CN1656226A - Methods for the production of unsaturated fatty acids - Google Patents

Methods for the production of unsaturated fatty acids Download PDF

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CN1656226A
CN1656226A CNA038050625A CN03805062A CN1656226A CN 1656226 A CN1656226 A CN 1656226A CN A038050625 A CNA038050625 A CN A038050625A CN 03805062 A CN03805062 A CN 03805062A CN 1656226 A CN1656226 A CN 1656226A
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A·伦茨
M·吉普曼斯
I·福伊斯纳
C－L·罗森菲尔德
D·C·克尼普尔
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Abstract

The invention relates to processes for the production of unsaturated fatty acids, preferably of conjugated polyunsaturated fatty acids such as conjugated linoleic acid (CLA), by the recombinant expression of desaturases from insects of the order Lepidoptera. Expression preferably takes place in an organism selected from the group of the plant organisms, yeasts, fungi and algae. Also according to the invention are recombinant expression cassettes for the recombinant expression of desaturases from insects of the order Lepidoptera, and the transgenic organisms transformed with these.

Description

Be used to produce the method for unsaturated fatty acids
Describe
The present invention relates to produce the method for unsaturated fatty acids, preferably produce for example conjugated linolic acid (CLA) of conjugation polyunsaturated fatty acid by the desaturase in recombinant expressed lepidopteran (Lepidoptera) insect.Express and preferably in the organism that is selected from plant biological body, yeast, fungi and algae, carry out.In addition, the present invention also relates to can be used for recombinant expressed lepidopterous insects desaturase recombinant expression cassettes and by the transgenic organism of its conversion.
Lipid acid and triglyceride level are widely used in foodstuffs industry, Animal nutrition, makeup and pharmacy field.Especially valuable and unsaturated fatty acids that attempt to obtain is those conjugated polyunsaturated fatty acid.Conjugation polyunsaturated fatty acid other polyunsaturated fatty acids relatively is comparatively rare.The example of conjugated polyunsaturated fatty acid has conjugated linolic acid (CLA), the weak acid of α-rake (18:4 therapic acid), eleostearic acid (18:3 punicic acid), conjugate linolenic acid, dimorphecolic acid and calendulicacid (see figure 1)
Fig. 1: conjugation polyunsaturated fatty acid
Figure A0380506200051
calendulic?acid
The weak acid of α-rake
Figure A0380506200053
α-eleostearic acid
Figure A0380506200054
dimorphecolic?acid
CLA is the general name of all linolic acid positions and constitutional isomer, and its obvious characteristic is the conjugated double bond system from carbon atom 8,9,10 or 11 beginnings.Some examples as shown in Figure 2.
All there is geometrical isomer in each of these positional isomerss, and is promptly suitable-suitable, anti--suitable, suitable-anti-, anti--anti-four kinds of forms.
Fig. 2: four kinds of isomer of conjugated linolic acid
Figure A0380506200061
Figure A0380506200062
10t.12c-CLA
Figure A0380506200063
Figure A0380506200064
11c.13t-CLA
The CLA isomer (9Z, 11E)-CLA and (10E, 12Z)-CLA is known as has biologic activity.CLA mainly exists in animal derived food.The CLA that finds high density is present in the dairy products of meat and ruminating animal especially: approximately in 3-4mg CLA/g cattle and sheep fat people (1992) J Food Comp Anal 5:185-197 such as () Chin and the about 3-7mg CLA/g dairy products fat people (1999) J Dairy Sci 82:2146-56 such as () Dhiman, (9Z wherein, 11E)-concentration of CLA is almost up to 80%, all is main isomer in all cases.Only contain micro-CLA in the higher plant, and the CLA isomer of above-mentioned two kinds of tool biologic activity is not found in plant as yet so far.
The series of active effect of CLA is found.Therefore, use CLA and can reduce the humans and animals body fat, be increased in that feed conversion is speed (people (1997) the Lipids 32:853-858 such as Park of body weight in the animal; People such as Park (1999) Lipids 34:235-241; WO 94/16690; WO96/06605; WO 97/46230; WO 97/46118).Use CLA also to active effect being arranged such as allergy (WO 97/32008) or cancer (people (1999) Carcino genesis 20:1019-1024 such as Banni, people such as Thompson (1997) Cancer Res 57:5067-5072).The arteriosclerosis effectiveness of CLA also is identified (people (2000) Nutr Res 20:1795-1805 such as Wilson).Isomer and isomer mixture are studied.
CLA can be synthetic by linoleic alkali isomerization.The vegetables oil of high linoleic acid content is mainly used in industrial production, for example sunflower oil, Thistle oil.Under alkaline condition, be heated to two reactions below the catalysis more than 180 ℃:
(1) the fatty acid ester key hydrolysis of triglyceride level skeleton and free fatty acids discharge,
(2) has the non-conjugated unsaturated fatty acids conjugation of two or more pairs of keys.
Commercially available CLA oil is to contain multiple CLA isomer and other mixture saturated, unsaturated fatty acids.Because these abiologies are active and the existence of non-natural isomer, (9Z, 11E) CLA is with (10E, 12Z) CLA purifying process perhaps need to confirm isomer mixture healthy harmless to humans and animals to need heavy biologic activity isomer.Up to now, can't obtain single CLA isomer by alkali isomerization with comparatively economic method.Fractional crystallization makes concentrated respectively isomer, and (9Z, 11E) CLA is with (10E, 12Z) CLA becomes possibility.But the above-mentioned method of mentioning also is not enough to prepare high-quality individual isomer.In the above-mentioned method of mentioning, reaction product is converted into methyl or ethyl ester usually, so the CLA of natural form is that free fatty acids or triacylglycerol can not obtain.
The inferior position of these chemical conversions can transform the method that linolic acid generates CLA by biocatalysis and be overcome.For example the multiple microorganism in the ruminant tumor gastric can be converted into CLA with linolic acid in biological hydrogenation approach.This particularly realizes by the enzymatic activity of CLA isomerase.This enzymatic activity is Butyrivibrio fibrisolvens (Butyrivibrio fibrisolvens) (Kepler and Tovee (1966) J Biol Chem 241:1350), Propionibacterium (Propionibacterium acnes) (people such as Deng, the international symposium first time about CLA, 2001, Alesund, Norway), (WO 99/32604 for clostridium sporogenes (Clostridium sporogenes) and lactobacillus reuteri (Lactobacillus reuteri); WO 01/00846) in describe to some extent.So far, the CLA isomerase is described to use free fatty acids to make substrate.CLA isomerase encoding gene from lactobacillus reuteri and Propionibacterium is cloned, and obtains functional expression from the isomerase of propiono-bacterium in the allos microorganism.Compare the advantage that the alkali isomerization method has matter although utilize microorganism that linolic acid is converted into CLA, owing to the fermentation expense with can only produce free fatty acids but not triglyceride level is in economic inferior position.Yet free fatty acids because smell is not good in fact and be not suitable for food and feedstuff industry.Though further conversion free fatty acids-for example use lipase-catalyzed generation glycerine or glyceryl ester-possibility is also very complicated.
Method based on bacterium CLA isomerase also usually can be applied to other biology.Existing so far CLA isomerase transforms the report (Cepler and Tove (1967) JBiochem Chem 242:5686-5692) that free linoleic acid becomes CLA.Linolic acid mainly exists with the form of esterification in such as the higher organism of plant.Lipid such as triglyceride constitute the storage form of free fatty acids, and thioesters for example aliphatic alcohol constitute the activity form of lipid acid.
Lipid acid with trans double bond is extremely rare.Some plant seed oils contain the lipid acid that has two keys in trans position.Therefore, E5-lipid acid is found people (1971) J Amer Oil Chem Soc 48:700-701 such as () Rankoff in a lot of Thalictrum (Thalictrum) plant seed oils.And E5 desaturase activity is also described people (2000) Biochem Soc Trans 28:641-643 such as () Longman to some extent in common garden columbine (Aquilegia vulgaris).Yet, do not have the plant report to contain trans-isooleic acid (E11-octadecenoic acid) or E10-octadecenoic acid.
The desaturationization of having known lipid acid in plant can mainly take place based on two mechanism:
(1) in plastid, lipid acid ACP ester is mainly occurred in the 9th by solubility desaturase desaturation, and
(2) at endoplasmic reticulum, film fat, particularly phosphatidylcholine, preferably further at the 6th, 12,15 tunicle in conjunction with the desaturase desaturation.
In some plants, the generation of conjugated fatty acids is by the activity of conjugation enzyme (conjugase) (people (1984) J Chem Soc Chem Commun 15:953-955 such as Crombie; People such as Crombie (1985) J Chem Soc Perkin Trans 1:2425-2434; People such as Fritsche (1999) FEBSLetters 462:249-253; People such as Cahoon (2001) J Biol Chem 276:2637-2643; People such as Qiu (2001) Plant Physiol 125:847-855).The conjugated fatty acids for example biosynthesizing of calendulic acid, eleostearic acid or Trichosanoic acid is: pass through D12-desaturase catalysis desaturation oleic acid earlier to produce linolic acid, go to satisfy by the further catalysis of the special desaturase that forms conjugated triene (conjugation enzyme) again, and carry out Z9-or Z12-double-bond rearrangement formation conjugated triene lipid acid.Except that the generation of having described calendulic acid people (2001) Plant Physiol 125:847-855 such as () Qui, the enzymic activity by the conjugation enzyme is produced conjugated linolic acid also description.Yet the described second active inferior position is that enzymic activity causes undesired conjugated linolic acid 8, the generation of 10-isomer.
The pheromone desaturase of lepidopterous insects shows substrate specificity and desaturation mechanism (Roelofs and Wolf (1988) J Chem Ecol 14:2019-2031 widely; Roelofs (1995) ProcNat Acad Sci USA 92:44-49; People such as Tillman (1999) Insect Biochem29:481-514).These enzymic activitys are induced and are produced unusual unsaturated fatty acids coenzyme A derivative, have different chain length, different position of double bond and isomorphism type not.They are as the biosynthetic initial substance of pheromone.People such as Liu have described the E11-desaturase from moth (apple shallow brown volume moth (light brown applemoth)) pheromone gland, its (people (2002) Proc Natl Acad Sci USA 99 (2) such as Liu WT: 620-624) that may play an important role in the biosynthetic process of pheromone.
People such as Knipple (Genetics in December, 2002; 162 (4): 1737-52) to comparing from the isolated multiple different conformability film desaturase of the pheromone gland of moth and fly.The aliphatic alcohol desaturase fragment (PgosVASQ) of cotton pink bollworm (Pectinophora gossypiella) has been described.Its corresponding sequence leaves GeneBank Acc.No.:AF482921 in.Full length sequence and specific activity to this desaturase all are not described.It only be with the basis of the homology comparison of other desaturase on name.
An object of the present invention is to provide following novel method, described method produces and is rich in unsaturated fatty acids, preferably produces the triglyceride level that is rich in as the conjugation unsaturated acid of CLA.We find that this purpose reaches by the present invention.
First theme of the present invention relates to by recombinant expressed at least a fatty acid desaturase from lepidopterous insects, the method for producing the triglyceride level that comprises unsaturated fatty acids.
Preferably, this production method that comprises the triglyceride level of unsaturated fatty acids is included in recombinant expressed at least a fatty acid desaturase from lepidopterous insects in the organism that is selected from plant biological body, yeast, fungi and algae.
In a preferred embodiment, used the C8 in lipid acid, fatty acid coa A ester or other derivative of fatty acid, C9, C10, C11 or C12 potential energy produce the fatty acid desaturase of two keys.Especially preferred be those can be in the lipid acid with 16 or 18 carbon atom chain lengths, fatty acid coa A ester or other derivative of fatty acid the fatty acid desaturase of special generation cis or trans double bond.Those and SEQ ID NO:2,4,6,8,10,12,14 or 22 described a kind of fatty acid desaturases fatty acid desaturase more preferably with at least 65% homology.
In a preferred embodiment, this method is used to produce the conjugated linolic acid as unsaturated fatty acids.
And, the invention describes triglyceride level prepared according to the methods of the invention, and they are in the purposes of preparation food, feed, makeup or fine chemicals.
In a large amount of organisms, be included in the plant, these lipid acid mainly exist in kytoplasm with the form of coenzyme A ester.In yeast and animal, the desaturation of aliphatic alcohol lipid acid is the main route of synthesis of unsaturated fatty acids, and this mechanism rarely found in plant (people (2000) Plant Physiol.124:243-251 such as Cahoon EB).Surprisingly, recombinant expressedly can cause desaturationization saturated, unsaturated fatty acids coenzyme A ester from lepidopterous desaturase.Last result is that two kinds of active CLA isomer are all produced and are integrated in the storage fat.
The advantage of this method contains the possibility of the triglyceride level of CLA particularly in direct generation according to the present invention, produces and starts from organism, is preferably plant, yeast, fungi or algae, and described organism has high oleaginousness, such as Semen Brassicae campestris or Sunflower Receptacle.Use can be carried out good expression from the eucaryon enzyme of lepidopterous insects is feasible, does not produce the toxic action relevant with former nucleoprotein usually simultaneously.
" fatty acid desaturase " refers to for example preferably to introduce at lipid acid or their derivative the enzyme of two keys in fatty coenzyme A ester.If suitable, cofactor such as NADPH, NADH or oxygen also reach the extra needs of this purpose.Preferred those can utilize the desaturase of aliphatic alcohol lipid acid as substrate in this article, and wherein said aliphatic alcohol lipid acid has 14,16,18 or 20 carbon atom chain lengths, preferred 18 carbon atoms.
Preferably, this term of fatty acid desaturase comprises those and can for example be preferably the enzyme that produces two keys on C8, C9, C10, C11 or the C12 position of fatty acid coa A ester at the lipid acid or derivatives thereof.Also preferred those can produce the desaturase of specific structure isomer, promptly have cis or trans double bond specificity.The formation amount that is interpreted as the isomer of being discussed especially in this article is at least 60%, and preferably at least 80%, very especially preferably at least 90%, most preferably at least 95%.Desaturase very particularly preferably is those enzymes that can produce two keys at lipid acid or derivatives thereof such as preferred fatty acid coenzyme A ester, and the wherein said pair of key is by as being found at the CLA isomer.
The enzyme that refers to have at least a following properties from " the preferred fundamental characteristics " of the fatty acid desaturase of lepidopterid:
I) at C8, C9, C10, C11 or C12 position cis-double bonds or in the special generation of C8, C9, C10, C11 or C12 position trans double bond.
The substrate specificity that ii) has lipid acid, fatty acid coa A ester and other derivative of fatty acid of 16 and/or 18 carbon atom chain lengths.In this article, substrate specificity refers to that fatty acid desaturase transforms the substrate characteristic faster than the substrate of other different chain length of described chain length.In this article, compare with not preferred substrate, the transformation efficiency of preferred substrate can improve 50% at least, preferably improves 100% at least, very particularly preferably improves 200% at least, most preferably improves 500% at least.
Preferably have characteristic i in all cases at least) and characteristic ii) wherein a kind of.
This term of fatty acid desaturase comprises the enzyme that can introduce isolated double bond, and the conjugation enzyme that begins to produce the conjugated double bond system from two keys of substrate.In this article, first pair key that has preferably moved.
The example of preferred conjugation enzyme is:
A) the two keys of Z11 are changed into two E10 and the two keys of Z12 the conjugation enzyme (" Z11-(and E10, Z12)-the conjugation enzyme ").
B) the two keys of E11 are changed into two E10 and the two keys of Z12 the conjugation enzyme (" E11-(and E10, Z12)-the conjugation enzyme ")
C) the two keys of Z10 are changed into two E10 and the two keys of Z12 the conjugation enzyme (" Z10-(and E10, Z12)-the conjugation enzyme ").
In a word, these enzymes can be described as (E10, Z12)-the conjugation enzyme.These enzymes also can be described as the E10-desaturase.The fundamental characteristics of E10-desaturase very particularly preferably is the C-10 position introducing trans double bond that has the lipid acid or the fatty acid coa A ester of 18C atom fatty acid chain length therein.
In addition, preferred conjugation enzyme is
D) the two keys of Z10 are changed into two Z9 and the two keys of E11 the conjugation enzyme (" Z10-(and Z9, E11)-the conjugation enzyme "),
E) the two keys of E10 are changed into two Z9 and the two keys of E11 the conjugation enzyme (" E10-(and Z9, E11)-the conjugation enzyme "),
F) the two keys of Z11 are changed into two Z9 and the two keys of E11 the conjugation enzyme (" Z11-(and Z9, E11)-the conjugation enzyme ").
In a word, these enzymes can be described as (Z9, E11)-the conjugation enzyme.These enzymes are also referred to as the E11-desaturase.The fundamental characteristics of E11-desaturase very particularly preferably is the C11 position introducing trans double bond that has the lipid acid or the fatty acid coa A ester of 18C atom fatty acid chain length therein.
Most preferably such E10-, E11-, Z10-and Z11-desaturase, and Z11-(E10, Z12)-conjugation enzyme, E11-(E10, Z12)-conjugation enzyme, Z10-(Z9, E11)-conjugation enzyme and E10-(Z9, E11)-the conjugation enzyme, these conjugation enzymes to lipid acid with 18C atom fatty acid chain length or derivative of fatty acid for example fatty acid coa A ester have the substrate characteristic.
Fatty acid desaturase preferably comes from lepidopteran section, and these sections are selected from adjacent diamond-back moth section (Acrolepiidae), tiger moth section (Agaristidae), Arctiidae (Arctiidae), Bombycidae (Bombycidae), Carposinidae (Carposinidae), Cochylisspp section (Cochylidae), Cossidae (Cossidae), Eriocraniidae (Eriocraniidae), Gelechidae (Gelechiidae), Geometridae (Geometridae), Gracilariidae (Gracillariidae), Hepialidae (Hepialidae), clearwing butterfly section (Ithomiidae), Lasiocampidae (Lasiocampidae), Lycaenidae (Lycaenidae), Lymantriidae (Lymantriidae), lyonetid section (Lyonetiidae), Nepticulidae (Nepticulidae), Noctuidae (Noctuidae), Notodontiade (Notodontidae), Nymphalidae (Nymphalidae), Oecophoridae (Oecophoridae), Papilionidae (Papilionidae), Sulfur butterfly (Pieridae), Moth files (Psychidae), Pterophoridae (Pterophoridae), Pyralidae (Pyralidae), Saturniidae (Saturniidae), Aegeriidae (Sesiidae), Sphingidae (Sphingidae), Tortricidae (Tortricidae), Yponomeutidae (Yponomeutidae) and Leaf skeletonizer moth (Zygaenidae).Especially preferred is the fatty acid desaturase that is selected from the lepidopteran section of Tortricidae, Pyralidae, Papilionidae, Noctuidae and Geometridae.
