CN100413969C - Chimeric gene with delta 6 fatty acid dehydrogenase function and its use - Google Patents

Chimeric gene with delta 6 fatty acid dehydrogenase function and its use Download PDF

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CN100413969C
CN100413969C CNB2005100116085A CN200510011608A CN100413969C CN 100413969 C CN100413969 C CN 100413969C CN B2005100116085 A CNB2005100116085 A CN B2005100116085A CN 200510011608 A CN200510011608 A CN 200510011608A CN 100413969 C CN100413969 C CN 100413969C
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胡赞民
陆万香
胡军
陈宇红
尹维波
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Institute of Genetics and Developmental Biology of CAS
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Abstract

In the present invention, one segment RnD6A which is from ribes nigrum L. DNA and other two segments of RnD6A and RnD6B of about 208 bp are used for respectively constructing two chimeric genes of RnD6CA and RnD6CB with complete reading frames, and the RnD6CA and RnD6CB are expressed in a yeast expression system, GC-MS analysis indicates that protein coded in the two chimeric genes in yeast can catalyze external substrate linoleic acid to generate gamma-linoleic acid, and the gamma-linoleic acid has the function of delta <6> fatty acid dehydrogenase. The present invention can be used for adopting a genetic engineering technique to produce the gamma-linoleic acid by a yeast fermentation system and a plant expression system.

Description

Has Δ 6The mosaic gene of fatty acid dehydrogenase function and application thereof
Technical field
The present invention relates to plant code gene and application thereof.Particularly, relate to two mosaic gene RnD6CA and RnD6CB with entire reading frame frame, its respectively the origin three segment DNA sequence constructs that come from blackcurrant (Ribesnigrum L.) form.The invention still further relates to this mosaic gene coded have a Δ 6The polypeptide of fatty acid dehydrogenase function, and contain the Yeast expression carrier of this dna sequence dna and plant expression vector, host cell, and utilize the application of transformed yeast and plant production gamma-linolenic acid respectively of this gene.
Background technology
In the fatty acid metabolism process, Δ 6Fatty acid dehydrogenase (Δ 6-fatty aciddesaturase, D6D) catalysis linolic acid (linoleic acid, LA, 18:2 Δ 9,12) the 6th carbon atom dehydrogenation form gamma-linolenic acid (γ-linolenic acid, GLA, 18:3/ Δ 6,9,12) [1].Gamma-linolenic acid can further form arachidonic acid, Prostaglandins and Leukotrienes class physiologically active substance by carbochain prolongation and dehydrogenation again, and these products produce material impact in a series of physiological functions such as brain development, vision, anaphylaxis and cardiovascular motion of people.
As a kind of essential unsaturated fatty acids of human body, gamma-linolenic acid has reducing blood-fat, anti peroxidation of lipid, fat-reducing, inhibition ulcer, strengthens a series of biological functions [1,2] such as Regular Insulin, antithrombotic cardiovascular disorder human body.At present, gamma-linolenic acid has been used as a kind of new VITAMIN-vitamin F and has been studied utilization [3] in the world.Thereby, in producing as gamma-linolenic acid key enzyme---Δ 6 fatty acid dehydrogenases are also also more and more studied and are utilized.Up to now, the clone's acquisition from animal [4], plant [5], fungi [6] and nematode different biologies such as [7] of Δ 6 fatty acid dehydrogenase genes, and yeast saccharomyces cerevisiae [7,8], tomato [9], tobacco [5,10], successfully obtained expression in rape [6,8] and the soybean [11] etc.
In plant, only a few plants such as root of Redsepal Eveningprimrose (Oenothera sp.), Borrago officinalis (Borago officinalisL.) and blackcurrant contain GLA[12], the existing abroad research report of Δ 6 fatty acid dehydrogenase genes of Borrago officinalis [5] and root of Redsepal Eveningprimrose [13], and two fusion genes that this patent relates to are the reported first that derive from Δ 6 fatty acid dehydrogenase genes of blackcurrant.
Because what this genoid was coded is an albumen, can adopt the yeast saccharomyces cerevisiae expression system to carry out vivoexpression, but owing to this albumen is positioned on chloroplast(id) or the endoplasmic reticulum, and be a membranin, even in the vivoexpression system, express, also more difficult separation and purification.So general the employing investigated expressed albumen the method for the catalytic activity that adds substrate carried out Function Identification.
In the yeast saccharomyces cerevisiae expression system that is adopted, recipient bacterium is uridylic defective type INVSc I, itself do not contain LA (linolic acid) and GLA (gamma-linolenic acid), therefore, adding under the linoleic condition of substrate, if in Yeast engineering bacteria, detect the catalysate gamma-linolenic acid, just can determine that the coded albumen of foreign gene has obtained expression in yeast expression system, produced katalysis to adding substrate.So yeast saccharomyces cerevisiae is as the most frequently used, the effectively expressing system of Δ 6, the functional evaluation of Δ 12-fatty acid dehydrogenase gene of research external source, and existing many successful researchs reports [5,6,7].The detection of lipid acid mainly adopts GC/MS (gas-chromatography-matter coupling technique) to finish.Its principle is by GC (gas-chromatography) different fatty acid components to be separated, and respectively every kind of component is carried out the mass spectrometry analysis by MS (mass spectrum) detector then, thus the coupling analytical technology of the comparative maturity of the composition of definite each component and content.
In the plant, GLA exists only in a few plants such as root of Redsepal Eveningprimrose (Oenothera sp.), Borrago officinalis (Boragoofficinalis L.) and blackcurrant [12], and the seed oil production of these plants also becomes at present the primary commercial source of GLA in the world.Utilize the produce oil fungi fermentation also can extract and obtain GLA, but the GLA output that obtains by existing these modes of production is all very low, can not satisfy the growing market requirement [13] far away.Therefore, utilize yeast saccharomyces cerevisiae or transgenosis oil plant crop plant to express the external source Δ 6Fatty acid dehydrogenase is produced gamma-linolenic acid and is had important Research Significance and economic worth.
