CN103509817B - The construction process of a kind of rhodotorula glutinis height Lipid-producing and linolic acid genetic engineering bacterium and application - Google Patents
The construction process of a kind of rhodotorula glutinis height Lipid-producing and linolic acid genetic engineering bacterium and application Download PDFInfo
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- 229960004232 linoleic acid Drugs 0.000 title claims abstract description 23
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Abstract
The present invention relates to construction process and the application of a kind of rhodotorula glutinis height Lipid-producing and linolic acid genetic engineering bacterium; tung oil tree oleic acid dehydrogenase gene vfFAD2 gene and Diacrylglycerol acyl transferase vfDGAT2 vector construction hexose transport protein are mainly imported rhodotorula glutinis by the construction process of its genetic engineering bacterium, make vfFAD2 gene and vfDGAT2 gene obtain high expression in rhodotorula glutinis body.Compared with non-transgenosis rhodotorula glutinis, transgenosis rhodotorula glutinis transformant fat content of the present invention improves 2.76 times, and linoleic acid content improves 25.86%.
Description
Technical field
The invention belongs to bioengineering field.Specifically, the present invention relates to the construction process of a kind of rhodotorula glutinis height Lipid-producing and linolic acid genetic engineering bacterium.
Background technology
Rhodotorula glutinis (Rhodotorula glutinis (Fres.) Harrison) is a kind of important industrial strain, microbial oil and other active substances can be produced, comprise carotenoid, L-Phe, superoxide dismutase, be day by day subject to the attention of nutritionist and pharmacologist in recent years.
Report at present for the improvement of produce oil rhodotorula glutinis genetically engineered is few, particularly to improve for the purpose of linoleic acid content, and the genetically engineered improvement improving oleaginousness does not also have report simultaneously, and linolic acid is the indispensable amino acid of human body, particularly linoleic content is significant therefore to improve rhodotorula glutinis fat content.
Summary of the invention
The object of the present invention is to provide the construction process of a kind of rhodotorula glutinis height Lipid-producing and linolic acid genetic engineering bacterium, to improve rhodotorula glutinis grease and linoleic content.
Technical program of the present invention also lies in the construction process that have employed a kind of rhodotorula glutinis grease and linolic acid genetic engineering bacterium; tung oil tree oleic acid dehydrogenase gene vfFAD2 gene and Diacrylglycerol acyl transferase vfDGAT2 vector construction hexose transport protein are imported rhodotorula glutinis; make vfFAD2 gene and vfDGAT2 gene obtain high expression in rhodotorula glutinis body, the concrete steps of its construction of genetic engineering are as follows:
(1) adopt homologous sequence cloning process, with tung oil tree genomic dna for template carries out pcr amplification, the product obtained that increases carries out 1% agarose gel electrophoresis detection, gel reclaims test kit and reclaims purifying, carries out TA clone, and checks order, obtain goal gene vfFAD2, vfDGAT2;
(2) utilize and be coupled little peptide goal gene vfFAD2 and vfDGAT2 is coupled;
(3) vfFAD2-vfDGAT2 and expression vector pCAMBIA1301 is recombinated, acquisition recombinant vectors pCAMBIA1301-vfFAD2-vfDGAT2, PCR checking, enzyme are cut, sequence verification recombinant vectors;
(4) agrobacterium-mediated transformation is utilized, recombinant expression vector pCAMBIA1301-vfFAD2-vfDGAT2 transforms rhodotorula glutinis, millipore filtration is transferred to containing 60 μ gmL-1 Streptomycin sulphates, in SM I screening culture medium of 200 μ gmL-1 cephamycins and 100 μ gmL-1 kantlex, after cultivating 3-5d, the transformant obtained by primary dcreening operation and wild-type rhodotorula glutinis are containing 300 μ gmL-1 cephamycins simultaneously, the flat lining out of SM II of 150 μ gmL-1 kantlex, cultivate after 3-5d, can normal growth tentatively regard as positive transformant;
(5) extract genomic dna, PCR checking obtains positive transformant.
