CN108531491A - Incise edge green alga lysophosphatidate acyltransferase gene and its application - Google Patents

Abstract

The present invention relates to incise edge green alga lysophosphatidate acyltransferase (LPAAT) gene and its application.The present invention cloned incise edge green alga LPAAT this be considered as TAG synthesis key enzyme gene, and be named as MiLPAAT.Further construct the prokaryotic expression carrier of MiLPAAT, after inverted e. coli bl21 and Fiber differentiation, purifying obtains the MiLPAAT maturation proteins of recombinant expression, the thin layer chromatogram analysis of external Enzyme assay and enzymic catalytic reaction product is shown, LPA can be synthesized PA by the MiLPAAT maturation proteins of recombination, shown that the albumen of MiLPAAT codings has the function of LPAAT, can be catalyzed and synthesized LPA as PA, the latter can further be synthesized into TAG along Kennedy's approach in frustule.The present invention is to be overexpressed to lay a good foundation with the yield for increasing triacylglycerol in grain and oil crop using MiLPAAT genes.

Description

Incise edge green alga lysophosphatidate acyltransferase gene and its application

Technical field

The present invention relates to bioenergies and plant biotechnology field, specifically, being related to one kind incising edge green alga haemolysis The gene of phosphatidic acid acyltransferase (LPAAT) and its application.

Background technology

Currently, various mineral resources such as coal, oil, natural gas etc., gradually exhaust, people with continuous develop and utilize Class is badly in need of finding the new energy, especially regenerative resource.Scientist thinks that bio-ethanol and biodiesel are two important Liquid biofuel, and it is possible to substitute oil fuel by mass producing biomass.Biodiesel has due to it Have the advantages that source than it is wide, can degrade, calorific value is high and to environment it is more friendly, have become countries in the world and develop new energy One hot spot in source.Wherein, microalgae is because oil content is high, its cultivation can be not take up arable land and can be combined etc. with wastewater treatment special Point, preparing biodiesel using microalgae also becomes scientist's increasingly focus of attention.Variety classes microalgae synthesizes triacylglycerol The ability of (triacylglycerol, TAG) is different.The study found that the fat content of the eukaryons microalgae such as green alga, diatom is relatively high, Promise to be the exploitable resources of production biodiesel.Incise edge green alga (Myrmecia incisa Reisigl H4301) It is a kind of unicellular microalgae of fresh water, is subordinate to Chlorophyta (Chlorophyta).The algae can largely accumulate TAG, especially in nitrogen stress item It is potential production microalgae biodiesel algae under part.

The main component of plant and microalgae grease is fatty acid triglyceride, which is otherwise known as triacylglycerol, i.e., TAG, it is a kind of neutral fats, is played an important role in the growth and development of plant and algae.The de novo formation of TAG be along Kennedy (Kennedy) approach, the i.e. priority in endoplasm on the net through 3 kinds of acyltransferases act on, and aliphatic acid is gradually added sweet Formed on the skeleton of oil -3- phosphoric acid (Cao et al., 2006；Baral et al., 2012).This 3 kinds of acyltransferases Respectively glycerol-3-phosphate acyltransferase (glycerol-3-phosphate 1-acyltransferase, GPAT), haemolysis Phosphatidic acid acyltransferase (lysophosphatidic acid acyltransferase, LPAAT) and Diacrylglycerol acyl turn Move enzyme (diacylglycerol O-acyltransferase, DGAT).Wherein, aliphatic acid-CoA can be added to molten by LPAAT The positions sn-2 of serium inorganic phosphorus resin acid (lysophosphatidic acid, LPA) glycerol backbone, to generate phosphatidic acid (phosphatidic acid, PA).On the one hand its product PA can be used for continuing along Kennedy's approach synthesizing TAG, another party Face can be used for synthesizing membrane phospholipid substance (Weselake et al., 2009).Thus, LPAAT genes are the key that oil synthesis Gene.

《Zhongshan University》2010 Master's thesis " tentatively grind by the clone of Peanut lysophosphatidic acid acyltransferase gene gene and function Study carefully ", according to the amino acid sequence that vegetable blood stasis-dissolving phosphatidic acid acyltransferase is guarded, by comparing the higher region of homology, if The degenerate primer of meter improvement carries out PCR, is fished from peanut cDNA library and takes out a LPAAT gene cDNA fragment, further according to obtaining The partial sequence obtained designs gene-specific primer, and the 3 '-of the cDNA is obtained by the ends cDNA rapid amplifying technology (RACE) With 5 '-end sequences, to obtain the cDNA sequence that overall length is 1561 bases, analysis shows sequence tool is there are one length 1131bp encodes the open reading frame (ORF) of 376 amino acid, and bioinformatic analysis, which shows that the amino acid sequence has, to be protected The amino acid sequence of the gene is compared and finds itself and castor-oil plant LPAAT by the acyltransferase structural domain kept (Ricinus communis putative lysophosphatidic acid acyltransferase,LPAAT, GenBank accession number：ACB30546.1 amino acid identity) is 90% and tung oil tree (Vernicia fordii putative Glycerol-3-phosphate acyltransferase, GenBank accession number：ACT32030.1) homologous similitude is 90%, therefore it is named as AhLPAAT (Arachis hypogaea Lysophosphatidic Acid Acyltransferase).The DNA sequence dna (overall length 3822bp) of the gene is also cloned.

However, yet there are no clone incises edge green alga lysophosphatidate acyltransferase gene, especially using valence It is worth the high relevant report for incising edge green alga lysophosphatidate acyltransferase gene.

Invention content

The purpose of the present invention is being directed to deficiency in the prior art, provides and incise edge green alga lysophosphatidate acyltransferase Gene and its application.

In a first aspect, the present invention provides a kind of polypeptide, any of the amino acid sequence of the polypeptide in following Kind：

a)SEQ ID NO:Amino acid sequence shown in 2；

b)SEQ ID NO:Amino acid sequence shown in 52-331 of 2；

c)SEQ ID NO:Amino acid sequence shown in 3.

Second aspect, the present invention provides a kind of nucleotide sequence of separation, the nucleotide sequence coded institute as above The polypeptide stated.

Preferably, its sequence of the nucleotide sequence contains any one of following：

A) nucleotide sequence shown in SEQ ID NO.1；

b)SEQ ID NO:Nucleotide sequence shown in 140-1135 of 1；

c)SEQ ID NO:Nucleotide sequence shown in 293-1135 of 1；

D) SEQ ID NO are included at least:Nucleotide sequence shown in 293-1135 of 1, and with SEQ ID NO:1 tool Standby 70% or more homology；

E) with it is above a), b), c) or d) described in nucleotide sequence complementation nucleotide sequence.

It is highly preferred that its sequence of the nucleotide sequence includes at least SEQ ID NO:Shown in 293-1135 of 1 Nucleotide sequence, and with SEQ ID NO:1 has 80% or more homology.

