CN1570116A

CN1570116A - Rhizopus arrhizus delta[12]-fatty acid dehydrogenase nucleic acid sequence and its uses

Info

Publication number: CN1570116A
Application number: CNA2004100192332A
Authority: CN
Inventors: 李明春; 邢来君; 魏东盛; 张欣昕
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2004-05-13
Filing date: 2004-05-13
Publication date: 2005-01-26
Anticipated expiration: 2024-05-13
Also published as: CN1263857C

Abstract

The invention relates to nucleotides sequence coding delta 12- fatty acid dehydrogenase derived from R.arrhizus, which is a nucleotide sequence SEQ ID NO:1 indicated or the fragment, analogue or derivative of the nucleotide sequence. The invention comprises vector which codes connected delta 12- fatty acid dehydrogenase nucleotide sequence with exogenous regulating sequence, and express in functionality, cell organism and filial generation of the organism. The invention also comprises unsaturated fatty acid production method using the said nucleotide sequence, polypeptide sequence, cell organism and its filial generation, and use of SEQ ID NO:1 nucleotide sequence as probes for identifying the related sequence.

Description

Rhizopus arrhizus △ 12The nucleotide sequence of-fatty acid dehydrogenase and application thereof

Technical field

The invention belongs to biological technical field and field of genetic engineering, relate to clone △ the rhizopus arrhizus (Rhizopus arrhizus) from a kind of filamentous fungus-Rhizopus ¹²-fatty acid dehydrogenase gene specifically is rhizopus arrhizus △ ¹²The nucleotide sequence of-fatty acid dehydrogenase and application thereof.This gene directly or with different expression vectors is connected, be transferred in bacterium, yeast, plant or the animal, utilize its coding △ ¹²-fatty acid dehydrogenase produces unsaturated fatty acids.

Background technology

△ ¹²-fatty acid dehydrogenase belongs to a class film intergrase.Up to the present, owing to separate and identify difficulty very big [MaKeon, 1981, Enzymology method, the 12141-12147 of embrane-associated protein; Wang etc., 1988, plant physiology biological chemistry, 26:777-792], therefore be difficult to obtain comprising △ ¹²-fatty acid dehydrogenase is at the higher structure of interior various embrane-associated protein character desaturases, and can only carry out preliminary study by the nucleotide sequence to enzyme, perhaps expresses the substrate specificity of studying enzyme in allogenic acceptor.The result of sequence alignment shows, coding △ ¹²The nucleotide sequence of-fatty acid dehydrogenase has the common constitutional features: have three Histidine conserved regions His I district HECGH, His II district HXXHH and His III district HVXHH, form structure [the Kyte et al. that strides film for 4 times, 1982, J.Mol.Biol.157:105-132], these all are to keep necessary [the Napier JA of dehydrogenase activity, Sayanova O, Stobart AK, et al., 1997, Biochem.J., 328:717-720].Comprise in people's the mammalian body owing to lack △ ¹²-fatty acid dehydrogenase and can only tentatively synthesize saturated and monounsaturated fatty acids, can not synthesize polyunsaturated fatty acid (the polyunsaturated fatty acids that contains two keys more than two and two, PUFAs), these polyunsaturated fatty acids must obtain from food.Therefore, linolic acid (linoleic acid, LA) and alpha-linolenic acid (α-linoleic acid ALA) is essential fatty acid.Meals absorption linolic acid and alpha-linolenic acid are through △ ⁶-fatty acid dehydrogenase is catalytically converted into gamma-linolenic acid (γ-linoleic acid, GLA) and therapic acid (Octadecatetraenoic acid, OTA), they can further change into other long chain polyunsaturated fatty acids (long-chain polyunsaturated fatty acids again under the catalysis of other relevant enzyme, LC-PUFAs) [Horrobin DF, 1992, Prog Lipid Res., 31:163-194].These long chain polyunsaturated fatty acidss are body tissue microbial film moietys, play the effect of keeping the cell normal function and increasing body resistance, simultaneously also be precursor [the Napier JA etal. that prostaglandin(PG), prostacyclin and interleukin class etc. have self instrumentality of strong physiologically active, 1999, Curr.Opin.Plant Biol., 2:123-127], the polyunsaturated fatty acid that therefore comprises linolic acid health care and medical aspect very wide application prospect is arranged.The synthetic physiological action with it of research unsaturated fatty acids becomes one of current focus.

For the biological function of fatty acid dehydrogenase, many laboratories have been adopted the gene that the clone is relevant from various organisms and have been changed in the various acceptors, observe the method for the lipid acid change of acceptor in acceptor.At present, from different sourcess such as animal, plant, microorganism, be separated to many △ ¹²The homologous sequence of-lipid acid dehydrogenation gene, and proof has corresponding biological function.Especially in recent years along with difunctional △ ¹²The discovery of-fatty acid dehydrogenase (Edgar B.Cahoon etal, 2001, THE JOURNAL OF BIOLOGICAL CHEMISTRY., 26:2637-2643.) with the excavation of conjugation oleic acid at medical health care function, people's attention begins again to turn to △ ¹²In the research of-fatty acid dehydrogenase.