Technical scheme of the present invention additionally uses a kind of screening method of rhodotorula glutinis transformant, first 60 μ gmL-1 Streptomycin sulphates are used, after the SM I screening culture medium cultivation 3-5d of 200 μ gmL-1 cephamycins and 100 μ gmL-1 kantlex, the transformant obtained by primary dcreening operation again and wild-type rhodotorula glutinis are containing 300 μ gmL-1 cephamycins simultaneously, the flat lining out screening of SM II of 150 μ gmL-1 kantlex, finally extract genomic dna, carry out PCR checking.
Technical scheme of the present invention additionally uses a kind of cultural method of rhodotorula glutinis, concrete steps are as follows: be transferred on PDA substratum by rhodotorula glutinis preservation inclined-plane, after 28 DEG C of cultivation 48h, choosing 2 articulatings enters to be equipped with in the 250mL triangular flask of 50mL liquid seed culture medium, 28 DEG C of shaking culture 24h, then be equipped with in the 250mL triangular flask of 50mL fermention medium by the inoculum size access of 1:10, in 28 DEG C of oscillation and fermentation 72h, fermentation ends is through collected by centrifugation thalline; Seed culture medium: 15gL
-1glucose, 1gL
-1yeast powder, 2gL
-1(NH
4)
2sO
4, 7gL
-1kH
2pO
4, 2gL
-1na
2hPO
4, 1.5gL
-1mgSO
4, PH5.5; Fermention medium: 50gL
-1glucose, 1gL
-1yeast powder, 2.5gL
-1(NH
4)
2sO
4, 7gL
-1kH
2pO
4, 2gL
-1na
2hPO
4, 2gL
-1mgSO
4, 0.1gL
-1caCl
2, 0.07gL
-1feCl
3, PH5.5.
The present invention also aims to provide a kind of rhodotorula glutinis height Lipid-producing and the application of linolic acid genetic engineering bacterium (Rhodotorula glutinis) in production microbial oil.
The present invention also aims to provide a kind of rhodotorula glutinis height Lipid-producing and the application of linolic acid genetic engineering bacterium (Rhodotorula glutinis) in production functional grease.
The present invention also aims to provide a kind of rhodotorula glutinis height Lipid-producing and linolic acid genetic engineering bacterium (Rhodotorula glutinis) application in food.
Figure of description:
The acquisition of Fig. 1 .vfFAD2, vfDGAT2 gene;
Fig. 2. the PCR checking of recombinant plasmid pCAMBIA1301-FAD2-2A-DGAT2,1:FAD2; 2:DGAT2; 3:FAD2+2A+DGAT2; 4: unloaded contrast; 5:DNA Marker;
The antibiotic-screening of Fig. 3 .FAD2/DGAT2 transformant and PCR checking.A:SM I plate screening; B:SM II plate screening; C: use FAD2-F/R primer; D: use DGAT2-F/R primer; M:DNA marker2000; 1-3:FAD2/DGAT2 yeast transformant; 4: wild-type yeast;
Fig. 4. the expression analysis of FAD2 and DGAT2 gene in coexpression yeast.The expression of a:FAD2 gene; The expression of d:DGAT2 gene;
The fatty acid compositional analysis of Fig. 5 .FAD2/DGAT2 coexpression yeast.
Embodiment
The structure of embodiment 1, FAD2-DGAT2 recombinant expression vector
(1) acquisition of gene
According to the tung oil tree vfFAD2 sequence (Genbank accession number: AF525534) that NCBI has submitted to, molecular biological analysis software Oligo6.0 is utilized to design primer:
VF2a：5’TATCTTCAGGTTGGGCAACGA3’；
VF2b：5’CGATTGAACGGCCTACATTAT3’。
DGAT2-F：5’-CTTTCGTTCTGATCGTGCTT-3’
DGAT2-R：5’-TAGGCTGTGGATTTTGCTTCG-3’
PCR response procedures: 94 DEG C of denaturation 3min, 94 DEG C of sex change 45s, 60 DEG C of annealing 45s, 72 DEG C extend 1min, totally 35 circulations, and last 72 DEG C extend 10min.1% agarose electrophoresis result is as Fig. 1.