It is highly preferred that its sequence of the nucleotide sequence includes at least SEQ ID NO:Shown in 293-1135 of 1 Nucleotide sequence, and with SEQ ID NO:1 has 90% or more homology.

It is highly preferred that its sequence of the nucleotide sequence includes at least SEQ ID NO:Shown in 293-1135 of 1 Nucleotide sequence, and with SEQ ID NO:1 has 95% or more homology.

It is highly preferred that its sequence of the nucleotide sequence includes at least SEQ ID NO:Shown in 293-1135 of 1 Nucleotide sequence, and with SEQ ID NO:1 has 99% or more homology.

The third aspect, the present invention provides a kind of recombinant expression carrier, the recombinant expression carrier is by as described above Nucleotide sequence and plasmid or virus constructed by recombinant expression carrier.

Fourth aspect, the present invention provides a kind of genetically engineered host cell, the host cell is selected from down One kind in row：

A) host cell for being converted or being transduceed with nucleotide sequence as described above；

B) host cell for being converted or being transduceed with recombinant expression carrier as described above.

Preferably, the host cell be bacterial cell, fungal cell, the plant cell for not having cellular omnipotency or Do not have the zooblast of cellular omnipotency.

5th aspect, the present invention provides polypeptide as described above, nucleotide sequence as described above, weights as described above The purposes of group expression vector, host cell as described above in increasing grease yield.

Preferably, the increase grease yield, which refers to, increases phosphatide acid yield or triacylglycerol yield.

The invention has the advantages that：

It incises edge green alga and is rich in arachidonic acid (arachidonic acid, ArA, 20:4^Δ5,8,11,14), and in its TAG 2/3 aliphatic acid is ArA (Ouyang tinkling sound of metal striking against metal etc., 2016).It is present in frustule as how stored the form of fat to parse ArA In, based on Kennedy's approach of microalgae TAG de novo formations, the present invention is based on the research experiences that inventor is abundant, especially biological Bioinformatics analysis experience utilizes the ends cDNA rapid amplifying (rapid amplification of cDNA ends, RACE) skill Art cloned incise edge green alga LPAAT this be considered as TAG synthesis key enzyme gene.The cDNA full length sequences of the gene A length of 1375bp, open reading frame (ORF) a length of 996bp of sequence encode 331 amino acid residues；3 '-non-translational regions (UTR) The a length of 240bp of sequence, a length of 139bp of 5 '-UTR sequences.It has acyltransferase structural domain PlsC, with LPAAT (the GenBank accession number of Auxenochlorella protothecoides：XP_011396467) sequence has 59% Homology, thus, by the unnamed gene of the clone be MiLPAAT.It at least has there are one transmembrane region.In order to further clear MiLPAAT encodes the function of albumen, and the present invention constructs the prokaryotic expression carrier of MiLPAAT, inverted Escherichia coli For (Escherichia coli) BL21 simultaneously after Fiber differentiation, purifying obtains the MiLPAAT maturation proteins of recombinant expression.To recombination MiLPAAT maturation proteins carry out external Enzyme assay, and the thin layer chromatogram analysis of enzymic catalytic reaction product is shown, recombination LPA can be synthesized PA by MiLPAAT maturation proteins.This is the result shows that the albumen of MiLPAAT codings has the work(of LPAAT Can, LPA can be catalyzed and synthesized as PA, the latter can further be synthesized into TAG, this hair along Kennedy's approach in frustule The albumen of bright MiLPAAT and its coding has important application value.The present invention is using MiLPAAT genes in grain and oil crop Middle overexpression is laid a good foundation with the yield for increasing triacylglycerol.

Description of the drawings

Fig. 1 incise the agarose gel electrophoresis figure of-RACE amplified productions of edge green alga LPAAT genes 3 ' -/5 '.M：DL 2000DNA molecular weight standards (Tiangeng is biochemical)；Swimming lane 1：3 '-RACE second take turns nested primer pcr amplification product；Swimming lane 2： 5 '-RACE second take turns nested primer pcr amplification product.

Fig. 2 .pET28a plasmid maps (on) and multiple cloning sites region sequence (under).

The SDS-PAGE electrophoresis spectrum (left side) of Fig. 3 induced expression MiLPAAT maturation proteins utilizes anti-polyhistidyl tags Antibody carry out Western immunoblottings collection of illustrative plates (in) and purifying the soluble SDS- for recombinantly expressing MiLPAAT maturation proteins PAGE electrophoresis patterns (right side).M：Pre-dyed Protein Marker (Fermenbtas)；Swimming lane 1 and 4：Carry empty plasmid pET28a Escherichia coli soluble protein；Swimming lane 2：Turn the whole bacterial protein of MiLPAAT gene Escherichia coli；Swimming lane 3：Utilize anti-poly group The Western immunoblottings that the antibody of His tag carries out the whole bacterial protein for turning MiLPAAT genes；Swimming lane 5：Utilize 250mM The electrophoresis pattern of the soluble recombinant expression MiLPAAT maturation proteins of the imidazoles affinity purification of concentration.

The thin layer chromatography collection of illustrative plates of the enzymic catalytic reaction product of Fig. 4 recombinant expression MiLPAAT maturation proteins.PA and LPA：It is the standard items of phosphatidic acid and lysophosphatidic acid respectively；Recombinate MiLPAAT：Refer to and recombinant expression is added in the reaction system MiLPAAT maturation proteins；Control：Refer to the MiLPAAT maturation proteins that recombinant expression is not added in reaction system.

Specific implementation mode

It elaborates below in conjunction with the accompanying drawings to specific implementation mode provided by the invention.

The main operational steps that the present invention uses include：

1) temperature is 25 DEG C, intensity of illumination is 115 μm of ol photons/ (m²S), Light To Dark Ratio is the item of 12h/12h Under part, is cultivated in BG-11 culture mediums and incise edge green alga.Frustule is collected, and total serum IgE is extracted using TRIzol methods.

2) edge green alga transcript profile database (Ouyang et al., 2013) is incised based on what this laboratory obtained, passed through The BLASTx of NCBI is compared, and finds contig10781 and the parts C-169 glueballs algae (Coccomyxa subellipsoidea) LPAAT sequences (GenBank accession number：XP_005650277) there is 70% similarity.According to this section of primers, utilize The sequence of contig10781 is cloned and verified to reverse transcription PCR (reverse transcription-PCR, RT-PCR) first；So The ends cDNA rapid amplifying (rapid amplification of cDNA ends, RACE) technology, splicing is utilized to obtain it afterwards CDNA full length sequences；Finally, the full length sequence design primer based on splicing, to incise the cDNA of edge green alga reverse transcription as template pair The cDNA full length sequences of MiLPAAT are verified and are cloned, and the full length cDNA sequence of the gene is obtained.