E11-desaturase or their nucleotide sequence of encoding are preferably from the wild snout moth's larva (Diaphania hyalinata) of muskmelon thin,tough silk, Diaphania nitidalis, the wild snout moth's larva (Leucinodesorbonalis) of the white wing of eggplant, European corn borer (Ostrinia nubilalis), Sesamia grisescens, brachmia triannuella (Brachmia macroscopa), Paraargyresthia japonica, Mnesictenaflavidalis, Telorta edentata, Epiplema moza, Argyresthia chamaecypariae, some kind (Bradina sp.) that dark water snout moth's larva belongs to, Dichrocrocis punctiferalis, the jowar fringe conceals phycitid (Cryptoblabes gnidiella), Palpita unionalis, Sceliodes cordalis, Anisodes sp., the thin moth of tea (Caloptilia theivora), Phyllonorycter ringoniella, Deilephila elpenor, cigarette beetle (Manduca Sexta), Scirpophagaexcerpalis, white to borer (Scirpophaga nivella), three-way cluster caterpillar moth (Andracabipunctata), separate in the organism of these species of cotton pink bollworm or little sugarcane borer (Diatraea saccharalis).E11-desaturase or their nucleotide sequence of encoding especially are preferably from the pheromone gland of above-mentioned insect and separate.The example that can mention is the E11-desaturase from the pheromone gland of apple shallow brown volume moth (Epiphyas postvittana) (SEQ ID NO:2), European corn borer (SEQ ID NO:4) and Ostrinia furnicalis (SEQ ID NO:6).Particularly preferably be the E11-desaturase shown in SEQ ID NO:22 from the cotton pink bollworm.From the E11-desaturase of organism above-mentioned with plant aliphatic alcohol derivative of fatty acid as substrate.
Using these desaturases or their variant may be particularly advantageous to the two keys of generation E11 are become.If desaturase (for example can not accomplish preferably to transform C16 and/or C18 aliphatic alcohol substrate, desaturase shown in SEQ ID NO:22) from the cotton pink bollworm, then preferably optimize described desaturase, purpose is to make it can preferably transform C16 and/or C18 aliphatic alcohol substrate.Therefore another theme of the present invention relates to and has the active polypeptide of E11-desaturase, and described polypeptide preferably transforms C16 and/or C18 aliphatic alcohol lipid acid, and comprises and be selected from least one following sequence:
A) aminoacid sequence shown in SEQ ID NO:2,4,6 and 22 and
B) with SEQ ID NO:2,4,6 or 22 shown in wherein a kind of aminoacid sequence have at least 65% homology aminoacid sequence and
C) comprise the aminoacid sequence of at least 20 continuous amino acid residue segments in the sequence shown in SEQ ID NO:2,4,6 or 22.
Described polypeptide is particularly preferably described with the aminoacid sequence shown in the SEQ ID NO:22.Another theme of the present invention relates to the nucleic acid molecule of the above-mentioned desaturase of encoding.Sequence particularly preferably is as shown in SEQ ID NO:21.
E10-desaturase or their nucleotide sequence of encoding are preferably from Dichrocrocischlorophanta, dichocrocis punctiferalis (Dichrocrocis punctiferalis), mulberry tree mulberry wild silkworm (Bombyxmandarina), silkworm (Bombyx mori), Coloradia velda, Hemileucaeglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, separate in the wild snout moth's larva species organisms such as (Notarcha derogata) of Hemileuca nuttalli or lap leaf.E10-desaturase or their nucleotide sequence of encoding particularly preferably separate from the pheromone gland of above-mentioned insect.
E9-desaturase or their nucleotide sequence of encoding are preferably from Epiplema plaqifera, Phyllonorycter coryli, Phyllonorycter sylvella, Gelechiinae, Bryotrophasp., Bryotropha terrella, Gelechia betulae, Adoxophyes orana, Exartemaappendiceum, Zeiraphera canadensis, Loxostege neobliteralis, European corn borer, Dioryctria clarioralis, Dioryctria merkeli, Dioryctria resinosella, beet armyworm (Spodoptera exigua), separate in the species organisms such as Spodoptera triturata oder Polia grandis.E9-desaturase or their nucleotide sequence of encoding particularly preferably separate from the pheromone gland of above-mentioned insect.
E8-desaturase or their nucleotide sequence of encoding preferably separate from species organisms such as Phyllonoryctersaportella, Phyllonorycter sp. or Dichrocrocis punctiferalis.E8-desaturase or their nucleotide sequence of encoding particularly preferably separate from the pheromone gland of above-mentioned insect.
The Z11-desaturase that can advantageously be used for producing the Z11-18 carbon enoic acid has discovery at silkworm (Ando etc. (1988) Agric Biol Chem.52:473-478), cabbage looper (Trichoplusia ni) and Heliothis zea (Helicoverpa zea) (Knipple etc. (1998) Proc Nat Acad Sci USA95:15287-15292).More Z11-desaturase or their nucleotide sequence of encoding can preferably separate from cigarette beetle, southwestern corn stalk crambid (Diatraea grandiosella), Eariasinsulana, Earias vittella, small cabbage moth (Plutella xylostella), silkworm or Diaphanianitidalis.The example that can mention is the desaturase from Heliothis zea (SEQ ID NO:8), cabbage looper (SEQ ID NO:10) and argyrotaenia velutinana (Argyrotaenia velutinana) (SEQ IDNO:12).Z11-desaturase or their nucleotide sequence of encoding preferably separate from the pheromone gland of above-mentioned insect.
Z10-desaturase or their nucleotide sequence of encoding preferably separate from species organisms such as Ctenopseustisfilicis, Pseudexentera spoliana, Hemileuca eglanterina, Hemileucaelectra electra, Hemileuca electra mojavensis, Hemileuca nuttalli, Eurhodope advenella, Mamestra configurata or Dichrocrocis punctiferalis, particularly preferably separate from the pheromone gland of stating insect.The example that can mention is the desaturase from Planototrix octo (SEQ ID NO:14).
(E10, Z12)-conjugation enzyme or their nucleotide sequence of encoding be preferably from silkworm, Phyllonorycter crataegella, Amorbia cuneana, Notocelia incarnatana, Notocelia uddmanniana, mulberry tree mulberry wild silkworm, Coloradia velda, Hemileucaeglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, Hemileuca nuttalli, the wild snout moth's larva of lap leaf, mulberry tree mulberry yellow moth (Rondotia menciana), Amphion floridensis, Hyles gallii, Hyloicus pinastri, maduca sexta, Sphinxdrupiferarum, Earias insulana, separate among Nola confusalis or the Notarcha basipunctalis.(E10, Z12)-conjugation enzyme or their nucleotide sequence of encoding particularly preferably separate from the pheromone gland of above-mentioned insect.
(Z9, E11)-conjugation enzyme or their nucleotide sequence of encoding be preferably from little sugarcane borer, Xyrosarislichneuta, Dioryctria abietella, Stenoma cecropia, Phalonidia manniana, Pselnophorus vilis, Dioryctria abietella; Separate in little red tip phycitid (Dioryctria rubella), Myelopsis tetricella, Jodis lactearia, true Xiao Ji chi pretty young woman (Scopula personata), Spodoptera descoinsi, southern spodoptera (Spodoptera eridania), Spodopteralatifascia, spodoptera (Spodoptera littoralis) or the prodenia litura (Spodoptera litura).(Z9, E11)-conjugation enzyme or their nucleotide sequence of encoding particularly preferably separate from the pheromone gland of above-mentioned insect.
Z11-desaturase and Z11-conjugation enzyme can both be accepted with plant aliphatic alcohol derivative of fatty acid as substrate, and this two class causes from the association list Danone of desaturase in plant of lepidopteran class that (10E 12Z)-generation of CLA, and is incorporated in the storage fat.
Have several possible conjugation enzymes or desaturase each other combination or with the combination of plant enzyme, to produce purpose CLA isomer, particularly (9Z, 11E)-CLA and (10E, 12Z)-CLA.Understand these methods in the mode of giving an example rather than limit below:
1) begins trans-desaturation from stearic acid, for example, just can produce trans-isooleic acid (Fig. 3) by the E11-desaturase by the C11-position.
Fig. 3: from stearic acid begin to produce (9Z, 11E)-CLA and (10E, 12Z)-biosynthetic pathway of CLA
From palmitinic acid, by the trans-desaturation of C9 position, for example use the E9-desaturase, can produce Zoomeric acid (9E hexadecylenic acid), then, prolong Zoomeric acid and obtain trans-isooleic acid (Fig. 4) by extending the enzymatic activity of enzyme.
Fig. 4: from palmitinic acid begin to produce (9Z, 11E)-CLA and (10E, 12Z)-biosynthetic pathway of CLA
Trans-isooleic acid next can transform and obtain by the D9-desaturase (9Z, 11E)-the CLA isomer.Proved Mammals and human enzymatic activity by endogenous D9-desaturase, trans-isooleic acid is converted into CLA, and (WO 99/20123; (2000) J Nutr130:208-215 such as Santora JE; (2000) Lipids 35:131-135 such as Adlof RO).Equally, also verified insect cell of expressing the E11-desaturase can change into Z9 with the E11-lipid acid initiator that obtains, E11-lipid acid (people (2002) Proc Natl Acad Sci USA 99 (2): 620-624 such as Liu WT.Equally, plant D9-desaturase is can catalysis trans-isooleic acid be converted into (Z9, E11)-the CLA isomer.In an especially preferred embodiment, extra Z9-desaturase expression can further increase this conversion.Preferably has the active Z9-desaturase of kytoplasm herein, for example yeast Z9-desaturase.Thereby, trans by expressing-desaturase, in plant, produce (Z9, E11)-the CLA isomer is possible.As the result of plant Z9-desaturase effect, formed particularly advantageous (9Z, 11E)-the CLA isomer.
2) from stearic acid, corresponding E10-octadecenoic acid can be at first by trans-desaturation generation (Fig. 3) on the C10 position.But as selection scheme, the E8-hexadecylenic acid can begin to pass through the trans-desaturation of C8 position and produce from palmitinic acid.The E8-hexadecylenic acid then obtains E10-octadecenoic acid (Fig. 4) by the enzymatic activity prolongation of carbochain extension enzyme.This lipid acid will be transformed and obtain by the D12-desaturase (E10, Z12)-the CLA isomer.This reaction also can be by the catalysis of plant D12-desaturase.In an especially preferred embodiment, the expression of extra Z12-desaturase can further increase this transformation efficiency.
3) from the lepidopteran class corresponding (E10, Z12)-the conjugation enzyme also can use.For example Z11-octadecenoic acid (cis-isooleic acid), E11-octadecenoic acid (trans-isooleic acid) or the generation of Z10-octadecenoic acid of these conjugation enzymatic conversions (E10, Z12)-CLA.For this purpose, at first stearic acid is converted into the Z11-octadecenoic acid, wherein under the effect of Z11-desaturase, is converted into above-mentioned trans-isooleic acid or at first stearic acid is converted into the Z10-octadecenoic acid under the effect of Z10-desaturase.
4) from the lepidopteran class (Z9, E11)-the conjugation enzyme also can be further advantageously with (Z11)-, (Z10)-or (E10)-desaturase combination.From stearic acid, these desaturases produce Z11-octadecenoic acid (cis-isooleic acid), Z10-octadecenoic acid or E10-octadecenoic acids, they subsequently by the enzymatic conversion of (Z9/E11)-conjugation produce (Z9, E11)-CLA.
Zhi Bei CLA lipid acid or their derivative fatty acid coa A ester for example is stored in film fat and the triglyceride level like this.
In lepidopterous insects, especially in above-mentioned insect species, located desired enzymic activity.Identification system provided by the present invention is used to this purpose (embodiment 2,3 and 4).
Encode these active enzymes or their nucleotide sequence of encoding can separate in the mode that those skilled in the art were familiar with from the organism of being discussed or come out by the mutagenesis reasoning of corresponding known array.
For a kind of enzymatic activity, functional or phenotype, finding protein sequence or corresponding cDNA sequence is a biological chemistry and a molecular biological normal work to do.Those skilled in the art are familiar with multiple solution to this problem.For example these methods are the assay method (particularly seeing embodiment 3) that is used for determining desaturase or conjugation enzymic activity based on provided by the present invention.The given activity of the desaturase of being discussed/conjugation enzyme is for example determined by gc analysis lipid acid sample as described in embodiment 2,3 and 4.From determining to have the biomaterial of desaturase or conjugation enzymic activity, make with this assay method and to separate the protein of being discussed or this proteinic nucleic acid and analyze it and become possibility of encoding with desaturase or conjugation enzymic activity.
For example, cloning by expression can adopt this method.What this method was frequent is used for, from certain enzyme activity, specific functionality or special phenotype, separate correspondingly gene or the corresponding cDNA of this gene (Dalboge H (1997) FEMS Microbiology Reviews21 (1): 29-42, Simonsen H and Lodish HF (1994) Trends Pharmacol Sci15 (12): 437-441).Conventional cloning by expression method has description in a large amount of occasions.For example for membranin, excreted factor and transmembrane channel ((1987) Nature329 (6142): 836-838 such as Masu Y; (1985) Science 228 (4701): 810-815 such as Wong GG; LustigKD etc. (1996) Development 122 (12): 4001-4012; Smith WC and Harland RM (1992) Cell 70 (5): 829-840; (1995) Cell 81 (1): 85-94 such as Lemaire P; GillissenB etc. (2000) Plant Cell 12 (2): 291-300; Lotan T and Hirschberg J (1995) FEBSLetters 364:125-128).
Hereinafter in the universal method that is called cloning by expression of Miao Shuing, common reorganization and clone technology have been used, these technology are for example at Maniatis T, Fritsch EF and Sambrook J, MolecularCloning:A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, NY (1989) and at Silhavy TJ, Berman ML and Enquist LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, NY (1984) and at (1987) Current Protocols inMolecular Biology such as Ausubel FM is described among the Greene Publishing Assoc.and Wiley Interscience.
Generally, begin to produce expression library from organism, the cell or tissue that can produce desaturase and conjugation enzymic activity.For example, at first from described organism, cell or tissue with the mode that those skilled in the art were familiar with separate mRMA or, preferably, poly (A)-mRMA, on the basis of the mRNA of resulting separation, prepare cDNA (Gubler U, Hoffman BJ (1983) Gene25:263-269).Those skilled in the art are familiar with the multiple system that is used for separating mRNA or poly (A)-mRNA, and these systems have commercially available.For example, available " Quick Prep Micro mRNAPurification Kit " (Amersham Pharmacia Biotech) synthesizes.The first chain cDNA preferably uses oligomerization (dT) primer, adopts synthetic ((1992) the PCRMethods Appl 2:144-148 such as Borson ND of reversed transcriptive enzyme; (1994) CLONTECHniques9 (1) such as Chenchik A: 9-12).The many systems that make full-length cDNA produce are known and commercially available.The example that can mention is " SMART TMCDNA Library Construction Kit " (Clontech, Cat.#K1051-1).In this test kit, SMART TMTechnology (SMART=SwitchMechanism At the 5 ' end of RNA Templates) is used for producing cDNA library (HerrlerM (2000) J Mol Med 78 (7): B23).Specific oligonucleotide and the method that is called as long-range PCR have been used.This method is described in embodiment 4.
Joint with the outstanding chain of sub-thread can be connected to double-stranded cDNAs; These joints for example make that the clone becomes possibility in expression vector, and wherein said expression vector has been cut by Restriction Enzyme and had a compatible sticky end.Preferably, the clone is directed, and the direction of Yue Duing is a fixed like this, and the promotor of arbitrary existence produces the adopted RNA that has that starts from the cDNA insertion.Be used for SMART for example TMNGGCC) in Qie Ge the carrier.In all cases, a cDNA molecule is cloned into carrier molecule, last like this generation based on identical underlying carrier and according to the cDNA that integrates and different variety carrier and form expression library.This expression library also comprises can express desaturase or the genetically modified carrier of conjugation enzyme.The expression vector that is fit to be meant in principle all that can be in the inverting biological body desaturase of recombinant expressed activity form or the carrier of conjugation enzyme.The example that can mention has and is used for recombinant expressed λ TriplEX2 carrier in intestinal bacteria (E.coli) (after the excision of pTriplEX2 carrier; Manufacturer: Clontech) or be used for recombinant expressed carrier pYES2 (manufacturer: Invitrogen) (see embodiment 4) in yeast saccharomyces cerevisiae (S.cerevisiae).
As required, can proofread and correct with the difference of compensation genes of individuals on expression level, each is represented with similar copy number in expression library by the cDNA of expressing gene like this, and is irrelevant with actual expression level.Many systems are described to produce poly (A)-mRNA, cDNA and standard cDNA (US 5,482,845) ((1994) Proc Natl Acad Sci USA91:9228-9232 such as Soares MB; (2000) Genome Res 10:1617-1630 such as Carninci P; BonaldoMF etc. (1996) Genome Res 6:791-806).
Yet, also can set up the deduction expression library.In the method, have the cDNA of organism, cell or tissue of desaturase or conjugation enzyme and the cDNA that do not have this active organism, cell and a tissue relatively, work if possible compares the cDNA of same genus and kind.Those skilled in the art are familiar with having the method for the cDNA that the separation of target only expresses in the biomaterial with desaturase or conjugation enzymic activity.These class methods and system are described and have commercially available.The example that can mention is " PCR-Select TMCDNA Subtraction Kit " (manufacturer: Clontech) or " Subtractor TMKit " (manufacturer: Invitrogen), described test kit can surpass 1000 times of cDNA that concentrate rare and/or selective expression.Delete all cDNA that in two kinds of biomaterials, all occur ((1997) Proc Nat Acad Sci USA94:10057-10062 such as Chu ZL with subtractive hybridization; (1997) Cell 91:397-405 such as Hudson C; (1997) J Exp Med 186:655-663 such as Mueller CGF; (1997) Nucleic Acids Res25:2598-2602 such as von Stein OD; (1997) J Biol Chem 272:25190-25194 such as Wong BR; (1997) Nature 387:620-624 such as Yokomizo T; Zhicheng S and Jacobs-Lorena M (1997) J Biol Chem 272:28895-28900).
CDNA in expression library can be for example recombinant expressed in protokaryon or eukaryotic cell, and next these cells are measured desaturase or conjugation zymoprotein, for example measures the activity of desaturase or conjugation enzyme.
For the carrier of recombinant expressed desaturase or conjugation enzyme is separated from expression library, preferably expression library is transformed in the organism.Preferably, the separate cell of above-mentioned organism is in this way transformed: each cell or only transform with one type carrier molecule from each organism of this cell regeneration.In principle, all these can recombinant expressed active desaturases or the organism of conjugation enzyme or all be fit to conversion from the cells of these organisms.These organisms can be protokaryon with eucaryon.Preferably all plants, from their cell, but also can be other photosynthesis organism, for example algae.The example that can mention is, but not only be confined to these examples, those are based in bacterium such as intestinal bacteria, yeast such as yeast belong (Saccharomyces) or Pichia (Pichia), mammalian cell such as COS or Chinese hamster ovary celI, or have system recombinant expressed in the cell of photosynthetic activity organism such as synechocystis.Preferred organism is a most eukaryotes, very particularly preferably be can synthetic fatty acid or the most eukaryotes of aliphatic alcohol lipid acid.Preferably, the expression vector that is used for transforming comprises selected marker, antibiotics resistance or in order to selecting the amino acid synthetic gene of amino acid defective for example, and its makes to select and transforms successful cell and become possibility.Further system of selection will be described below.
In a preferred embodiment, cloning by expression can be implemented in yeast.For this purpose, can for example use yeast expression system based on the pYES2 carrier from Invitrogen.PYES2 is the episomal vector of a high copy, in order to abduction delivering recombinant protein in yeast saccharomyces cerevisiae.The yeast strain that transforms can be based on uridylic defective selected come out (pYES2 comprises the ura3 gene)
The transfection method of further preferred expression vector and generation inverting biological body is consistent with the method that is commonly used to produce transgenic organism.
From the inverting biological body of all numbers or come from their cell, concentrating and separate those can recombinant expressed desaturase or conjugation enzyme and/or have corresponding desaturase or the inverting biological body of conjugation enzymic activity or come from their cell.
