifies in purpose band, Fig. 3 (B) and be shown in 13000bp or so, in Fig. 3 amplifies purpose
Band.
Recombinant plasmid is imported into rhodotorula glutinis competent cell by electrotransformation, the cell pyrolysis liquid of rhodotorula glutinis is entered
Performing PCR is detected, is entered performing PCR amplification with target gene D6D specific primer, is analyzed by agarose gel electrophoresis, Fig. 5 is shown
1st, 2,3 passages (recombinant bacterial strain) amplify purpose band in 13000bp or so, and negative control (empty carrier bacterial strain, i.e., it is wild viscous red
Yeast strain GM4, without recombinant plasmid transformed) then without band.Illustrate that recombinant plasmid pGK1Z-rD-D6D is successfully transferred to viscous red ferment
Mother is simultaneously inserted into rhodotorula glutinis genome.
Recombinant bacterium GM4-D6D extracts plasmid after culture, is imaged by EcoRI and HindIII double digestions rear electrophoresis,
As a result such as Fig. 4, it can be seen that have band (passage 1) in 2100bp and 3100bp or so, the clip size after digestion and inserted
Target gene clip size is consistent, and illustrates that the direction of insertion of target gene fragment on recombinant plasmid is correct.
2.3.2 recombinant plasmid stability is detected
In order to detect recombinant plasmid pPGK1Z-rD-D6D genetic stability, we are to conversion bacterial strain in non-selection pressure
Lower Shaking culture 60 from generation to generation, picks bacterium solution in being coated with, cultivating in Zeocine resistant panels, calculates clump count.As a result such as table 2-1
It is shown, the rhodotorula glutinis bacterial strain of conversion is shown after continuously 60 generations of culture, and stability still can reach 99.31%, show
Under non-selection pressure, the plasmid in rhodotorula glutinis has good genetic stability.
The Detection of Stability of table 2-1 recombinant plasmids
2.3.3 rhodotorula glutinis bacterial strain D6D expression analysis is converted
The result of real-time fluorescence quantitative PCR analysis D6D gene transcription levels is shown in Fig. 6, and the D6D expressions of conversion bacterial strain are
2 times or so of wild strain, wild strain D6D transcriptional levels drop to reduced levels after cultivating 72 hours, and convert bacterial strain and exist
It grows and gamma-Linolenic acid expression phase, and its D6D transcriptional level is all higher than wild strain always.D6D high transcriptional level can
With can for gamma-Linolenic acid and synthesis provide needed for enough enzymes so that the synthetic quantity of gamma-Linolenic acid maintain it is higher
Level.
2.3.4 the accumulation research of gamma-Linolenic acid
In order to which the rhodotorula glutinis for being overexpressed D6D genes converts whether bacterial strain can be produced in the GLA of higher level, experimentation
Its fatty acid composition is determined and analyzed, it is found that the GLA contents of conversion bacterial strain significantly increase, is carried by original 3.12%
It is high to 10.36%, while the content of the substrate linoleic acid of GLA synthesis is substantially reduced, it can thus be seen that with wild strain phase
Than conversion bacterial strain can accumulate more GLA.