Summary of the invention
The invention provides two mosaic gene RnD6CA and RnD6CB, its nucleotide sequence is respectively shown in SEQ ID NO:1 and SEQ ID NO:2, these two mosaic genes are dna fragmentation RnD6C and the RnD6A that are used to come from blackcurrant (Ribes nigrum L.), reach RnD6C and RnD6B structure and form, encoded polypeptide is respectively shown in SEQ ID NO:6 and SEQ ID NO:7 (Fig. 6 and 7).
The invention provides three dna fragmentation RnD6C, RnD6A, RnD6B that derive from blackcurrant (Ribes nigrum L.), its nucleotide sequence is respectively shown in SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:5.
Another object of the present invention provides the Yeast expression carrier that contains mosaic gene RnD6CA, RnD6CB, and utilizes this expression vector transformed yeast cells.Polypeptide expressed in transformed yeast cells has Δ 6Fatty acid dehydrogenase function can catalysis add substrate linolic acid generation gamma-linolenic acid.
Another purpose of the present invention provides a kind of plant expression vector that contains mosaic gene RnD6CA, RnD6CB.Can use any expression vector that can guide foreign gene in plant, to express.These plant expression vectors include but not limited to: the double base agrobacterium vector, and pBIN19 for example, pBI121, pB221 contains the evoked promoter of DRE and/or ABRE element and the plant expression vector that is used for the monocotyledons micropellet bombardment.
Carrier of the present invention also can contain suitable promotor.Can use any strong promoter in the present invention.These promotors include but not limited to cauliflower mosaic virus (CaMV 35S), Ubiqutin, Actin etc. and plant tissue position specific expression promoter etc.It can use separately or be used in combination with other plant promoter.
Expression vector of the present invention can be by the use Ti-plasmids, the Ri plasmid, and plant viral vector, directly DNA transforms, microinjection, modes such as electroporation import vegetable cell.
Can be used for plant transformed host of the present invention and be dicotyledons, described dicotyledons is selected from the group of being made up of plants such as tobacco, rape, Sunflower Receptacle, soybean, tomato, castor-oil plant, sesame and peanuts.
The invention still further relates to mosaic gene RnD6CA, the RnD6CB application in producing gamma-linolenic acid.
Description of drawings:
Fig. 1. the nucleotide sequence of mosaic gene RnD6CA, SEQ ID NO:1.
Fig. 2. the nucleotide sequence of mosaic gene RnD6CB, SEQ ID NO:2.
The segmental nucleotide sequence of Fig. 3 .RnD6A, SEQ ID NO:3.
The segmental nucleotide sequence of Fig. 4 .RnD6B, SEQ ID NO:4.
The segmental nucleotide sequence of Fig. 5 .RnD6C, SEQ ID NO:5.
Fig. 6. mosaic gene RnD6CA encoded polypeptide, SEQ ID NO:6, wherein line part is respectively Cytb5 functional domain and three His Box functional domains.
Fig. 7. mosaic gene RnD6CB encoded polypeptide, SEQ ID NO:7, wherein line part is respectively Cytb5 functional domain and three His Box functional domains.
Fig. 8. the Yeast expression carrier figure of mosaic gene RnD6CA/B.
Fig. 9. the plant expression vector figure of mosaic gene RnD6CB/B.
Figure 10. the total fatty acids GC-MS that mosaic gene RnD6CA/B expresses in yeast saccharomyces cerevisiae analyzes
A) GC-MS of INVSc I yeast strain lipid acid composition;
B) GC-MS of LA (linolic acid), GLA (gamma-linolenic acid) standard model is formed and be added with to INVSc I yeast strain lipid acid;
C) contain unloaded pYES2 expression plasmid zymic total fatty acids GC-MS;
D) contain the total fatty acids GC-MS of unloaded pYES2 expression plasmid yeast after adding the LA substrate for induction;
E) the total fatty acids GC-MS of yeast saccharomyces cerevisiae after adding the LA substrate for induction of expression RnD6CA gene, the red arrow indication is the product peak;
F) product peak partial enlarged drawing (X10);
G) the total fatty acids GC-MS behind the yeast saccharomyces cerevisiae interpolation LA substrate for induction of expression RnD6CB gene, the arrow indication is the product peak.
Figure 11. MS collection of illustrative plates during RnD6CA/B genetic expression catalysate GC-MS analyzes in the yeast saccharomyces cerevisiae, the MS figure of the yeast expression catalysate of a:RnD6CA/B; B: the MS figure of gamma-linolenic acid standard substance.
Embodiment
Describe the present invention in detail below with reference to embodiment and accompanying drawing.What those having ordinary skill in the art will appreciate that is, following embodiment is illustrational purpose, and it should not be interpreted as limitation of the present invention by any way.Protection scope of the present invention is limited by accompanying Claim.
The structure of embodiment 1. mosaic genes
1. the preparation of blackcurrant genomic dna
With blackcurrant (Ribes nigrum L.) the live body plant planted in the Beijing Botanical Garden is material, get its young tender leaf agreement that contracts a film or TV play to an actor or actress 100mg, in 7mL Ep pipe, add steel ball (diameter 5mm), place the freezing 20-30min of liquid nitrogen, high-speed breakage on the whirlpool device, repeatable operation 2-3 time, broken fully to material.The CTAB extract of adding 1-2mL preheating (referring to " fine works molecular biology experiment guide " 2001, Science Press, Yan Ziying, Wang Hailin is translated).Mixing.65 ℃ of water-bath 30min.Add the equal-volume chloroform, the soft about 5min of extracting.In the centrifugal 10min of 12000rpm room temperature.Get supernatant, add 1/2 volume Virahol mixing, room temperature is placed 10min deposit D NA.With the Tip head DNA that is settled out is chosen, with washed twice in 70% ethanol, room temperature is placed 30min in 70% ethanol.Remove 70% ethanol, dry up in the air.Be dissolved in an amount of sterilization ddH 2O ,-20 ℃ of preservations.