Object fragment reclaims and is connected with pMD18-T simple vector:
The vfFAD2 object fragment obtained by pcr amplification carries out gel recovery, reclaims product and is connected with pMD18-T simple vector:
16 DEG C of reaction 1h.
4) qualification of transformation of E. coli and positive colony:
Connect product pMD-FAD25 μ L transformation of E. coli, the hickie that the blue hickie of picking filters out carries out bacterium colony PCR qualification, after identifying that the clone be positive shakes bacterium, and order-checking qualification.
(2) structure of plant expression vector pCAMBIA1301-vfFAD2-vfDGAT2
1) primer is designed:
The polycistronic feature of external structure FMDV-2A is: connect with 2A sequence between gene, and the terminator codon simultaneously removing upstream gene is to form a long opening code-reading frame.Therefore, during design primer, while introducing restriction enzyme site, the termination codon subsequence (table 1) of FAD2 gene also should be removed.
Table 1 primer sequence
(note: underscore is restriction enzyme site, black matrix is promotor and terminator sequence.）
2) structure of carrier pCAMBIA1301-2A:
Vector plasmid pCAMBIA1301, after BamH I/Xba I double digestion, connects with the 2A sequence 16 DEG C with same restriction enzyme site, connects product 5 μ L transformation of E. coli DH5 α competent cell, by sequence verification positive colony.
3) structure of carrier pCAMBIA1301-2A-DGAT2:
Take pMD-DGAT2 as template, 3-F/3-R is primer, pcr amplification goal gene DGAT2.Reaction system 25 μ L, reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 68 DEG C of annealing 40s, and 72 DEG C extend 1min, totally 35 circulations, and last 72 DEG C extend 7min.
DGAT2PCR reclaims product and is connected conversion with pCAMBIA1301-2A by after Xba I/Pst I double digestion, obtains pCAMBIA1301-2A-DGAT2 recombinant vectors.
4) structure of carrier pCAMBIA1301-FAD2-2A-DGAT2:
Take pMD-FAD2 as template, primer 1-F/1-R amplifying target genes FAD2.Reaction system 25 μ L, reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 45s, 66 DEG C of annealing 45s, and 72 DEG C extend 1min, totally 35 circulations, and last 72 DEG C extend 7min.
Reclaim product and carrier pCAMBIA1301-2A-DGAT2 to the PCR of FAD2 gene to carry out KpnI/BamH I enzyme and cut, then connect, transformation of E. coli DH5 α competent cell, checking positive colony (Fig. 2).
Case study on implementation 2 recombinant expression vector is to the genetic transformation of rhodotorula glutinis
(1) agriculture bacillus mediated pCAMBIA1301-2A-DGAT2 gene transformation yeast
This research is carried out with reference to the agrobacterium-mediated transformation of (1996) such as Piers.First picking rhodotorula glutinis mono-clonal is inoculated in the YPD substratum of 10mL, 30 DEG C, 180rpm shaking culture 18h, then be diluted in YPD fresh culture by 1:10 and cultivate 6h, collected by centrifugation thalline, with aseptic water washing twice, resuspended with liquid IM substratum, dilution yeast is about 0.7 (cell concn about 1 × 10 to OD660
8mL-1); Agrobacterium tumefaciens containing plasmid pBI121-FAD2 and pBI121 to be rule on LB solid medium activation, in substratum, add 50 μ gmL
-1kantlex and 50 μ gmL
-1rifampin, picking Agrobacterium mono-clonal is inoculated in 10mL LB liquid medium and (adds 50 μ gmL
-1kantlex, 50 μ gmL
-1rifampin), 28 DEG C, 180rpm shaking culture 18h, collected by centrifugation thalline, resuspended (containing 50 μ gmL with 10mL liquid IM substratum
-1kantlex, 50 μ gmL
-1rifampin), continue to cultivate 6h.Collected by centrifugation thalline subsequently, by IM substratum dilution Agrobacterium OD600 value about 0.5, (cell concn is 1 × 1010mL about
-1).Respectively get yeast that 50 μ L have diluted and Agrobacterium joins in 10mL liquid IM substratum (Syringylethanones containing 200 μMs), 28 DEG C, after 180rpm shaking culture is spent the night, get 150 μ L nutrient solutions to coat and be covered with (Syringylethanones containing 200 μMs) on the IM solid medium of millipore filtration, 25 DEG C, be inverted, dark culturing 48h.