Summary of the invention

An object of the present invention is to provide separated coding △ from the rhizopus arrhizus of filamentous fungus Rhizopus ¹²The nucleotide sequence of-fatty acid dehydrogenase or the fragment of its nucleotide sequence, analogue or derivative.Clone △ from a kind of rhizopus arrhizus (Rhizopus arrhizus) of filamentous fungus-Rhizopus ¹²-fatty acid dehydrogenase gene specifically is rhizopus arrhizus △ ¹²The nucleotide sequence of-fatty acid dehydrogenase and application thereof.This gene directly or with different expression vectors is connected, be transferred in bacterium, yeast, plant or the animal, utilize its coding △ ¹²-fatty acid dehydrogenase produces the methods and applications of unsaturated fatty acids.

Another object of the present invention provides the coded △ of this nucleotide sequence ¹²-fatty acid dehydrogenase polypeptide or its fragment, analogue or derivative.

Another object of the present invention provides and contains this gene nucleotide series and allos and regulate sequence and be connected, and carries out the recombinant vectors of functional expression.

Another object of the present invention provides and contains this gene nucleotide series or this gene nucleotide series and allos and regulate host cell and the offspring thereof that recombinant vectors that sequence is connected transforms or transduces.

Another object of the present invention provides a kind of usefulness and contains this gene nucleotide series or this gene nucleotide series and allos and regulate host cell and progeny cell or the coded △ of this nucleotide sequence that recombinant vectors that sequence is connected transforms or transduces ¹²-fatty acid dehydrogenase polypeptide prepares the method for unsaturated fatty acids.

A first aspect of the present invention provides fragment, analogue and derivative with the nucleotide sequence shown in the SEQ ID NO:1 or this nucleotide sequence.

Isolating △ ¹²The nucleotide sequence of-fatty acid dehydrogenase gene, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 65% homology with a kind of nucleotides sequence that is selected from down group: (1) has the nucleotide sequence of the active polypeptide of coding SEQ ID NO:2 aminoacid sequence; (2) with nucleotide sequence (1) complementary nucleotide sequence.Exactly, this nucleotide sequence has the nucleotide sequence shown in the SEQ ID NO:1.More accurately, this nucleotide sequence is to be selected from down a kind of in the group: (a) have the sequence of 1-1315 in the SEQ ID NO:1 sequence: and (b) have the sequence of 65-1234 among the SEQ ID NO:1.

In a second aspect of the present invention, isolating this nucleotide sequence encoded polypeptide is provided, this polypeptide comprises: polypeptide or its fragment or its conservative property variation polypeptide or its derived analogs with SEQ ID NO:2 aminoacid sequence.Exactly, this polypeptide is that polypeptide or its amino acid variation with SEQ ID NO:2 aminoacid sequence is no more than 30% derivative.

A third aspect of the present invention provides the recombinant vectors that contains above-mentioned nucleotide sequence, and by above-mentioned nucleotide sequence or recombinant vectors transformed host cells and progeny cell thereof.

A fourth aspect of the present invention provides a kind of usefulness to contain this gene nucleotide series or this gene nucleotide series and allos and has regulated recombinant vectors that sequence is connected transform or transduce host cell and progeny cell thereof or with the coded △ of this nucleotide sequence ¹²-fatty acid dehydrogenase polypeptide prepares the method for unsaturated fatty acids.

Other aspects of the present invention are because disclosing of this paper technology is conspicuous to those skilled in the art.

As used herein, " isolating " are meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.For example, nucleotide sequence and polypeptide under the native state in the active somatic cell do not have separation and purification, but same nucleotide sequence or polypeptide as from native state with separating in other material that exists, then be separation and purification.

As used herein, " isolating nucleotide sequence " is meant and is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be with the DNA purification technique purifying of standard.

The invention provides a kind of new isolating nucleotide sequence---coding rhizopus arrhizus △ ¹²The nucleotide sequence of-fatty acid dehydrogenase, it is made up of nucleotide sequence shown in the SEQ ID NO:1 basically, it is characterized in that: this sequence is long to be 1315bp (base), and wherein 65bp-1234bp is coding △ ¹²The open reading frame of-fatty acid dehydrogenase mature polypeptide SEQ ID NO:2; 1bp-64bp is the non-region sequence of translating of 5`, and 1235bp-1315bp is the non-region sequence of translating of 3`.

The invention provides isolating nucleotide sequence, this nucleotide sequence is made up of the nucleotide sequence that coding has an active polypeptide of SEQ ID NO:2 aminoacid sequence substantially.Particularly, nucleotide sequence of the present invention has the nucleotide sequence of SEQ ID NO:1.

The nucleotide sequence of coding SEQ ID NO:2 active polypeptide comprises: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " coding rhizopus arrhizus △ ¹²The nucleotide sequence of-fatty acid dehydrogenase " be meant comprise coding rhizopus arrhizus △- ¹²The nucleotide sequence of fatty acid dehydrogenase polypeptide and comprise additional code and/or noncoding nucleotide sequence.

Nucleotide sequence of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can with the identical or varient of degeneracy of coding region sequence shown in the SEQ ID NO:2.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The present invention also comprises coding rhizopus arrhizus △ ¹²The fragment of the nucleotide sequence of-fatty acid dehydrogenase, analogue or derivative.As used herein, term " fragment ", " analogue " are meant to encode with " derivative " and keep the natural identical biological function of the present invention or the nucleotide sequence of active polypeptide basically.

By the elaboration of this paper, such fragment, analogue or derivative are considered within those skilled in the art's ken.