2.RnD6A, the clone of RnD6B, RnD6C dna fragmentation
The design primer is cloned RnD6A, RnD6B, RnD6CDNA fragment (Fig. 3,4 and 5) respectively from the DNA that extract the front.Used reaction system is as follows:
PCR reaction system (50ul):
Figure C20051001160800071
(RnD6A, RnD6B, three segmental sizes of purpose of RnD6C are respectively 0.22kb to cut glue recovery purpose fragment behind the PCR product electrophoresis (1% gel strength), 0.22kb and 1.1kb), be connected into pMD18-T carrier (Takara Biotech. (Dalian) Co.Ltd) sequence verification.
The primer is as follows respectively:
1) the segmental clone's the primer of .RnD6ADNA
Forward primer: 5 '-TTGAGCATCATTTGTTCCCTCGATTGCCTCGATGCC-3 ' (SEQ ID NO:8)
Reverse primer: Xba I
5′-GCGG TCTAGATCACCCCGGGCAGGTGACAGCTT-3′(SEQ?IDNO：9)
2) clone's the primer of .RnD6B dna fragmentation
Forward primer: 5 '-TTGAGCATCATTTGTTCCCAAGATTGCCTCGATGCC-3 ' (SEQ ID NO:10)
Reverse primer: Xba I
5′-GCGG TCTAGATCAGCCATAGGTGTTGACAGCTTCCC-3′(SEQID?NO：11)
3) clone's the primer of .RnD6C dna fragmentation
Forward primer: Hind III
5′-CGCG AAGCTTATGGCTAATGCAATCAAGAAGTACATG-3′(SEQ?ID?NO：12)
Reverse primer: 5 '-GGGAACAAATGATGCTCAATTTGG-3 ' (SEQ ID NO:13)
3. the structure of mosaic gene RnD6CA and RnD6CB
RnD6A, RnD6B, RnD6C fragment that be cloned into and sequence verification are template above utilizing, utilize round pcr to obtain mosaic gene RnD6CA and RnD6CB sequence, and, be connected into pMD18-T in two ends introducing HindIII and XbaI enzyme cutting site, pass through sequence verification.
Concrete operations are as follows:
1). the used RnD6A of preparation mosaic gene, RnD6B, RnD6C fragment
The 2nd identical PCR reaction system of joint and PCR program among the embodiment 1 obtains RnD6A, RnD6B, RnD6C fragment respectively above utilizing, and reclaims by gel electrophoresis (1% gel strength).
Wherein:
The RnD6A fragment utilizes that primer sequence obtains by pcr amplification shown in SEQ ID NO:8 and the SEQ ID NO:9;
The RnD6B fragment utilizes that primer sequence obtains by pcr amplification shown in SEQ ID NO:10 and the SEQ ID NO:11;
The RnD6C fragment utilizes that primer sequence obtains by pcr amplification shown in SEQ ID NO:12 and the SEQ ID NO:13.
2) mosaic gene preparation
A) preparation of RnD6CA mosaic gene
RnD6A (fragment 1) and RnD6C (fragment 2) fragment are merged formation RnD6CA mosaic gene (Fig. 1).The primer sequence is:
Forward primer: Hind III
5′-CGCG AAGCTTATGGCTAATGCAATCAAGAAGTACATG-3′(SEQ?IDNO：12)
Reverse primer: Xba I
5′-GCGG TCTAGATCACCCCGGGCAGGTGACAGCTT-3′(SEQ?ID?NO：9)
B) preparation of RnD6CB mosaic gene
RnD6B (fragment 1) and RnD6C (fragment 2) fragment are merged formation RnD6CB mosaic gene (Fig. 2).The primer sequence is:
Forward primer: Hind III
5′-CGCG AAGCTTATGGCTAATGCAATCAAGAAGTACATG-3′(SEQ?IDNO：12)
Reverse primer: Xba I
5′-GCGG TCTAGATCAGCCATAGGTGTTGACAGCTTCCC-3′(SEQ?IDNO：11)
The PCR reaction system is carried out the PCR fusion below utilizing, to obtain mosaic gene:
Merge PCR reaction system (20ul):
Figure C20051001160800091
Total length mosaic gene PCR reaction system (50ul):
Figure C20051001160800092
Cutting glue behind the PCR product electrophoresis reclaims, obtain RnD6CA and RnD6CB mosaic gene full length fragment (about 1.3Kb) respectively, (left end has the HindIII restriction enzyme site to be connected into the pMD18-T carrier, right-hand member has the BamHI restriction enzyme site), transformed into escherichia coli DH-5 α (concrete source please be provided) preserves, simultaneously sequence verification.
The structure of embodiment 2. mosaic gene yeast vectors
From the pMD18-T carrier that contains mosaic gene RnD6CA and RnD6CB, utilize HindIII and XbaI enzyme cutting site, two enzyme enzymes are cut the back and are obtained RnD6CA and RnD6CB gene, directed cloning is in Yeast expression carrier pYES2.0 (available from Invitrogen company), obtain expression plasmid of yeast pYRnD6CA and pYRnD6CB, transformed into escherichia coli DH-5 α preserves.Its carrier collection of illustrative plates is seen Fig. 8.
The expression of embodiment 3. mosaic genes in yeast
1. zymic transforms
With reference to the described method of the pYES2Kit of Invitrogen company (Cat#V285-20), expression plasmid of yeast pYRnD6CA and pYRnD6CB (plasmid figure is referring to Fig. 8) with above-mentioned mosaic gene, adopt Lithium Acetate mediated transformation yeast saccharomyces cerevisiae auxotrophic strain INVSc I (with reference to the described method for transformation of the pYES2Kit of Invitrogen company) (purchasing company) in Invitrogen, with empty carrier pYES2.0 plasmid is contrast, obtains to contain the yeast cell of each expression plasmid.
2. the abduction delivering of yeast conversion cell
Get and contain the yeast list bacterium colony that the mosaic gene expression plasmid of yeast transforms, be inoculated in 50ml and contain in the SC-U nutrient solution of 2% silk floss sugar (with reference to the described prescription of the pYES2Kit of Invitrogen company) 250rpm on shaking table, 28 ℃ of overnight incubation.Add Nuo Nade P-40 (NP-40 then, available from BBI company) (final concentration 1%), external source linolic acid substrate (available from Sigma company) (final concentration is 0.003%), and yeast expression inductor D-semi-lactosi (available from Amresco company) (final concentration is 2%) 250rpm, cultivate the 48h abduction delivering for 22 ℃.