Generally, the desaturase activity can be by cultivating the inverting biological body or from its cell, with it/they place suitable damping fluid or solvent to digest, digest is contacted with lipid acid or aliphatic alcohol lipid acid, and, as required, add cofactor such as NADH or NADPH or oxygen, measure desaturation lipid acid or the aliphatic alcohol lipid acid that obtains thus then.If organism or come from its cell itself can synthetic fatty acid or aliphatic alcohol lipid acid, the organism self that lipid acid or aliphatic alcohol lipid acid are preferably hung oneself and transformed.If yet can not synthesize, also can add lipid acid or aliphatic alcohol lipid acid.
As required, after for example ethyl acetate was extracted from mixtures incubated with solvent, lipid acid or the aliphatic alcohol lipid acid crossed by desaturase or conjugation enzyme modification can detect by the ordinary method that those skilled in the art are familiar with.Separation method such as high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), tlc (TLC) and detection method such as mass spectroscopy (MS or MALDI), ultraviolet spectral analysis method (UV) or radioautography can be used for this purpose.Preferably, the detection method of vapor-phase chromatography is implemented as embodiment 3,4 and 5 is described.
Can recombinant expressed desaturase or the inverting biological body of conjugation enzyme or can for example be divided into subgroup by cell all quantity from their cell, cultivate these subgroups, if separate suitablely, measure the activity of each independent subgroup desaturase or conjugation enzyme and separate.The subgroup that comprises recombinant expressed desaturase of a kind of energy or the proteic cell type of conjugation enzyme functionally active form has enhanced desaturase or conjugation enzymic activity.These subgroups are further divided and are repeated this process up to obtaining the mono-clonal culture, and promptly culture only comprises a kind of carrier of the specific cDNA of coding desaturase or conjugation enzyme.
The carrier that comprises the nucleic acid of coding desaturase or conjugation enzyme can reclaim from inverting biological body or cell.This can be undertaken by for example polymerase chain reaction (PCR).For this purpose, used the carrier sequence complementary Oligonucleolide primers that inserts the fragment flank with desaturase or conjugation enzyme cDNA.Do not need to know desaturase or conjugation enzymatic nucleic acid sequence in this article.As selection scheme, if carrier is not incorporated in the host genome, can from cell, reclaim, transform in the intestinal bacteria, propagation and order-checking are so that obtain the nucleotide sequence of coding desaturase or conjugation enzyme.According to above-mentioned method, desaturase or conjugation enzymatic nucleic acid sequence also can be separated from isolated vectors with the method for PCR, and do not need to know its sequence order.And making directed cloning become possible joint can be added in the amplicon after the PCR reaction.Yet the clone also can implement by the method that flat terminal connection of the usefulness that those skilled in the art were familiar with or T-protruding terminus connect.With the desaturase of PCR method selective amplification or conjugation enzyme cDNA thereby can directly clone yet and into be fit to produce in the carrier of the transgenic organism of expressing desaturase or conjugation enzyme, and do not need to illustrate in advance sequence.This clone's process can be implemented by the flat terminal method of for example being familiar with the technician of cloning.Be used to this purpose as above-cited conventional reorganization and clone technology.
Cloning by expression method above-mentioned can particularly preferably be implemented in yeast saccharomyces cerevisiae.In order to reach this purpose, also can use some such systems, in these systems, recombinant expressed cDNA is integrated into integration platform ((2000) Applied and Environmental Microbiology 66 (1) such as Lagarde D: 64-72) that has produced by homologous recombination
As alternatives, the method for cloning by expression also can be implemented at the above-mentioned non-amplification expression library of external use.Suitable system has description and has commercially available.The example that can mention is " TNT TMCoupled Reticulocyte Lysate System " (Promega; Promega Notes Number67,1998, p.02).People such as Kirschner have have researched and developed a kind of in vitro method (IVEC=" vivoexpression clone "), this method do not need viable cell (US 5,654,150; (1997) Science 277,973 such as King RW).This system successfully applies to especially enzyme and kinases.Kirschner etc. have identified kinase substrate and proteolytic enzyme ((1997) Meth Enzymol 283:83 such as Lustig KD; (1997) Curr Biol 7:338 such as Stukenberg PT; (1997) Science278:294 such as Kothakota S)." vivoexpression clone " (IVEC) in, used the small portion expression library.In each case, about 50 to 100 cDNA clone is present in the plasmid, and by transcribe based on the external coupling of net matter erythrocyte splitting thing/the translation system is translated as their corresponding proteins matter.For reaching this purpose, make up oligomerization (dT)-primer cDNA library as mentioned above, and clone in the high copy expression plasmid with T3, T7 or SP6 promotor.This plasmid library transforms in the intestinal bacteria then.In all cases, about 50 to 100 independently transformant have the bacterium colony of cultivating into about 1mm on the antibiotic agar plate of corresponding selection, collect then and concentrate.Plasmid DNA is separated from this bacterium pond of part.These plasmid DNA that are used as template are directly transcribed and are translated in the reticulocyte system.Concrete working method can be at for example manufacturer's handbook (TNT TMCoupledReticulocyte Lysate Systems Technical Bulletin#TB126 finds in PromegaCorporation).According to the number of full length cDNA clone in the library, nearly 30 to 50 protein synthesis of each independent mixture.Test these proteinic desaturase that has produced and conjugation enzymic activitys.Independent mixture with enhanced desaturase or conjugation enzymic activity is further segmented.The segmentation of mixture can be found the cDNA of coding desaturase or conjugation enzyme separately.The nucleic acid of coding desaturase or conjugation enzyme can be from the method amplification of the carrier being present in this prepared product with PCR, if it is suitable, do not need to know sequence and analyze it, use described nucleic acid to produce the organism of recombinant expressed desaturase or conjugation enzyme as mentioned above.But as the selection scheme of rabbit net matter red corpuscle extract, what the wheat germ extract also can be used to unite transcribes/translation steps.
In having more favourable application, cDNA can be cloned in the retroviral vector.This allows high efficiency cell transformation.Retrovirus expression vector and expression system are existing to be described (KitamuraT, International Journal of Hematology 1998,67:351-359) and have commercially available (for example from Clontech; New Retroviral﹠amp; ClonCapture Expression Libraries; CLONTECHniques, in October, 1998, XIII (4): 22-23).Transfection is to carry out (EcoPack for example in package cell line at first TM-293 or RetroPack TMPT67 clone is from Clontech).With the virus that results from this mode, but the target cell that transfection is discussed system and selection purpose activity.Then, inserting fragment can obtain and analyze, and for example passes through PCR.In addition, they can directly be cloned in the suitable expression vector, even do not need in advance they to be carried out sequential analysis, and these expression vectors can be used to produce the organism of recombinant expressed desaturase or conjugation enzyme then.
Modify known cis-desaturase by mutagenesis and can obtain such E10-or E11-desaturase, promptly they have the desat specificity in trans position.For reaching this purpose, the nucleotide sequence of for example following coding cis desaturase can carry out mutagenesis then as homing sequence:
I) from the nucleotide sequence of the coding Z11-desaturase of Heliothis zea, SEQ ID NO:7
Ii) from the nucleotide sequence of the coding Z11-desaturase of cabbage looper, SEQ ID NO:9
Iii) from the nucleotide sequence of the coding Z11-desaturase of argyrotaenia velutinana, SEQ ID NO:11 or
Iv) roll up the nucleotide sequence of moth (Planotortrix octo) coding Z10-desaturase, SEQ ID NO:13 from green head
And, can make it can transform longer chain fatty acid more effective or fully by mutagenesis E11-desaturase.Be operable homing sequence below:
A) from the shallow brown nucleotide sequence of rolling up moth coding E11-desaturase of apple, SEQ ID NO:1,
B) from the nucleotide sequence of European corn borer coding E11-desaturase, SEQ ID NO:3, or
C) from the nucleotide sequence of Ostrinia furnacalis (Ostrinia furnacalis) coding E11-desaturase, SEQ ID NO:5.
Method by mutagenesis changes desaturase above-mentioned, and to introduce the specificity of position of double bond also be conceivable.
Go to change characteristic by the method for mutagenesis, for example the method for the substrate specificity of desaturase is known (Cahoon etc. (1997) Proc Nat Acad Sci USA 94:4872-4877; WO98/06735).Further suitable method is used for having described other lipid metabolism enzyme, and for example lipoxidase, and energy is used (Hornung etc. (2000) Biochem Soc Trans28:825-826 similarly; Schwarz etc. (2001) J Biol Chem 276:773-779).
The nucleotide sequence that is used for the coding desaturase/conjugation enzyme of the inventive method also can separate from lepidopteran species above-mentioned cDNA prepared product or library separately with polymerase chain reaction, and described polymerase chain reaction adopts suitable degeneracy oligonucleotide primer.Those are preferably adopted be the Oligonucleolide primers in SEQ IDNO:15 and 16, described to or the Oligonucleolide primers in SEQ ID NO:17 or 18, described right.
Be used for the inventive method from the nucleotide sequence of lepidopteran fatty acid desaturase preferably:
A) in its sense strand, comprise SEQ ID NO:15 or 17 described sequence motifs, or
B) in its antisense strand, comprise SEQ ID NO:16 or 18 described sequence motifs.
In a further preferred embodiment, the wherein a kind of fatty acid desaturase sequence preference ground of using in the methods of the invention from describing among the protein sequence of lepidopterous fatty acid desaturase and the SEQ ID NO:2,4,6,8,10,12 or 14 has at least 65%, preferably at least 70%, particularly preferably at least 80%, at least 90% homology very particularly preferably, and randomly and preferably have at least a in the preferred fundamental characteristics of desaturase or conjugation enzyme.These protein also comprise their the natural or artificial mutation of one of homologue that belongs to kind from desaturase nucleotide sequence above-mentioned and from other animal or plant.Sudden change comprises alternative, interpolation, disappearance, inversion or inserts one or more nucleotide residues.When inserting, lack or substituting, for example base conversion and transversion where applicable can adopt original known technology, for example vitro mutagenesis, primer reparation, restriction enzyme digestion effect and ligation.Can provide segmental complementary end to be used for connecting, by for example using restriction enzyme digestion, returning to cut (chewing-back) or fill and lead up overhang and obtain flat terminal.Similarly the result also can obtain by the polymerase chain reaction (PCR) that adopts specific oligonucleotide primers.
At one further in the embodiment preferred, nucleotide sequence and the SEQ ID NO:1 that are used for the inventive method from lepidopterous coding fatty acid desaturase, 3,5,7,9, wherein a kind of fatty acid desaturase nucleotide sequence described in 11 or 13 preferably has at least 65%, preferably at least 70%, particularly preferably at least 80%, very particularly preferably at least 90% homology, and its such protein of randomly and preferably encoding, described protein have at least a in desaturase or the preferred fundamental characteristics of conjugation enzyme.
Article two, the homology between nucleic acid or the polypeptide is interpreted as the identity of the nucleotide sequence of full sequence length in all cases, this identity is calculated (Wisconsin Package Version 10.0 by means of the GAP programmed algorithm by sequence alignment, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), following parameter is set:
Breach weight: 12 length weights: 4
Average coupling: 2.912 on average do not mate :-2.003
In a preferred embodiment, the inventive method adopted from the nucleotide sequence of lepidopterous coding fatty acid desaturase preferably under standard conditions, with one of them of the nucleotide sequence of coding desaturase above-mentioned, preferably with as SEQ ID NO:1,3,5,7, sequence hybridization shown in 9,11 or 13.
The terminology standard hybridization conditions should be broadly interpreted as strict or more undemanding hybridization conditions.This type of hybridization conditions is described in the following book especially: Sambrook J, Fritsch EF, Maniatis T etc., Molecular Cloning (A Laboratory Manual), the 2nd edition, Cold SpringHarbor Laboratory Press, 1989, pages 9.31-9.57 page or leaf) or Current Protocolsin Molecular Biology, John Wiley﹠amp; Sons, NY (1989), 6.3.1-6.3.6.For example, the condition of washing step can be selected from the condition in the certain limit, and these conditions are divided into low stringency condition (about 0.2X SSC is under 50 ℃) and high stringent condition (about 0.2X SSC is under 50 ℃, preferably under 65 ℃) (20X SSC:0.3M Trisodium Citrate, 3M NaCl, pH 7.0).And, can be in the temperature of washing stage from low stringency condition at room temperature, about 22 ℃, be elevated to about 65 ℃ high stringent condition.Can change simultaneously salt concn and these two parameters of temperature, perhaps one of these two parameters remain unchanged and only change another parameter.During hybridization, also can use denaturing agent such as methane amide or SDS.When having 50% methane amide, hybridization preferably carries out at 42 ℃.Details are as follows to be used to hybridize some examples with the condition of washing step:
(1) hybridization conditions, for example,
A) 4X SSC is under 65 ℃
B) 6X SSC is under 45 ℃
C) 6X SSC is under 68 ℃, 100 μ g/ml sex change fish sperm DNAs
D) 4X SSC, 50% methane amide is under 42 ℃
E) 2X or 4X SSC under 50 ℃ (low stringency condition), or
F) 2X or 4X SSC, 30 to 40% methane amides are under 42 ℃ (low stringency condition).
(2) washing step, for example:
A) 0.1X SSC, under 65 ℃, or
B) 0.1X SSC, 0.5%SDS, at 68 ℃, or
C) 0.1X SSC, 0.5%SDS, 50% methane amide, under 42 ℃, or,
D) 0.2X SSC, 0.1%SDS, under 42 ℃, or
E) 2X SSC is under 65 ℃ (low stringency condition).
For the object of the invention, " nucleotide sequence " refers to, for example, and genome or complementary DNA (cDNA) sequence or RNA sequence and their semi-synthetic or complete synthesis analogue.These sequences can exist with wire or cyclic form, and it is outer or be integrated in the genome to be present in karyomit(e).The nucleotide sequence of expression cassette of the present invention or nucleic acid can synthesize and produces or obtain natively, or comprises synthetic and natural DNA component mixture, and is made up of the different heterologous gene fragments from different organisms.And artificial sequence nucleic acid is exactly suitable as long as they have the purpose basic characteristics.For example, can produce synthesizing ribonucleotide sequence with the codon that is transformed plant optimization.These can be used for determining by making based on the codon from the codon with high protein frequency by the codon of plant optimization usually in a usual manner.Specially suitable is codon use the coding nucleotide sequence by peptide sequence reverse translation obtained special according to host plant.In order to prevent the undesired plants regulation mechanism, can be for example from aminoacid sequence from the lepidopterous insects desaturase, and the codon of considering plant uses, thus reverse translation dna fragmentation and produce the external source desaturase sequence of complete optimization in plant, to use with this result.
All nucleotide sequences above-mentioned can pass through known mode chemosynthesis itself from nucleotide units, for example, and by the double-helical independent overlapping complementary nucleic acid unit of fragment condensation.Oligonucleotide can be by chemosynthesis, for example with known phosphinylidyne imines (phosphoamidite) method (Voet, Voet, the 2nd edition, Wiley Press New York, 896-897 page or leaf) itself.When the preparation nucleic acid construct, can operate different dna fragmentations, to obtain to have the nucleotide sequence of correct reading direction and proper reading frame.Joint or adapter can join in the fragment so that nucleic acid fragment is interconnection.The interpolation of synthetic oligonucleotide, by means of the breach of archaeal dna polymerase Klenow fragment fill and lead up, ligation and general cloning process be by (1989) Molecular cloning:Alaboratory manual such as Sambrook, describe in Cold Spring Harbor Laboratory Press one book.
When referring to, for example, when nucleotide sequence, expression cassette, carrier or organism, " transgenosis "/" reorganization " refers to those gene components that produces with recombination method, or refers to their purposes, wherein
A) coding desaturase nucleotide sequence or
B) may be operably coupled to gene regulating sequence promotor for example on the nucleotide sequence of coding desaturase, or
C) (a) and (b)
Not in their natural genotypic environments or modify by recombination method, this modification is for example taked to substitute, is added, disappearance, inversion or insert the form of one or more nucleotide residues.The natural genotypic environment of term refers to karyomit(e) seat natural in initial organism or refers to existence in genomic library.At genomic library in this case, the natural genotypic environment of nucleotide sequence preferably keeps to small part.The monolateral at least nucleotide sequence flank that is positioned at of described environment has the sequence length of 50bp at least, 500bp at least preferably, 1000bp at least particularly preferably, very particularly preferably 5000bp at least.Naturally occurring expression cassette, for example naturally occurring molectron with coding delta 8 desaturase genes sequence of its natural promoter is worked as by non-natural, synthetic (" artificial ") method, and for example mutagenesis is modified, and just becomes recombinant expression cassettes.
The recombinant expressed use that refers to the recombinant expression cassettes that is used for the express nucleic acid sequence.
The present invention relates to the recombinant expression cassettes of the nucleotide sequence that comprises the desaturase of encoding in addition and comprises the carrier of these expression cassettes.
The nucleic acid molecule of coding desaturase preferably is operably connected with at least a gene regulating element (for example promotor) in above-mentioned recombinant expression cassettes, and described controlling element can guarantee that above-mentioned desaturase is organism, preferably recombinant expressed in plant, plant tissue, algae, yeast or fungi (transcribe and/or translate).If recombinant expression cassettes is desired directly in the importing plant, and desaturase desires to express in plant, and the special gene regulating element (for example promotor) of plant is preferred.Yet desaturase also can be in other organism or vivoexpression.Here, preferred protokaryon or eukaryotic gene controlling element (for example promotor) are that those can allow to produce expression promoter under the various situations in selected organism.
Therefore the present invention also relates to such recombinant expression cassettes, and described recombinant expression cassettes comprises at least a from lepidopterous insects, the nucleotide sequence of the coding fatty acid desaturase under the promoter regulation that plays a role in plant, algae, yeast or fungi.Preferably, the polypeptide of the nucleic acid sequence encoding in the expression cassette comprises at least a following sequence that is selected from:
A) aminoacid sequence as shown in SEQ ID NO:2,4,6,8,10,12,14 or 22, and
B) with one of aminoacid sequence as shown in SEQ ID NO:2,4,6,8,10,12,14 or 22 have at least 65% homology aminoacid sequence and
C) comprise the aminoacid sequence of at least 20 continuous amino acid sequence fragments of sequence as shown in SEQ ID NO:2,4,6,8,10,12,14 or 22.
Fatty acid desaturase particularly preferably is the aminoacid sequence shown in SEQ ID NO:22 in this article.
The present invention relates to the recombinant expression vector that comprises at least a expression cassette of the present invention in addition, and relates to the transgenic organism that comprises at least a recombinant expression cassettes of the present invention or at least a recombinant expression vector of the present invention.Preferably, transgenic organism is the plant biological body, particularly preferably is selected to be used for the plant that oil plant is produced, for example Sunflower Receptacle, sesame, safflower, olive, soybean, Semen Lini, peanut, castor-oil plant, oil palm, corn, wheat, cocoa tree and nut kind.
Exercisable connection is interpreted as, promotor and the desire desaturase nucleotide sequence of expressing and as required for example, comprise for example being arranged in order of terminator of further controlling element, make that at nucleotide sequence each controlling element can both be realized its function when recombinant expressed.Not necessarily need the direct connection on the chemical sense to reach this purpose.The gene regulating sequence for example enhancer sequence also can to target sequence from away from the position or in fact from other dna molecular performance function.Preferably be arranged as the recombinant expressed nucleotide sequence of desire and be positioned at after the sequence as promotor, thereby two sequences covalently are connected to each other.Distance is preferably in 200 base pairs between the recombinant expressed nucleotide sequence of promoter sequence and desire, more preferably in 100 base pairs, very particularly preferably in 50 base pairs.