The fragment of nucleotide sequence of the present invention, derived sequence or similar sequence can be that wherein one or more Nucleotide replace, the nucleotide sequence of disappearance, insertion, inversion, i.e. the artificial mutant of original separation sequence, and it also has required enzymatic functions.It can also be other gene Fusion sequence of described nucleotide sequence and fatty acid biological synthetic.

The allelic variant that derivative shown in SEQ ID NO:1 or functional derivatives are represented, it has at least 75% homology at the deutero-amino acid levels, preferred at least 80% homology, preferred especially 85% homology, very special preferably 90% homology.Described homology is based on complete amino acid fragment calculating.Shown in SEQ ID NO:2, homology is represented identity by described nucleotide sequence coded aminoacid sequence, and aminoacid sequence at least 70% is identical in other words.Described new nucleotide sequence has 65% homology at least on nucleic acid level, preferred at least 70% homology, preferred especially 75% homology, very special preferably 80% homology.

Derivative is also represented the homologue of sequence shown in the SEQ ID NO:1, as the sequence of eucaryon homologue, brachymemma, but still have required function, have this proteic enzymatic activity in other words, therefore, functionally equivalent comprise above-mentioned sequence natural variant and artificial sequence oligodeoxynucleotide.

Noncoding derivative also represents to can be used for suppressing the biosynthetic antisense DNA of described novel protein.Described antisense DNA belongs to no function derivative of the present invention.As not possessing the derivative of enzymatic activity.Available those skilled in the art knows other method of producing no function derivative has common inhibition, the use of ribozyme and intron.

The invention still further relates to and the nucleotide sequence of above-mentioned sequence hybridization (has at least 50% homology between two sequences, preferred at least 70% homology), and interfertile nucleotide sequence coded polypeptide has identical biological function with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, the length of " nucleic acid fragment " contain 10 Nucleotide at least, 20-30 Nucleotide at least preferably, and 50-60 Nucleotide at least especially preferably is very especially preferably more than at least 100 Nucleotide.Nucleotide fragments also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the separation nucleotide sequence coding.

Polypeptide among the present invention and nucleotide sequence preferably provide with isolating form, more preferably are purified to homogeneous.

Specific nucleotide sequence can obtain with several different methods among the present invention.For example, with hybridization technique separating nucleotide sequence well known in the art.These technology including, but not limited to: (1) with probe and genome or the hybridization of cDNA library to detect the homologous nucleotide sequence; (2) antibody screening of expression library is to detect the nucleotide sequence fragment of the clone with common structure feature.

Sequence dna fragment of the present invention also can obtain with following method: (1) separates double chain DNA sequence from genomic dna; (2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of dna sequence dna.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, also can obtain from commercial channels with test kit.And the construction cDNA library also is a usual method.When in conjunction with polymerase chain reaction technology (PCR), even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries, these methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or disappearance; (3) level of the transcript of Ce Dinging; (4) measure biologic activity by immunological technique, detect the protein product of genetic expression.Aforesaid method can singly be used, but also several different methods combined utilization.In (1) in the method, hybridize used probe and be any a part of homology with nucleotide sequence of the present invention, at least 10 Nucleotide of its length, preferably at least 30 Nucleotide, especially preferably at least 50 Nucleotide, very especially preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.The normally dna sequence dna of chemosynthesis on the basis of gene order information of the present invention of probe that gets used herein.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

The method of application round pcr DNA amplification/RNA (Saiki et al., 1985, Science 230:1350-1354) is optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can be according to nucleotide sequence information appropriate selection of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also provides a kind of new peptide sequence-rhizopus arrhizus △ ¹²The aminoacid sequence of-fatty acid dehydrogenase, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology (for example, to produce in bacterium, yeast, higher plant, insect and the mammalian cell from protokaryon or eucaryon host.

The △ that polypeptide of the present invention comprises ¹²The fragment of-fatty acid dehydrogenase, derivative and analogue.As used herein, term " fragment ", " analogue " are meant with " derivative " and keep natural identical biological function of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) a kind of like this, wherein one or more amino-acid residues are by conservative or non-conservative amino acid residues (preferably conservative amino acid residues) replacement, inversion, insertion or disappearance; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, and wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).

The present invention also relates to contain coding rhizopus arrhizus △ ¹²The nucleotide sequence of-fatty acid dehydrogenase and exogenous adjusting sequential element are in conjunction with the recombinant expression vector that carries out functional expression.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carrier.The sequential element that can influence gene expression product includes replication origin, promotor, marker gene and translational control element.

Available method well-known to those having ordinary skill in the art makes up and contains coding rhizopus arrhizus △ ¹²The expression vector of the nucleotide sequence of-fatty acid dehydrogenase and suitable transcribing/translational control element.These methods comprise [Sambroook, et al., Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory, New York, 1989] such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described coding rhizopus arrhizus △ ¹²The nucleotide sequence of-fatty acid dehydrogenase can effectively be connected on the appropriate promotor of expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The P of lambda particles phage _LPromotor: eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and common nearly 10-300bp acts on promotor transcribing with enhancing gene.As the adenovirus enhanser.

