CN103397007B - CeDGAT1 (Chlorella ellipsoidea Diacylglycerol Acyltransferase 1) gene and application thereof - Google Patents
CeDGAT1 (Chlorella ellipsoidea Diacylglycerol Acyltransferase 1) gene and application thereof Download PDFInfo
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Abstract
The invention provides a CeDGAT1 (Chlorella ellipsoidea Diacylglycerol Acyltransferase 1) gene and an application thereof for increasing the fatty acid content of cells. A nucleotide sequence of the CeDGAT1 is as shown in SEQ ID NO: 1. The CeDGAT1 gene is derived from Chlorella ellipsoidea, and encoded diacylglycerol acyltransferase is a key enzyme in the TAG (triacylglycerol) synthesis process in a biological body. The CeDGAT1 gene can be used for transforming yeast cells, plant cells and microalgae cells, so that the total fatty acid content of the cells can be greatly increased.
Description
Technical field
The present invention relates to a kind of diacylglycerol acyltransferase gene DGAT1 and application thereof.Particularly, relate to the acquisition of this gene order and the structure of Yeast expression carrier, and it can increase substantially the research of yeast fat acid content aspect.
Background technology
Micro-algae is the plant plankton of water body of spreading all over the world, and has large, pollution-free, renewable, the easy cultivation of production capacity, containing advantages such as more lipid materials, in Conversion of energy and carbon circulation, has important effect.There is algae slightly that photosynthate is converted into lipid acid, synthesize triacylglycerol (TAG) further, get up with the storage of the form of oil droplet in cell.Triacylglycerol in microalgae cell can reach the 20%-50% of dry weight, can change fatty acid methyl ester into, i.e. biofuel after extraction after transesterification.In addition, micro-algae also has larger edible and chemical utilization value.Based on above many-sided advantage, micro-algae is considered to one of energy source raw material having exploitation future now most
[1-3].Chlorella (Chlorella) is the class in micro-algae, belongs to Chlorophyta Chlorella, is the unicellular micro-algae of a kind of eucaryon, the about 3-8 micron of diameter.Chlorella reproduction speed is very fast, and containing abundant nutritive element, such as protein, grease, polyunsaturated fatty acid, VITAMIN, mineral substance, foodstuff fibre, nucleic acid and chlorophyll etc., also such as, containing various bioactivators, glycoprotein, polysaccharide.Chlorella can carry out the cultivation of light autotrophy, also can carry out the Heterotrophic culture of unglazed photograph, and is that a few can be used for micro-algae of large scale culturing.So chlorella carries out the potential ideal material of of energy microalgae research
[4].
Triacylglycerol (TAG) is all topmost storage lipid in most of organism, comprises vertebrates, oil crops, fungi and micro-algae.In micro-algae, TAG generally accumulates in oil body under adverse environmental factor
[5].First, in plastid, acetyl-CoA forms CoA malonyl CoA-CoA under the effect of acetyl-CoA carboxylase (ACCase); CoA malonyl CoA-CoA carries out condensation reaction progressively; from two carbon monoacyl carrier proteinss (ACP); successively two carbosilane units are brought into acyl chain; the acyl-acp of different carbon chain lengths is formed after several circulating polymerization and desaturation reaction; then synthesizing acyl coenzyme A under the effect of acyl-CoA synthetase (ACS); and transfer to endoplasmic reticulum or kytoplasm from plastid, become the substrate of eukaryote endoplasmic reticulum triacylglycerol synthesis.After the esterified generation ester acyl coenzyme A (CoA) of free fatty acids; at membrane-bound GPAT (GPAT); lysophosphatidate acyltransferase (LPAT); under Diacrylglycerol acyl transferase (DGAT) 3 kinds of acyltransferase effects; successively glycerol backbone first is synthesized triacylglycerol to the 3rd through acylations, this biosynthetic process is exactly usually said Kennedy approach
[6-7].
The final step of Diacrylglycerol acyl transferase (DGAT) catalysis TAG route of synthesis and Kennedy approach is also the unique rate-limiting enzyme of this approach.DGAT is considered to be in the enzyme played a major role in TAG building-up process, is prevalent in all eukaryotes studied.Up to the present, find 3 class dgat gene families altogether, comprise DGAT1, DGAT2 and DGAT3 gene family
[8-9].Cases etc.
[10]cloned first DGAT1 gene in mouse after, Hobbs etc.