Exercisable connection and transgene expression cassette all can produce by conventional reorganization and clone technology, these technology are for example at Maniatis T, Fritsch EF and Sambrook J (1989) MolecularCloning:A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor (NY), at Silhavy TJ, Berman ML und Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor (NY), at (1987) Current Protocols inMolecular Biology such as Ausubel FM, Greene Publishing Assoc.and Wiley Interscience and in (1990) Plant Molecular Biology Manual such as Gelvin, describing.Yet, other sequence, the joint or the signal peptide that for example have the special cutting of Restriction Enzyme point of contact also can be between these two sequences.And insertion sequence can cause Expression of Fusion Protein.Preferably, comprise the recombinant expression cassettes that is connected to the promotor on the desire express nucleic acid sequence and can have and be inserted into Plant Genome with the form of vector integration, for example insert by transforming.
Yet transgene expression cassette also can be understood as such construct, and the nucleotide sequence of the desaturase of wherein encoding places after the endogenous promotor, and in this way, the latter regulates and control the expression of desaturase.Fusion by the endogenesis promoter that insert to produce and desaturase nucleotide sequence is the recombinant expression cassettes for the object of the invention.
The specific promotor of plant is interpreted as any energy regulatory gene, particularly foreign gene expression promoter in a part, vegetable cell, plant tissue or the plant culture of plant or plant.In this article, express for example composing type, induction type or grow dependent form.Following is preferred promotor:
A) constitutive promoter
Preferred carrier be those make in plant constitutive expression become possible carrier (Benfey etc. (1989) EMBO J.8,2195-2002), " composing type " promotor refers to such promotor, they be can guarantee in a large number, whole tissue preferably, and between development of plants quite long-term, preferably in the whole growth period expression promoter of plant.Especially preferably use plant promoter and from the promotor of plant virus.CaMV (cauliflower mosaic virus) 35S transcript (Franck etc. (1980) Cell 21:285-294; Odell etc. (1985) Nature 313:810-812; Shewmaker etc. (1985) Virology 140:281-288; Gardner etc. (1986) Plant Mol Biol 6:221-228) promotor or 19 S CaMV promotors (US 5,352,605; WO 84/02913; People such as Benfey (1989) EMBO J 8:2195-2202) be particularly preferred.Another suitable constitutive promoter is that (US 4 for Rubisco small subunit (SSU) promotor, 962,028), legumin B promotor (GenBank Acc.No.X03677), rouge alkali synthetase promoter, TR double-promoter, OCS (octopine synthase) promotor, ubiquitin promoter ((1995) Plant MolBiol 29:637-649 such as Holtorf S), ubiquitin 1 promotor (Christensen etc. (1992) Plant Mol Biol18:675-689 from Agrobacterium from Agrobacterium; Bruce etc. (1989) Proc Natl Acad Sci USA 86:9692-9696), the Smas promotor, (US 5 for the cinnamyl-alcohol dehydrogenase promotor, 683,439), vacuole ATP enzyme subunit promotor, or the proteinic promotor (WO 91/13991) of proline rich in the wheat, and other promotor of the gene of constitutive expression is as well known to those skilled in the art in plant.
B) organizing specific type promotor
It further preferably is promotor with pollen, ovary, flower, leaf, stem, root and seed-specific.
Seed specific promoters
For example, and the phaseolin promotor (US 5,504, and 200; Bustos MM etc. (1989) PlantCell 1 (9): 839-53), 25 albumin gene promotors (Joseffson LG etc. (1987) J BiolChem 262:12196-1220 1), legumin promotor (Shirsat A etc. (1989) Mol GenGenet 215 (2): 326-331), USP (unknown seed albumen) promotor ((1991) Mol Gen Genet 225 (3) such as B  umlein H: 459-67), (US 5 for the napin gene promoter, 608,152; (1996) L Planta 199:515-519 such as Stalberg K), sucrose-binding proteins promotor (WO00/26388) or legumin B4 promotor (LeB4; (1991) Mol GenGenet 225:121-128 such as B  umlein H; (1992) Plant Journal 2 (2): 233-9 such as B  umlein; (1995) Biotechnology (NY) 13 (10) such as Fiedler U: 1090f), Arabidopsis oleosin promotor (WO 98/45461) and rape Bce4 promotor (WO 91/13980).Further suitable seed specific promoters is the promotor of those coding high molecular weight glutenins, gliadine, branching enzyme, ADP glucose Pyrophosphate phosphohydrolase (AGPase) or starch synthase gene.Preferred in addition promotor is that those allow for example to carry out the promotor of seed-specific expression in corn, barley, wheat, naked barley, the rice etc. in monocotyledons.(WO 95/15389, and the promotor of describing WO95/23230) or in WO 99/16890 (promotor of hordein gene, glutenin gene, oryzin gene, prolamine gene, gliadine gene, zein spirit-soluble gene, casirin gene or secaline gene) preferably adopts for the promotor of lpt2 or lpt1 gene.
Stem tuber-, storage root-or root-specificity promoter, for example I class patatin promotor (B33) and from cathepsin D's inhibitor promotor of potato.
Leaf-specificity promoter
For example potato kytoplasm FBPase promotor (WO 97/05900), Rubisco (ribulose 1.5 bisphosphate carboxylases) SSU promotor (small subunit) or potato ST-LSI promotor (Stockhaus etc. (1989) EMBO J 8:2445-2451).
Flower-specificity promoter
For example phytoene synthase promotor (WO 92/16635) or P-rr gene promoter (WO98/2259 3).
Flower pesticide-specificity promoter
For example 5126 promotors (US 5,689,049, and US 5,689,051), glob-l promotor and g-zein promotor.
C) chemical inducible promoter
Recombinant expression cassettes also can comprise chemical inducible promoter (survey article: Gatz etc. (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108), in this way, the exogenous gene expression in plant can be regulated and control at specified point timely.Such promotor for example, PRP1 promotor (Ward etc. (1993) Plant Mol Biol 22:361-366), salicylic acid inducible promotor (WO 95/19443), benzsulfamide inducible promoter (EP 0 388 186), tsiklomitsin inducible promoter (Gatz etc. (1992) Plant J 2:397-404), dormin inducible promoter (EP 0 335528) or ethanol-or pimelinketone inducible promoter (WO 93/21334) can be utilized equally.
D) stress or pathogen-inducible promoter
Other preferably promotor be that those are by biology or abiotic stress inductive promotor, for example, the pathogen-inducible promoter of PRP1 gene (Ward etc. (1993) Plant Mol Biol22:361-366), (US 5 from the thermal induction type hsp70 of tomato or hsp80 promotor, 187,267), from the pinII promotor (EP375091) of low temperature induction type α-Dian Fenmei promotor (WO 96/12814), photoinduction type PPDK promotor or the wound-induced of potato.
Pathogen-inducible promoter comprises gene for example PR albumen, SAR albumen, the b-1 that is induced generation as the pathogenic agent attack result, (people (1983) Neth J Plant Pathol 89:245-254 such as Redolfi for example such as the promotor of 3-dextranase, chitinase etc.; People such as Uknes (1992) The PlantCell 4:645-656; Van Loon (1985) Plant Mol Viral 4:111-116; People such as Marineau (1987) Plant Mol Biol 9:335-342; People such as Matton (1987) MolecularPlant-Microbe Interactions 2:325-342; People such as Somssich (1986) Proc NatlAcad Sci USA 83:2427-2430; People such as Somssich (1988) Mol Gen Genetics2:93-98; People such as Chen (1996) Plant J 10:955-966; Zhang and Sing (1994) ProcNatl Acad Sci USA 91:2507-2511; People such as Warner (1993) Plant J 3:191-201; People such as Siebertz (1989) Plant Cell 1:961-968 (1989).
In being also included within is wound-induced type promotor, for example pinII gene (Ryan (1990) AnnRev Phytopath 28:425-449; People such as Duan (1996) Nat Biotech 14:494-498), (US 5 for wun1 and wun2 gene promoter, 428,148), the promotor of win1 and win2 gene (people (1989) Mol Gen Genet 215:200-208 such as Stanford), systemin promotor (people (1992) Science 225:1570-1573 such as McGurl), WIP1 gene promoter (people (1993) Plant Mol Biol 22:783-792 such as Rohmeier; People such as Eckelkamp (1993) FEBS Letters323:73-76), MPI gene promoter (people (1994) The Plant J6 (2) such as Corderok: 141-150) etc.
E) grow the dependent form promotor
Other suitable promotor is that for example, the ripe specificity promoter of fruit is as the fruit maturation specificity promoter (WO 94/21794, and EP 409 625) from tomato.Because some tissues form naturally along with the carrying out that grows, thereby growth dependent form promotor comprises some tissue-specific promoters.
Other is suitably in that expression promoter is described in the plant (people (1987) Methin Enzymol 153:253-277 such as Rogers; People such as Schardl (1987) Gene 61:1-11; People such as Berger (1989) Proc Natl Acad Sci USA 86:8402-8406).Especially preferred group moulding and seed specific type promotor.
And, make to be expressed in other plant tissue or for example to become other possible promotor in the intestinal bacteria and can operationally be connected with the nucleotide sequence that desire is expressed other organism.Suitable plant promoter is all above-mentioned promotors in principle.
The nucleotide sequence that is present in recombinant expression cassettes of the present invention or the carrier can operationally be connected with other gene regulating sequence except that operon, interpreted in its broadest sense, ie wanted in this term of gene regulating sequence, and it refers to that all that is to expression cassette foundation of the present invention or the influential sequence of function.Gene regulating sequence Change Example is as transcribing and translating in protokaryon or most eukaryotes.Recombinant expression cassettes of the present invention preferably comprise have want recombinant expressed nucleotide sequence 5 ' upstream can be specifically at the embryo skin and/or spend middle expression promoter, with 3 ' catchment terminator sequence as extra gene regulating sequence, and, as required, other conventional controlling element that operationally is connected in all cases with the recombinant expressed nucleotide sequence of desire.
The gene regulating sequence comprises that also other can change the promotor of expression regulation characteristic, promoter element or minimal promoter.Thereby the gene regulating sequence can for example cause and additionally depend on the tissue specific expression that some coerces factor.These factors can be described as, and for example coerce for water and dormin (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131-17135) and heat stress (people (1989) Mol Gen Genetics 217 (2-3) such as Schoffl F: 246-53).
Other favourable regulating and controlling sequence is for example in Gram-positive promotor amy and SPO2 and in yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
In principle, as those promotors, all natural promoters with regulating and controlling sequence all can be used for method of the present invention.And, also can advantageously use synthetic promoter.
The gene regulating sequence also comprises 5 ' non-translational region, intron or the noncoding 3 ' district of gene further, for example, Actin muscle-1 intron or Adh1-S introne 1,2 and 6 are (for general introduction property reference, see: The Maize Handbook, 116 chapters, Freeling and Walbot edit, Springer, New York (1994)).Verified these regulating and controlling sequences play remarkable effect in regulatory gene is expressed.Thereby, proved that 5 ' non-translated sequence can strengthen the transient expression of heterologous gene.The translational enhancer that can mention by way of example is tobacco mosaic virus (TMV) 5 ' leader sequence (people (1987) Nucl Acids Res 15:8693-8711 such as Gallie) etc.They can improve tissue specificity (people (1998) Plant J 15:435-440 such as Rouster) in addition
Recombinant expression cassettes advantageously can comprise the one or more known enhancer sequence that can be operatively connected with promotor, and these enhancer sequence make the recombinant expressed enhancing of nucleotide sequence become possibility.The sequence that other is favourable such as other controlling element or terminator also can be inserted into 3 ' end of the recombinant expressed nucleotide sequence of desire.The recombinant expressed nucleotide sequence of desire that in gene construct, can have one or more copies.
The polyadenylic acid signal sequence that is fit to do regulating and controlling sequence is a plant polyadenylic acid signal, preferably those correspond essentially to the gene 3 (octopine synthase) (people (1984) EMBO J 3:835 such as Gielen and following or the like) of the sequence, the particularly T-DNA of Ti-plasmids pTiACHS of agrobacterium tumefaciens (Agrobacterium tumefaciens) T-DNA polyadenylic acid signal or the sequence of its function equivalent.Particularly suitable terminator sequence is OCS (octopine synthase) terminator and NOS (nopaline synthase) terminator.
Regulating and controlling sequence can be interpreted as further that those make homologous recombination or insert the host organisms genome or remove and become possible sequence from genome.Method such as cre/lox technology allow tissue specificity ground, may can remove recombinant expression cassettes (Sauer B (1998) Methods, 14 (4): 381-92) inductively from the host organisms genome.Here, some flanking sequence is added into target sequence (lox sequence), and making to remove by the time point of cre recombinase method after becomes possibility.
Recombinant expression cassettes and can comprise other functional element by its its deutero-recombinant vectors.This term of functional element will be from broadly understanding, it refer to all that to the generation of recombinant expression cassettes of the present invention, carrier or transgenic organism, duplicate or the influential element of function.The example that can mention but be not limited only to this is:
A) to metabolic poison for example 2-deoxyglucose-6-phosphoric acid (WO 98/45456), microbiotic or biocide, preferred weedicide, for example kantlex, G 418, bleomycin, Totomycin, phosphinothricin etc. are given the selected marker of resistance.Particularly preferred selected marker is the selected marker of those conferring herbicide resistances.The example of can way of example mentioning is: the dna sequence dna (bar and pat gene) of coding phosphinothricin acetyl transferase (PAT) and deactivation glutamine synthase inhibitor; the 5-enol pyruvoyl shikimic acid-3-phosphate synthase gene (epsp synthase gene) of conferring glyphosate (N-((phosphonomethyl)) glycine) resistance; the gox gene of coding glyphosate degrading enzyme (glyphosate oxidoreductase); deh gene (dehalogenase of coding deactivation dalapon); sulfonylurea-and the acetolactate synthestase of imidazolone deactivation; bxn gene with the nitrilase of coding degraded bromoxynil; give the aasa gene of microbiotic apectinomycin resistance; allow streptomycin phosphotransferase (SPT) gene of anti-streptomycin; give neomycin phosphotransferase (NPII) gene of kantlex or Geneticin resistance; give hygromix phosphotransferase (HPT) gene of hygromycin resistance; give the acetolactic acid sy nthase gene ALS of sulfonylurea herbicide resistance) (Tu Bian ALS variant for example, it has for example S4 and/or Hra sudden change).
B) but the quantitative protein and allow by its color or enzymic activity assessment transformation efficiency or express site or the reporter gene of expression time of being easy to encode.Herein very particularly preferably be " green fluorescent protein " (GFP) (people (1995) Plant Journal 8 (5) such as Sheen: 777-784) for example; People such as Haseloff (1997) Proc Natl Acad Sci USA 94 (6): 2122-2127; People such as Reichel (1996) Proc Natl Acad Sci USA 93 (12): 5888-5893; People such as Tian (1997) PlantCell Rep 16:267-271; WO 97/41228; People (1996) Curr Biol6:325-330 such as Chui WL; People such as Leffel SM (1997) Biotechniques.23 (5): 912-8), CAT, luciferin enzyme (people (1986) Science 234:856-859 such as Ow; People such as Millar (1992) Plant Mol Biol Rep 10:324-414), aequorin gene (people (1985) Biochem Biophys Res Commun 126 (3) such as Prasher: 1259-1268), beta-galactosidase enzymes, R locus gene (encoded protein is regulated the cyanin pigment (showing red) in the plant tissue, but thereby make the direct analysis promoter activity and do not need extra utility appliance or chromophoric substrate; People such as Dellaporta: Chromosome Structure and Function:Impact of New Concepts, 18 ThStadler Genetics Symposium, 11:263-282,1998), reporter protein (Schenborn E, the GroskreutzD.Mol Biotechnol.1999 of beta-glucuronidase (people (1987) EMBO J 6:3901-3907 such as Jefferson); 13 (1): 29-44).
C) replication orgin that allows recombinant expression cassettes of the present invention or carrier in intestinal bacteria for example, to duplicate.The example that can mention is ORI (starting point of dna replication dna), pBR322 ori or P15A ori (people such as Sambrook: Molecular Cloning.A Laboratory Manual, the 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
D) the agriculture bacillus mediated needed element of Plant Transformation, for example, the right margin of T-DNA or left margin, vir district.
Select successfully to experience the cell of homologous recombination or by successful cell transformed, need usually extra importing can give biocide (for example weedicide), metabolic poison for example the selective marker of 2-deoxyglucose-6-phosphoric acid (WO 98/45456) or antibiotics resistance to successfully in the cell of experience reorganization people (1986) Plant Cell Reports 5:81-84 such as () McCormick.
By way of example, but be not limited only to this, recombinant expression cassettes of the present invention can have following array structure:
A) 5 '-plant specificity promoter/desaturase/terminator-3 '
Recombinant expression cassettes of the present invention preferably has following array structure:
A) 5 '-35S promoter/desaturase/OCS/ terminator 3 ', or
B) 5 '-legumin B promotor/desaturase/NOS terminator 3 '
Use more than above-mentioned example a) or b) one of common conversion of recombinant expression cassettes have the CLA biosynthetic means that is beneficial to advantage of the present invention.And transform with one or more carriers, every kind of carrier comprises the combination of recombinant expression cassettes above-mentioned, has advantage.
In addition, described recombinant expression cassettes can comprise such nucleotide sequence, and the recombinant expressed fatty acid biological that causes of described nucleotide sequence synthesizes increase (being called proOIL hereinafter).This can be selected from down example but be not limited to encode aliphatic alcohol carboxylase (ACCase), glycerol-3-phosphate salt acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT), diacylglycerol acyltransferase (DAGAT) and phosphatide by extra recombinant expressed proOIL nucleotide sequence: the nucleic acid of triglyceride-acyltransferase (PDAT).
The proOIL nucleotide sequence also comprises such nucleotide sequence, and the recombinant expressed generation of described nucleotide sequence can cause antisense-RNA or the double-stranded RNA that lipid acid output increases.
Preferred example comprises the carrier that contains following recombinant expression cassettes:
A) 5 '-35S promoter/desaturase/OCS terminator/legumin B promotor/proOIL/NOS terminator 3 ';
B) 5 '-35S promoter/preceding-desaturase or conjugation enzyme/OCS terminator/legumin B promotor/proOIL/NOS terminator 3 ';
Construct a) and b) allow with desaturase with can increase fatty acid biological synthetic proOIL sequence and transform plant simultaneously.
Use above-cited reorganization and clone technology, desaturase of the present invention or proOIL nucleic acid or recombinant expression cassettes can be cloned in the suitable carrier, and these carriers allow them for example breeding in the intestinal bacteria.The cloning vector that is fit to especially is pBR332, pUC series, M13mp series and pACYC184.Specially suitable is the binary vector that can both duplicate in intestinal bacteria and Agrobacterium.
Desaturase of the present invention or proOIL nucleic acid or recombinant expression cassettes are preferably inserted in the suitable conversion carrier.Suitable carriers especially at " molecular biology of plants and biotechnological means " (CRCPress) is described in 6/7 chapter, 71-119 page or leaf (1993).It is as mentioned below to transform example and method for transformation.