The invention still further relates to containing the present invention rhizopus arrhizus △ that encodes ¹²The recombinant vectors of the nucleotide sequence of-fatty acid dehydrogenase or directly with coding rhizopus arrhizus △ ¹²The host cell that the nucleotide sequence of-fatty acid dehydrogenase produces through genetically engineered.Among the present invention, coding rhizopus arrhizus △ ¹²The nucleotide sequence of-fatty acid dehydrogenase or the recombinant vectors that contains this nucleotide sequence can transform or transduce into host cell, contain the genetically engineered host cell of this nucleotide sequence or recombinant vectors with formation.Term " host cell " refers to prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.The representative example of host cell has: intestinal bacteria; Fungal cell such as yeast; Vegetable cell such as rape, tobacco, soybean; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

Can carry out with method well-known to those having ordinary skill in the art with nucleotide sequence of the present invention or the recombinant vectors transformed host cell that contains nucleotide sequence.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be collected thalline in exponential phase of growth, uses CaCl ₂Method is handled, and used step is well known in the art.Also available MgCl ₂, methods such as electroporation are carried out.When the host is an eukaryote, can select methods such as DNA infection protocol, microinjection, electroporation, liposome packing for use.

The invention still further relates to above-mentioned genetically modified host cell and produce lipid acid, according to the difference of host cell, with those skilled in the art method growth or cultivation in common knowledge.Normally at 0-100 ℃, preferred 10-60 ℃, also want oxygen simultaneously such as microorganism cells.Contain carbon source in the substratum, as glucose, nitrogenous source, the form of organonitrogen normally, as yeast extract, amino acid, or salt, as ammonium sulfate, trace element as iron, magnesium salts, also has VITAMIN if necessary.The pH of the substratum value that can be maintained fixed in other words, is controlled in the training period or is not controlled during this period.Cultivation can batch culture, half discontinuous cultivation or cultured continuously form are carried out.After cultivating, collecting cell is smashed to pieces or directly use, extracts lipid acid by method well known to those skilled in the art from cell.

The invention still further relates to a kind of method for preparing unsaturated fatty acids, this method is achieved in that cultivates the triglyceride level with saturated or unsaturated fatty acids with SEQ ID NO:2.This method is preferably carried out under by the compound existence condition that can absorb or discharge the reductibility equivalent.Then, can release fat acid from triglyceride level.Aforesaid method preferably can synthesize the lipid acid with 12 two keys.

The invention still further relates to method for preparing unsaturated fatty acids (rhizopus arrhizus △ particularly ¹²And be applied to produce human food prods, animal-feed, makeup or medicine purposes the linolic acid that-fatty acid dehydrogenase catalysis oleic acid produces).

Description of drawings

The rhizopus arrhizus △ that Fig. 1, demonstration are inferred ¹²The hydrophobic figure of-fatty acid dehydrogenase, two solid lines represent two hydrophobic domains inferring.

Fig. 2, demonstration rhizopus arrhizus △ of the present invention ¹²-fatty acid dehydrogenase and volume branch Mucor △ ¹²The amino acid sequence homology comparison diagram of-fatty acid dehydrogenase (AB052087).

Fig. 3, the constructed yeast saccharomyces cerevisiae expression vector pYRAD12 of demonstration.

The gas chromatogram of Fig. 4 A, demonstration linolic acid methyl ester standard substance.

Fig. 4 B, contain the gas chromatogram of the transgenic yeast of pYES2.0 empty carrier.

Fig. 4 C, contain the gas chromatogram of the transgenic yeast of recombinant plasmid pYRAD12.

Embodiment

Further set forth the present invention below in conjunction with specific embodiment.Should understand these embodiment only is used to the present invention is described rather than limit the scope of the invention.

Embodiment 1 separates △ from rhizopus arrhizus ¹²The nucleotide sequence of-fatty acid dehydrogenase

According to (Liu Xiaoyong etc., 1997, the SDS-CTAB improved method of extraction plant and microbial DNA, Beijing Forestry University's journal, 100-103 such as Liu Xiaoyong.) method that provides, from cultivate 48 hours rhizopus arrhizus mycelium, extract total DNA, getting 5 μ g is that template is carried out the polymerase chain reaction.According to the △ that is delivered ¹²The Histidine conserved regions I of-fatty acid dehydrogenase homologous sequence and III region amino acid sequence design degenerate primer (primer 1 and primer 2), DNA with said extracted is that template increases at the enterprising performing PCR of T-Gradient PCR instrument (Biometra company), and reaction the primer, component and amplification condition are as follows: primer 1:5-CA (TC) GA (AG) TG (TC) GG (I) CA (TC) CA (CAG)-3`

Primer 2: 5` (AG) TG (AG) TGIGCIAC (GA) TGIGT-3`

Reactive component add-on final concentration

Template DNA 5 μ l

Damping fluid (10 *)