Embodiment 4. mosaic gene yeast expression products are to Δ 6The GC-MS of fatty acid dehydrogenase substrate catalysate analyzes
1. the extraction of lipid acid and esterification.
Yeast thalline after centrifugal collection is got and induced, deionized water wash, 50 ℃ of oven dry.Get 0.1g yeast powder (in example 3.2, induce obtained yeast promptly) and fully grind, add 5ml 5%KOH-CH through 50 ℃ of oven dry 3OH (available from Aldtich company), behind 70 ℃ of water-bath 5h, adding HCl is acidified to its pH value and reaches 2.0.Add 4ml 14%BF again 3-CH 3OH (available from Aldtich company) solution, 70 ℃ of water-bath 1.5h.Add 2ml 0.9%NaCl solution, static a moment behind the mixing.Add the 2ml chloroform again: normal hexane (V/V 1: 4) extracting, draw extract, N 2Dry up.Be dissolved in 100 μ l ethyl acetate at last.
2. end product GC-MS check and analysis experiment.
Used GC/MS instrument is TurboMass (a PerkinElmer company), pillar: BPX-70,30m * 0.25mm * 0.25vm, 120 ℃ of column temperatures, 230 ℃ of vaporizer temperature.Get sample on the 1 μ l end product, splitting ratio 10: 1.
3.GC-MS interpretation of result
Comparison diagram 10a, 10b and 10c, show and do not adding under the linolic acid substrate condition, used yeast engineering strain and contain and new product peak do not occur occur in the unloaded pYES2 expression plasmid of inductive yeast illustrates not contain in used yeast engineering strain and the unloaded pYES2 expression plasmid zymic total fatty acids to add substrate linolic acid (LA) and catalysate gamma-linolenic acid (GLA).
When adding the linolic acid substrate, and when carrying out abduction delivering, contrast (unloaded pYES2.O) does not also have the appearance (referring to Figure 10 d) of product peak gamma-linolenic acid (GLA).And after the yeast expression bacterium of pYRnD6CA/B induced, add substrate linolic acid (LA) and can be generated gamma-linolenic acid (GLA) (referring to Figure 10 e and 10g) by catalysis
Comparison diagram 10e, 10f and 10g can find when retention time is 7.6 minutes, have a new material to generate., its appearance time and peak figure and the mass spectrum consistent (referring to Figure 11 a and 11b) of mass spectrum with standard substance gamma-linolenic acid (available from Sigma company) are defined as gamma-linolenic acid.
Above result's proof derives from two mosaic gene RnD6CA/B successful expression in yeast saccharomyces cerevisiae of blackcurrant, and expressed albumen can catalysis add substrate linolic acid (LA) generation gamma-linolenic acid (GLA).
The structure of embodiment 5. mosaic gene RnD6CA/B plant expression vectors
The structure of the plant expression vector of RnD6CA/B molecular biology method routinely carries out (referring to " fine works molecular biology experiment guide " 2001, Science Press, Yan Ziying, Wang Hailin is translated).With RnD6CA/B DNA is template, introduce Sac I site (Xba I is changed to Sac I site) in BamHI site (Hind III is changed to BamH I site) and the reverse primer (SEQ ID NO:9 (RnD6CA) and SEQ ID NO:11 (RnD6CB)) by in forward primer (SEQ ID NO:12), introducing, be inserted into the CaMV 35S promoter back in the pBI121 binary expression vector [14], obtain a binary expression vector pRnD6CA/B, its collection of illustrative plates as shown in Figure 9.Through BamH I and SacI enzyme cut, PCR identify to insert fragment errorless after, change Agrobacterium LBA4404[15 over to], through extracting plasmid, enzyme is cut, PCR confirms.This expression vector can be directly used in the conversion (referring to Fig. 9) of plant.
Reference:
1.Gunstone?FD(1992)Gamma?linolenic?acid-occurrence?and?physicaland?chemical?properties.Prog?Lipid?Res?31：145-161.
2.Horrobin?DF(1992)Nutritional?and?medical?importance?ofgamma-linolenic?acid.Prog?Lipid?Res?31：163-194.
3.Huang?Y-S，Milles?DE(1996)Gamma-Linolenic?Acid：Metabolismand?Its?Roles?in?Nutrition?and?Medicine.AOCS?Press，Champaign，IL.
4.Hyekyung?P.Cho，Manabu?T.Nakamura，and?Steven?D.Clarke(1999)Cloning，Expression，and?Nutritional?Regulation?of?the?MammalianΔ 6-Desaturase.JBC?274(1)：471-477.
5.Sayanova?O，Smith?MA，Lapinskas?P，Stobart?AK，Dobson?G，ChristieWW，Shewry?PR，Napier?JA(1997)Expression?of?a?borage?desaturasecDNA?containing?an?N-terminal?cytochrome?b5?domain?results?in?theaccumulation?of?high?levels?ofΔ 6-desaturated?fatty?acids?in?transgenictobacco.PNAs?94：4211-4216.
6.Haiping?Hong，Nagamani?Datla，Darwin?W.Reed，Patrick?S.Covello，Samuel?L.MacKenzie，and?Xiao?Qiu(2002)High-Level?Production?ofγ-Linolenic?Acid?in?Brassica?juncea?Using?aΔ 6Desaturase?from?Pythiumirregulare.Plant?Physiology?129：354-362.
7.Napier?JA，Hey?SJ，Lacey?DJ，Shewry?PR(1998)Identification?of?aCaenorhabditis?elegans?Δ 6-fatty-acid-desaturase?by?heterologousexpression?in?Saccharomyces?cerevisiae.Biochem?J?330：611-614.
8.Qiu?X，Hong?HP，Datla?N，MacKenzie?SL，Tayler?CD，Thomas?LT(2002)Expression?of?borageΔ 6-desaturase?in?Saccharomyces?cerevisiaeand?oilseed?crops.Can?J?Bot?80：42-49.