The invention further relates to the cell, cell culture, tissue, plant part of the transgenic organism that transforms with at least a recombinant expression cassettes of the present invention or carrier of the present invention, plant biological body-for example leaf, root etc., or come from the reproductive material of these organisms.
Organism, initial organism or these terms of host organisms are interpreted as protokaryon or most eukaryotes, for example, and microorganism or plant biological body.
Preferred microorganism is bacterium, yeast, algae or fungi.
Preferred bacterium is from Escherichia (Escherichia), corynebacterium (Corynebacterium), bacillus (Bacillus), fusobacterium (Clostrridium), propiono-bacterium (Proionibacterium), Butyrivibrio (Butyrivibrio), eubacterium (Eubacterium), genus lactubacillus (Lactobacillus), her Wen's Bacillaceae (Erwinia), Agrobacterium, Flavobacterium (Flavobacterium), Alkaligenes (Alcaligenes), Phaeodactylum, Colpidium (Colpidium), genus mortierella (Mortierella), entomophthora belongs to (Entomophthora), Mucor (Mucor), Crypthecodinium cohnii belongs to (Crypthecodinium) or cyanobacteria, for example cytoalgae (Synechocystis).
Particularly preferably be energy infection plant thereby the microorganism that can shift construct of the present invention.Preferred microorganism is an Agrobacterium, particularly the microorganism of agrobacterium tumefaciens kind.
Preferred yeast is mycocandida (Candida), yeast belong, Hansenula (Hansenula) or Pichia.Particularly preferred yeast is that those lipid acid/derivative of fatty acid account for cell dry-matter at least 20%, preferred 40%, preferred especially 60% yeast, particularly for example crooked Cryptococcus (Cryptococcus the curvatus) (biotechnology of Ratledge (1989) oil ﹠ fat,: Microbiol lipids Vol.2, Academic Press, London, 567-668 page or leaf).
Preferred fungi is Aspergillus (Aspergillus), Trichoderma (Trichoderma), AshbyaCif (Ashbya), Neurospora (Neurospora), fusarium (Fusarium), Beauveria (Beauveria), phytophthora infestans (Phytophthora infestans) or in IndianChem Engr.B part, the 37th volume, No 1,2 (1995), 15 pages, other fungi of describing in the table 6.Particularly preferably be that those fat/derivative of fatty acid account for cell dry-matter at least 10%, preferred 20% fungi, particularly volume branch Mucor (Mucor circinelloides) and Mortierella alpina (Mortierella alpina) people (2001) Microbiology 147:2857-2864 such as () Wynn
Preferred transgenic organism is the plant biological body particularly." plant biological body " this term comprises any organism that can carry out the light compositing effect and from cell, tissue, the part reproductive material (as seed and fruit) of this organism.The high kind with the lower plant genus of all vegitabilias all is included in the purpose of the present invention.Annual, perennial, monocotyledons and dicotyledons and gymnosperm are preferred.What included has maturation plant, seed, sprout and seedling and the part, reproductive material (for example stem tuber, seed or fruit), plant organ, tissue, protoplastis, callus culture and other culture, for example cell culture that come from them." maturation plant " refers to any plant above the seedling development stage.This speech of seedling refers to be in the immature immature plant of early development stage.
" plant " comprises all annual and perennial unifacial leaf and dicotyledonss, comprises example down, but is not limited to.Cucurbita; rose belongs to (Rosa); Vitis; white walnut; Fragaria; Nelumbo; Medicago; sainfoin belongs to (Onobrychis); Trifolium; frenugreek spp; Vigna; both citrus; linum; Pelargonium; cassava; Daucus; Arabidopsis; Btassica; radish; sinapis; Atropa; Capsicum; instrument belongs to; poison tobacco; tomato belongs to; Nicotiana; Solanum; the Pittosporum of leading a cow; Digitalis; marjoram (Majorana); witloof; Helianthus; Lactuca; Bromus; Asparagus; antirrhinum; Hemericallis; Nemesia belongs to; Pelargonium; Panicum; Pennisetum; Ranunculus; Senecio; salpiglossis belongs to; melon; Browaalia; Wei Shi soybean (Glycine); pea; Phaseolus; lolium; rice; corn; oat; Hordeum; rye; Triticum; jowar belongs to; Picea and Populus.
Preferred plant is from following plant section: Amaranthaceae, composite family (Asteraceae), rape, Caryophyllaceae, Chenopodiaceae, composite family (Compositae), Cruciferae, Curcurbitaceae, Labiatae, pulse family, Papilionidae, Liliaceae, flax family (Linaceae), Malvaceae, the Rosaceae, Rubiaceae, Saxifragaceae, scrophulariaceae, Solanaceae, Sterculiaceae, Aizoaceae, Theaceae, umbelliferae.
Preferred monocotyledons is selected from the monocot crops plant especially, for example, Gramineae, as clover, rice, corn, wheat or other cereal class such as barley, broomcorn millet and jowar, rye, triticale or oat, and sugarcane and all draft classes.
The present invention extremely is particularly advantageously applied to the dicotyledons organism.Preferred dicotyledons is selected from the dicotyledonous crops plant especially, for example,
-composite family (Asteraceae), as Helianthus, Tagetes, calendulin etc.,
-composite family (Compositae), particularly Lactuca, particularly lettuce etc.,
-Cruciferae, particularly Btassica, rape, beet, wild cabbage, Cauliflower and blue and white willow dish and other wild cabbage very in particular; And Arabidopsis, mouseearcress and Lepidium apetalum or canola etc. very in particular.
-Curcurbitaceae, for example muskmelon, pumpkin or summer squash etc.
-pulse family, Glycine, green soy bean, soya bean and alfalfa, pea, French beans or peanut etc. very in particular in particular.
-Rubiaceae, preferably extra large shin longicorn subfamily (Lamiidae), for example fruitlet coffee (Coffeaarabica) or big fruit coffee (Coffea liberica) etc.
-Solanaceae, tomato belongs in particular, and ten minutes is tomato in particular, Solanum, potato and eggplant and tobacco or capsicum etc. very in particular.
-Sterculiaceae, Dilleniidae, for example Theobroma etc. in particular.
-Theaceae, Dilleniidae in particular, for example Camellia, Camellia or etc.
-umbelliferae, in particular Daucus (very Radix Dauci Sativae) in particular and apium (ten minutes is celery (graveolens dulce) etc. in particular; And Capsicum, Piper etc. very in particular, and Semen Lini, soybean, cotton, hemp, flax, cucumber, spinach, Radix Dauci Sativae, beet and various tree, nut and grape kind, particularly banana and A.chinensis Planch..
In being also contained in is ornamental plant, useful or ornamental tree, flower, cut-flower, shrub or turf.By way of example, but the plant that is not limited only to this is angiosperm, bryophyte, for example Hepaticae (Hepaticae) and moss guiding principle (Musci); Pteridophyte, for example fern, scouring rush and lycopod; Gymnosperm, for example coniferals, cycad, ginkgo and Gnetopsida; The Rosaceae; rose for example; Ericaceae; for example Rhododendron (rhododendron) and Rhododendron (azalea); Euphorbiaceae (Euphorbiaceae); for example poinsettia belongs to (poinsettias) and crotons belong to (croton); Caryophyllaceae (Caryophyllaceae); dianthus (pinks) for example; Solanaceae (Solanaceae); petunia Pittosporum (petunias) for example; Gesneriaceae (Gesneriaceae); for example African violet belongs to (African violet); Balsaminaceae (Balsaminaceae); impatiens (touch-me-not) for example; the orchid family (Orchidaceae); for example orchid belongs to (orchids), Iridaceae (Iridaceae), for example gladiolus (gladioli); Jris (iris); Freesia (freesia); crocus (crocus); composite family; calendulin for example, Mang ox seedling section (Geraniaceae), for example Pelargonium; Liliaceae; dracaena (dracena) for example, Moraceae (Moraceae), for example Ficus (ficus); Araeceae (Araceae), for example Philodendron (philodendron) and many other classes.
In addition, being suitable for the object of the invention plant biological body further is the organism that photosynthetic activity can be arranged, for example, and algae, cyanobacteria and mosses.Preferred algae is a green alga, and for example haematococcus (Haematococcus), Phaeodactylum tricornutum (Phaedactylum tricornatum), volvox (Volvox) or Dunaliella salina belong to (Dunaliella).
More preferably be fit to the plant that oils is produced, for example rape, Sunflower Receptacle, sesame, safflower (Carthamus tinctorius), olive, soybean, Semen Lini, peanut, castor-oil plant, oil palm, corn, wheat, cocoa or various nuts plant, for example English walnut, coconut or apricot.Most preferably Arabidopsis, cotton, flax and Semen Lini in addition.
Preferred algae is a green alga, and for example, haematococcus, Phaeodactylum tricornutum, volvox or Dunaliella salina belong to.Other preferably can be should be mentioned that protozoon as quilt, for example the dinoflagellate class.
Preferred special in the organism above-mentioned, it is the organism of the natural synthetic a great deal of oils of those energy, Mycophyta for example, be volume branch Mucor, Mortierella alpina, fantastic corruption mould (Pythiuminsidiosum) for instance, the yeast class, for example yeast saccharomyces cerevisiae or crooked Cryptococcus, or plant, for example, soybean, Semen Lini, rape, coconut, oil palm, safflower, castor-oil plant, peanut, cocoa tree or Sunflower Receptacle particularly preferably are soybean, rape, Sunflower Receptacle, volume branch Mucor, Mortierella alpine mould, mould, the crooked Cryptococcus of fantastic corruption or yeast saccharomyces cerevisiae.
More the example of wide region store oil organism can be at Kyle and Ratledge (1992) Industrial applications of Single Cell Oils, American Oil Chemists ' Society, and Champaign finds in Illinois one book.
Decide on host organisms, the mode that the organism that is used for present method is understood with the technician is grown or is cultivated.Usually, in the microorganism growth liquid medium within, substratum comprises carbon source, nitrogenous source, trace elements and also contains VITAMIN as required, wherein carbon source is generally the form of sugar, nitrogenous source is generally organic nitrogen source form (for example yeast extract) or salt (for example ammonium sulfate), and trace elements is molysite, manganese salt, magnesium salts for example, and culture temperature is between 0 ℃ to 100 ℃, preferably between 10 ℃ and 60 ℃, and pass to oxygen.In this process, the pH value of liquid nutrient media can keep stable, just can adjust in culturing process or not adjust.In batches, semi-batch or cultured continuously all are feasible.Nutrition can be in when beginning fermentation supply or semicontinuous or provide continuously.
By using the carrier that wherein has recombinant expression cassettes, recombinant expression cassettes of the present invention can advantageously import in organism or its cell, tissue, organ, part or the seed and (preferably import in plant or vegetable cell, tissue, organ, part or the seed).Recombinant expression cassettes can import in the carrier (for example plasmid) by suitable restricted cleavage site.The plasmid that obtains at first imports in the intestinal bacteria.Selected, the growth of the correct intestinal bacteria that transform, recombinant plasmid can obtain by the method that the technician is familiar with.Restriction analysis and order-checking can be used for confirming clone's step.
Use the carrier can be advantageously in transfered cell, the preferred plant cell with expression cassette of the present invention.The example of carrier is plasmid, clay, phage, virus or Agrobacterium.In a preferred embodiment, expression cassette imports by plasmid vector.Preferred carrier is that those make expression cassette stably be integrated into host gene to consist of possible carrier.
Produce inverting biological body (or transformant or tissue) and DNA, RNA or the protein of being discussed need be imported the host cell of being discussed.Process at being called as conversion (or transduction or transfection) (Keown etc. (1990) Enzymology method 185:527-537) has several different methods to use.Therefore, for example, can DNA or RNA directly be imported by the method for microinjection mode or the microparticle bombardment by being enclosed with DNA.In addition, cell can for example be used polyoxyethylene glycol by chemosmosis, and DNA can be by diffusing into cell like this.DNA can also be by for example minicell, cell, lysosome or liposome carry out protoplasma and merge and import with other unit that contains DNA.The method of the importing DNA that another is suitable is an electroporation, by electricimpulse cell is reversibly penetrated.These methods are at (people (1991) Gene 100:247-250 such as Bilang; People such as Scheid (1991) Mol Gen Genet228:104-112; People such as Guerche (1987) Plant Science 52:111-116; People such as Neuhause (1987) Theor Appl Genet 75:30-36; People such as Klein (1987) Nature 327:70-73; People such as Howell (1980) Science 208:1265; People such as Horsch (1985) Science227:1229-1231; People such as DeBlock (1989) Plant Physiology 91:694-701; Molecular biology of plants method (Weissbach and Weissbach edit) Academic Press Inc. (1988); With molecular biology of plants method (Schuler and Zielinski edit) Academic Press Inc. (1989)) in describe to some extent.
Can use desaturase or conjugation enzyme that a large amount of organisms is preferably carried out recombinant modified to plant, organism becomes one or more derived from the product of the fat from the beginning producer of two kinds of CLA isomer for example like this.Usually, the initial regeneration of plant occurs in after step of converting and cultivation in succession or the growth.
Be used for the cloning vector of ciliates and algae and gene manipulation techniques and be familiar with by the technician that (WO 98/01572; People such as Falciatore (1999) Marine Biotechnology 1 (3): 239-251; People such as Dunahay (1995) J Phycol 31:10004-1012).
Be used for gene transfered plant genome neutralization is used for several different methods and known (molecular biology of plants and biotechnology (the CRC Press of carrier from plant tissue or vegetable cell aftergrowth body, BocaRaton, Florida), 6/7 chapter, 71-119 (1993); White FF (1993) is used for the carrier that higher plant gene shifts; : Transgenic Plants, Bd.1, Engineering and Utilization, Hrsgb.:Kung und R.Wu, Academic Press, 15-38; People (1993) gene transfer techniques such as Jenes B: Transgenic Plants, Bd.1, Engineering and Utilization, Hrsgb.:Kung und R.Wu, Academic Press, S.128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225; Halford NG, Shewry PR (2000) Br Med Bull 56 (1): 62-73).They comprise for example above-mentioned those.In plant, being used for of having described transforms and the method for aftergrowth is used to instantaneous or stable conversion from plant tissue or vegetable cell.Protoplast transformation, biological trajectory gunning (being also referred to as the particle bombardment method), electroporation, dried embryo hatching and microinjection in dna solution that suitable method is particularly absorbed by the polyoxyethylene glycol inducing DNA with particle gun.In these " directly " method for transformation, used plasmid does not need to satisfy any special requirement.Available simple plasmid, for example those are from pUC series, pBR322, M13mp series, pACYC184 etc.If desire is from the complete plant of cell transformed regeneration, plasmid must comprise extra selected marker.
Except that these " directly " transformation technologies, transform also and may realize by the infectation of bacteria method of agrobacterium tumefaciens or Agrobacterium rhizogenes (Agrobacterium rhizogenes).These bacterial strains comprise the plasmid (being respectively Ti and Ri) that can shift along with agroinfection in the plant into.The part of described plasmid is transfer DNA (T-DNA), is integrated in the vegetable cell genome.Scheme as an alternative, Agrobacterium also can shift binary vector (mini-Ti plasmid) and enter in the plant, and these vector integrations advance in the Plant Genome then.Agriculture bacillus mediated conversion is best suited for dicots diplont cell, and directly transformation technology is applicable to any cell type.Agriculture bacillus mediated method for transformation is described by people (1985) Science 225:1229f such as for example Horsch RB.If use Agrobacterium, expression cassette is integrated in the specific plasmid, promptly or be integrated into shuttle vectors (intermediate carrier) or be integrated into binary vector.If Ti or Ri plasmid are used for transforming, the right margin at least of Ti or Ri plasmid T-DNA, but right in the case and left margin is connected to the expression cassette of desire insertion as flanking region.
Binary vector is preferably used for using the conversion of Agrobacterium.Binary vector can both duplicate in intestinal bacteria and Agrobacterium.Usually, they comprise selected marker and the joint or the polylinker that are positioned at right and left T-DNA border sequence side.They can directly be transformed into Agrobacterium (people (1978) Mol Gen Genet 163:181-187 such as Holsters).The selected marker is a nptII gene of for example giving kalamycin resistance, and its permission is selected the Agrobacterium that has transformed.In the case, the Agrobacterium as host organisms should comprise the plasmid that has the vir district.It is necessary that the latter enters vegetable cell for transfer T-DNA.The Agrobacterium of Zhuan Huaing can be used for transformed plant cells by this way.The purposes of the T-DNA that is used for transformed plant cells has been carried out further investigation and described that (EP 120 516; Hoekema: binary plant vector system, Offsetdrukkerij KantersB.V., Alblasserdam, V chapter; People such as An (1985) EMBO J 4:277-287).Known multiple binary vector, wherein some have had commercially available, for example pBI101.2 or pBIN19 (ClontechLaboratories, InC.USA; People such as Bevan (1984) Nucl Acids Res 12:8711), pBinAr, pPZP200 or pPTV.
So the Agrobacterium that transforms with examples of such carriers can be used to transform for example Semen Brassicae campestris of plant, particularly crop plants in known manner, for example is bathed in the Agrobacterium solution by leaf or the blade that will cut off, cultivates in suitable medium subsequently.With the Agrobacterium-mediated Transformation plant at (White FF, higher plant gene transfer vector; : Transgenic Plants, the 1st volume, Engineering andUtilization, S.D.Kung and R.Wu edit, Academic Press, 1993,15-38 page or leaf; People (1993) gene transfer techniques such as Jenes B: Transgenic Plants, the 1st volume, Engineering and Utilization, S.D.Kung and R.Wu edit, Academic Press, 128-143 page or leaf; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol42:205-225).The transgenic plant that contain above-mentioned desaturase of the present invention or conjugation enzyme, desaturation proenzyme or conjugation proenzyme or proOIL nucleic acid or recombinant expression cassettes or carrier can regenerate in known manner from the transformant of cutting off leaf or blade.
When selected marker is when inserting DNA a part of, the cell of stable conversion, promptly those comprise the cell of the insertion DNA that is integrated into host cell DNA, can choose from non-transformed cell.For example, any gene that can give microbiotic or weedicide (for example kantlex, G 418, bleomycin, Totomycin or phosphinothricin etc.) resistance can serve as a mark (on seeing).The transformant of expressing this marker gene can kill under the microbiotic of non-conversion wild-type or the weedicide concentration in existence survives.Example is mentioned in the above, preferably include conferring herbicide phosphinothricin resistance the bar gene (people (1993) Plant Mol Biol 21 (5) such as RathoreKS: 871-884), give kalamycin resistance the nptII gene, give the hpt gene of hygromycin resistance or the EPSP gene of conferring herbicide glyphosate resistance.Selected marker makes and transformant can be chosen people (1986) Plant Cell Reports 5:81-84 such as () McCormick from non-transformed cell.The plant that obtains can be cultivated and hybridizes in common mode.Preferred cultivate 2 generations or many generations so that guarantee that genome conformity is stable and can heredity.
When the plant transformed cell produces, complete plant just can obtain with the mode that the technician is familiar with.The callus culture thing is an example of parent material.The growth of spray and root can be induced generation in known manner in this undifferentiated so far cellular biomass.The seedling that obtains can be planted and grow outside.Suitable method is at (people (1992) Plant Cell Rep.11:567-570 such as Fennell; People such as Stoeger (1995) Plant Cell Rep.14:273-278; Jahne etc. (1994) Theor ApplGenet 89:525-533) in description is arranged.