3) after utilizing TRIzol methods extraction RNA, the first chains of cDNA are synthesized through reverse transcription reagent box.According to opening for MiLPAAT The primers for putting reading frame (ORF) sequence and pET28a vector multiple cloning sites utilize PCR amplification by template of cDNA Technology clone MiLPAAT does not contain the ORF sequences for leading peptide and builds pMD-19T/MiLPAAT plasmids.

4) it after carrying out double digestion (HindIII/XhoI) respectively to pET28a carriers and pMD-19T/MiLPAAT plasmids, returns Target fragment is received, then uses T₄Ligase connects to obtain recombinant vector pET28a-MiLPAAT, is converted, coated plate, sequence verification After extract plasmid.

5) competence that the recombinant plasmid pET28a-MiLPAAT of extraction is converted to e. coli bl21 using heat shock method is thin Born of the same parents.Then, the Escherichia coli of conversion are coated on the LB solid mediums containing kanamycins (Kan), screening is turned The Escherichia coli of pET28a-MiLPAAT.

6) target gene will be turned, turn the BL21 Escherichia coli of pET28a empty carriers through 1mM isopropylthiogalactosides (IPTG) at 37 DEG C after Fiber differentiation 4h, Escherichia coli is collected, are preserved in -80 DEG C of refrigerators after liquid nitrogen multigelation 3 times.

7) ultrasonication is carried out to the Escherichia coli of multigelation, with the MiLPAAT maturation proteins of extraction recombinant expression, then Utilize His₆- tag nickel column purifies the MiLPAAT maturation proteins of pronuclear recombination to specifications, with dodecyl sodium sulfate-poly- third Acrylamide gel electrophoresis (SDS-PAGE) technology detects prokaryotic recombinant protein expression, and utilizes anti-His₆The antibody pair of-tag The albumen of expression carries out Western immune-blotting methods.

8) the pronuclear recombination MiLPAAT maturation proteins of purifying are added in the reaction system with LPA and aliphatic acid-CoA, Reaction after a certain period of time, utilizes the generation of thin layer chromatography (TLC) method detection phosphatidic acid (PA), it was confirmed that pronuclear recombination LPA can be synthesized PA by MiLPAAT maturation proteins in vitro, to illustrate to incise the MiLPAAT being cloned into edge green alga certainly Coded albumen has the function of LPAAT.

Specific experiment process is as follows：

1, material

1) the edge green alga (Myrmecia incisa Reisigl H4301) of incising in this laboratory is looked into from Prague, CZE Li Si universities algae culture center (Culture Collection of Algae of Charles University of Prague, CAUP).Temperature is 25 DEG C, intensity of illumination is 115 μm of ol photons/ (m²S), light dark ratio is 12h/12h Under conditions of cultivate, culture medium BG-11.Every 2 weeks update a BG-11 culture medium in incubation, and daily not Periodically rock 3-4 times.

2) plastic recovery kit, Escherichia coli (Escherichia coli) DH5 α and BL21 competent cell, His₆- Tag antibody, HRP labels goat anti-rabbit antibody, enhanced HRP-DAB colour reagents box is purchased from Tiangeng biochemical technology (Beijing) has Limit company；PMD-19T plasmids, pET-28a plasmids, reverse transcription reagent box are purchased from precious bioengineering (Dalian) Co., Ltd (TaKaRa)；TRIzol is purchased from Invitrogen companies；SMART^TMRACE cDNA amplification kits are purchased from Clontech companies； Bio-Scale^TM Mini Profinity^TMIMAC Cartridges nickel columns are purchased from Bio-Rad companies；Other reagents are state Production or import, analysis are pure.

2, method

1) it waits for that frustule was grown to exponential phase of growth, is centrifuged with the rotating speed of 5500revolutions/min (rpm) at 4 DEG C Frustule is equally collected by centrifugation after being used in combination sterile deionized water to wash 3 times in 10min again.100mg is taken just to be collected by centrifugation fresh scarce It carves edge chlorella cell to be placed in the mortar of precooling, liquid nitrogen is added and sufficiently rapidly grinds 1.5min.

2) with reference to the specification of TRIzol methods, the total serum IgE of edge green alga is incised in extraction, and -20 DEG C save backup.

3) based on incising the contig10781 sequences obtained in edge green alga transcript profile database, design primer 10781 (L) (CTATCACTCTTCCCACCTACAGC, SEQ ID NO:And 10781 (R) 4)

(ATGTAGTATGGATCTGCGATCAGG, SEQ ID NO:5) contig10781 sequences are verified.According to 3 '/the 5 '-RACE gene-specific primers of contig10781 sequence designs having verified that, including gene used in 5 '-RACE is special twice 5 ' RACE2 of specific primer (R)

(CAGGGTGAAGGTCCATAGGTAGAAA, SEQ ID NO:6, nested primer) and 5 '

RACE2 (L) (TGGTCTCTGCTCTATGCCTTTCTGG, SEQ ID NO:7), and twice gene used in 3 '-RACE 3 ' RACE2 of specific primer (R)

(AACCGCTCGTTCAAGTTCATCTCCA, SEQ ID NO:And 3 ' RACE2 (L) 8)

(CACAACCTGCCCAAGAATGATGAGC, SEQ ID NO:9, nested primer).Utilize Clontech companies SMART^TMRACE cDNA amplification kits carry out PCR amplification.The PCR reaction conditions of the 5 '-RACE first round are：94 DEG C of pre-degenerations 5min；25 cycles include 94 DEG C of denaturation 30s, 62.5 DEG C of annealing 30s, 72 DEG C of extension 2min；Last 72 DEG C of extensions 10min.Institute It is 5 ' RACE2 (L) and UPM (in kit band primer) with primer；Second wheel PCR reactions take 1 μ L first round PCR product conducts Reaction template, 5 ' RACE2 of the primer (R) and NUP (institute's band primer in kit), reaction condition is the same as first round PCR.3’- RACE reaction conditions are the same as 5 '-RACE.But, the PCR primer of the first round is 3 ' RACE2 (R) and UPM；Second wheel PCR, which reacts, is also Take 1 μ L first round PCR product as reaction template, but the primer is 3 ' RACE2 (L) and NUP.