(contain 20mmol/LMgCl ₂) 5 μ l 1 *

dNTP(2.5mmol/L) 4μl 0.4mmol/L

Primer 1 (10 μ mol/L) 1 μ l 0.2 μ mol/L

Primer 2 (10 μ mol/L) 1 μ l 0.2 μ mol/L

Taqase (5u/ μ l) 0.5 μ l 2.5u/ reaction

H ₂O 33.5μl

Cumulative volume 50 μ l

Amplification condition: 94 ℃ of sex change 3min, use 94 ℃ of 1min again; 55 ℃ of 1min → 72 ℃ 1min carry out 30 circulations, last 72 ℃ of 10min.The agarose gel electrophoresis detected result shows, amplification obtains the fragment of the about 750bp of size, reclaim with UNIQ-10 pillar PCR product purification test kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product), reclaiming the fragment subclone in sequencing vector pGEM-T (Promega company product); The connection product is transformed into uses CaCl ₂The bacillus coli DH 5 alpha that method is handled, overnight incubation on the LB solid medium that contains penbritin (final concentration is 100 μ g/ml); The white colony of growing on the picking flat board, access contains overnight incubation in the LB liquid nutrient medium of penbritin (final concentration is 100 μ g/ml), centrifugal collection thalline is pressed alkaline lysis [Sambroook, et al., 1989, Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory, New York, p19-21] extract plasmid, identify through NcoI and SacI double digestion and pcr amplification correct, order-checking (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).Sequencing result shows that the clip size that obtains that increases is 730bp, in the Genbank database, use Blast program (Basic local Alignment seatch tool) [Altschul SFet al by its amino acid sequence coded, 1997, Nucleic Acids Res.25:3389-3402] carry out the homology retrieval, result for retrieval shows that the homologous fragment the most similar to this fragment is △ ¹²-fatty acid dehydrogenase, (and contain a 46bp intron), but and incomplete same, prove that institute's amplified fragments is the fragment of new potential dehydrogenase gene.

According to the partial sequence design gene-specific primer (primer 3 and primer 4) that is obtained, (Rapid Amplification of cDNA ends, RACE) acquisition comprises the 3` and the 5` end sequence of above-mentioned segmental gene to utilize the terminal amplification technique of cDNA.Extract cell total rna earlier, use SMR ^TMArticle one cDNA is synthesized in RACE cDNA Amplification Kit (Clontech company product) reverse transcription, with the joint primer that gene-specific primer and test kit provide, carry out the PCR of 3` and 5` end sequence respectively by pcr amplification test kit (Clontech company product).

Primer 3 (3`RACE): 5`-CTGCTCTTACCGTTGACCG-3`, (SEQ ID NO:3)

Primer 4 (5`RACE): 5`-CATCAGCTTGAGGGTCTTTGICGCG-3` (SEQ ID NO:4)

Template cDNA 2.5 μ l, damping fluid (10 *) 5 μ l, dNTP (50 *) 1 μ l, primer 3 (10 μ mol/L) 1 μ l, primer 4 (10 μ mol/L) 1 μ l, Advantage2 Polymeerase Mix 1 μ l, H ₂O 34.5 μ l amplification conditions: 95 ℃ of sex change 2min of elder generation enter 95 ℃ of 30sec; 68 ℃ of 3min carry out 30 circulations altogether.Pcr amplification product adopts above-mentioned identical method to identify, check order.Be total to the sequence information of 328bp before The sequencing results demonstration 3`RACE amplification obtains from primer 3 sequences to polyA, and 5`RACE obtains from primer 4 sequences to the sequence information that is total to 567bp the end fitting sequence.With sequence analysis software (DNAMAN Version4.0, Lynnon BioSoft) three fragments is spliced and analyze, obtain the nucleotide sequence shown in SEQ ID NO:1, wherein 65bp-1234bp (ATG----TAA) is the potential open reading frame, 389 amino acid of encoding.Both sides are respectively non-district (64bp) and the non-district (81bp) that translates of 3` of translating of 5`.According to two end sequences, design gene-specific primer (primer 5 and primer 6) press 3` and 5`RACE condition pcr amplification, order-checking, the result with to splice the result consistent.

Primer 5:5`-GGTACCTCACCTCTCTCCCTTCTCT-3` (SEQ ID NO:5);

Primer 6:5`-GAATTCGAAATTGTATACATTTTATTG-3` (SEQ ID NO:6);

Embodiment 2: the homology search of institute's separating nucleotide sequence

To infer △ ¹²The aminoacid sequence of-fatty acid dehydrogenase carries out the homology search on Genbank, the gained homologous sequence is most of to be coding △ ¹²The sequence of-fatty acid dehydrogenase, minority coding △ ¹⁵-fatty acid dehydrogenase and other desaturase sequence, wherein the homology with the sequence that derives from volume branch Mucor is the highest: homogeny 80.05%, (accompanying drawing 2 shows rhizopus arrhizus △ of the present invention ¹²-fatty acid dehydrogenase and volume branch Mucor △ ¹²The amino acid sequence homology comparison diagram of-fatty acid dehydrogenase (AB052087).RAD12: rhizopus arrhizus △ of the present invention ¹²-fatty acid dehydrogenase, MCD12: volume branch Mucor (Mucor circinelloides) △ ¹²-fatty acid dehydrogenase; Identical amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+").This illustrates that the coded enzyme of new nucleotide sequence of the present invention has potential △ ¹²-fatty acid dehydrogenase function.