Recombinant expressed expression of nucleic acids effect can for example be used wherein a kind of above-mentioned system of selection, determines by the breeding of seedling meristematic tissue external.And the character of desaturase or proOIL nucleotide sequence and changes of expression level and they can be tested with test plants in the greenhouse the influence of CLA and/or fatty acid biological synthesis rate.
Those transgenic organisms of comparing the CLA output with raising with unconverted wild-type are preferred.The CLA output that improves means for the object of the invention, for example, not genetically modified initial organism is compared, and manually in transgenic organism has obtained to improve for example ability of the biosynthesizing speed of at least a compound in coenzyme A ester or the glyceryl ester of CLA, its fat.In this article, compare with not genetically modified organism, the output of CLA in transgenic organism has preferably improved 10%, has particularly preferably improved 50%, has very particularly preferably improved 100%.Improve the quality also referred to advantageously change the CLA mixture and form, promptly compare with initial organism, (9Z, 11E)-CLA and/or (10E, 12Z)-raising on the CLA content.
Under the situation that is the transgenic plant organism, also consistent with the present invention is its cell, cell culture, partly-for example root, leaf etc., and the rotaring gene breeding material that comes from above-mentioned transgenic organism for example seed or fruit.
The invention further relates to the purposes of the above-mentioned transgenic organism of the present invention, and, be used for for example particularly production of CLA of free fatty acids of food or feed, makeup or fine chemicals for relating to its cell, its cell culture, its part-for example root, leaf etc.-and the rotaring gene breeding material that comes from them for example seed or fruit under the situation of transgenic plant organism.Particularly preferably be the fat that is used to contain CLA, the production purposes of preferably glycerine three esters.
Can also can for example directly or according to known processing method own be used as food and feed by the genetically modified plant of the present invention that the human and animal consumes.
After organism was cultivated, lipid obtained in a usual manner.For this purpose, organism can at first digestion or directly use after collection.Lipid is with suitable solvent non-polar solvent for example, and for example for example hexane/isopropyl alcohol, phenol/chloroform/primary isoamyl alcohol of hexane or ethanol, Virahol or following mixture between 0 ℃ and 80 ℃, preferably advantageously extracts between 20 ℃ and 50 ℃.Usually biomass is with excessive solvent extraction, for example with respect to biomass with excessive solvent extraction in 1: 4.Solvent is removed subsequently, for example by distillation.Use postcritical CO 2Extract and also can finish extraction.After the extraction, the residue of biomass is removed, for example by filtering.
The crude oil that obtains by this way can then be further purified, for example with for example acetone or chloroform processing of polar solvent, subsequent filtration or centrifugal to remove muddiness.Also can cross pillar is further purified.
In order from triglyceride level, to obtain free fatty acids, with triglyceride level with common mode hydrolysis.
The invention further relates to such vegetables oil, fatty acid mixt and/or triglyceride mixture, the preferred CLA content of unsaturated fatty acids that wherein has increase, and it is by the inventive method production above-mentioned, also relates to their purposes on food, animal-feed, makeup or pharmaceuticals are produced.For this purpose, their amounts with custom are joined in food, animal-feed, makeup or the pharmaceuticals.
Sequence
1.SEQ ID NO:1: coding is from the nucleotide sequence of the aliphatic alcohol E11-desaturase of the shallow brown volume moth of apple.
2.SEQ ID NO:2: coding is from the protein sequence of the aliphatic alcohol E11-desaturase of the shallow brown volume moth of apple.
3.SEQ ID NO:3: coding is from the nucleotide sequence of the aliphatic alcohol Z/E11-desaturase of European corn borer.
4.SEQ ID NO:4: coding is from the protein sequence of the aliphatic alcohol Z/E11-desaturase of European corn borer.
5.SEQ ID NO:5: coding is from the nucleotide sequence of the aliphatic alcohol Z/E11-desaturase of Ostrinia furnacalis
6.SEQ ID NO:6: coding is from the protein sequence of the aliphatic alcohol Z/E11-desaturase of Ostrinia furnacalis.
7.SEQ ID NO:7: coding is from the nucleotide sequence of the aliphatic alcohol Δ 11-desaturase of Heliothis zea.
8.SEQ ID NO:8: coding is from the protein sequence of the aliphatic alcohol Δ 11-desaturase of Heliothis zea.
9.SEQ ID NO:9: coding from cabbage looper the nucleotide sequence of aliphatic alcohol Δ 11-desaturase.
10.SEQ ID NO:10: coding from cabbage looper the protein sequence of aliphatic alcohol Δ 11-desaturase.
11.SEQ ID NO:11: coding is from the nucleotide sequence of the aliphatic alcohol Δ 11-desaturase of argyrotaenia velutinana.
12.SEQ ID NO:12: coding is from the protein sequence of the aliphatic alcohol Δ 11-desaturase of argyrotaenia velutinana.
13.SEQ ID NO:13: coding is from the nucleotide sequence of the aliphatic alcohol Z10-desaturase of green head volume moth.
14.SEQ ID NO:14: coding is from the protein sequence of the aliphatic alcohol Z10-desaturase of green head volume moth.
15.SEQ?ID?NO：15：5‘-ATYACHGCCGGKKMYCAYMG-3‘
16.SEQ?ID?NO：16：5‘-GGRAABDYGTGRTGGWAGTT-3‘
17.SEQ?ID?NO：17：5‘-CCCCAYCRNCTSTGGWCNCA-3’
18.SEQ?ID?NO：18：5‘-CCCTCTAGARTGRRWARTTRTGRWA-3’
19.SEQ?ID?NO：19：5‘-TAATACGACTCACTATAG-3‘
20.SEQ?ID?NO：20：5‘-ACATAACTAATTACATGAT-3‘
21.SEQ ID NO:21: coding is from the nucleotide sequence of the aliphatic alcohol Z/E11-desaturase of cotton pink bollworm.
22.SEQ ID NO:22: coding is from the aminoacid sequence of the aliphatic alcohol Z/E11-desaturase of cotton pink bollworm.
Embodiment
The present invention is described in detail with the purposes embodiment with reference to accompanying drawing subsequently.The abbreviation of using has following meaning:
Universal method
Clone's step of implementing for the object of the invention, for example, restriction enzyme digestion, agarose gel electrophoresis, dna fragmentation purifying, with nucleic acid transfer to that nitrocellulose filter is connected with nylon membrane, dna fragmentation, Bacillus coli cells conversion, microbial culture and press of people (1989) cold spring harbor laboratory such as recombinant DNA sequence analysis such as Sambrook; Carrying out described in the ISBN 0-87969-309-6.Oligonucleotide can chemosynthesis, for example uses phosphinylidyne imines method (Voet, Voet, second edition, Wiley Press, New York, 896-897 page or leaf) in known manner.Clone's step of in the present invention, implementing, for example restricted cutting, agarose gel electrophoresis, dna fragmentation purifying, with nucleic acid transfer to that nitrocellulose filter is connected with nylon membrane, dna fragmentation, press of people (1989) cold spring harbor laboratory such as Bacillus coli cells conversion, microbial culture, phage breeding, recombinant DNA sequence analysis such as Sambrook; Carry out described in the ISBN0-87969-309-6.(MWG Biotech sells the laser fluorescence dna sequencing instrument of recombinant DNA molecules from Licor, Ebersbach) with Sanger method people (1977) Proc Natl Acad Sci USA 74:5463-5467 such as () Sanger order-checking.
Embodiment 1: the raising of lepidopteran class insect:
Insect maintenance is in container on the appropriate host plant.Growth conditions is: 27 ℃, and day-circadian rhythm: illumination in 14 hours, 10 hours dark.Insect and larva per week are transferred to fresh plant for twice.Collect pupa (Puppae), male and female in raising container maintenance separate up to adult insects and hatch.About 1 to 2 day of insect hatching back, separable pheromone gland.
Embodiment 2: separate desaturase or conjugation enzyme cDNA by degenerated primer
The pheromone body of gland is separated from the belly of the moth that grows up, is frozen in the liquid nitrogen to separate until carrying out RNA, for example by TRIzol (Gibco/BRL) method, according to the operation instruction isolation of RNA of manufacturer.Experience shows that total RNA of about 60 to 80 μ g can separate from about 30mg flesh tissue.The total RNA of about 5 μ g is used to produce the first chain cDNA, uses oligomerization (dT) primer therebetween.This can for example carry out according to the operation instruction of manufacturer with SMART RACE cDNA amplification kit (Clontech).The first chain cDNA is as pcr template, and the important area of desaturase in PCR/conjugation enzyme cDNA is amplified.Designed two degenerated primers,, be used for following PCR operation then so that they can increase from the important conservative region of lepidopterous desaturase/conjugation enzyme:
The first chain cDNA of 5 μ l dilution
0.2mM??????????????dATP，dTTP，dGTP，dCTP
0.5 μ M 5 ' primer (SEQ ID NO:8)
0.5 μ M 3 ' primer (SEQ ID NO:9)
10 μ l 5X Advantage 2 reaction buffers (Clontech)
1 μ l 50X Advantage, 2 archaeal dna polymerase mixtures (Clontech)
Add water to 50 μ l
But as selection scheme, the degenerated primer of SEQ ID NO:10 and SEQ ID NO:11 is right as primer, amplification desaturase important area.
PCR carries out under following cycling condition:
94 ℃ (5 minutes);
94 ℃ (30 seconds), 56 ℃ (30 seconds), 72 ℃ (3 minutes) carry out 35 circulations;
72 ℃ (10 minutes);
4 ℃ of placements
The PCR product is directly connected to and for example arrives linearizing TOPO TA PCR 2.1 carriers (Invitrogen), and subsequent transformation is gone in the competence intestinal bacteria TOP10 cell (Invitrogen).The positive bacterium colony that increases again, plasmid DNA purification (Qiagen Plasmid Mini Kit), order-checking subsequently.
Based on the sequence information that is obtained, can derive gene-specific primer, and 5 ' and the 3 ' district (SMART RACE cDNA Amplification Kit (Clontech)) that is used for increasing.Once more these PCR products are connected in TOPO TA PCR 2.1 carriers, subsequent transformation is gone in competence TOP 10 cells (Invitrogen).The positive bacterium colony that increases again, plasmid DNA purification (Qiagen Plasmid MiniKit), order-checking subsequently.
From the fragment that in this way produces, the full sequence of desaturase/conjugation enzyme can couple together by the standard clone technology, for example transfers in the pYES2 carrier (Invitrogen), so that express in yeast in order to describe purpose.Yeast INVSc1 (Invitrogen) transforms by improved PEG/ Lithium Acetate method people (1996) Current Protocols inMolecular Biology.John Wiley and Sons such as (, New York) Ausubel with corresponding pYES2 expression vector.After the selection that contains on the CMdum agar plate of 2% glucose, select 4 pYES2DESAT transformants (pYES2DESATa-d) and a pYES2 transformant to be used for further cultivating and functional expression.
Embodiment 3: the desaturase/functional expression of conjugation enzyme in yeast
The pre-cultivation: (pYES2DESATa-d, pYES2), cultivation is 3 days under 30 ℃, 200 rev/mins conditions, up to 600nm (OD for inoculation transgenic yeast clone in the 20ml CMdum liquid nutrient medium that appends 2% (w/v) raffinose 600) optical density(OD) located reaches 1.5-2.
The main cultivation: in order to express, for example stearic acid, Zoomeric acid or tetradecanoic acid are modulated the ultimate density that reaches 0.003% (w/v) with corresponding substrate to have appended the 20ml CMdum liquid nutrient medium of 2% raffinose, 1% (v/v) Tergitol NP-40.The pre-culture of inoculation makes OD on the substratum 600Reach 0.05.At OD 600Be 0.2 o'clock, used 2% (w/v) semi-lactosi abduction delivering 16 hours, thereafter culture OD 600Reach 0.8 to 1.2.
Fatty acid analysis: from yeast culture, extract all fatty acids, and pass through gc analysis.For this purpose, by centrifugal (1000 * g, 10 minutes, 4 ℃),, with 100mM pH value 8.0 NaHCO with the 5ml cell culture in order to remove lipid acid and substratum resistates 3Wash once.Be preparation fatty acid methyl ester (FAMES), the cell precipitation thing is freezing rapidly in liquid nitrogen, then in 30 ℃ of N 2Lyophilize under the gas.Throw out is homogenization in the methyl alcohol that contains 1% sodium methylate, and this homogenate was in incubated at room 20 minutes then.Add isopyknic 1M NaCl and normal heptane subsequently, biased sample is transferred to mixed solution in the GC pipe.Sample is at DB-wax capillary column (30m, 0.25mm, the 0.25 μ m of HewlettPackard 6890 gas chromatographs of being furnished with flame ionic detector; Agilent J﹠amp; W) separate in.The speed that furnace temperature is set with 20 ℃/minute rises to 200 ℃ (keeping 20 minutes) from 60 ℃ (keeping 5 minutes), and last speed with 20 ℃/minute rises to 250 ℃ (keeping 30 minutes).The carrier gas of using is nitrogen (1.6ml/ minute).Lipid acid is by identifying with the retention time comparison of FAME standard substance (SIGMA).For this purpose, following lipid acid uses preferably as standard substance: c9-16:1, t9-16:1,18:0, c9-18:1, t9-18:1, c11-18:1, t11-18:1, c9, c12-18:2, t9, t12-18:2, c9, t11-18:2, t10, c12-18:2.(nomenclature of standard substance is corresponding to traditional nomenclature of lipid acid, and at first points out configuration (c=cis, t=is trans) and the position or the fatty acid chain length of two keys).
Use other different lipid acid (for example lauric acid, trans isooleic acid, cis isooleic acid, E10-octadecenoic acid or Z10-octadecenoic acid) to carry out the feed supplement experiment further, to be used for the optionally deeply checking of described desaturase/conjugation enzyme substrates.
Embodiment 4: from the lepidopterous desaturase/clonal expression of conjugation enzyme in yeast saccharomyces cerevisiae.
A) generation in cDNA library
Isolate the pheromone gland from the belly of ripe moth, freezing up to carrying out total RNA separation, for example by using TRIzol (Gibco/BRL) method to separate in liquid nitrogen according to manufacturer's operation instruction.Experience shows that the total RNA of about 60 to 80 μ g can separate from about 30mg flesh tissue.The total RNA of about 5 μ g is used for producing the cDNA library.This can realize by for example using SMART cDNA LibraryConstruction Kit (Clontech).Isolating double-stranded cDNA is connected in linearizing pYES2 (Invitrogen) Yeast expression carrier at last.For this purpose, at first pTriplEx2 carrier (Clontech) multiple clone site is inserted into pYES2.Double-stranded cDNA is cloned into modified carrier in direct mode then with SfiIA and SfiIB digestion.
B) yeast conversion
According to manufacturer's working instructions, about 1.5 μ g plasmid DNA are transformed among the INVSc1 (Invitrogen) with yeast saccharomyces cerevisiae EASY COMP conversion reagent box (Invitrogen).The two crowdes 50 μ l are applied to big square plate (the selection substratum in 245 * 245mm) and 30 ℃ of incubations 3 days.
C) yeast culture in the microtiter plate
Use Pick Roboter single colony lift in microtiter plate (MTP).Cultivate in advance at 200 μ l substratum [1 * CSM-Ura; 1 * YNB of no amino acid and sugar; 0.5% raffinose; 5% glycerine; The 40mg/l adenine sulfate; 0.5% ammonium sulfate] in carried out 72 hours in 30 ℃, per minute 250 changes.In main the cultivation, adjust to OD by adding the pre-culture of mean vol 600Be 0.2.The main cultivation at 1ml substratum [1 * CSM-Ura; 1 * YNB of no amino acid and sugar; 0.5% raffinose; 2% semi-lactosi; 0.2%Tergitol NP-40; The 40mg/l adenine sulfate; 0.5% ammonium sulfate; 0.3mM lipid acid substrate] in 16 ℃ carried out for 2 to 3 weeks, per minute 250 changes, up to reaching OD 600Be 3 to 4.
D) fatty acid analysis
Precipitation yeast cell (1000g, 10 minutes, 4 ℃) is-80 ℃ of preservations, up to further processing.In order to prepare fatty acid methyl ester (FAMES), the cell precipitation thing is freezing rapidly in liquid nitrogen, and in 30 ℃ N 2Freeze-drying under the gas.Throw out homogenization in the methyl alcohol that contains 1% sodium methylate was also at room temperature hatched 20 minutes.Add isopyknic 1M NaCl and normal heptane, biased sample, supernatant liquor are transferred in the GC pipe.Sample is at DB-wax capillary column (30m, 0.25mm, the 0.25 μ m of Hewlett Packard 6890 gas chromatographs of being furnished with flame ionic detector; Agilent J﹠amp; W) separate in.The speed that furnace temperature is set with 20 ℃/minute rises to 200 ℃ (keeping 20 minutes) from 60 ℃ (keeping 5 minutes), and last speed with 20 ℃/minute rises to 250 ℃ (keeping 30 minutes).The carrier gas of using is nitrogen (1.6ml/ minute).Lipid acid is by relatively identifying with the retention time of FAME standard substance (SIGMA).The same standard product that use is listed in embodiment 3.Use other different lipid acid (for example lauric acid, trans isooleic acid, cis isooleic acid, E10-octadecenoic acid or Z10-octadecenoic acid) to carry out the feed supplement experiment further, to be used for the optionally deeply checking of described saturated enzyme/conjugation enzyme substrates.
E) positive yeast plasmid preparation:
Verified as male list bacterium colony again at minimal medium [1 * CSM-Ura of 10ml; 1 * YNB w/o AA, sugar; 2% glucose; The 40mg/l adenine sulfate] the middle cultivation 24 hours, temperature is 28 ℃, works as OD 600Surpass at 3 o'clock, by centrifugation.The yeast sedimentation thing is at SCE damping fluid [the 1.2M sorbyl alcohol of 400 μ l; 0.1M Trisodium Citrate, pH 7.0; 10mM EDTA; 1mg/ml Lytikase] in 37 ℃ of incubations 20 minutes.Then, the STE damping fluid [2%SDS that adds 400 μ l; 50mM Tris/HCl, pH 8.0; 10mM EDTA] in cell pyrolysis liquid, its mixed solution was hatched 10 minutes at normal temperatures.For sedimentation cell protein, add 200 μ l 5M sodium-acetates, its mixture was shelved on ice 30 minutes.After centrifugal, shift supernatant liquor also with 2.5 times of volume of ethanol precipitations.Precipitation is washed with 70% ethanol, is dissolved in the sterilized water at last.As required, the sequence of desaturase/conjugation enzyme can use carrier specificity primer (SEQ ID NO:12 and 13) to determine by order-checking.
Embodiment 5: the biosynthetic operation of vegetable fatty acid
A) be used in the generation of transgenic plant expression from the DNA construct of lepidopterous desaturase/conjugation enzyme
In order to produce the chimeric DNA construct, be used to produce transgenic arabidopsis or colea (B.napus) plant of expression, used pBinAR carrier (H fgen and Willmitzer (1990) Plant Sci 66:221-230) from lepidopterous desaturase/conjugation enzyme.This carrier comprises CaMV (cauliflower mosaic virus) 35S promoter (people (1980) Cell 21 (1) such as Franck: 285-294) and the termination signal of octopine synthase gene people (1984) EMBO J 3:835-846 such as () Gielen.After desaturase/conjugation enzyme clone is gone into this carrier, transgenic arabidopsis and colea plant have been produced.