[11]in Arabidopis thaliana, cloned DGAT1 gene, this gene only has a copy in Arabidopis thaliana.The overexpression research in wildtype Arabidopsis thaliana of AtDGAT1 gene finds, DGAT1 transcriptional level and activity significantly improve (10%-70%), and oil and fat accumulation increases, and seed weight in average increases
[12].Subsequently, in the plant such as rape, castor-oil plant, tobacco, soybean, corn, olive, safflower, Sunflower Receptacle, flame winged euonymus and Root or stem of Littleleaf Indianmulberry, DGAT1 gene is also cloned in succession
[13-16].In recent years, Lock
[17]find Deng by a BnDGAT1 inverted defined gene is transformed in rape DH12075, the expression amount of DGAT1 gene, the overall enzymic activity of DGAT albumen and the oleaginousness of seed all significantly decrease, simultaneously, the output of seed and germination rate also reduce than before conversion, and seed development severe deformities.These results show, DGAT is except playing an important role in being formed at total grease, and it also affects the normal development of seed, but concrete regulatory mechanism is not clear at present.At present, in one slightly algae, be also cloned into DGAT1 gene, the people such as Boyle have cloned a DGAT1 gene in chlamydomonas
[18], Guiheneuf etc. are also cloned into DGAT1 gene in Phaeodactylum tricornutum
[5].In Chlamydomonas reinhardtii and Phaeodactylum tricornutum, the function of DGAT1 gene has been verified and has specifically set forth.The member of DGAT2 gene family, animal, exists in plant and yeast, and does not have obvious homology with DGAT1 gene family.Less to the research of DGAT2 at present, only clone out in Arabidopis thaliana (the GenBank number of including NM115011), rape (the GenBank number of including AY916129), castor-oil plant minority plant.Nearly 2 years at Chlamydomonas reinhardtii
[19-20] and Ostreococcus tauri
[21]in also have report.Saha
[8]clone from developmental peanut cotylcdon tenuigenin obtain DGAT3 gene Deng people, the similarity of this gene and DGAT1 and DGAT2 gene family is less than 10%, but albumen of its coding has similar DGAT protein function motif, and synthesizes closely related with TAG.In addition, evidence suggests that the formation of TAG also exists an other approach, namely by phosphatide: triglyceride acyltransferase (PDAT) utilizes phosphatide catalyzing glycerol diester (DAG) to synthesize the reaction of TAG and lysophospholipid.No matter be in plant or micro-algae, all few to the research of PDAT gene.
In recent years, the widespread demand of Vegetable oil lipoprotein is facilitated greatly to the research of DGAT genes involved, especially get a larger development utilizing gene engineering method to improve in high-quality quality.2008, Zheng
[22]by finding the research of high oil corn DGAT1, being the important determinative improving oil-contg and oleic acid content in the insertion of a phenylalanine of the F469 site of DGAT1-2 albumen, illustrating the molecular basis of oil-contg and composition difference.
After the present invention finds that the CeDGAT1 gene deriving from chlorella ellipsoidea is proceeded to yeast saccharomyces cerevisiae INVSC1, the content of its main fatty acid component can be increased substantially, comprise palmitinic acid (C16:0) and improve 1.86 times, Zoomeric acid (C16:1) improves 1.08 times, stearic acid (C18:0) improves 1.86 times, and oleic acid (C18:1) improves 1.48 times.The content of yeast cell total fatty acids also improves about 1.4 times.The research of existing species DGAT1 gene substantially all have selected yeast DGAT1 mutant and carries out complementary function checking to it, very rare wild-type yeast.The present invention selects wild-type yeast while carrying out functional verification to chlorella ellipsoidea DGAT1 gene, also found its remarkable effect to oil synthesis.Study in yeast and show, CeDGAT1 gene turns yeast strains compared with control group (turning pYES2 zero load), total fatty acid content improves about 1.4 times, and the content of its main fatty acid component is also all significantly improved, wherein palmitinic acid (C16:0) improves 1.86 times, Zoomeric acid (C16:1) improves 1.08 times, and stearic acid (C18:0) improves 1.86 times, and oleic acid (C18:1) improves 1.48 times.The raising of this amplitude of content of total fatty acids and variant lipid acid have not been reported.
Summary of the invention
The invention provides a DGAT1 gene, its cDNA nucleotide sequence is as shown in SEQ ID NO:1, and this gene source is in chlorella ellipsoidea, and this gene cDNA total length is 2142bp.
Present invention also offers the Yeast expression carrier containing gene of the present invention (CeDGAT1).