Embodiment 3: the structure of yeast saccharomyces cerevisiae recombinant expression vector

According to coding region sequence shown in the SEQ ID NO:1, design a pair of gene specific amplimer (primer 7 and primer 8) and separate its potential open reading frame sequence:

Primer 7:5`-CAC GGTACCATGGCAACCAAGAGAAATATCAGT-3` (SEQ ID NO:7);

Primer 8:5`-GGT GAATTCATTATTTTTGTAAAACACAACATC-3` (SEQ ID NO:8);

The 5` end black matrix of these two primers contains KpnI and EcoR1 restriction enzyme site respectively.Used amplification condition is identical with 5`RACE with 3` with reactive component, and the sequencing result of amplified production shows consistent with the sequence of 64bp-1230bp shown in the SEQ ID NO:1.Get 50 μ l PCR products then and 3 μ l pYES2.0 carry out double digestion respectively, reaction system is as follows:

PCR product double digestion reaction system pYES2.0 reaction system

Buffer 10μl 1μ1

BSA 10μl 1μl

Triton 10μl 1μl

Substrate 50 μ l 3 μ l

KpnI 2μl 2μl

EcoR1 2μl 2μl

H ₂O 16μl

Cumulative volume 100 μ l 10 μ l

0.8% sepharose reclaims enzyme and cuts big fragment, and connects with the T4 ligase enzyme.Connect product transformed into escherichia coli DH5 α, extract and the PCR screening positive clone by plasmid, and the evaluation of checking order.Plasmid construction the results are shown in accompanying drawing 3, the constructed △ that contains ¹²The yeast expression called after pYRAD12 of-fatty acid dehydrogenase gene.This plasmid is to be made up by the PCR product of pYES2.0 (Invitrogen company) and primer 7 and primer 8 to form.Plasmid and respectively behind KpnI and EcoR1 double digestion, electrophoresis reclaims purifying, connects through T4DNALigase, connects product transformed into escherichia coli DH5 α, the Screening and Identification matter example that goes out to recombinate, called after pYRAD12.

Embodiment 4: recombinant expression vector transformed saccharomyces cerevisiae cell

The single bacterium colony of picking Wine brewing yeast strain INVSc1 in 10ml YEPD liquid nutrient medium, 30 ℃ of shaking table incubated overnight, detect the OD of bacterium liquid ₆₀₀Value is got an amount of bacterium liquid and is diluted in 50ml, makes OD ₆₀₀Be 0.4; Continue to cultivate 2 behind 4h, the 2500rpm centrifugation cell, with the resuspended thalline of 40ml 1 * TE, the 2500rpm centrifugation cell with 2ml1 * LiAc/0.5 * TE suspension cell, and is at room temperature placed 10min, and this cell is competent cell; Get the yeast competent cell that 100 μ l prepare, add 1 μ g recombinant plasmid pYRAD12 and 100 μ g sex change salmon sperm DNAs (Sigma), mixing adds 700 μ l, 1 * LiAc/40%PEG-4000/1 * TE and mixing, in 30 ℃ of incubation 30min; Then, add 88 μ l DMSO mixings, heat shock 7min in 42 ℃ of water-baths; Centrifugal 10s sedimentation cell adds 1ml1 * TE suspension cell and recentrifuge sedimentation cell, adds 100 μ l re-suspended cells at last, is laid on SC-Ura (no uridylic) entirely and selects culture medium flat plate, puts 30 ℃ and cultivates 48-72h.

Embodiment 5: the abduction delivering of Yeast engineering bacteria

The positive that picking SC-Ura (no uridylic) selects to occur on the culture medium flat plate transforms, be inoculated in 10ml SC-Ura and select substratum (containing 2% glucose), 28 ℃ are shaken the bacterium incubated overnight, inoculum size with 5% adds the 100ml SC-Ura substratum that contains 2% semi-lactosi, and 28 ℃ are continued to cultivate 72h; 5000rpm, 10min collects thalline, uses deionized water wash three times, and 50 ℃ of oven dry grind, and get the KOH-CH that 100mg adds 5ml 5% ₃OH solution, 70 ℃ of reaction 4h; Reaction finishes, and cool to room temperature is with the pH value to 2.0 of the hydrochloric acid conditioning solution of 6mol/L, adding 4ml 14%BF ₃-CH ₃OH, 70 ℃ of reaction 1.5h, synthesizing fatty acid methyl ester; Add saturated NaCl solution 10ml again, the concuss mixing, and transfer in the separating funnel, use 1: 4 chloroform of 8ml: hexane extracting twice, united extraction liquid; Add an amount of anhydrous Na ₂SO ₄Dry extraction liquid leaves standstill 1h, removes Na ₂SO ₄, the supernatant liquor that contains fatty acid methyl ester is dried up with nitrogen, return molten sample with the normal hexane of 200 μ L, the back is with the filtering with microporous membrane of 0.45mm.

Embodiment 6: the lipid acid gas chromatographic analysis

Undertaken by following condition:

Instrument is Tianjin, island GC-7A, pillar: fused-silica capillary column, 0.32 * 30m, solid support: Dienthyeneglycol succinate (Poly-diethylene glycol succinate, DEGS) plated film thing: polyimide.Carrier gas: N ₂, linear speed: 10cm/s.Splitting ratio: 100: 1, the vaporizer temperature: 250 ℃, column temperature: 180 ℃, tail blew: 50ml/min, detector: flame ionization ditector.The LA methyl esters of producing with Sigma company is standard substance, the sample of the methyl esterification of fatty acid of method for preparing, carries out GC and analyzes, and applied sample amount is 1 μ l; Analysis software: Anstar, the star color spectrum workstation of analysis.