4) illustrate to recycle PCR using the plastic recovery kit of TIANGEN Biotech's production and according to it Product.PCR product is stayed overnight and is connected to pMD-19T loads by the explanation then provided according to precious bioengineering (Dalian) Co., Ltd Body.Linked system is：The solution I (kit carrying) of 5 μ L, the glue recovery product of 4 μ L, the pMD-19T carriers of 1 μ L.So The pMD-19T for carrying target fragment is converted to bacillus coli DH 5 alpha competent cell by heat shock method afterwards.Conversion process is as follows： Connection reaction solution is added in the bacillus coli DH 5 alpha competent cell of 100 μ L, the content for the concussion centrifuge tube that is gently vortexed； Centrifuge tube is fully embedded ice bath 30min in ice；After 42 DEG C of heat shock 90s, 1min is placed on ice；Each centrifuge tube adds 890 μ L to contain Then the LB liquid medium of ampicillin sodium (Amp) is preserved with rotating speed 2~3h of shake culture of 180rpm at 37 DEG C In 4 DEG C of refrigerators.Picking positive colony bacterium colony is screened by blue hickie.Method is as follows：100 μ L conversion fluids are taken to be laid in containing 5- Bromo- 4 chloro- 3- indoles-β-D- galactosides (X-gal, 400 μ L), isopropyl-β-D-thiogalactoside (IPTG, 200 μ L), On the LB Agar Platings of Amp (200 μ L), 37 DEG C are inverted 10~14h of culture, form single bacterium colony；Picking 3~5 is single Hickie bacterium colony, in LB (2~3mL) fluid nutrient medium containing Amp, at 37 DEG C with the rotating speed shake culture 10 of 180rpm~ 16h.Whether the thalline that positive colony is verified using bacterium colony PCR contains target gene.Its reaction system is：2×RT Primer The 9.5 μ L of water of Mix 12.5 μ L, no RNase, target gene fragment clone identical each 1 μ L of upstream and downstream primer, bacterium solution mould 1 μ L of plate；Bacterium colony PCR response procedures are：94 DEG C of pre-degeneration 3min, 30 cycles include 94 DEG C of denaturation 50s, 64 DEG C of annealing 45s, 72 DEG C extend 2min, it is last 72 DEG C extension 10min.The positive colony bacterium solution that packing 1mL has verified that is sent to Shanghai life work bioengineering Co., Ltd is sequenced, and obtains the sequence of target gene.

5) based on the MiLPAAT full length cDNA sequences cloned and spliced using RACE technologies, design primer is cloned simultaneously Verify the full length cDNA sequence of MiLPAAT.Edge green alga total serum IgE is incised in extraction, and cDNA is obtained using RT-PCT reverse transcription reagent box. To incise edge green alga cDNA as template, design primer 2cDNA (L)

(CAGTGGTATCAACGCAGAGTACAT, SEQ ID NO:And 2cDNA (R) 10)

(ACACAGAAGTCAGAGACTCTACAAC, SEQ ID NO:11) sequence is verified.PCR response procedures are： 94 DEG C of pre-degeneration 5min, 30 cycles include 94 DEG C of denaturation 45s, 60 DEG C of annealing 45s, 72 DEG C of extension 1.5min, and last 72 DEG C are prolonged Stretch 10min.Glue recycling, connection, conversion, bacterium colony PCR verifications and sequencing etc. are carried out to PCR product by method 4).

6) in order to recombinantly express MiLPAAT in Escherichia coli and determine its function, in above-mentioned MiLPAAT full-length cDNAs sequence On the basis of Lek is grand and bioinformatic analysis, the primer with restriction enzyme site is designed, clone MiLPAAT is without containing leading opening for peptide Frame frame (ORF) sequence.PCR sense primers RcF is aagcttATGCACATGAAGCCCTGCT (SEQ ID NO:12, small letter Mother is HindIII restriction enzyme sites), downstream primer RcR is ctcgagTCACTCCGTCTGCCTGCGCT (SEQ ID NO:13, it is small It is XhoI restriction enzyme sites to write female).PCR response procedures：94 DEG C of pre-degeneration 3min, 30 cycles comprising 94 DEG C of denaturation 50s, 64 DEG C Anneal 45s, 72 DEG C of extensions 2min, last 72 DEG C of extensions 10min.According to method 4) to PCR product carry out glue recycling, connection, Conversion, bacterium colony PCR verifications and sequencing etc..

7) plasmid construction of MiLPAAT is recombinantly expressed.Extracting carries MiLPAAT from 6) the middle bacillus coli DH 5 alpha obtained Without containing the pMD-19T plasmids for leading peptide ORF, double digested reaction is carried out with restriction enzyme HindIII and XhoI；Together When, same double digestion reaction is also carried out to pET28a plasmids.Endonuclease reaction system is：10 × M buffer solutions of 2 μ L, Plasmid DNA About 2 each 1 μ L of μ g, HindIII and XhoI, add no RNase water to 20 μ L.It is placed in PCR instrument, the digestion that 4h is carried out at 37 DEG C is anti- It answers.Then, glue recycling is carried out to the target fragment after digestion according to method 4), T is used in combination₄DNA ligase returns glue after digestion The target fragment of receipts connect to obtain recombinant expression carrier pET28a-MiLPAAT with pET28a segments.Coupled reaction system is：2.5 The T of μ L₄Buffer solution, MiLPAAT pieces segment DNA about 0.3pmol, pET28a carrier DNAs about 0.03pmol, the T of 1 μ L₄DNA ligase, Add no RNase water to 25 μ L.16 DEG C of connections are overnight.The recombinant expression carrier pET28a-MiLPAAT conversions obtained after connection is big Enterobacteria DH5 α competent cells carry out Bacteria Culture, bacterium colony PCR verifications and sequencing according to method 4).

8) recombinant expression plasmid pET28a-MiLPAAT is extracted from bacillus coli DH 5 alpha, is turned the plasmid using heat shock method Change into e. coli bl21 competent cell.Picking positive colony bacterium colony is screened by blue hickie and is passed through according to method 4) After bacterium colony PCR verifications, the sequencing of Hai Shenggong bioengineering Co., Ltd is served.In addition, in same way as described above by empty plasmid PET28a is converted into e. coli bl21 competent cell.