B) generation of transgenic arabidopsis plant
Wild-type arabidopsis thaliana (Colombia's type) agrobacterium tumefaciens bacterial strain (GV3101[pMP90]) transform, based on improvement vacuum infiltration method (Clough S and Bent A (1998) Plant J 16 (6): 735-43; People (1993) such as Bechtold N: carry out the agriculture bacillus mediated transgenosis of plant by soaking into sophisticated arabidopsis thaliana, CRAcad Sci Paris 1144 (2): 204-212).Used agrobacterium tumefaciens cell transforms with plasmid in advance.The seed of elementary transformant can be selected antibiotic resistance based on them.The seedling cultivation that antibiosis is have resistance is used for biochemical analysis with the plant after reaching full growth in soil.
C) generation of transgenosis colea plant
The transgenic rapeseed plant according to Bade JB and Damm B (in Gene Transfer toPlants one book, Potrykus I and Spangenberg G (editor) Springer Lab Manual, Springer Verlag, 1995,30-38) described method produces, and has wherein also described the substratum of use and the composition of damping fluid.
With agrobacterium tumefaciens bacterial strain GV3101[pMP90] implement to transform.The pBinAR-TkTP/VitE-AT plasmid is used for transforming.The seed of colea mutation Westar is with 70% ethanol (v/v) surface sterilization, in 55 ℃ of water, washed 10 minutes, be to hatch 20 minutes in 1% the hypochlorite solutions (25%v/v Teepol, 0.1%v/v Tween 20) in concentration, use aseptic washing six times in each case.Seed on filter paper dry 3 days, 10 to 15 seeds allow its germination in the glass flask that 15ml germination substratum is housed.Remove root and the terminal bud of a few strain seedling (the about 10cm of size), remaining hypocotyl is cut into the long piece of about 6mm.About 600 explants that obtain are like this washed 30 minutes with the 50ml basic medium, and are transferred to the 300ml flask.Behind the callus inducing medium that adds 100ml, culture was hatched 24 hours in 100 rev/mins.
The overnight culture of agrobacterium strains results from the Luria nutrient solution that is added with kantlex (20mg/l) under 29 ℃, the Luria nutrient solution that the above-mentioned culture of 2ml is not had kantlex at 50ml was hatched 4 hours in 29 ℃, up to OD 600Reach 0.4 to 0.5.Culture in 2000 rev/mins the precipitation 25 minutes after, cell precipitation is resuspended in the 25ml basic medium.Bacterium enriched material in solution makes its OD by adding more basic medium 600Be 0.3.
Remove callus inducing medium with aseptic straw from the Semen Brassicae campestris explant, add 50ml Agrobacterium solution, careful mixed culture was hatched 20 minutes.Remove agrobacterium suspension,, add the callus inducing medium of 100ml subsequently with the callus inducing medium washing oil vegetable seed explant of 50ml 1 minute.On orbital shaker, cultivated altogether 24 hours with 100 rev/mins.Stop common cultivation by reclaiming callus inducing medium, under 100 commentaries on classics/per minutes, explant cleans substratum with 25ml and washes twice, and each one minute, cleaned then twice 60 minutes, each 100ml cleans substratum.Clean substratum and explant and transfer to together in the 15cm plate, remove substratum with aseptic transfer pipet then.
In order to regenerate, will add in the plate of branch inducing culture of kantlex with 20 to 30 25ml that accommodate that transfer to 90mm for a collection of explant.Plate is equipped with 2 layers of Leukopor, 25 ℃ and 2000 luxs, cultivates under 16 hours illumination/8 hour no optical condition.Every 12 days, developmental callus was transferred in the fresh plate that the branch inducing culture is housed.All further steps in order to the complete plant materials of regenerating all are as Bade, J.B and Damm, B. (: Gene Transfer to Plants, Potrykus I and Spangenberg G (editor) Springer LabManual, Springer Verlag, 1995,30-38) described carrying out.
D) analysis of lipid acid form in the transgenic plant
The seed of transgenic plant is homogenate in 1% the methanol solution at sodium methylate content, and homogenate was hatched 20 minutes at normal temperatures.Add isopyknic 1M NaCl and normal heptane subsequently, biased sample, supernatant liquor are transferred in the GC pipe.These samples are at DB-wax capillary column (30m, 0.25mm, the 0.25 μ m of HewlettPackard 6890 gas chromatographs of being furnished with flame ionic detector; Agilent J﹠amp; W) separate in.The speed that furnace temperature is set with 20 ℃/minute rises to 200 ℃ (keeping 20 minutes) from 60 ℃ (keeping 5 minutes), and last speed with 20 ℃/minute rises to 250 ℃ (keeping 30 minutes).The carrier gas of using is nitrogen (1.6ml/ minute).Lipid acid is by relatively determining with the retention time of FAME standard substance (SIGMA).Use is at the same standard substance described in the embodiment 3.
Embodiment 6, from the functional expression of lepidopteran cotton pink bollworm desaturase in yeast
Isolating desaturase is cloned into Yeast expression carrier pYES2 (Invitrogen) by means of BamHI and EcoRI from cotton pink bollworm (SEQ ID NO:21 and 22) body of gland.Consequent construct is named as pYES2::DesPgos.PYES2::DesPgos and the contrast pYES2 all use S.c.Easy Comp conversion reagent box (Invitrogen) be transformed into yeast cell (yeast saccharomyces cerevisiae INVSc1[MAT, his3-1, leu2, trp1-289, ura3-52]; Invitrogen).These yeast cell have also comprised construct pESC::ACS in addition, to be used to express colea aliphatic alcohol synthase.Single bacterium colony of pYES2::DesPgos and contrast pYES2 is from selecting substratum and separate and at 50 milliliters of pre-culture mediums (1 *CSM-Leu/-Ura; 1 *YNB w/o AA; 0.5% raffinose; 5% glycerine and 0.5% ammonium sulfate) in change at 30 ℃ of per minutes 200 and to cultivate 2 days to produce pre-culture.From these pre-cultures, will OD0.06 with the yeast of pYES2::DesPgos with the absorbancy, be that OD0.1 is inoculated in the main medium with the absorbancy with the yeast of pYES2,3 bottles of every kind of inoculations are incubated at 16 ℃ and 30 ℃ simultaneously.Main medium (1 *CSM-Leu/-Ura; 1 *YNB w/o AA; 0.5% raffinose; 2% semi-lactosi; 0.2%Tergitol NP-40 and 0.5% ammonium sulfate) handle with the 0.3mM stearic acid.Cultivated 4 days down for 16 ℃, cultivate after 2 days down for 30 ℃, the absorbancy of master culture is adjusted to OD1.2,10 milliliters of cultures at room temperature with 17500g centrifugal 30 minutes.The throw out aqueous suspension, heating is centrifugal with similarity condition after 10 minutes down at 90 ℃.Throw out washes with water, and post precipitation is used the liquid nitrogen quick freezing, subsequently freeze-drying under nitrogen.In order to extract lipid and to prepare fatty acid methyl ester, be deposited in the methanol solution of 300 μ l1% sodium methylates homogenate and hatched at room temperature 20 minutes.In all cases, add 300 μ l 1M sodium-chlor and normal heptanes subsequently, biased sample is also transferred to supernatant in the GC pipe.Sample is at DB-wax capillary column (30m, 0.25mm, the 0.25 μ m of Hewlett Packard 6890 gas chromatographs of being furnished with flame ionic detector; Agilent J﹠amp; W) separate in.The speed that furnace temperature is set with 20 ℃/minute rises to 200 ℃ (keeping 20 minutes) from 60 ℃ (keeping 5 minutes), and last speed with 20 ℃/minute rises to 250 ℃ (keeping 30 minutes).The carrier gas of using is nitrogen (1.6ml/ minute).Lipid acid is by identifying with the retention time comparison of FAME standard substance (SIGMA).
Table 1 derives from the FAME and the relative distribution compared of contrast pYES2 of the lipid of pYES2::DesPgos when being presented at 16 ℃.As seen the expression of cotton pink bollworm desaturase causes the accumulation of following monounsaturated fatty acids: C16:1D11, C18:1D11 and C18:1D13.And the heterogenous expression of cotton pink bollworm desaturase cause (9Z, 11E)-formation of CLA.
Table 2 derives from the FAME and the relative distribution compared of contrast pYES2 of the lipid of pYES2::DesPgos when being presented at 30 ℃.Expression of results in the time of 30 ℃ with conform to 16 ℃ the time: the expression at yeast middle cotton premium earworm desaturase causes monounsaturated fatty acids C16:1D11, the accumulation of C18:1D11 and C18:1D13.And the heterogenous expression of cotton pink bollworm desaturase cause (9Z, 11E)-formation of CLA.
When showing 1:16 ℃ at the heterogenous expression of yeast saccharomyces cerevisiae middle cotton premium earworm desaturase
Numerical value is represented the relative quantity of single fat acid in all fatty acids spectrum
????????????????????????????????pYES2 ???????????????????????????pYES2::DesPgos
Sample 1 Sample 2 Sample 3 Average Standard deviation Sample 1 Sample 2 Sample 3 Average Standard deviation
?C16:0 ????18.24 ????18.84 ????18.29 ????18.46 ????0.27 ????20.14 ????20.31 ????20.44 ????20.30 ????0.13
?C16:1Δ9 ????43.97 ????43.75 ????43.52 ????43.74 ????0.18 ????41.09 ????41.32 ????41.70 ????41.37 ????0.25
?C16:1Δ11 ????0.02 ????0.13 ????0.25 ????0.13 ????0.09 ????1.60 ????1.54 ????1.62 ????1.59 ????0.03
?C18:0 ????8.49 ????8.52 ????8.62 ????8.54 ????0.06 ????8.11 ????8.19 ????7.95 ????8.08 ????0.10
?C18:1Δ9 ????29.17 ????28.60 ????29.19 ????28.99 ????0.27 ????22.32 ????22.319 ????21.81 ????22.15 ????0.24
?C18:1Δ11 ????0.03 ????0.04 ????0.03 ????0.04 ????0.59 ??? *10E-2 ????6.28 ????6.01 ????6.16 ????6.15 ????0.11
?C18:1Δ13 ????0 ????0 ????0 ????0 ????0 ????0.16 ????0.15 ????0.15 ????0.15 ????0.38 ??? *10E-2
?C18:2-Z9，E11 ????0.01 ????0.01 ????0.01 ????0.01 ????0.21 ??? *10E-2 ????0.05 ????0.04 ????0.04 ????0.05 ????0.26 ??? *10E-2
?C18:2-Z9，Z11 ????0.01 ????0.01 ????0.01 ????0.01 ????0.10 ??? *10E-2 ????0.04 ????0.04 ????0.04 ????0.04 ????0.19 ??? *10E-2
When showing 2:30 ℃ at the heterogenous expression of yeast saccharomyces cerevisiae middle cotton premium earworm desaturase
Numerical value is represented the relative quantity of single fat acid in all fatty acids spectrum
??????????????????????????pYES2 ??????????????????????pYES2::DesPgos
Sample 1 Sample 2 Sample 3 Average Standard deviation Sample 1 Sample 2 Sample 3 Average Standard deviation
??C16:0 ??17.34 ??17.49 ??17.30 ??17.38 ??0.10 ??14.01 ??13.71 ??13.63 ??13.78 ??0.20
??C16:1Δ9 ??40.31 ??41.24 ??41.14 ??40.90 ??0.51 ??37.06 ??37.12 ??38.51 ??37.56 ??0.82
??C16:1Δ11 ??0.16 ??0.17 ??0.17 ??0.17 ??0.60 ? *10E-2 ??3.17 ??3.32 ??3.19 ??3.23 ??0.08
??C18:0 ??8.00 ??7.74 ??8.04 ??7.92 ??0.16 ??5.16 ??4.96 ??4.96 ??5.03 ??0.12
??C18:1Δ9 ??32.40 ??31.71 ??31.68 ??31.93 ??0.41 ??25.49 ??25.24 ??25.22 ??25.32 ??0.15
??C18:1Δ11 ??1.57 ??1.57 ??1.53 ??1.56 ??0.02 ??14.32 ??14.90 ??14.00 ??14.41 ??0.45
??C18:1Δ13 ??0.04 ??0.04 ??0.06 ??0.05 ??0.01 ??0.32 ??0.33 ??0.37 ??0.34 ??0.03
??C18:2-Z9，E11 ??0.01 ??0.01 ??0.01 ??0.01 ??0.06 ? *10E-2 ??0.08 ??0.09 ??0.08 ??0.08 ??0.75 ? *10E-2
??C18:2-Z9，Z11 ??0.04 ??0.03 ??0.04 ??0.04 ??0.31 ? *10E-2 ??0.14 ??0.14 ??0.15 ??0.15 ??0.55 ? *10E-2
Sequence table
＜110〉BASF Plant Science AG
＜120〉be used to produce the method for unsaturated fatty acids
<130>AE20030075-PCT
<140>
<141>
<160>22
＜170〉PatentIn version 2 .1
<210>1
<211>999
<212>DNA
＜213〉the shallow brown volume moth of apple (Epiphyas postvittana)
<220>
<221>CDS
<222>(1)..(996)
＜223〉aliphatic alcohol E11 desaturase
<400>1
atg?gct?cca?aac?gta?gaa?gaa?att?gaa?act?gat?tta?aca?gaa?act?gaa????48
Met?Ala?Pro?Asn?Val?Glu?Glu?Ile?Glu?Thr?Asp?Leu?Thr?Glu?Thr?Glu
1???????????????5??????????????????10??????????????????15
gag?aaa?tgg?gaa?aaa?tta?gtt?gca?ccc?cag?gct?gct?ccc?aga?aag?cat????96
Glu?Lys?Trp?Glu?Lys?Leu?Val?Ala?Pro?Gln?Ala?Ala?Pro?Arg?Lys?His
20??????????????????25??????????????????30
gaa?ata?tta?tac?acg?aac?ctg?cta?atc?ttc?ggc?tac?ggg?cat?ctc?gct????144
Glu?Ile?Leu?Tyr?Thr?Asn?Leu?Leu?Ile?Phe?Gly?Tyr?Gly?His?Leu?Ala
35??????????????????40??????????????????45
gga?ctg?tac?ggt?tta?tac?ctg?tgc?ttc?act?tct?gct?cga?ttg?caa?act????192
Gly?Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Arg?Leu?Gln?Thr
50??????????????????55??????????????????60
att?ata?ctt?gct?ttc?atc?ctt?cac?gca?atg?gca?atc?ttg?ggc?ata?aca????240
Ile?Ile?Leu?Ala?Phe?Ile?Leu?His?Ala?Met?Ala?Ile?Leu?Gly?Ile?Thr
65?????????????????70??????????????????75??????????????????80
gcc?ggc?gct?cac?aga?ctc?tgg?aca?cac?aga?agc?tac?aaa?gcg?aca?atg????288
Ala?Gly?Ala?His?Arg?Leu?Trp?Thr?His?Arg?Ser?Tyr?Lys?Ala?Thr?Met
85??????????????????90??????????????????95
cct?ctt?caa?atc?atc?ctt?ata?att?ttc?aac?tcg?ctg?tca?ttc?caa?aac????336
Pro?Leu?Gln?Ile?Ile?Leu?Ile?Ile?Phe?Asn?Ser?Leu?Ser?Phe?Gln?Asn
100?????????????????105?????????????????110
agt?gcc?att?aat?tgg?gtc?aga?gac?cac?cga?tcg?cac?cac?aag?tat?tgt????384
Ser?Ala?Ile?Asn?Trp?Val?Arg?Asp?His?Arg?Ser?His?His?Lys?Tyr?Cys
115?????????????????120?????????????????125
gat?acg?gac?gcc?gac?cct?cac?aac?gcc?gcc?aga?gga?ctc?ttc?tac?tcc????432
Asp?Thr?Asp?Ala?Asp?Pro?His?Asn?Ala?Ala?Arg?Gly?Leu?Phe?Tyr?Ser
130?????????????????135?????????????????140
cat?atc?ggt?tgg?ctc?ttg?gtg?aag?aag?cac?cct?gaa?gtc?aag?aag?aga????480
His?Ile?Gly?Trp?Leu?Leu?Val?Lys?Lys?His?Pro?Glu?Val?Lys?Lys?Arg
145?????????????????150?????????????????155?????????????????160
gga?aag?atg?acc?gac?atg?tcc?gat?gtc?tac?agg?aac?ccc?gtc?ttg?cgg????528
Gly?Lys?Met?Thr?Asp?Met?Ser?Asp?Val?Tyr?Arg?Asn?Pro?Val?Leu?Arg
165?????????????????170?????????????????175
ttt?caa?aag?aag?tat?gca?gtg?cct?ttc?ata?ggc?acg?ata?tgt?ttc?gta????576
Phe?Gln?Lys?Lys?Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Ile?Cys?Phe?Val
180?????????????????185?????????????????190
ctg?cca?acg?ata?ata?cct?atg?tat?ttc?tgg?gga?gaa?tct?ttg?aac?aac????624