9.Cook?D，Grierson?D，Jones?C，Wallace?A，West?G，Tucker?G(2002)Modification?of?fatty?acid?composition?in?tomato(Lycopersiconesculentum)by?expression?of?a?borageΔ 6-desaturase.Mol?Biotechnol.21(2)：123-128.
10.Olga?Sayanova，Mark?A.Smith，Peter?Lapinskas，A.Keith?Stobart，Gary?Dobson，William?W.Christie，Peter?R.Shewry，And?Johnathan?A.Napier(1997)Expression?of?a?borage?desaturase?cDNA?containing?anN-terminal?cytochrome?b5?domain?results?in?the?accumulation?of?highlevels?of?Δ 6-desaturated?fatty?acids?in?transgenic?tobacco.PNAs94：4211-4216.
11.Shirley?Sato，Aiqiu?Xing，Xingguo?Ye，Bruce?Schweiger，AnthonyKinney，George?Graef?and?Tom?Clemente(2004)Production?of?γ-LinolenicAcid?and?Stearidonic?Acid?in?Seeds?of?Marker-Free?Transgenic?Soybean.Genomics?Molecular?Genetics?&?Biotechology?44：646-652.
12.Ucciani?E(1995)Nouveau?Dictionnaire?des?Huiles?Végétales-Composition?en?Acides?Gras.Lavoisier，Paris?pp?3-596.
13.Emilio?Mendoza?de?Gyves，Caroline?A.Sparks，Olga?Sayanova，PaulLazzeri，Johnathan?A.Napier?and?Huw?D.Jones(2004)Geneticmanipulation?of?γ-linolenic?acid(GLA)synthesis?in?a?commercial?varietyof?evening?primrose(Oenothera?sp.)Plant?Biotechnology?2(4)：351-357.
14.Chen?PY，Wang?CK，Soong?SC，To?KY(2003)Complete?sequence?ofthe?binary?vector?pBI121?and?its?application?in?cloning?T-DNA?insertionfrom?transgenic?plants.Molecular?Breeding?11：287-293.
15.Hoekema?A，Hirsch?PR，Hooykaas?PJJ，Schilperoort?RA(1983)Abinary?plant?vetor?strategy?based?on?separation?of?vir-and?T-region?of?theAgrobacterium?tumefaciens?Ti-plasmid.Nature?303：179-180.
The IB051489-sequence table
SEQUENCE?LISTING
＜110〉Inst. of Genetics and Development Biology, CAS
＜120〉have the mosaic gene and the application thereof of Δ 6 fatty acid dehydrogenase functions
<130>IB051489
<160>13
<170>PatentIn?version?3.1
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＜213〉artificial sequence
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tctgggcatt?atcagacaat?gtctactcct?ttttatacca?atttggctca?aattcttact 540
ggaaattgcc?ttagtggtat?tagtatggct?tggtggaaat?ggacccacaa?tgctcaccac 600
gttgctgtta?atagtattga?ccatgaccct?gatcttcagt?acatgccatt?ttttgtcctc 660
tctcccaaat?tgttcaatgg?cataacttct?cgcttttatg?ggaggaagtt?ggagtttgat 720
gcttttgcta?ggttcatggt?aagttatcag?cattggacat?tttatccggt?gatgattttt 780
gcacgatttt?atatgtttgt?gcccacgttt?tttatgttgc?tttcaaagaa?aaaagtaccc 840
aacagacctt?tgaatatagc?gggaattatt?gtgttctgga?tttggtttcc?tcttcttgtt 900
tcatgccttc?ccaattggac?tgagaggatg?atgtttgtgc?tagtgagctt?tacagttact 960
tcaattcaac?acgttacatt?ttgtttgaat?cactactcgg?ctgataatta?tatgggacat 1020
cctactggga?acgattggtt?tgagaagcag?acaagtggga?ctttggacat?ttcgtgcccg 1080
tcttggatgg?attggtttca?tggtgggctg?caattccaaa?ttgagcatca?tttgttccct 1140
cgattgcctc?gatgccagct?taggacggtc?tctccttttg?cgatggagct?atgcaagaag 1200
cacaatttgc?cttataggag?tttgtccttt?ttggaggcca?atgtcgcgac?cattatgacg 1260
ttgaggactg?cagccctgca?agcgcgcgac?ttgtccaacc?cggttccgaa?gaacttgctc 1320
tgggaagctg?tcacctgccc?ggggtga 1347
<210>2
<211>1347
<212>DNA
＜213〉artificial sequence
<400>2
atggctaatg?caatcaagaa?gtacatgaca?gttgaggagc?taaaagtgca?taacaagcca 60
gaggatttgt?ggatctctat?tcagggtaag?gtttacaatg?tcactgattg?gaaaaaggaa 120
cacccaggtg?gggatagctc?tctcctgaat?ctaggtggtc?aagatgtcac?agatgcattc 180
attgcttatc?atccaggtac?tgcttggcaa?tatcttgaca?ggttctttac?tgggtattat 240
ctcaaagatt?tcaatgtctc?agatgtatcc?aaagattata?gaaaacttgc?atctgagttt 300
accaaaatgg?gtctttttgc?aaagaaaggg?catggtgttt?tctactcctt?ttgtcttggg 360
gcattcttgt?ttgctgtttg?tgtttatggt?gttttgtatt?ctcaaagttt?gtttgtgcat 420
ttgtgttgtg?gtgggatttt?ggggttttta?tggatgcaaa?gtggttatgc?tggtcacgat 480
tctgggcatt?atcagacaat?gtctactcct?ttttatacca?atttggctca?aattcttact 540
ggaaattgcc?ttagtggtat?tagtatggct?tggtggaaat?ggacccacaa?tgctcaccac 600