The present invention provide in one aspect comprise gene of the present invention yeast, plant or algae, it is selected from yeast, algae, Arabidopis thaliana, tobacco, rape, Sunflower Receptacle, soybean, castor-oil plant, cotton, olive, Flos Carthami etc.Preferably, described algae is selected from chlorella.More specifically, described yeast is selected from yeast saccharomyces cerevisiae, and chlorella is selected from chlorella ellipsoidea (Chlorella ellipsoidea).
Chlorella ellipsoidea DGAT1 gene provided by the invention, can be applicable to utilize gene engineering method to improve the aspects such as yeast, plant or algae grease.
More specifically, the invention provides the following:
1.CeDGAT1 albumen, its aminoacid sequence is as shown in SEQ ID NO:2.
2. the gene of the CeDGAT1 albumen of coding according to 1.
3. the gene according to 2, its nucleotide sequence is as shown in SEQ ID NO:1.
4. carrier, it comprises the gene according to 2 or 3, described carrier can be pYES2.0 (can purchased from Invitrogen company), pBIN19, pBI121, pBI221 (can purchased from Clontech), pCambia1300 (can purchased from Cambia company) or pGreen (can purchased from the John Innes Centre).
5. host cell, it comprises CeDGAT1 albumen, the gene according to 2 or 3 or the carrier according to 4 according to 1.
6. prepare there is high palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content for one kind, or there is the method for cell of the yeast of high total fatty acid content, plant or algae, described method comprises in the cell gene according to 2 or 3 or the carrier according to 4 being proceeded to described yeast, plant or algae, described yeast is preferably yeast saccharomyces cerevisiae, is more preferably uracil-deficient type yeast saccharomyces cerevisiae INVSC1; Described algae is preferably chlorella, is more preferably chlorella ellipsoidea (Chlorellaellipsoidea); Described plant optimization is selected from Arabidopis thaliana, tobacco, rape, Sunflower Receptacle, soybean, tomato, castor-oil plant, cotton, sesame or peanut.
7. the CeDGAT1 albumen according to 1, the gene according to 2 or 3 or the carrier according to 4 have the purposes in the cell of the yeast of high total fatty acid content, plant or algae in preparation, described yeast is preferably yeast saccharomyces cerevisiae, is more preferably uracil-deficient type yeast saccharomyces cerevisiae INVSC1; Described algae is preferably chlorella, is more preferably chlorella ellipsoidea (Chlorella ellipsoidea); Described plant optimization is selected from Arabidopis thaliana, tobacco, rape, Sunflower Receptacle, soybean, tomato, castor-oil plant, cotton, sesame or peanut, described in there is the yeast of high total fatty acid content, yeast that the cell of plant or algae preferably has high palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content, plant or algae cell.
8. the CeDGAT1 albumen according to 1, the gene according to 2 or 3 or the carrier according to 4 be palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content in the cell improving yeast, plant or algae, or the purposes in total fatty acid content, described yeast is preferably yeast saccharomyces cerevisiae, is more preferably uracil-deficient type yeast saccharomyces cerevisiae INVSC1; Described algae is preferably chlorella, is more preferably chlorella ellipsoidea (Chlorella ellipsoidea); Described plant optimization is selected from Arabidopis thaliana, tobacco, rape, Sunflower Receptacle, soybean, tomato, castor-oil plant, cotton, sesame or peanut.
9. what utilize method according to 6 to prepare has high palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content, or has the cell of the yeast of high total fatty acid content, plant or algae.
10. the purposes of cell in production biofuel of the yeast according to 9, plant or algae.
Accompanying drawing explanation
Fig. 1. containing the structure of the Yeast expression carrier of chlorella ellipsoidea CeDGAT1 gene.
(A turns a unloaded control group to the peak figure of Fig. 2 .GC-MS method mensuration yeast fat acid, and B is the yeast fat acidity test peak figure turning CeDGAT1 gene.Arrows goes out the fatty acid component of each peak representative).
The each fatty acid component content of yeast that Fig. 3 .GC-MS method measures and total fatty acid content (CK is contrast, and pYES-DGAT1 is the yeast fat acidity test turning CeDGAT1 gene).
Embodiment
The present invention is described in detail below with reference to embodiment and accompanying drawing.Those having ordinary skill in the art will appreciate that, following embodiment is illustrational object, and it should not be interpreted as limitation of the present invention by any way.Protection scope of the present invention limited by accompanying.