(2) qualification of vfFAD2-vfDGAT2 gene rhodotorula glutinis is turned
Millipore filtration is transferred to containing 60 μ gmL
-1streptomycin sulphate, 200 μ gmL
-1cephamycin and 100 μ gmL
-1in SM I screening culture medium of kantlex, after cultivating 3-5d, the transformant obtained by primary dcreening operation and wild-type rhodotorula glutinis are containing 300 μ gmL simultaneously
-1cephamycin, 150 μ gmL
-1the flat lining out of SM II of kantlex, cultivates after 3-5d, can normal growth tentatively regard as positive transformant.After SM I plate screening (Fig. 3 a), obtain 3 yeast transformants, these 3 transformants and wild-type yeast are screened (Fig. 3 b) at the flat lining out of SM II simultaneously, FAD2-F/R and DGAT2-F/R two pairs of primer pair transformants are utilized to carry out PCR qualification, wherein FAD2-F/R the fragment length that expands be 584bp, DGAT2-F/R the fragment length that expands be 542bp, Fig. 3 c shows, band is had in object fragment with 2 yeast transformants during FAD2-F/R primer amplification, and (Fig. 3 d shows to have band with 3 yeast transformants during DGAT2-F/R primer amplification in object fragment, ), wherein wild-type yeast is all without band, therefore by transformant FAD2/DGAT2-1, FAD2/DGAT2-2 is considered as the transgenic yeast successfully proceeding to DGAT2.
The expression analysis of FAD2 in case study on implementation 3 turns of vfFAD2-vfDGAT2 gene yeasts
In order to verify the expression of tung oil tree vfFAD2, vfDGAT2 gene in transgenic yeast, we are extracted the total serum IgE of 3 transgenic yeast bacterial strains, detect after its concentration through micro-spectrophotometer, by its integrity of 1% agarose electrophoretic analysis, obtain complete RNA, according to
reverse transcription is carried out in the explanation of III First-Strand Synthesis System test kit.
Verify with PCR through screening, obtain the yeast of FAD2/DGAT2 gene co-expressing.In order to verify the expression of FAD2 and DGAT2 gene in transgenic yeast, utilize quantitative real-time PCR to detect the expression of each gene in transgenic yeast, with FAD2 and DGAT2 respectively overexpression yeast and turn the unloaded yeast of pBI121 for contrasting.Fig. 4 a shows, in the yeast of FAD2/DGAT2 coexpression, FAD2 gene all has expression, but the mean level (ML) that expression amount comparatively turns FAD2 single-gene yeast is low.Carry out detection to the expression level of DGAT2 in FAD2/DGAT2 coexpression yeast simultaneously and find (Fig. 4 b), in double gene coexpression yeast, the expression of DGAT2 is with to turn DGAT2 single-gene yeast expression amount relatively low, without noticeable change.