9) the errorless bacterium solution containing recombinant expression plasmid pET28a-MiLPAAT will be sequenced, by 1:1000 ratio is inoculated in LB liquid medium containing kanamycins (Kan), with the rotating speed shake culture of 180rpm overnight with activation at 37 DEG C.Take 2mL Activated bacterium solution is seeded to LB liquid mediums of the 200mL containing Kan, expands under similarity condition and cultivates, after about 2~3h, bacterium is thin The OD value that born of the same parents detect at 600nm is 0.6~1.0 or so.At this point, the IPTG of 1mM is added, under similarity condition culture 4h with Induce the expression of recombinant protein MiLPAAT.Then culture solution is placed in cooled on ice 15min makes cell stop growing.4 DEG C of conditions Under Escherichia coli are collected with the rotating speed of 5500rpm centrifugation 5min, with the sterile water washing cell 2 times of 20mL precoolings, similarity condition It is lower that thalline were collected by centrifugation；1 × PBS (0.137mM NaCl, 2.7mM KCl, the 10mM Na being pre-chilled again with 20mL₂HPO₄、2mM KH₂PO₄) solution washing cell 2 times, it is subsequently placed in 1 × PBS solution of 15mL precoolings.Take the appropriate cell just collected by 1:1 Ratio be added albumen sample-loading buffer mixing after boiling water boiling 10min.Gel electrophoresis is carried out using SDS-PAGE and is gathered using anti- The antibody of histidine tag carries out Western immunoblottings, and analysis turns in the bacterium of recombinant expression plasmid pET28a-MiLPAAT The expression of recombinant protein MiLPAAT.Western western blotting methods are as follows：The filter paper of transferring film, sponge and nitric acid is fine The plain film (NC films) of dimension and the good protein gel of electrophoresis are immersed in transferring film buffer solution (3.03g Tris, 14.4g glycine, 200mL Methanol is settled to 1000mL with water) in, the electrophoresis 45min under 100V voltages, 200mA current conditions, by protein from gel Electricity is gone on NC films；The NC films that electricity turns first are dyed with Ponceaux, judge whether protein is successfully transferred to NC films；Then it spends Ionized water cleans film 5min, to wash away the dye liquor of film surface；TBST (0.137M NaCl, 2.7mM KCl, 0.025M are used again Tris, pH 7.4, with preceding addition 0.05%Tween 20) solution washs three times, each 5min；With confining liquid, (TBST is prepared 5% skimmed milk power) the NC film 1h washed are closed, it is then washed 3 times with TBST solution again, each 5min；Addition confining liquid is dilute Release the anti-His of (extension rate 1/2000)₆- tag antibody gently shakes 1h with decolorization swinging table at normal temperatures, then with TBST solution Washing 3 times, each 10min；Addition confining liquid dilutes the goat-anti rabbit secondary antibody of the HRP labels of (extension rate 1/6000), same It is anti-to be equally incubated 1h, it washes 3 times, each 10min；The specification of enhanced HRP-DAB substrates colour reagent box is finally pressed in darkroom In prepare developing solution, then in developing solution rinsing handle well NC films until purpose band occur, place into ultra-pure water and rinse Remaining developing solution, preservation of taking pictures after drying on clean film.

10) by above-mentioned SDS-PAGE detect after it is remaining turn target gene Escherichia coli by liquid nitrogen multigelation 3 times after It is preserved in -80 DEG C of refrigerators.When experiment, the Bacillus coli cells for being stored in -80 DEG C of refrigerators are placed in 37 DEG C of metal baths to melt, Using the ultrasonication machine ultrasound 5s of 160W power, interval 5s to be crushed thalline, until clarification (about 1h).Under the conditions of 4 DEG C with The rotating speed of 12000rpm centrifuges 15min, collects supernatant respectively and precipitates two parts, wherein sediment fraction again with supernatant same volume Long-pending 1 × PBS dissolvings.Utilize the expression of recombinant protein MiLPAAT in SDS-PAGE analysis supernatant precipitations.

11) in supernatant soluble recombinant expression protein MiLPAAT purifying.New 5mL is taken to pre-install nickel column first with 2 times of columns 20% ethyl alcohol of bed volume is washed with the flow velocity of 2mL/min, then with the precooling sterile waters of 2 times of bed volumes with identical flow velocity Washing, nickel column reach balance, can be used for protein purification；With wash buffer (imidazoles containing 5mM, the 50mM of 5 times of bed volumes KH₂PO₄With 300mM KCl, pH 8.0) balance nickel column, flow control is in 2mL/min or so；Soluble protein in supernatant is used 0.22 μm of filter filtering, prevents the impurity such as cell fragment from blocking nickel column, after being then added to balance with the flow velocity of 0.5mL/min In nickel column；Acquisition mesh can be purified with determination by cleaning nickel column with the imidazoles of the various concentrations such as 5mM, 10mM, 20mM, 250mM respectively Albumen ideal wash-out concentration, collect eluent, utilize the purifying of SDS-PAGE testing goal recombinant expression proteins MiLPAAT Situation.

12) the enzymic catalytic reaction product of thin layer chromatography method detection MiLPAAT is utilized.Enzyme is added in glass reaction tube Reaction system [lysophosphatidic acid (LPA) of the destination protein MiLPAAT, 30 μ L of 400 μ L after purification, the ArA-CoA of 10 μ L, 50 μ The KCl (0.4M) of the Tris-HCl (25mM, pH 7.6) of L, 50 μ L], after reacting 10min in 30 DEG C of water-baths, it is added 1.33mL methanol:Chloroform:Acetic acid (50:50:1, v/v) it terminates and reacts and be vortexed, then add 133 μ L aqua sterilisas, be vortexed, with The rotating speed of 5500rpm centrifuges 15min.It draws lower layer's organic phase to be transferred in new reaction tube, 660 μ L chloroforms, identical item is added It is centrifuged under part, extracts organic phase from water phase again.The organic phase extracted twice is mixed, is then dried up with nitrogen, 20 μ L are added Chloroform dissolves.Capillary syring point sample is used on thin plate, is then unfolded, and solvent used is ethyl acetate:Isopropanol:Chloroform:Nothing Water methanol:0.25%KCl (25:25:25:10:9, v/v), development distance 12.5cm, time about 1h.Then by thin plate sulphur Sour copper solution (20g CuSO₄、80mL H₃PO₄It is dissolved in 1L water) colour developing, it is placed in 180 DEG C of baking ovens and bakes 20min.Compare LPA and PA standard specimens check the generation situation of PA on thin plate.

3, result

1) the gene order clone of MiLPAAT.Using RACE technologies from incise expanded in edge green alga the gene 5 '-and 3 '-end sequences, 3 ' -/5 '-RACE second wheel nest-type PRC the primers are respectively 3 ' RACE2 (L) and 5 ' RACE2 (R), amplification The results are shown in Figure 1.3 '-terminal sequencing results show that gained target fragment size is 719bp, and have apparent polyA tails Bar；5 '-terminal sequencing results show that gained target fragment size is 407bp.By 5 '-ends and 3 '-end sequences and verification After contig8647 sequences are spliced, the cDNA sequence that a length is 1404bp is obtained.One is redesigned to the cDNA sequence PCR reactions and sequence verification are carried out to primer 2 cDNA (R) and 2cDNA (L), it is that the accurate cDNA of 1375bp are complete to obtain a length Long sequence (SEQ ID NO:1).Through Bioinformatics Prediction, its 5 '-non-translational region (UTR) long 139bp, 3 '-UTR is long 240bp (is free of polyA tails), open reading frame (ORF) long 996bp of sequence, and coding one is formed and divided by 331 amino acid The precursor protein that son amount is 37.7kD.The amino acid sequence is positioned with prediction in Auxenochlorella protothecoides There is 59% homology in the sequence of chloroplaset LPAAT (XP_011396467), thus be named as MiLPAAT.It is right Coded by the ORF of MiLPAAT albumen carry out bioinformatic analysis after, it is understood that MiLPAAT aminoterminal exist by What 51 amino acid formed leads peptide, illustrates in incising edge chlorella cell, and preceding 51 amino acid of the precursor protein aminoterminal will It is removed, the maturation protein only formed with 280 amino acid functions, and the maturation protein molecular weight of prediction is 31.78kD.