Table 2-2 converts the aliphatic acid composition of bacterial strain and wild strain
In the present invention, Δ 6- fatty acid desaturases are the rate-limiting enzymes of synthesis of long-chain polyunsaturated fatty acids, respectively in n-3
Oleic acid (LA) dehydrogenation generation GLA in ALA dehydrogenations generation SDA and n-6 approach is catalyzed in approach.Closed in the biology of gamma-Linolenic acid
During, LA the 6th carbon atom dehydrogenation is changed into GLA by △ 6- fatty acid desaturases.Constantly exerted by researchers
Power, the encoding gene for having had many GLA key enzymes is cloned and realizes functional verification, and some genes are also utilized
In the modification of fatty acid metabolism approach, and obtain ideal effect.Such as Laoteng is successfully separated the Δ 6- fat in Mucor rouxii
Fat acid desaturase, it is found that it has the similitude of height in the Δ 6- fatty acid desaturases of plant, and make it in saccharomyces cerevisiae
It is middle successfully to be expressed；He Lu et al. have found that substantial amounts of GLA is had in the fermentation process of fermented soya bean to be produced, therefore isolate height
GLA rhizopus is produced, and therefrom clone obtains Δ 6- desaturase genes, being expressed in saccharomyces cerevisiae can cause GLA's
Accumulation；Δ 6- fatty acid desaturases in cunninghamella echinulata bacterium are expressed in tangerine woods saccharomyces oleaginosus by Wang Ping et al., are made
Its GLA content accounts for the 1.2% of total fatty acids.And in this experiment, by fatty acid analysis, it is found that the LA in conversion bacterial strain is obvious
Less than wild strain, this can also reflect LA from side and further synthesize GLA through D6D catalysis, so that in conversion bacterial strain
LA content reduction, GLA content increases.
Rhodotorula glutinis is the blast resistance of a plant height Lipid-producing, standing to be used for producing microbial grease and unsaturated fat
Acid, carotenoid etc..This laboratory early stage screens one plant of stronger rhodotorula glutinis of production fat ability, and its fat content can be up to
22.54%.In this experiment, we make use of the rhodotorula glutinis expression plasmid construction work of this laboratory early stage, successfully build
Two rDNA fragments containing rhodotorula glutinis and saccharomyces cerevisiae is strong on integrating expression vector pPGK1Z-rD-D6D, the carrier
Promoter PGK1, the stable high copy expression of target gene is realized by integration site of rDNA, so that external source is pierced into small gram of silver of spore
The D6D gene integrations of Chinese mould are on the chromosome of rhodotorula glutinis, can stablizing, high efficient expression, as a result shows and pass on 60
Dai Hou, plasmid remains to be stable in the presence of in transformant, and rhodotorula glutinis conversion bacterial strain D6D expressions apparently higher than wild
Bacterial strain, its GLA yield is also significantly improved.
Generally speaking, the present invention successfully constructs foreign gene D6D expression vector pPGK1Z-rD-D6D, is reflected through double digestion
Fixed, the purpose fragment direction of insertion on recombinant plasmid is correct；Recombinant plasmid is imported in rhodotorula glutinis, inverted bacterial strain PCR mirror
Fixed, recombinant plasmid is successfully imported in rhodotorula glutinis and is inserted into the genome of rhodotorula glutinis, realizes D6D stabilization, permanent
Expression；Real-time fluorescence quantitative PCR shows that D6D expression improves 2 times or so；Through gas chromatography analysis, bacterial strain is converted
In GLA improved compared with wild strain more than 3.3 times.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent replacement mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of high yield unrighted acid recombinant strain, it is characterised in that：Work is arrived into the restructuring of Δ 6- delta 8 desaturase genes
In the genome of journey bacterium.
2. a kind of high yield unrighted acid recombinant strain according to claim 1, it is characterised in that：The engineering
Bacterium is saccharomycete, and the saccharomycete is saccharomyces cerevisiae, and the saccharomyces cerevisiae is CGMMCC 2.1445.
3. a kind of high yield unrighted acid recombinant strain according to claim 1, it is characterised in that：The yeast
Bacterium is rhodotorula glutinis.
4. a kind of high yield unrighted acid recombinant strain according to claim 1, it is characterised in that：The Δ 6-
Desaturase is obtained by the separation of the D6D genes of cunninghamella echinulata with amplification.
5. according to a kind of construction method of high yield unrighted acid recombinant strain described in claim 1, it is characterised in that
The step of it includes is as follows：
(1) expression vector pPGK1Z-rD-ME extracting；
(2) connection PGK1 genes and D6D genes；
(3) PGK1-D6D fragments are reacted with the double digestion of expression vector carrier, are connected.