gttgctgtta?atagtattga?ccatgaccct?gatcttcagt?acatgccatt?ttttgtcctc 660
tctcccaaat?tgttcaatgg?cataacttct?cgcttttatg?ggaggaagtt?ggagtttgat 720
gcttttgcta?ggttcatggt?aagttatcag?cattggacat?tttatccggt?gatgattttt 780
gcacgatttt?atatgtttgt?gcccacgttt?tttatgttgc?tttcaaagaa?aaaagtaccc 840
aacagacctt?tgaatatagc?gggaattatt?gtgttctgga?tttggtttcc?tcttcttgtt 900
tcatgccttc?ccaattggac?tgagaggatg?atgtttgtgc?tagtgagctt?tacagttact 960
tcaattcaac?acgttacatt?ttgtttgaat?cactactcgg?ctgataatta?tatgggacat 1020
cctactggga?acgattggtt?tgagaagcag?acaagtggga?ctttggacat?ttcgtgcccg 1080
tcttggatgg?attggtttca?tggtgggctg?caattccaaa?ttgagcatca?tttgttccca 1140
agattgcctc?gatgccagct?taggaaggtc?tctccttttg?tgaaagagct?ttgcaagaaa 1200
cacaatttgc?cttataggag?tttatcattt?tttgaggcca?atgttgcaac?cattaagacg 1260
ttgaggactg?ccgccttgca?agctcgcgac?ctatccaacc?ctatttcaaa?gaatttgcta 1320
tgggaagctg?tcaacaccta?tggctga 1347
<210>3
<211>227
<212>DNA
＜213〉artificial sequence
<400>3
ttgagcatca?tttgttccct?cgattgcctc?gatgccagct?taggacggtc?tctccttttg 60
cgatggagct?atgcaagaag?cacaatttgc?cttataggag?tttgtccttt?ttggaggcca 120
atgtcgcgac?cattatgacg?ttgaggactg?cagccctgca?agcgcgcgac?ttgtccaacc 180
cggttccgaa?gaacttgctc?tgggaagctg?tcacctgccc?ggggtga 227
<210>4
<211>227
<212>DNA
＜213〉artificial sequence
<400>4
ttgagcatca?tttgttccca?agattgcctc?gatgccagct?taggaaggtc?tctccttttg 60
tgaaagagct?ttgcaagaaa?cacaatttgc?cttataggag?tttatcattt?tttgaggcca 120
atgttgcaac?cattaagacg?ttgaggactg?ccgccttgca?agctcgcgac?ctatccaacc 180
ctatttcaaa?gaatttgcta?tgggaagctg?tcaacaccta?tggctga 227
<210>5
<211>1139
<212>DNA
＜213〉artificial sequence
<400>5
atggctaatg?caatcaagaa?gtacatgaca?gttgaggagc?taaaagtgca?taacaagcca 60
gaggatttgt?ggatctctat?tcagggtaag?gtttacaatg?tcactgattg?gaaaaaggaa 120
cacccaggtg?gggatagctc?tctcctgaat?ctaggtggtc?aagatgtcac?agatgcattc 180
attgcttatc?atccaggtac?tgcttggcaa?tatcttgaca?ggttctttac?tgggtattat 240
ctcaaagatt?tcaatgtctc?agatgtatcc?aaagattata?gaaaacttgc?atctgagttt 300
accaaaatgg?gtctttttgc?aaagaaaggg?catggtgttt?tctactcctt?ttgtcttggg 360
gcattcttgt?ttgctgtttg?tgtttatggt?gttttgtatt?ctcaaagttt?gtttgtgcat 420
ttgtgttgtg?gtgggatttt?ggggttttta?tggatgcaaa?gtggttatgc?tggtcacgat 480
tctgggcatt?atcagacaat?gtctactcct?ttttatacca?atttggctca?aattcttact 540
ggaaattgcc?ttagtggtat?tagtatggct?tggtggaaat?ggacccacaa?tgctcaccac 600
gttgctgtta?atagtattga?ccatgaccct?gatcttcagt?acatgccatt?ttttgtcctc 660
tctcccaaat?tgttcaatgg?cataacttct?cgcttttatg?ggaggaagtt?ggagtttgat 720
gcttttgcta?ggttcatggt?aagttatcag?cattggacat?tttatccggt?gatgattttt 780
gcacgatttt?atatgtttgt?gcccacgttt?tttatgttgc?tttcaaagaa?aaaagtaccc 840
aacagacctt?tgaatatagc?gggaattatt?gtgttctgga?tttggtttcc?tcttcttgtt 900
tcatgccttc?ccaattggac?tgagaggatg?atgtttgtgc?tagtgagctt?tacagttact 960
tcaattcaac?acgttacatt?ttgtttgaat?cactactcgg?ctgataatta?tatgggacat 1020
cctactggga?acgattggtt?tgagaagcag?acaagtggga?ctttggacat?ttcgtgcccg 1080
tcttggatgg?attggtttca?tggtgggctg?caattccaaa?ttgagcatca?tttgttccc 1139
<210>6
<211>448
<212>PRT
＜213〉artificial sequence
<400>6
Met?Ala?Asn?Ala?Ile?Lys?Lys?Tyr?Met?Thr?Val?Glu?Glu?Leu?Lys?Val
1 5 10 15
His?Asn?Lys?Pro?Glu?Asp?Leu?Trp?Ile?Ser?Ile?Gln?Gly?Lys?Val?Tyr
20 25 30
Asn?Val?Thr?Asp?Trp?Lys?Lys?Glu?His?Pro?Gly?Gly?Asp?Ser?Ser?Leu
35 40 45
Leu?Asn?Leu?Gly?Gly?Gln?Asp?Val?Thr?Asp?Ala?Phe?Ile?Ala?Tyr?His
50 55 60
Pro?Gly?Thr?Ala?Trp?Gln?Tyr?Leu?Asp?Arg?Phe?Phe?Thr?Gly?Tyr?Tyr
65 70 75 80
Leu?Lys?Asp?Phe?Asn?Val?Ser?Asp?Val?Ser?Lys?Asp?Tyr?Arg?Lys?Leu
85 90 95
Ala?Ser?Glu?Phe?Thr?Lys?Met?Gly?Leu?Phe?Ala?Lys?Lys?Gly?His?Gly
100 105 110
Val?Phe?Tyr?Ser?Phe?Cys?Leu?Gly?Ala?Phe?Leu?Phe?Ala?Val?Cys?Val
115 120 125
Tyr?Gly?Val?Leu?Tyr?Ser?Gln?Ser?Leu?Phe?Val?His?Leu?Cys?Cys?Gly