Stratographic analysis the results are shown in accompanying drawing 4, and accompanying drawing 4 shows methyl linoleate standard substances (4A), contain the transgenic yeast (4B) of pYES2.0 empty carrier and contain the gas chromatogram of the transgenic yeast (4C) of recombinant plasmid pYRAD12.Compare by retention time and to identify each peak with known fatty acid methyl ester standard substance.Retention time is that the peak of 14.637min correspondence is rhizopus arrhizus △ among Fig. 4 C ¹²The linolic acid that-fatty acid dehydrogenase catalysis oleic acid produces.

Sequence list

SEQUENCE?LISTING

＜110〉Nankai University

＜120〉nucleotide sequence and the application thereof of rhizopus arrhizus △ 12-fatty acid dehydrogenase

<130>2004.4.28

<160>8

<170>PatentIn?version?3.1

<210>1

<211>1315

<212>DNA

＜213〉rhizopus arrhizus (Rhizopus arrhizus)

<400>1

tcacctctct?cccttctctt?ttaataactt?ttctctttct?agaagaaaga?cataattagg 60

gataatggca?accaagagaa?atatcagttc?caatgaacca?gaaaataagc?ctgttatcga 120

cgaagcagta?gcaagaaact?gggagattcc?tgattttacc?atcaaagaaa?ttcgtgatgc 180

tattccttct?cactgcttcc?gtcgagacac?attcagatca?ttcacttatg?ttattcatga 240

ttttgctatt?atcgccgtct?tgggttattt?agctacttac?attgatcaag?ttcattctgc 300

tgctcttcgc?ttgcttttat?ggtccttgta?ttggactgct?caaggtattg?ttggtactgg 360

tgtttgggtt?gttggtcacg?aatgtggaca?tcaagctttc?agtccatcca?aggccgtcaa 420

taacagtgtc?ggctttgtcc?ttcatacact?cttattagtt?ccttatcact?cttggagatt 480

ctctcactct?aagcatcata?aagctactgg?tcacatgtca?aaagaccaag?ttttccttcc 540

caagacaaga?gaaaaggttg?gtttaccacc?tcgcgacaaa?gaccctcaag?ctgatggtcc 600

tcatgatgtt?cttgacgaaa?cacctattgt?tgtactttac?cgtatgtttc?ttatgttctt 660

gtttggctgg?ccattatacc?ttttcaccaa?tgtcaccggt?caagattacc?ctggctgggc 720

ctctcacttc?aacccatcct?gcgacattta?cgaagagggc?caatattggg?atgtcgtcag 780

ttcctctgtt?ggtgttgttg?gcatggtagg?tcttttaggt?tactgtggtc?aaatctttgg 840

ttccttaaac?atgatcaaat?actatgttat?tccttacttg?tgtgttaact?tttggcttgt 900

cttgattact?tatttgcaac?acactgaccc?caaattgcct?cactaccgcg?agaatgtctg 960

gaacttccaa?cgtggtgctg?ctcttaccgt?tgaccgttct?tacggtgccc?ttattaatta 1020

tttccaccat?cacatttccg?acacccacgt?cgcccaccac?ttcttttcta?ctatgcctca 1080

ctatcatgct?gaagaagcta?ctgttcatat?caagaaagct?cttggtaagc?attaccactg 1140

tgataatact?cctattccca?tcgctctttg?gaaagtttgg?aagagctgta?gatttgttga 1200

aagtgaagga?gatgttgtgt?tttacaaaaa?ttaatttcca?ttacaccctc?ttttcatttt 1260

gatatataat?actttattct?accatctttc?cattcaataa?aatgtataca?atttc 1315

<210>2

<211>389

<212>PRT

＜213〉Roryzae (Rhizopus arrhizus)