2) purifying of the structure and albumen of pronuclear recombination MiLPAAT expression plasmids.According to above-mentioned MiLPAAT maturation proteins The sequence (Fig. 2) of gene order and expression plasmid pET28a multiple cloning sites, in order to use His₆The affinity chromatography sides-Tag Method more easily purifies the MiLPAAT maturation proteins recombinantly expressed to carry out Function Identification, and the present invention devises a pair of of band The primer RcF and RcR of restriction enzyme site are cloned into MiLPAAT and do not contain the ORF for leading peptide sequence, and the sequence is connected to On the pET28a of HindIII and XhoI digestions.On the MiLPAAT maturation protein recombinant expression plasmids built, because being inserted into The upstream of target fragment there are the sequence (Fig. 2) for being applicable in other restriction enzyme sites carried on 6 His coded sequences and plasmid, Increase 44 amino acid, thus the prediction of recombinant expression protein point again on the basis of 280 amino acid of maturation protein in this way Son amount is 36.57kD.Recombinant expression plasmid pET28a-MiLPAAT is converted to e. coli bl21, IPTG inductions are recycled Protein expression.By carrying out SDS-PAGE detections to recombinant expression protein MiLPAAT and utilizing commercialization His₆- Tag is general anti- Body carries out Western blot analysis, only occurs the list of a molecular size range about 36.57kD on immunoblotting collection of illustrative plates (in Fig. 3) One signal, its molecular size range and induction expression protein electrophoresis result (Fig. 3 left) with prediction unanimously, illustrate successfully to recombinate It expresses without containing the MiLPAAT for leading peptide, the i.e. maturation protein of MiLPAAT.Utilize His₆- tag nickel column simultaneously uses various concentration Imidazoles purifying pronuclear recombination expression ripe MiLPAAT, the imidazoles of SDS-PAGE testing results (Fig. 3 right) display 250mM is dense Degree is conducive to the purifying of ripe MiLPAAT, shows that the imidazoles using the concentration carries out affinity chromatography and can obtain relative components ratio More single, molecular size range is the recombinant expression MiLPAAT of 36.57kD.And empty plasmid pET28a is being contained only as negative There is not the band of the size on the swimming lane of the Escherichia coli supernatant soluble protein of control, further demonstrates that the present invention has become It recombinantly expresses to work(and has purified ripe MiLPAAT.

3) the thin-layer chromatography detection of enzymatic reaction product.The ripe MiLPAAT of above-mentioned purifying is added to containing reactant Lysophosphatidic acid (LPA) and ArA-CoA and buffer solution 0.4M KCl, 25mM Tris-HCl (pH 7.6) reaction system in, Reaction is terminated after 10min, and thin-layer chromatography is carried out to the product of reaction.Thin layer chromatogram analysis (Fig. 4) is shown, in the weight of addition purifying The generation of phosphatidic acid (PA) can be detected in the reaction system of group expression MiLPAAT maturation proteins, and is not adding the recombination But the generation of PA is not detected in the reaction system of albumen.Show that this purified recombinant mature MiLPAAT has in vitro There is the activity of LPAAT.Illustrate that it can carry out the synthesis of phosphatidic acid further to synthesize in frustule along Kennedy approach TAG。

It is the special instruction about partial sequence of the present invention below：

SEQ ID NO:1 --- (underscore is starting or terminator codon, shadow part to the cDNA full length sequences of MiLPAAT It is divided into the cDNA sequence led corresponding to peptide)

AAGCAGTGGTATCAACGCAGAGTACATGGGGACATTTGCGTTGATCAAGGTCACTGCAGCACGCGTCCT GCAAACGACAAGCAGCGCCCTGTACGCTGCCTTTGTGTAGCAGCTGTGCAGTTTCACTTGCAGTTCAGGCATGCACA TGAAGCCCTGCTATCACTCTTCCCACCTACAGCACCATTCCTGCGGACTGCAGCCTGGCAACCAGCCCATCTACAGA GTTCAAAGCCGTTTAGCGCCCACAGCGCCCAGCCCAAGGCCATGCCAGCACAGGCGGCAAACTTGCAGGCTGTCCTC AACACCAAGCCTGCCAAGGCGTACAAGGAAGCAAATGGCTTGCTCAGCATCCTCCTTAGCTCTGGCTGTTGACCCAC CGTCCCCAGAAAGGCATAGAGCAGAGACCATACTAGCAAGCGTTAAGGGGCTGCTTTTCTACCTATGGACCTTCACC CTGTCCCTGCCGCTGTTCGTGACCATGCTCGTCATTGCACCCTTCATGGCCGTCCTTGACAAGCATAGACGGCTTGC ACAGCACTTTGTGAACAACATCTGGGCAAAGGTGTCAACCTGGCCCTTCTACCGAGTACGGATTGATGGGCGACACA ACCTGCCCAAGAATGATGAGCCAGCTGTCTATGTAGCCAATCATGCGAGTTTCCTGGACATCTATACGCTGTTCCAT CTCAACCGCTCGTTCAAGTTCATCTCCAAGACCAGCAACTTCCTCATCCCAATCATTGGCTGGAGCATGTTCCTGAC AGGGCATGTCCGGCTGAACCGCGTGGACCGCAAGAGCCAGATCAAGTGCCTGTCCAAGTGCCGCGAGCTGCTTTCGG AGGGCGCCTCCGTGCTGTTCTTCCCCGAGGGCACGCGCAGCAAGGACGGGAAGATCCAGGACTTCAAAAAGGGCGCG TTCAGCGTGGCTGCCAAGGCCAAGGTGCCGGTTGTGCCCATCACGCTGGTGGGCACCGGACGCATCATGCCCAACAA GCAGGAGTCCAAAATGTTCCCGGGCAATGTGCATGTGATTGTCCATCCGCGCGTGGAGCCGAAGGATCCTGACGCCA TGATGGCTGAGGCATACGACCTGATCGCAGATCCATACTACATCGAGCGCAGGCAGACGGAGTGATGATATCAGATA AGCATGTTCAAGAATAAAGGGTGTTGCAAGGCATGCCCCCAAGCGATGCGCCAGGATTGCTGGTTTTGTTTCGTGAC CCAGGCACAGGTTGGCATGCTTGGGTAACACACGCTTGGTTGCGGTAGCAAGGGCTGTGTTGGGGTGCAAACTGTGA ATGCTTACAGGTGACTGCTTCAGTTGTAGAGTCTCTGACTTCTGTGTACTGACATGTAATGTGTGGGATTTCTGAAA AAAAAAAAAAAAAA

SEQ ID NO：The amino acid sequence of albumen coded by the ORF of 2-MiLPAAT (dash area is to lead peptide sequence)