130 135 140
Gly?Ile?Leu?Gly?Phe?Leu?Trp?Met?Gln?Ser?Gly?Tyr?Ala?Gly?His?Asp
145 150 155 160
Ser?Gly?His?Tyr?Gln?Thr?Met?Ser?Thr?Pro?Phe?Tyr?Thr?Asn?Leu?Ala
165 170 175
Gln?Ile?Leu?Thr?Gly?Asn?Cys?Leu?Ser?Gly?Ile?Ser?Met?Ala?Trp?Trp
180 185 190
Lys?Trp?Thr?His?Asn?Ala?His?His?Val?Ala?Val?Asn?Ser?Ile?Asp?His
195 200 205
Asp?Pro?Asp?Leu?Gln?Tyr?Met?Pro?Phe?Phe?Val?Leu?Ser?Pro?Lys?Leu
210 215 220
Phe?Asn?Gly?Ile?Thr?Ser?Arg?Phe?Tyr?Gly?Arg?Lys?Leu?Glu?Phe?Asp
225 230 235 240
Ala?Phe?Ala?Arg?Phe?Met?Val?Ser?Tyr?Gln?His?Trp?Thr?Phe?Tyr?Pro
245 250 255
Val?Met?Ile?Phe?Ala?Arg?Phe?Tyr?Met?Phe?Val?Pro?Thr?Phe?Phe?Met
260 265 270
Leu?Leu?Ser?Lys?Lys?Lys?Val?Pro?Asn?Arg?Pro?Leu?Asn?Ile?Ala?Gly
275 280 285
Ile?Ile?Val?Phe?Trp?Ile?Trp?Phe?Pro?Leu?Leu?Val?Ser?Cys?Leu?Pro
290 295 300
Asn?Trp?Thr?Glu?Arg?Met?Met?Phe?Val?Leu?Val?Ser?Phe?Thr?Val?Thr
305 310 315 320
Ser?Ile?Gln?His?Val?Thr?Phe?Cys?Leu?Asn?His?Tyr?Ser?Ala?Asp?Asn
325 330 335
Tyr?Met?Gly?His?Pro?Thr?Gly?Asn?Asp?Trp?Phe?Glu?Lys?Gln?Thr?Ser
340 345 350
Gly?Thr?Leu?Asp?Ile?Ser?Cys?Pro?Ser?Trp?Met?Asp?Trp?Phe?His?Gly
355 360 365
Gly?Leu?Gln?Phe?Gln?Ile?Glu?His?His?Leu?Phe?Pro?Arg?Leu?Pro?Arg
370 375 380
Cys?Gln?Leu?Arg?Thr?Val?Ser?Pro?Phe?Ala?Met?Glu?Leu?Cys?Lys?Lys
385 390 395 400
His?Asn?Leu?Pro?Tyr?Arg?Ser?Leu?Ser?Phe?Leu?Glu?Ala?Asn?Val?Ala
405 410 415
Thr?Ile?Met?Thr?Leu?Arg?Thr?Ala?Ala?Leu?Gln?Ala?Arg?Asp?Leu?Ser
420 425 430
Asn?Pro?Val?Pro?Lys?Asn?Leu?Leu?Trp?Glu?Ala?Val?Thr?Cys?Pro?Gly
435 440 445
<210>7
<211>448
<212>PRT
＜213〉artificial sequence
<400>7
Met?Ala?Asn?Ala?Ile?Lys?Lys?Tyr?Met?Thr?Val?Glu?Glu?Leu?Lys?Val
1 5 10 15
His?Asn?Lys?Pro?Glu?Asp?Leu?Trp?Ile?Ser?Ile?Gln?Gly?Lys?Val?Tyr
20 25 30
Asn?Val?Thr?Asp?Trp?Lys?Lys?Glu?His?Pro?Gly?Gly?Asp?Ser?Ser?Leu
35 40 45
Leu?Asn?Leu?Gly?Gly?Gln?Asp?Val?Thr?Asp?Ala?Phe?Ile?Ala?Tyr?His
50 55 60
Pro?Gly?Thr?Ala?Trp?Gln?Tyr?Leu?Asp?Arg?Phe?Phe?Thr?Gly?Tyr?Tyr
65 70 75 80
Leu?Lys?Asp?Phe?Asn?Val?Ser?Asp?Val?Ser?Lys?Asp?Tyr?Arg?Lys?Leu
85 90 95
Ala?Ser?Glu?Phe?Thr?Lys?Met?Gly?Leu?Phe?Ala?Lys?Lys?Gly?His?Gly
100 105 110
Val?Phe?Tyr?Ser?Phe?Cys?Leu?Gly?Ala?Phe?Leu?Phe?Ala?Val?Cys?Val
115 120 125
Tyr?Gly?Val?Leu?Tyr?Ser?Gln?Ser?Leu?Phe?Val?His?Leu?Cys?Cys?Gly
130 135 140
Gly?Ile?Leu?Gly?Phe?Leu?Trp?Met?Gln?Ser?Gly?Tyr?Ala?Gly?His?Asp
145 150 155 160
Ser?Gly?His?Tyr?Gln?Thr?Met?Ser?Thr?Pro?Phe?Tyr?Thr?Asn?Leu?Ala
165 170 175
Gln?Ile?Leu?Thr?Gly?Asn?Cys?Leu?Ser?Gly?Ile?Ser?Met?Ala?Trp?Trp
180 185 190
Lys?Trp?Thr?His?Asn?Ala?His?His?Val?Ala?Val?Asn?Ser?Ile?Asp?His
195 200 205
Asp?Pro?Asp?Leu?Gln?Tyr?Met?Pro?Phe?Phe?Val?Leu?Ser?Pro?Lys?Leu
210 215 220
Phe?Asn?Gly?Ile?Thr?Ser?Arg?Phe?Tyr?Gly?Arg?Lys?Leu?Glu?Phe?Asp
225 230 235 240
Ala?Phe?Ala?Arg?Phe?Met?Val?Ser?Tyr?Gln?His?Trp?Thr?Phe?Tyr?Pro
245 250 255
Val?Met?Ile?Phe?Ala?Arg?Phe?Tyr?Met?Phe?Val?Pro?Thr?Phe?Phe?Met
260 265 270
Leu?Leu?Ser?Lys?Lys?Lys?Val?Pro?Asn?Arg?Pro?Leu?Asn?Ile?Ala?Gly
275 280 285
Ile?Ile?Val?Phe?Trp?Ile?Trp?Phe?Pro?Leu?Leu?Val?Ser?Cys?Leu?Pro
290 295 300
Asn?Trp?Thr?Glu?Arg?Met?Met?Phe?Val?Leu?Val?Ser?Phe?Thr?Val?Thr
305 310 315 320
Ser?Ile?Gln?His?Val?Thr?Phe?Cys?Leu?Asn?His?Tyr?Ser?Ala?Asp?Asn
325 330 335
Tyr?Met?Gly?His?Pro?Thr?Gly?Asn?Asp?Trp?Phe?Glu?Lys?Gln?Thr?Ser
340 345 350
Gly?Thr?Leu?Asp?Ile?Ser?Cys?Pro?Ser?Trp?Met?Asp?Trp?Phe?Hi?s?Gly
355 360 365
Gly?Leu?Gln?Phe?Gln?Ile?Glu?Hi?s?His?Leu?Phe?Pro?Arg?Leu?Pro?Arg
370 375 380
Cys?Gln?Leu?Arg?Lys?Val?Ser?Pro?Phe?Val?Lys?Glu?Leu?Cys?Lys?Lys
385 390 395 400
His?Asn?Leu?Pro?Tyr?Arg?Ser?Leu?Ser?Phe?Phe?Glu?Ala?Asn?Val?Ala
405 410 415
Thr?Ile?Lys?Thr?Leu?Arg?Thr?Ala?Ala?Leu?Gln?Ala?Arg?Asp?Leu?Ser
420 425 430
Asn?Pro?Ile?Ser?Lys?Asn?Leu?Leu?Trp?Glu?Ala?Val?Asn?Thr?Tyr?Gly