The acquisition of embodiment 1. chlorella ellipsoidea CeDGAT1 gene cDNA total length
Get logarithmic phase chlorella ellipsoidea (coming from Inst. of Hydrobiology, Chinese Academy of Sciences) the algae liquid cultivated 3-4 days, collected by centrifugation frond is placed in liquid nitrogen and fully grinds.Total RNAs extraction and purifying is carried out with reference to Ai Delai EASYspinRNA extraction test kit specification sheets and Takara DNase I specification sheets.Carry out reverse transcription to be with the primer of polyT and obtain cDNA, and carry out RT-PCR (TOYOBO ReverTra Ace-α test kit is shown in concrete operations).
The synthetic reaction condition of cDNA is as follows:
With RNase Free H
2o benefit is 20 μ L to cumulative volume.Flick mixing and carry out reverse transcription reaction by following program after brief centrifugation:
Get appropriate above-mentioned reverse transcription product and carry out pcr amplification with forward primer (ATGCCAGATGATGCCAGCA) and reverse primer (TCAGCTGCCATTTGCGAG).
PCR reaction conditions is as follows:
Cut glue after PCR primer electrophoresis (1% sepharose concentration) and reclaim object fragment (CeDGAT1, about 2.1kb), be connected into pEASY-Blunt carrier (purchased from Transgen company), sequence verification obtains the full-length cDNA (SEQ ID NO:1) of CeDGAT1.
The structure of the Yeast expression carrier of embodiment 2. containing CeDGAT1 gene
According to the cds sequence of CeDGAT1, design the primer (two sequence of primer is respectively: cgccAAGCTTATGCCAGATGATGCCAGCA and ccggGAATTCTCAGCTGCCATTTGCGAG) with Hind III and EcoR I restriction enzyme site.From the pEASY-Blunt carrier containing CeDGAT1, adopt high-fidelity EasyPfu DNA Polymerase (purchased from Transgen company), amplify the fragment containing Hind III and EcoR I restriction enzyme site, double digestion is carried out with Hind III and EcoR I (purchased from Takara company), connect with the Yeast expression carrier pYES2 (purchased from Invitrogen company) of same double digestion, sequence verification, and by its called after pYES-DGAT1, its carrier figure is shown in Fig. 1.
Embodiment 3. Yeast expression carrier pYES-DGAT1 transformed saccharomyces cerevisiae
Inoculation uracil-deficient type yeast saccharomyces cerevisiae INVSC1 (purchased from Invitrogen company) is in 10mLYPD substratum, and 30 DEG C shake overnight incubation.Bacterium liquid is inoculated in 50mL YPD substratum by next day, is diluted to OD
600=0.4, continue to cultivate 2-4h to OD
600between 0.5-0.6,5,000rpm frozen centrifugation 1min, with 40mL1 × TE (10mM Tris, pH7.5,1mM EDTA) suspend precipitation, 5,000rpm frozen centrifugation, to suspend precipitation with 2mL1 × LiAc (10mM lithium acetate, pH7.5)/0.5 × TE, room temperature places 10min.100 μ L yeast suspensions and 1ug Yeast expression carrier pYES-DGAT1 and 100ug denaturated salmon essence DNA are mixed, then adds 700 μ L1 × LiAc/40%PEG-3350/1 × TE, mixing.Cultivate 30min, add 88 μ L DMSO for 30 DEG C, mixing, 42 DEG C of heat shock 7min.The centrifugal 10s of 10000rpm, removes supernatant, suspends and precipitates, the centrifugal 10s of 10,000rpm, remove supernatant with 1mL1 × TE.Suspend with 50-100 μ L1 × TE and precipitate, coat on SC-U minimum medium, cultivate 2 days for 30 DEG C.
After 2 days, picking colony from culture medium flat plate, with reference to Bo Maide yeast high purity plasmid rapid extraction test kit specification sheets in a small amount, carries out plasmid extraction, then the plasmid of extraction is carried out PCR checking as template.Finally the yeast plasmid and corresponding bacterium colony that are verified as the positive are preserved.
The abduction delivering of embodiment 4.CeDGAT1 in yeast saccharomyces cerevisiae
Conversion there is single colony inoculation of the yeast saccharomyces cerevisiae INVSC1 of pYES-DGAT1 in 5mLSC-U substratum, 200rpm, 30 DEG C of overnight incubation.The bacterium liquid getting incubated overnight is transferred to 50mL and contains in the SC-U nutrient solution of 1% raffinose and 2% yeast expression inductor D-semi-lactosi, makes its OD
600be about 0.1, add NP-40 (final concentration is 1%, is conducive to yeast cell and suspends), 200rpm, cultivates 72h abduction delivering by 20 DEG C.There is the yeast transformant of pYES2 zero load to be set to contrast conversion, yeast transformant is in contrast operated equally.