MHMKPCYHSSHLQHHSCGLQPGNQPIYRVQSRLAPTAPSPRPCQHRRQTCRLSSTPSLPRRTRKQMACS ASSLALAVDPPSPERHRAETILASVKGLLFYLWTFTLSLPLFVTMLVIAPFMAVLDKHRRLAQHFVNNIWAKVSTWP FYRVRIDGRHNLPKNDEPAVYVANHASFLDIYTLFHLNRSFKFISKTSNFLIPIIGWSMFLTGHVRLNRVDRKSQIK CLSKCRELLSEGASVLFFPEGTRSKDGKIQDFKKGAFSVAAKAKVPVVPITLVGTGRIMPNKQESKMFPGNVHVIVH PRVEPKDPDAMMAEAYDLIADPYYIERRQTE

SEQ ID NO：3 --- (dash area is carrier to the amino acid sequence of pronuclear recombination expression MiLPAAT maturation proteins In the entrained encoded amino acid sequence of base for genetic engineering operation)

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELRRQASMLSSTPSLPRRTRKQMACSASSLALA VDPPSPERHRAETILASVKGLLFYLWTFTLSLPLFVTMLVIAPFMAVLDKHRRLAQHFVNNIWAKVSTWPFYRVRID GRHNLPKNDEPAVYVANHASFLDIYTLFHLNRSFKFISKTSNFLIPIIGWSMFLTGHVRLNRVDRKSQIKCLSKCRE LLSEGASVLFFPEGTRSKDGKIQDFKKGAFSVAAKAKVPVVPITLVGTGRIMPNKQESKMFPGNVHVIVHPRVEPKD PDAMMAEAYDLIADPYYIERRQTE

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as Protection scope of the present invention.

SEQUENCE LISTING

<110>Shanghai Ocean University

<120>Incise edge green alga lysophosphatidate acyltransferase gene and its application