<400>2

Met?Ala?Thr?Lys?Arg?Asn?Ile?Ser?Ser?Asn?Glu?Pro?Glu?Asn?Lys?Pro

1 5 10 15

Val?Ile?Asp?Glu?Ala?Val?Ala?Arg?Asn?Trp?Glu?Ile?Pro?Asp?Phe?Thr

20 25 30

Ile?Lys?Glu?Ile?Arg?Asp?Ala?Ile?Pro?Ser?His?Cys?Phe?Arg?Arg?Asp

35 40 45

Thr?Phe?Arg?Ser?Phe?Thr?Tyr?Val?Ile?His?Asp?Phe?Ala?Ile?Ile?Ala

50 55 60

Val?Leu?Gly?Tyr?Leu?Ala?Thr?Tyr?Ile?Asp?Gln?Val?His?Ser?Ala?Ala

65 70 75 80

Leu?Arg?Leu?Leu?Leu?Trp?Ser?Leu?Tyr?Trp?Thr?Ala?Gln?Gly?Ile?Val

85 90 95

Gly?Thr?Gly?Val?Trp?Val?Val?Gly?His?Glu?Cys?Gly?His?Gln?Ala?Phe

100 105 110

Ser?Pro?Ser?Lys?Ala?Val?Asn?Asn?Ser?Val?Gly?Phe?Val?Leu?His?Thr

115 120 125

Leu?Leu?Leu?Val?Pro?Tyr?His?Ser?Trp?Arg?Phe?Ser?His?Ser?Lys?His

130 135 140

His?Lys?Ala?Thr?Gly?His?Met?Ser?Lys?Asp?Gln?Val?Phe?Leu?Pro?Lys

145 150 155 160

Thr?Arg?Glu?Lys?Val?Gly?Leu?Pro?Pro?Arg?Asp?Lys?Asp?Pro?Gln?Ala

165 170 175

Asp?Gly?Pro?His?Asp?Val?Leu?Asp?Glu?Thr?Pro?Ile?Val?Val?Leu?Tyr

180 185 190

Arg?Met?Phe?Leu?Met?Phe?Leu?Phe?Gly?Trp?Pro?Leu?Tyr?Leu?Phe?Thr

195 200 205

Asn?Val?Thr?Gly?Gln?Asp?Tyr?Pro?Gly?Trp?Ala?Ser?His?Phe?Asn?Pro

210 215 220

Ser?Cys?Asp?Ile?Tyr?Glu?Glu?Gly?Gln?Tyr?Trp?Asp?Val?Val?Ser?Ser

225 230 235 240

Ser?Val?Gly?Val?Val?Gly?Met?Val?Gly?Leu?Leu?Gly?Tyr?Cys?Gly?Gln

245 250 255

Ile?Phe?Gly?Ser?Leu?Asn?Met?Ile?Lys?Tyr?Tyr?Val?Ile?Pro?Tyr?Leu

260 265 270

Cys?Val?Asn?Phe?Trp?Leu?Val?Leu?Ile?Thr?Tyr?Leu?Gln?His?Thr?Asp

275 280 285

Pro?Lys?Leu?Pro?His?Tyr?Arg?Glu?Asn?Val?Trp?Asn?Phe?Gln?Arg?Gly

290 295 300

Ala?Ala?Leu?Thr?Val?Asp?Arg?Ser?Tyr?Gly?Ala?Leu?Ile?Asn?Tyr?Phe

305 310 315 320

His?His?His?Ile?Ser?Asp?Thr?His?Val?Ala?His?His?Phe?Phe?Ser?Thr

325 330 335

Met?Pro?His?Tyr?His?Ala?Glu?Glu?Ala?Thr?Val?His?Ile?Lys?Lys?Ala

340 345 350

Leu?Gly?Lys?His?Tyr?His?Cys?Asp?Asn?Thr?Pro?Ile?Pro?Ile?Ala?Leu

355 360 365

Trp?Lys?Val?Trp?Lys?Ser?Cys?Arg?Phe?Val?Glu?Ser?Glu?Gly?Asp?Val

370 375 380

Val?Phe?Tyr?Lys?Asn

385

<210>3

<211>19

<212>DNA

＜213〉rhizopus arrhizus (Rhizopus arrhizus)

<400>3

ctgctcttac?cgttgaccg 19

<210>4

<211>25

<212>DNA

＜213〉rhizopus arrhizus (Rhizopus arrhizus)

<400>4

catcagcttg?agggtctttg?tcgcg 25

<210>5

<211>25

<212>DNA

＜213〉rhizopus arrhizus (Rhizopus arrhizus)

<400>5

ggtacctcac?ctctctccct?tctct 25

<210>6

<211>27

<212>DNA

＜213〉rhizopus arrhizus (Rhizopus arrhizus)

<400>6

gaattcgaaa?ttgtatacat?tttattg 27

<210>7

<211>33

<212>DNA

＜213〉rhizopus arrhizus (Rhizopus arrhizus)

<400>7

cacggtacca?tggcaaccaa?gagaaatatc?agt 33

<210>8

<211>33

<212>DNA

＜213〉rhizopus arrhizus (Rhizopus arrhizus)

<400>8

ggtgaattca?ttatttttgt?aaaacacaac?atc 33

Claims

1, a kind of rhizopus arrhizus Δ ¹²The nucleotide sequence of-fatty acid dehydrogenase is characterized in that it is to have the nucleotide sequence shown in the SEQ IDNO:1, or the fragment of this nucleotide sequence, analogue or derivative.

2, according to the nucleotide sequence of claim 1, it is characterized in that described nucleotide sequence is to be selected from coding to have the aminoacid sequence shown in the SEQ ID NO:2, or with this nucleotide sequence complementary nucleotide sequence; Or the aforementioned nucleotide sequence that at least 65% homogeny is arranged.

3, a peptide species is characterized in that it is that polypeptide or its amino acid variation with the aminoacid sequence shown in the SEQ ID NO:2 is no more than 30%.

4, a kind of recombinant expression vector is characterized in that it is by the described nucleotide sequence of claim 1 and plasmid, virus or the constructed recombinant vectors of vehicle expression vector.

5,, it is characterized in that it is pYRAD12 according to the described recombinant expression vector of claim 4.

6, a kind of genetically engineered host cell is characterized in that it is to be selected from following a kind of host cell:

(a) it is the host cell that transforms or transduce with the described nucleotide sequence of claim 1;

(b) it is the host cell that transforms or transduce with the described recombinant expression vector of claim 4.

7, according to the described host cell of claim 6, it is characterized in that described host cell is bacterial cell, fungal cell, vegetable cell or zooblast, or the offspring of these host cells.

8,, it is characterized in that described host cell is a brewing yeast cell according to the described host cell of claim 6.

9, claim 1-8 each be used to produce the application of unsaturated fatty acids.

10,, it is characterized in that described unsaturated fatty acids is a linolic acid according to the application of the described unsaturated fatty acids of claim 9.