6. a kind of construction method of high yield unrighted acid recombinant strain according to claim 5, its feature exists
In：The step of method of expression vector pPGK1Z-rD-ME extracting, is as follows：
(1) bacillus coli DH 5 alpha containing pPGK1Z-rD plasmids is inoculated into equipped with 5mL LB-amp culture mediums, at 37 DEG C
250rpm shaken cultivations are stayed overnight；
(2) draw 1.5mL and stay overnight bacterium solution in centrifuge tube, 12000rpm is centrifuged 1 minute at 4 DEG C, abandons supernatant；
(3) fluid nutrient medium in centrifuge tube is blotted with filter paper, bacterial sediment is suspended in 200 μ L STET buffer solutions, whirlpool is used
Rotation blender is fully mixed；
(4) lysozyme soln that 4mL is newly prepared is added, mixed after standing 5 minutes at room temperature；
(5) centrifuge tube is lived with boom frame, be placed in boiling water bath, accurately clocked 45 seconds, 12000rpm centrifuges 5 points at once after taking-up
Clock；
(6) discarded with the sediment in sterile toothpick picking centrifuge tube, 8mL 5%CTAB are added in the supernatant of centrifuge tube, used
After blender is mixed, 13000rpm is centrifuged 5 minutes, abandons supernatant, the liquid in centrifuge tube is blotted with filter paper；
(7) 1.2M NaCl 300mL are added, sediment is fully dissolved, adds 750mL pre-cooled ethanol, after fully mixing,
13000rpm is centrifuged 15 minutes, abandons supernatant；
(8) the cold ethanol of 1mL 70% is taken, slow elution centrifugation inside pipe wall, 13000rpm centrifugation 5min abandon supernatant, inhaled with filter paper
Liquid on main wall, putting makes nucleic acid precipitation spontaneously dry 5-10min in room temperature；
(9) sediment is dissolved in 50mL TE buffer solutions, and blender is mixed, and is saved backup in -20 DEG C.
7. a kind of construction method of high yield unrighted acid recombinant strain according to claim 6, its feature exists
In：Connection PGK1 genes and the primers of D6D genes are：
PGK1-Bam HIprimer1：
5′-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3′
PGK1-XhoIprimer 2：
5′-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAGATGTCGCCTATTATT-3′D6D-
XhoIprimer1：
5′-TCTGCTTTCTTCGCTCCGCTCGAGATGTCAGGGCAAACTCGAG-3′
D6D-XhoIprimer2：
5′-CATGCCATGGATCATCTAAAACATCTTTTGAGAG-3′。
8. a kind of construction method of high yield unrighted acid recombinant strain according to claim 6, its feature exists
In：Engineering bacteria and the mol ratio of D6D genetic fragments will be controlled 1:3-10.
9. a kind of high yield unrighted acid recombinant strain according to claim 1, it is characterised in that：For conduct
The carrier that the medicine of external source lipophilic quasi-molecule, polypeptide or compound is introduced.
10. a kind of high yield unrighted acid recombinant strain according to claim 1, it is characterised in that：For making
The carrier of human body is introduced for the functional food of external source lipophilic quasi-molecule, polypeptide or compound.
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| CN107400635A (en) * | 2017-04-27 | 2017-11-28 | 广州弘宝元生物科技有限公司 | Import rhodotorula glutinis recombinant bacterial strain of α MSH polypeptides and its preparation method and application |
| WO2020082221A1 (en) * | 2018-10-23 | 2020-04-30 | 利时雨 | Live cell liposome of rhodotorula glutinis carrying exogenous polypeptide of recombinant phosphocholine cytidine transferase and use thereof |
| CN114891822A (en) * | 2022-06-29 | 2022-08-12 | 山东理工大学 | Construction method of high-yield gamma-linolenic acid mucor circinelloides recombinant bacteria, recombinant bacteria constructed by method and application |
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