<130> /

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 1392

<212> DNA

<213> Myrmecia incisa

<400> 1

aagcagtggt atcaacgcag agtacatggg gacatttgcg ttgatcaagg tcactgcagc 60

acgcgtcctg caaacgacaa gcagcgccct gtacgctgcc tttgtgtagc agctgtgcag 120

tttcacttgc agttcaggca tgcacatgaa gccctgctat cactcttccc acctacagca 180

ccattcctgc ggactgcagc ctggcaacca gcccatctac agagttcaaa gccgtttagc 240

gcccacagcg cccagcccaa ggccatgcca gcacaggcgg caaacttgca ggctgtcctc 300

aacaccaagc ctgccaaggc gtacaaggaa gcaaatggct tgctcagcat cctccttagc 360

tctggctgtt gacccaccgt ccccagaaag gcatagagca gagaccatac tagcaagcgt 420

taaggggctg cttttctacc tatggacctt caccctgtcc ctgccgctgt tcgtgaccat 480

gctcgtcatt gcacccttca tggccgtcct tgacaagcat agacggcttg cacagcactt 540

tgtgaacaac atctgggcaa aggtgtcaac ctggcccttc taccgagtac ggattgatgg 600

gcgacacaac ctgcccaaga atgatgagcc agctgtctat gtagccaatc atgcgagttt 660

cctggacatc tatacgctgt tccatctcaa ccgctcgttc aagttcatct ccaagaccag 720

caacttcctc atcccaatca ttggctggag catgttcctg acagggcatg tccggctgaa 780

ccgcgtggac cgcaagagcc agatcaagtg cctgtccaag tgccgcgagc tgctttcgga 840

gggcgcctcc gtgctgttct tccccgaggg cacgcgcagc aaggacggga agatccagga 900

cttcaaaaag ggcgcgttca gcgtggctgc caaggccaag gtgccggttg tgcccatcac 960

gctggtgggc accggacgca tcatgcccaa caagcaggag tccaaaatgt tcccgggcaa 1020

tgtgcatgtg attgtccatc cgcgcgtgga gccgaaggat cctgacgcca tgatggctga 1080

ggcatacgac ctgatcgcag atccatacta catcgagcgc aggcagacgg agtgatgata 1140

tcagataagc atgttcaaga ataaagggtg ttgcaaggca tgcccccaag cgatgcgcca 1200

ggattgctgg ttttgtttcg tgacccaggc acaggttggc atgcttgggt aacacacgct 1260

tggttgcggt agcaagggct gtgttggggt gcaaactgtg aatgcttaca ggtgactgct 1320

tcagttgtag agtctctgac ttctgtgtac tgacatgtaa tgtgtgggat ttctgaaaaa 1380

aaaaaaaaaa aa 1392

<210> 2

<211> 331

<212> PRT

<213> Myrmecia incisa

<400> 2

Met His Met Lys Pro Cys Tyr His Ser Ser His Leu Gln His His Ser

1 5 10 15

Cys Gly Leu Gln Pro Gly Asn Gln Pro Ile Tyr Arg Val Gln Ser Arg

20 25 30

Leu Ala Pro Thr Ala Pro Ser Pro Arg Pro Cys Gln His Arg Arg Gln

35 40 45

Thr Cys Arg Leu Ser Ser Thr Pro Ser Leu Pro Arg Arg Thr Arg Lys

50 55 60

Gln Met Ala Cys Ser Ala Ser Ser Leu Ala Leu Ala Val Asp Pro Pro

65 70 75 80

Ser Pro Glu Arg His Arg Ala Glu Thr Ile Leu Ala Ser Val Lys Gly

85 90 95

Leu Leu Phe Tyr Leu Trp Thr Phe Thr Leu Ser Leu Pro Leu Phe Val

100 105 110

Thr Met Leu Val Ile Ala Pro Phe Met Ala Val Leu Asp Lys His Arg

115 120 125

Arg Leu Ala Gln His Phe Val Asn Asn Ile Trp Ala Lys Val Ser Thr

130 135 140

Trp Pro Phe Tyr Arg Val Arg Ile Asp Gly Arg His Asn Leu Pro Lys

145 150 155 160

Asn Asp Glu Pro Ala Val Tyr Val Ala Asn His Ala Ser Phe Leu Asp

165 170 175

Ile Tyr Thr Leu Phe His Leu Asn Arg Ser Phe Lys Phe Ile Ser Lys

180 185 190

Thr Ser Asn Phe Leu Ile Pro Ile Ile Gly Trp Ser Met Phe Leu Thr

195 200 205

Gly His Val Arg Leu Asn Arg Val Asp Arg Lys Ser Gln Ile Lys Cys

210 215 220

Leu Ser Lys Cys Arg Glu Leu Leu Ser Glu Gly Ala Ser Val Leu Phe

225 230 235 240

Phe Pro Glu Gly Thr Arg Ser Lys Asp Gly Lys Ile Gln Asp Phe Lys

245 250 255

Lys Gly Ala Phe Ser Val Ala Ala Lys Ala Lys Val Pro Val Val Pro

260 265 270

Ile Thr Leu Val Gly Thr Gly Arg Ile Met Pro Asn Lys Gln Glu Ser

275 280 285

Lys Met Phe Pro Gly Asn Val His Val Ile Val His Pro Arg Val Glu

290 295 300

Pro Lys Asp Pro Asp Ala Met Met Ala Glu Ala Tyr Asp Leu Ile Ala

305 310 315 320

Asp Pro Tyr Tyr Ile Glu Arg Arg Gln Thr Glu

325 330

<210> 3

<211> 324

<212> PRT

<213> Myrmecia incisa

<400> 3

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Ser Met Leu Ser Ser Thr

35 40 45

Pro Ser Leu Pro Arg Arg Thr Arg Lys Gln Met Ala Cys Ser Ala Ser

50 55 60

Ser Leu Ala Leu Ala Val Asp Pro Pro Ser Pro Glu Arg His Arg Ala

65 70 75 80

Glu Thr Ile Leu Ala Ser Val Lys Gly Leu Leu Phe Tyr Leu Trp Thr

85 90 95

Phe Thr Leu Ser Leu Pro Leu Phe Val Thr Met Leu Val Ile Ala Pro

100 105 110

Phe Met Ala Val Leu Asp Lys His Arg Arg Leu Ala Gln His Phe Val

115 120 125

Asn Asn Ile Trp Ala Lys Val Ser Thr Trp Pro Phe Tyr Arg Val Arg

130 135 140

Ile Asp Gly Arg His Asn Leu Pro Lys Asn Asp Glu Pro Ala Val Tyr

145 150 155 160

Val Ala Asn His Ala Ser Phe Leu Asp Ile Tyr Thr Leu Phe His Leu

165 170 175

Asn Arg Ser Phe Lys Phe Ile Ser Lys Thr Ser Asn Phe Leu Ile Pro

180 185 190

Ile Ile Gly Trp Ser Met Phe Leu Thr Gly His Val Arg Leu Asn Arg

195 200 205

Val Asp Arg Lys Ser Gln Ile Lys Cys Leu Ser Lys Cys Arg Glu Leu

210 215 220

Leu Ser Glu Gly Ala Ser Val Leu Phe Phe Pro Glu Gly Thr Arg Ser

225 230 235 240

Lys Asp Gly Lys Ile Gln Asp Phe Lys Lys Gly Ala Phe Ser Val Ala

245 250 255

Ala Lys Ala Lys Val Pro Val Val Pro Ile Thr Leu Val Gly Thr Gly

260 265 270

Arg Ile Met Pro Asn Lys Gln Glu Ser Lys Met Phe Pro Gly Asn Val

275 280 285

His Val Ile Val His Pro Arg Val Glu Pro Lys Asp Pro Asp Ala Met

290 295 300

Met Ala Glu Ala Tyr Asp Leu Ile Ala Asp Pro Tyr Tyr Ile Glu Arg

305 310 315 320

Arg Gln Thr Glu

<210> 4

<211> 23

<212> DNA

<213>Artificial sequence

<400> 4

ctatcactct tcccacctac agc 23

<210> 5

<211> 24

<212> DNA

<213>Artificial sequence

<400> 5

atgtagtatg gatctgcgat cagg 24

<210> 6

<211> 25

<212> DNA

<213>Artificial sequence

<400> 6

cagggtgaag gtccataggt agaaa 25

<210> 7

<211> 25

<212> DNA

<213>Artificial sequence

<400> 7

tggtctctgc tctatgcctt tctgg 25

<210> 8

<211> 25

<212> DNA

<213>Artificial sequence

<400> 8

aaccgctcgt tcaagttcat ctcca 25

<210> 9

<211> 25

<212> DNA

<213>Artificial sequence

<400> 9

cacaacctgc ccaagaatga tgagc 25

<210> 10

<211> 24

<212> DNA

<213>Artificial sequence

<400> 10

cagtggtatc aacgcagagt acat 24

<210> 11

<211> 25

<212> DNA

<213>Artificial sequence

<400> 11

acacagaagt cagagactct acaac 25

<210> 12

<211> 25

<212> DNA

<213>Artificial sequence

<400> 12

aagcttatgc acatgaagcc ctgct 25

<210> 13

<211> 26

<212> DNA

<213>Artificial sequence

<400> 13

ctcgagtcac tccgtctgcc tgcgct 26

Claims (10)

1. a kind of polypeptide, which is characterized in that the amino acid sequence of the polypeptide is selected from any one of following：

a)SEQ ID NO:Amino acid sequence shown in 2；

b)SEQ ID NO:Amino acid sequence shown in 52-331 of 2；

c)SEQ ID NO:Amino acid sequence shown in 3.

2. a kind of nucleotide sequence of separation, which is characterized in that described is nucleotide sequence coded described in claim 1 more Peptide.

3. nucleotide sequence according to claim 2, which is characterized in that its sequence of the nucleotide sequence contains following Any one of：

A) nucleotide sequence shown in SEQ ID NO.1；

b)SEQ ID NO:Nucleotide sequence shown in 140-1135 of 1；

c)SEQ ID NO:Nucleotide sequence shown in 293-1135 of 1；

D) SEQ ID NO are included at least:Nucleotide sequence shown in 293-1135 of 1, and with SEQ ID NO:1 has 70% or more homology；

E) with it is above a), b), c) or d) described in nucleotide sequence complementation nucleotide sequence.

4. nucleotide sequence according to claim 3, which is characterized in that its sequence of the nucleotide sequence includes at least SEQ ID NO:Nucleotide sequence shown in 293-1135 of 1, and with SEQ ID NO:1 have 80% or more it is homologous Property.

5. nucleotide sequence according to claim 4, which is characterized in that its sequence of the nucleotide sequence includes at least SEQ ID NO:Nucleotide sequence shown in 293-1135 of 1, and with SEQ ID NO:1 have 90% or more it is homologous Property.

6. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier is by the nucleotide described in claim 2 Sequence and the recombinant expression carrier constructed by plasmid or virus.

7. a kind of genetically engineered host cell, which is characterized in that the host cell is selected from one of the following：

A) host cell of the nucleotide sequence conversion or transduction described in claim 2 is used；

B) host cell of the recombinant expression carrier conversion or transduction described in claim 6 is used.

8. host cell according to claim 7, which is characterized in that the host cell is that bacterial cell, fungi are thin Born of the same parents, the plant cell for not having cellular omnipotency or the zooblast for not having cellular omnipotency.

9. the recombination described in any nucleotide sequence of polypeptide described in claim 1, claim 2-5, claim 6 Purposes of the host cell in increasing grease yield described in expression vector, claim 7 or 8.

10. purposes according to claim 9, which is characterized in that the increase grease yield, which refers to, increases phosphatide acid yield Or triacylglycerol yield.