Case study on implementation 4 turns of vfFAD2-vfDGAT2 gene yeast greases and Analysis of Fatty Acids Composition
In order to detect the regulating and controlling effect that the acid of tung oil tree FAD2 gene pairs yeast fat may have, we utilize acid heat method to be extracted the grease of yeast.First activated yeast strain on YPD substratum, cultivates after 3d, is inoculated in 50mL seed culture medium by transgenic yeast bacterial strain respectively with contrasting, 30 DEG C, after 24h cultivated by 180rpm shaking table, culture transferring is in 500mL fermentation culture, 30 DEG C, 72h cultivated by 180rpm shaking table.Acid heat method is adopted to carry out the extraction of grease the fermented liquid of acquisition.Concrete steps:
1) collected by centrifugation thalline, bacterial sediment is weighed, the resuspended thalline of ratio adding 6ml4molL-1 hydrochloric acid in about every gram of bacterium, vibration mixing;
2) mixed solution room temperature is placed 30min, boiling water bath 3min, puts into refrigerator ,-20 DEG C of cold 30min of speed;
3) take out mixed solution, add the chloroform of 2 times of volumes: methyl alcohol (1:1) extracting solution, mixing of fully vibrating;
4) the centrifugal 5min of 5000rpm, gets chloroform layer (lower phase) to new centrifuge tube;
5) add equal-volume 0.1% sodium chloride solution, mixing, the centrifugal 5min of 5000rpm, gets chloroform layer (lower phase);
6) chloroform layer is put into revolving bottle rotary evaporation, namely removing chloroform obtains grease.The revolving bottle that grease is housed is put into vacuum drying oven 60 DEG C, 30min.
Carry out chromatography of gases analytical results to grease to show, each component reference standard product go out peak situation and carry out qualitative, calculate the relative content of each lipid acid, and T-test significance analysis is carried out to linoleic acid content, P < 0.01, for extremely remarkable, is designated as " * * "; 0.01 < P < 0.05 is significantly, is designated as " * "; P > 0.05, for not remarkable.To adopt in gc analysis yeast the lipid acid that five kinds main, comprise palmitinic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linolic acid (C18:2) and linolenic acid (C18:3).The acid of FAD2/DGAT2 coexpression yeast fat is analyzed, with FAD2 and DGAT2 respectively overexpression yeast and turn the unloaded yeast fat acid of pBI121 and compare.To adopt in gc analysis yeast the lipid acid that five kinds main.
Fat content detected result shows, the fatty acid total amount of FAD2/DGAT2-1, FAD2/DGAT2-2 transformant improves 1.63 times, 2.76 times than contrast respectively,
Lipid acid ratio analysis is shown (Fig. 5), change to some extent compared with each component of transgenic yeast bacterial strain contrasts with zero load, wherein larger with oleic acid, linolic acid rangeability, FAD2/DGAT2-1, the oleic acid content of FAD2/DGAT2-2 transformant have dropped 7.83%, 5.44% respectively, linoleic acid content is the highest improves 25.86%, shows that FAD2/DGAT2 coexpression increases to yeast linoleic acid content.Comprehensive analysis results shows, and in FAD2/DGAT2 coexpression yeast, grease total amount and linoleic acid content all increase.
Claims (4)
1. the construction process of a rhodotorula glutinis height Lipid-producing and linolic acid genetic engineering bacterium; it is characterized in that; tung oil tree oleic acid dehydrogenase gene vfFAD2 gene and Diacrylglycerol acyl transferase vfDGAT2 vector construction hexose transport protein are imported rhodotorula glutinis; make vfFAD2 gene and vfDGAT2 gene obtain high expression in rhodotorula glutinis body, the concrete steps of its construction of genetic engineering are as follows:
(1) rhodotorula glutinis carries out activating and cultivating: be transferred on PDA substratum by rhodotorula glutinis preservation inclined-plane, after 28 DEG C of cultivation 48h, choosing 2 articulatings enters to be equipped with in the 250mL triangular flask of 50mL liquid seed culture medium, 28 DEG C of shaking culture 24h, then be equipped with in the 250mL triangular flask of 50mL fermention medium by the inoculum size access of 1:10, in 28 DEG C of oscillation and fermentation 72h, fermentation ends is through collected by centrifugation thalline; Seed culture medium: 15gL