435 440 445
<210>8
<211>36
<212>DNA
＜213〉artificial sequence
<400>8
ttgagcatca?tttgttccct?cgattgcctc?gatgcc 36
<210>9
<211>33
<212>DNA
＜213〉artificial sequence
<400>9
gcggtctaga?tcaccccggg?caggtgacag?ctt 33
<210>10
<211>36
<212>DNA
＜213〉artificial sequence
<400>10
ttgagcatca?tttgttccca?agattgcctc?gatgcc 36
<210>11
<211>36
<212>DNA
＜213〉artificial sequence
<400>11
gcggtctaga?tcagccatag?gtgttgacag?cttccc 36
<210>12
<211>37
<212>DNA
＜213〉artificial sequence
<400>12
cgcgaagctt?atggctaatg?caatcaagaa?gtacatg 37
<210>13
<211>24
<212>DNA
＜213〉artificial sequence
<400>13
gggaacaaat?gatgctcaat?ttgg 24

Claims (12)

1. mosaic gene RnD6CA, its nucleotide sequence is shown in SEQ ID NO:1.
2. mosaic gene RnD6CB, its nucleotide sequence is shown in SEQ ID NO:2.
3. the mosaic gene RnD6CA encoded polypeptide of claim 1, its aminoacid sequence is shown in SEQID NO:6.
4. the mosaic gene RnD6CB encoded polypeptide of claim 2, its aminoacid sequence is shown in SEQID NO:7.
5. an expression vector is characterized in that comprising claim 1 or 2 described mosaic genes.
6. the expression vector of claim 5, it is Yeast expression carrier or plant expression vector.
7. the expression vector of claim 6, wherein said plant expression vector is selected from pBIN19, pBI121, or contain the evoked promoter of DRE and/or ABRE element and the plant expression vector that is used for the monocotyledons micropellet bombardment.
8. the expression vector of claim 5, it is characterized in that also comprising promotor, described promotor is selected from the promotor of cauliflower mosaic virus CaMV 35S, the promotor of ubiquitin (Ubiqutin), the promotor or the plant tissue position specific expression promoter of Actin muscle (Actin), and described promotor is used in combination separately or with other plant promoter.
9. host cell is characterized in that comprising the expression vector of claim 5, and described host cell is yeast cell or vegetable cell.
10. the host cell of claim 9, wherein said expression vector are that the mode by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection or electroporation imports.
11. the host cell of claim 9, wherein said yeast cell are brewing yeast cell, described vegetable cell is the cell of tobacco, rape, Sunflower Receptacle, soybean, tomato, castor-oil plant, sesame or peanut.
12. the mosaic gene RnD6CA of claim 1 or the mosaic gene RnD6CB of claim 2 application in producing gamma-linolenic acid.
CNB2005100116085A 2005-04-21 2005-04-21 Chimeric gene with delta 6 fatty acid dehydrogenase function and its use Expired - Fee Related CN100413969C (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101353661B (en) * 2007-07-25 2012-05-09 中国科学院遗传与发育生物学研究所 Genes having delta 6 fatty acid dehydrogenase function and use thereof
CN102839134B (en) * 2012-09-24 2014-04-16 山东大学 Yeast strain for producing alpha-linolenic acid, culture method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020170090A1 (en) * 1997-02-18 2002-11-14 Washington State University Research Foundation Omega-3 fatty acid desaturase
CN1570116A (en) * 2004-05-13 2005-01-26 南开大学 Rhizopus arrhizus delta[12]-fatty acid dehydrogenase nucleic acid sequence and its uses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020170090A1 (en) * 1997-02-18 2002-11-14 Washington State University Research Foundation Omega-3 fatty acid desaturase
CN1570116A (en) * 2004-05-13 2005-01-26 南开大学 Rhizopus arrhizus delta[12]-fatty acid dehydrogenase nucleic acid sequence and its uses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
$6_脂肪酸脱氢酶的分子生物学研究进展. 张琦等.生物工程学报，第20卷第3期. 2004 *

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