The extraction of embodiment 5. yeast fat acid and detection
1. the extraction of yeast fat acid
Get the yeast juice that inducing culture is good, the centrifugal 5min of 4000rpm, room temperature collects thalline; Repeatedly suspend through deionized water, room temperature centrifuge washing three times, 50 DEG C of oven dry; Get 100mg yeast dry powder fully to grind, add 3mL7.5%KOH-CH
3oH, adds the d17:0 (purchased from sigma company, concentration 27mg/mL) of 15-20 μ L, 70 DEG C of water-bath 3-5h; Add 2mL HCl to be acidified to its pH value and to reach 2.0; Add 2mL14%BF
3-CH
3oH (purchased from Aldtich company) solution, 70 DEG C of water-bath 1.5h; Add 1mL0.9%NaCl solution, the extracting of 4mL normal hexane once, N
2dry up; 300 μ L acetic acid ethyl dissolutions.The each each sample parallel of this experiment does two parts, altogether in triplicate.
2. end product GC-MS detects analysis design mothod
GC/MS instrument used is TurboMass (PerkinElmer company); GC condition: chromatographic column: BPX-70,30m × 0.25mm × 0.25um.Column temperature: 120 DEG C, vaporizer temperature 230 DEG C.Get 1 μ L end product loading, splitting ratio 10: 1.
3.GC-MS interpretation of result
Research shows, CeDGAT1 gene turns yeast strains compared with control group (turning pYES2 zero load), total fatty acid content improves about 1.4 times, and the content of its main fatty acid component is also all significantly improved, wherein palmitinic acid (C16:0) improves 1.86 times, Zoomeric acid (C16:1) improves 1.08 times, and stearic acid (C18:0) improves 1.86 times, and oleic acid (C18:1) improves 1.48 times.The peak figure of the yeast fat acid that GC-MS measures is shown in Fig. 2 (A is control group, and B is the yeast strains turning CeDGAT1 gene), and each fatty acid component of yeast and assay the results are shown in Figure 3 (notes: * * represents: P < 0.01).
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Claims (13)
1.CeDGAT1 albumen, its aminoacid sequence is as shown in SEQ ID NO:2.
2. the gene of coding CeDGAT1 albumen according to claim 1.
3. gene according to claim 2, its nucleotide sequence is as shown in SEQ ID NO:1.
4. carrier, it comprises the gene according to Claims 2 or 3.
5. prepare there is high palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content for one kind, or there is the method for cell of yeast of high total fatty acid content, described method comprises and proceeds in the cell of described yeast by the gene according to Claims 2 or 3 or carrier according to claim 4, and described yeast is yeast saccharomyces cerevisiae.
6. method according to claim 5, wherein said yeast is uracil-deficient type yeast saccharomyces cerevisiae INVSC1.
7. CeDGAT1 albumen according to claim 1, gene according to Claims 2 or 3 or carrier according to claim 4 are preparing the purposes had in the cell of the yeast of high total fatty acid content, and described yeast is yeast saccharomyces cerevisiae.
8. purposes according to claim 7, wherein said yeast is uracil-deficient type yeast saccharomyces cerevisiae INVSC1.
9. purposes according to claim 7, the wherein said cell with the yeast of high total fatty acid content is the cell of the yeast with high palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content.
10. CeDGAT1 albumen according to claim 1, gene according to Claims 2 or 3 or carrier according to claim 4 palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content in the cell improving yeast, or the purposes in total fatty acid content, described yeast is yeast saccharomyces cerevisiae.
11. purposes according to claim 10, wherein said yeast is uracil-deficient type yeast saccharomyces cerevisiae INVSC1.
12. utilize method according to claim 5 or 6 to prepare there is high palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid content, or there is the cell of yeast of high total fatty acid content.
The purposes of cell in production biofuel of 13. yeast according to claim 12.
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| CN108330114B (en) * | 2018-03-21 | 2020-11-03 | 北京大学 | EPA-utilizing diacylglycerol acyltransferase and application thereof |
| CN108794611B (en) * | 2018-06-28 | 2021-11-16 | 中国农业科学院棉花研究所 | Plant seed oil content related protein GhPDAT1d, and coding gene and application thereof |
| CN110564759B (en) * | 2019-08-09 | 2021-08-27 | 中国科学院遗传与发育生物学研究所 | Application of increasing content of fatty acid in leguminous plants in promoting root nodule formation |
| CN110499320B (en) * | 2019-08-26 | 2021-09-28 | 山西大学 | Dunaliella salina diacylglycerol acyltransferase gene DsDGAT and application thereof |
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