-1glucose, 1gL
-1yeast powder, 2gL
-1(NH
4)
2sO
4, 7gL
-1kH
2pO
4, 2gL
-1na
2hPO
4, 1.5gL
-1mgSO
4, PH 5.5; Fermention medium: 50gL
-1glucose, 1gL
-1yeast powder, 2.5gL
-1(NH
4)
2sO
4, 7gL
-1kH
2pO
4, 2gL
-1na
2hPO
4, 2gL
-1mgSO
4, 0.1gL
-1caCl
2, 0.07gL
-1feCl
3, PH 5.5;
(2) homologous clone method is utilized, according to known array on GenBank, with tung oil tree genomic dna for template, carry out pcr amplification, the product obtained that increases carries out 1% agarose gel electrophoresis detection, and gel reclaims test kit and reclaims purifying, carries out TA clone, and check order, obtain goal gene vfFAD2 and vfDGAT2;
PCR response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 58 DEG C of annealing 40s, 72 DEG C extend 50s, 35 circulations; 72 DEG C extend 10min; Pcr amplification product 1% agarose gel electrophoresis detects;
(3) utilization is coupled little peptide, is coupled by vfFAD2 and vfDGAT2, then recombinates with expression vector pCAMBIA1301, and obtain recombinant vectors pCAMBIA1301-vfFAD2-vfDGAT2, enzyme is cut, sequence verification recombinant vectors;
(4) utilize agrobacterium-mediated transformation, recombinant vectors pCAMBIA1301-vfFAD2-vfDGAT2 is transformed rhodotorula glutinis, then transfer to containing the millipore filtration transforming rhodotorula glutinis containing 60 μ gmL
-1streptomycin sulphate, 200 μ gmL
-1cephamycin and 100 μ gmL
-1in SM I screening culture medium of kantlex, after cultivating 3-5d, the transformant obtained by primary dcreening operation and wild-type rhodotorula glutinis are containing 300 μ gmL simultaneously
-1cephamycin, 150 μ gmL
-1the flat lining out of SM II of kantlex, cultivates after 3-5d, can normal growth tentatively regard as positive transformant; Extract genomic dna, PCR checking obtains positive transformant;
(5) vapor-phase chromatography detected result shows, compared with non-transgenosis rhodotorula glutinis, transgenosis rhodotorula glutinis transformant fat content improves 2.76 times, and linoleic acid content improves 25.86%;
Wherein, amplimer is:
FAD2-F：5’-TGTCCTCCGTTCATTCTCAT-3’；
FAD2-R：5’-GCAAGACGGTAGAGACCAAA-3’
DGAT2-F：5’-CTTTCGTTCTGATCGTGCTT-3’
DGAT2-R：5’-TAGGCTGTGGATTTTGCTTCG-3’。
2. a rhodotorula glutinis height Lipid-producing as described in claim 1 and linolic acid genetic engineering bacterium are producing the application in microbial oil.
3. a rhodotorula glutinis height Lipid-producing as described in claim 1 and linolic acid genetic engineering bacterium are producing the application in functional grease.
4. a rhodotorula glutinis height Lipid-producing as described in claim 1 and linolic acid genetic engineering bacterium are producing the application in food.
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| CN101993881A (en) * | 2010-04-08 | 2011-03-30 | 中国林业科学研究院亚热带林业研究所 | Method for constructing engineered strain of filamentous fungi with high oleic acid yield |
| CN102796675A (en) * | 2012-06-29 | 2012-11-28 | 南阳奇伟微生态基因科技开发有限公司 | Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof |
| CN103509725A (en) * | 2013-09-16 | 2014-01-15 | 中国林业科学研究院亚热带林业研究所 | Construction method of rhodotorula glutinis high-yield linoeic acid genetically engineered bacterium and application thereof |
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| CN102796675A (en) * | 2012-06-29 | 2012-11-28 | 南阳奇伟微生态基因科技开发有限公司 | Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof |
| CN103509725A (en) * | 2013-09-16 | 2014-01-15 | 中国林业科学研究院亚热带林业研究所 | Construction method of rhodotorula glutinis high-yield linoeic acid genetically engineered bacterium and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| "FAD2-DGAT2 genes coexpressed in endophytic Aspergillus fumigatus derived from tung oilseeds";Yi-Cun Chen 等;《The Scientific World Journal》;20120731;第2012卷;摘要、第2.3-2.5节和图1 * |
| "汪阳东简介";http://skltgb.caf.ac.cn/content/yanjiuduiwu/yanjiurenyuan/201306;《中国林科院林木遗传育种国家重点实验室》;20130702;"学术成就"部分第5-8行 * |
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