Leu?Pro?Thr?Ile?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Ser?Leu?Asn?Asn
195?????????????????200?????????????????205
gct?tgg?cac?atc?acg?ctg?cta?cgc?tat?atc?ttt?agc?atg?cac?acg?ata????672
Ala?Trp?His?Ile?Thr?Leu?Leu?Arg?Tyr?Ile?Phe?Ser?Met?His?Thr?Ile
210?????????????????215?????????????????220
ttc?ctt?gtg?aac?agc?gta?gcc?cat?cta?tgg?ggc?aac?agg?cct?tac?gac????720
Phe?Leu?Val?Asn?Ser?Val?Ala?His?Leu?Trp?Gly?Asn?Arg?Pro?Tyr?Asp
225?????????????????230?????????????????235?????????????????240
aaa?aac?att?ttg?cca?gcg?gac?aac?aga?aca?tta?tca?atc?gca?acg?tta????768
Lys?Asn?Ile?Leu?Pro?Ala?Asp?Asn?Arg?Thr?Leu?Ser?Ile?Ala?Thr?Leu
245?????????????????250?????????????????255
gga?gaa?gcc?agc?cac?aac?tac?cat?cac?acg?ttt?cct?tgg?gac?tat?aga????816
Gly?Glu?Ala?Ser?His?Asn?Tyr?His?His?Thr?Phe?Pro?Trp?Asp?Tyr?Arg
260?????????????????265?????????????????270
tct?aca?gaa?cta?ggg?tat?tta?cca?act?aac?ttt?act?acg?aac?ttc?att????864
Ser?Thr?Glu?Leu?Gly?Tyr?Leu?Pro?Thr?Asn?Phe?Thr?Thr?Asn?Phe?Ile
275?????????????????280?????????????????285
gat?ttc?ttc?gct?tgg?atc?ggc?tgg?gca?tac?gac?ttg?aaa?aca?aca?tcg????912
Asp?Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Thr?Ser
290?????????????????295?????????????????300
gga?gaa?att?att?aac?agc?agg?ata?caa?aga?act?ggc?gac?ggg?act?cat????960
Gly?Glu?Ile?Ile?Asn?Ser?Arg?Ile?Gln?Arg?Thr?Gly?Asp?Gly?Thr?His
305?????????????????310?????????????????315?????????????????320
tca?agg?agc?aag?aaa?aat?ata?tct?acg?caa?gat?gag?taa????????????????999
Ser?Arg?Ser?Lys?Lys?Asn?Ile?Ser?Thr?Gln?Asp?Glu
325?????????????????330
<210>2
<211>332
<212>PRT
＜213〉the shallow brown volume moth of apple
<400>2
Met?Ala?Pro?Asn?Val?Glu?Glu?Ile?Glu?Thr?Asp?Leu?Thr?Glu?Thr?Glu
1???????????????5??????????????????10??????????????????15
Glu?Lys?Trp?Glu?Lys?Leu?Val?Ala?Pro?Gln?Ala?Ala?Pro?Arg?Lys?His
20??????????????????25??????????????????30
Glu?Ile?Leu?Tyr?Thr?Asn?Leu?Leu?Ile?Phe?Gly?Tyr?Gly?His?Leu?Ala
35??????????????????40??????????????????45
Gly?Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Arg?Leu?Gln?Thr
50??????????????????55??????????????????60
Ile?Ile?Leu?Ala?Phe?Ile?Leu?His?Ala?Met?Ala?Ile?Leu?Gly?Ile?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Gly?Ala?His?Arg?Leu?Trp?Thr?His?Arg?Ser?Tyr?Lys?Ala?Thr?Met
85??????????????????90??????????????????95
Pro?Leu?Gln?Ile?Ile?Leu?Ile?Ile?Phe?Asn?Ser?Leu?Ser?Phe?Gln?Asn
100?????????????????105?????????????????110
Ser?Ala?Ile?Asn?Trp?Val?Arg?Asp?His?Arg?Ser?His?His?Lys?Tyr?Cys
115?????????????????120?????????????????125
Asp?Thr?Asp?Ala?Asp?Pro?His?Asn?Ala?Ala?Arg?Gly?Leu?Phe?Tyr?Ser
130?????????????????135?????????????????140
His?Ile?Gly?Trp?Leu?Leu?Val?Lys?Lys?His?Pro?Glu?Val?Lys?Lys?Arg
145?????????????????150?????????????????155?????????????????160
Gly?Lys?Met?Thr?Asp?Met?Ser?Asp?Val?Tyr?Arg?Asn?Pro?Val?Leu?Arg
165?????????????????170?????????????????175
Phe?Gln?Lys?Lys?Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Ile?Cys?Phe?Val
180?????????????????185?????????????????190
Leu?Pro?Thr?Ile?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Ser?Leu?Asn?Asn
195?????????????????200?????????????????205
Ala?Trp?His?Ile?Thr?Leu?Leu?Arg?Tyr?Ile?Phe?Ser?Met?His?Thr?Ile
210?????????????????215?????????????????220
Phe?Leu?Val?Asn?Ser?Val?Ala?His?Leu?Trp?Gly?Asn?Arg?Pro?Tyr?Asp
225?????????????????230?????????????????235?????????????????240
Lys?Asn?Ile?Leu?Pro?Ala?Asp?Asn?Arg?Thr?Leu?Ser?Ile?Ala?Thr?Leu
245?????????????????250?????????????????255
Gly?Glu?Ala?Ser?His?Asn?Tyr?His?His?Thr?Phe?Pro?Trp?Asp?Tyr?Arg
260?????????????????265?????????????????270
Ser?Thr?Glu?Leu?Gly?Tyr?Leu?Pro?Thr?Asn?Phe?Thr?Thr?Asn?Phe?Ile
275?????????????????280?????????????????285
Asp?Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Thr?Ser
290?????????????????295?????????????????300
Gly?Glu?Ile?Ile?Asn?Ser?Arg?Ile?Gln?Arg?Thr?Gly?Asp?Gly?Thr?His
305?????????????????310?????????????????315?????????????????320
Ser?Arg?Ser?Lys?Lys?Asn?Ile?Ser?Thr?Gln?Asp?Glu
325?????????????????330
<210>3
<211>990
<212>DNA
＜213〉European corn borer (Ostrinia nubilalis)
<220>
<221>CDS
<222>(1)..(987)
＜223〉aliphatic alcohol E/Z11 desaturase
<400>3
atg?gtt?cca?tac?gct?acc?aca?gca?gat?gga?cat?cca?gaa?aaa?gat?gag????48
Met?Val?Pro?Tyr?Ala?Thr?Thr?Ala?Asp?Gly?His?Pro?Glu?Lys?Asp?Glu
1???????????????5??????????????????10??????????????????15
tgc?ttt?gaa?gat?aat?gaa?atc?aaa?tcg?aat?tcc?ttg?ccg?aaa?ctg?gaa????96
Cys?Phe?Glu?Asp?Asn?Glu?Ile?Lys?Ser?Asn?Ser?Leu?Pro?Lys?Leu?Glu
20??????????????????25??????????????????30
ata?cta?tac?ttc?aac?gtt?atg?aca?ttc?acg?ttc?tta?cat?cta?tct?gcg????144
Ile?Leu?Tyr?Phe?Asn?Val?Met?Thr?Phe?Thr?Phe?Leu?His?Leu?Ser?Ala
35??????????????????40??????????????????45
ctt?tat?ggg?ctg?tat?ttg?gga?ttt?aca?tca?gtt?aaa?tgg?gca?act?ata????192
Leu?Tyr?Gly?Leu?Tyr?Leu?Gly?Phe?Thr?Ser?Val?Lys?Trp?Ala?Thr?Ile
50??????????????????55??????????????????60
gga?ctt?gga?att?ata?ttt?tat?ttt?ttt?gct?gag?att?gga?atc?act?gct????240
Gly?Leu?Gly?Ile?Ile?Phe?Tyr?Phe?Phe?Ala?Glu?Ile?Gly?Ile?Thr?Ala
65??????????????????70??????????????????75??????????????????80
ggt?gcc?cat?aga?tta?tgg?agc?cac?aga?agc?tac?aaa?gcg?aaa?ctc?ccc????288
Gly?Ala?His?Arg?Leu?Trp?Ser?His?Arg?Ser?Tyr?Lys?Ala?Lys?Leu?Pro
85??????????????????90??????????????????95
ctg?gaa?ata?ctt?ctc?atg?gtg?ttt?aac?agc?atg?gca?ttt?caa?aat?act????336
Leu?Glu?Ile?Leu?Leu?Met?Val?Phe?Asn?Ser?Met?Ala?Phe?Gln?Asn?Thr
100?????????????????105?????????????????110
gcg?ctc?tcg?tgg?gcc?aga?gac?cat?cgt?gtg?cac?cat?aaa?tgt?cct?gac????384
Ala?Leu?Ser?Trp?Ala?Arg?Asp?His?Arg?Val?His?His?Lys?Cys?Pro?Asp
115?????????????????120?????????????????125
acc?aat?ggt?gat?cct?cac?aat?gcg?aat?cga?gga?ttc?ttc?tat?tca?cat????432
Thr?Asn?Gly?Asp?Pro?His?Asn?Ala?Asn?Arg?Gly?Phe?Phe?Tyr?Ser?His
130?????????????????135?????????????????140
gta?gga?tgg?cta?atg?acc?aaa?aaa?tct?gat?gaa?gtc?atc?aaa?cag?gga????480
Val?Gly?Trp?Leu?Met?Thr?Lys?Lys?Ser?Asp?Glu?Val?Ile?Lys?Gln?Gly
145?????????????????150?????????????????155?????????????????160
aaa?ttg?tgt?gat?gtg?gct?gat?tta?tat?agt?aac?cct?gtg?tta?cgt?ttc????528
Lys?Leu?Cys?Asp?Val?Ala?Asp?Leu?Tyr?Ser?Asn?Pro?Val?Leu?Arg?Phe
165?????????????????170?????????????????175
cag?aaa?aaa?tac?gca?gtg?ccg?ttt?att?gga?acg?ctt?tgt?ttc?gtt?ctc????576
Gln?Lys?Lys?Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Leu?Cys?Phe?Val?Leu
180?????????????????185?????????????????190
ccg?act?ctt?atc?ccg?atg?tac?ttc?tgg?ggc?gaa?act?tta?aac?aat?gcc????624
Pro?Thr?Leu?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Thr?Leu?Asn?Asn?Ala
195?????????????????200?????????????????205
tgg?cat?ttt?aac?atg?ttt?cgt?tac?gtc?att?aac?ctt?aac?gca?acg?ttc????672
Trp?His?Phe?Asn?Met?Phe?Arg?Tyr?Val?Ile?Asn?Leu?Asn?Ala?Thr?Phe
210?????????????????215?????????????????220
tgc?gtc?aac?agc?gtc?gtc?cat?aag?tgg?ggc?tac?aag?ccg?tac?gac?aaa????720
Cys?Val?Asn?Ser?Val?Val?His?Lys?Trp?Gly?Tyr?Lys?Pro?Tyr?Asp?Lys
225?????????????????230?????????????????235?????????????????240
aat?att?tgt?ccg?aca?caa?aac?gtt?ctt?ctg?aat?ctt?gct?gtg?ctt?ggc????768
Asn?Ile?Cys?Pro?Thr?Gln?Asn?Val?Leu?Leu?Asn?Leu?Ala?Val?Leu?Gly
245?????????????????250?????????????????255
gaa?gcg?ttc?cac?aac?tac?cac?cat?gtg?ttc?cca?tgg?gac?tac?agg?gcg????816
Glu?Ala?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Ala
260?????????????????265?????????????????270
gcg?gaa?tta?ggc?aac?caa?aaa?atg?aac?ccc?acg?act?ctg?ttc?ata?gac????864
Ala?Glu?Leu?Gly?Asn?Gln?Lys?Met?Asn?Pro?Thr?Thr?Leu?Phe?Ile?Asp
275?????????????????280?????????????????285
ttc?ttc?gct?tgg?att?gga?tgg?gct?tat?gat?ctc?aag?aca?gcg?tct?aaa????912
Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser?Lys
290?????????????????295?????????????????300
gaa?atg?ata?aag?agt?agg?tcg?gag?aga?act?ggc?gac?ggc?acg?gac?tta????960
Glu?Met?Ile?Lys?Ser?Arg?Ser?Glu?Arg?Thr?Gly?Asp?Gly?Thr?Asp?Leu
305?????????????????310?????????????????315?????????????????320
tgg?ggt?cac?agt?gcc?gat?aaa?cta?aaa?taa????????????????????????????990
Trp?Gly?His?Ser?Ala?Asp?Lys?Leu?Lys
325
<210>4
<211>329
<212>PRT
＜213〉European corn borer
<400>4
Met?Val?Pro?Tyr?Ala?Thr?Thr?Ala?Asp?Gly?His?Pro?Glu?Lys?Asp?Glu
1???????????????5??????????????????10??????????????????15
Cys?Phe?Glu?Asp?Asn?Glu?Ile?Lys?Ser?Asn?Ser?Leu?Pro?Lys?Leu?Glu
20??????????????????25??????????????????30
Ile?Leu?Tyr?Phe?Asn?Val?Met?Thr?Phe?Thr?Phe?Leu?His?Leu?Ser?Ala
35??????????????????40??????????????????45
Leu?Tyr?Gly?Leu?Tyr?Leu?Gly?Phe?Thr?Ser?Val?Lys?Trp?Ala?Thr?Ile
50??????????????????55??????????????????60
Gly?Leu?Gly?Ile?Ile?Phe?Tyr?Phe?Phe?Ala?Glu?Ile?Gly?Ile?Thr?Ala
65???????????????????70??????????????????75??????????????????80
Gly?Ala?His?Arg?Leu?Trp?Ser?His?Arg?Ser?Tyr?Lys?Ala?Lys?Leu?Pro
85??????????????????90??????????????????95
Leu?Glu?Ile?Leu?Leu?Met?Val?Phe?Asn?Ser?Met?Ala?Phe?Gln?Asn?Thr
100?????????????????105?????????????????110
Ala?Leu?Ser?Trp?Ala?Arg?Asp?His?Arg?Val?His?His?Lys?Cys?Pro?Asp
115?????????????????120?????????????????125
Thr?Asn?Gly?Asp?Pro?His?Asn?Ala?Asn?Arg?Gly?Phe?Phe?Tyr?Ser?His
130?????????????????135?????????????????140
Val?Gly?Trp?Leu?Met?Thr?Lys?Lys?Ser?Asp?Glu?Val?Ile?Lys?Gln?Gly
145?????????????????150?????????????????155?????????????????160
Lys?Leu?Cys?Asp?Val?Ala?Asp?Leu?Tyr?Ser?Asn?Pro?Val?Leu?Arg?Phe
165?????????????????170?????????????????175
Gln?Lys?Lys?Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Leu?Cys?Phe?Val?Leu
180?????????????????185?????????????????190
Pro?Thr?Leu?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Thr?Leu?Asn?Asn?Ala
195?????????????????200?????????????????205
Trp?His?Phe?Asn?Met?Phe?Arg?Tyr?Val?Ile?Asn?Leu?Asn?Ala?Thr?Phe
210?????????????????215?????????????????220
Cys?Val?Asn?Ser?Val?Val?His?Lys?Trp?Gly?Tyr?Lys?Pro?Tyr?Asp?Lys
225?????????????????230?????????????????235?????????????????240
Asn?Ile?Cys?Pro?Thr?Gln?Asn?Val?Leu?Leu?Asn?Leu?Ala?Val?Leu?Gly
245?????????????????250?????????????????255
Glu?Ala?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Ala
260?????????????????265?????????????????270
Ala?Glu?Leu?Gly?Asn?Gln?Lys?Met?Asn?Pro?Thr?Thr?Leu?Phe?Ile?Asp
275?????????????????280?????????????????285
Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser?Lys
290?????????????????295?????????????????300
Glu?Met?Ile?Lys?Ser?Arg?Ser?Glu?Arg?Thr?Gly?Asp?Gly?Thr?Asp?Leu
305?????????????????310?????????????????315?????????????????320
Trp?Gly?His?Ser?Ala?Asp?Lys?Leu?Lys
325
<210>5
<211>990
<212>DNA
＜213〉Ostrinia furnacalis (Ostrinia furnacalis)
<220>
<221>CDS
<222>(1)..(987)
＜223〉aliphatic alcohol E/Z11 desaturase
<400>5
atg?gtt?cca?tac?gct?acc?aca?gca?gat?gga?cat?cca?gaa?aaa?gat?gag????48
Met?Val?Pro?Tyr?Ala?Thr?Thr?Ala?Asp?Gly?His?Pro?Glu?Lys?Asp?Glu
1???????????????5??????????????????10??????????????????15
tgc?ttt?gaa?gat?aat?gaa?atc?aaa?tcg?aat?tcc?ttg?ccg?aaa?ctg?gaa????96
Cys?Phe?Glu?Asp?Asn?Glu?Ile?Lys?Ser?Asn?Ser?Leu?Pro?Lys?Leu?Glu
20??????????????????25??????????????????30
ata?cta?tac?ttc?aac?gtt?atg?aca?ttc?acg?ttc?tta?cat?cta?tct?gcg????144
Ile?Leu?Tyr?Phe?Asn?Val?Met?Thr?Phe?Thr?Phe?Leu?His?Leu?Ser?Ala
35??????????????????40??????????????????45
ctt?tat?ggg?ctg?tat?ttg?gga?ttt?aca?tca?gtt?aaa?tgg?gca?act?ata????192
Leu?Tyr?Gly?Leu?Tyr?Leu?Gly?Phe?Thr?Ser?Val?Lys?Trp?Ala?Thr?Ile
50??????????????????55??????????????????60
gga?ctt?gga?att?ata?ttt?tat?ttt?ttt?gct?gag?att?gga?atc?act?gct????240
Gly?Leu?Gly?Ile?Ile?Phe?Tyr?Phe?Phe?Ala?Glu?Ile?Gly?Ile?Thr?Ala
65??????????????????70??????????????????75??????????????????80
ggt?gcc?cat?aga?cta?tgg?agc?cac?aga?agc?tac?aaa?gcg?aaa?ctc?ccc????288
Gly?Ala?His?Arg?Leu?Trp?Ser?His?Arg?Ser?Tyr?Lys?Ala?Lys?Leu?Pro
85??????????????????90??????????????????95
ctg?gaa?ata?ctt?ctc?atg?gtg?ttt?aac?agc?atg?gca?ttt?caa?aat?act????336
Leu?Glu?Ile?Leu?Leu?Met?Val?Phe?Asn?Ser?Met?Ala?Phe?Gln?Asn?Thr
100?????????????????105?????????????????110
gcg?ctc?tcg?tgg?gcc?aga?gac?cat?cgt?gtg?cac?cat?aaa?tgt?cct?gac????384
Ala?Leu?Ser?Trp?Ala?Arg?Asp?His?Arg?Val?His?His?Lys?Cys?Pro?Asp
115?????????????????120?????????????????125
acc?aat?ggt?gat?cct?cac?aat?gcg?aat?cga?gga?ttc?ttc?tat?tcg?cac????432
Thr?Asn?Gly?Asp?Pro?His?Asn?Ala?Asn?Arg?Gly?Phe?Phe?Tyr?Ser?His
130?????????????????135?????????????????140
gta?gga?tgg?cta?atg?acc?aag?aaa?tct?gat?gaa?gtc?atc?aaa?cag?gga????480
Val?Gly?Trp?Leu?Met?Thr?Lys?Lys?Ser?Asp?Glu?Val?Ile?Lys?Gln?Gly
145?????????????????150?????????????????155?????????????????160
aaa?ttg?tgt?gat?gtg?gct?gat?tta?tac?agt?aac?cct?gtg?tta?cgt?ttc????528
Lys?Leu?Cys?Asp?Val?Ala?Asp?Leu?Tyr?Ser?Asn?Pro?Val?Leu?Arg?Phe
165?????????????????170?????????????????175
cag?aaa?aaa?tac?gca?gtg?ccg?ttt?att?gga?acg?ctt?tgt?ttc?gtt?ctc????576
Gln?Lys?Lys?Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Leu?Cys?Phe?Val?Leu
180?????????????????185?????????????????190
ccg?act?ctt?atc?ccg?atg?tac?ttc?tgg?ggc?gaa?act?tta?aac?aat?gcc????624
Pro?Thr?Leu?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Thr?Leu?Asn?Asn?Ala
195?????????????????200?????????????????205
tgg?cat?ttt?aac?atg?ttt?cgt?tac?gtc?att?aac?cta?aac?gca?acg?ttc????672
Trp?His?Phe?Asn?Met?Phe?Arg?Tyr?Val?Ile?Asn?Leu?Asn?Ala?Thr?Phe
210?????????????????215?????????????????220
tgc?gtc?aac?agc?gtc?gtc?cat?aag?tgg?ggc?tac?aag?ccg?tac?gac?aaa????720
Cys?Val?Asn?Ser?Val?Val?His?Lys?Trp?Gly?Tyr?Lys?Pro?Tyr?Asp?Lys
225?????????????????230?????????????????235?????????????????240
aat?att?tgt?ccg?aca?caa?aac?gtt?ctt?ctg?aat?ctt?gct?gtg?ctt?ggc????768
Asn?Ile?Cys?Pro?Thr?Gln?Asn?Val?Leu?Leu?Asn?Leu?Ala?Val?Leu?Gly
245?????????????????250?????????????????255
gaa?gcg?ttc?cac?aac?tac?cac?cat?gtg?ttc?cca?tgg?gac?tac?agg?gcg????816
Glu?Ala?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Ala
260?????????????????265?????????????????270
gcg?gaa?tta?ggc?aac?caa?aaa?atg?aac?ccc?acg?act?ctg?ttc?ata?gac????864
Ala?Glu?Leu?Gly?Asn?Gln?Lys?Met?Asn?Pro?Thr?Thr?Leu?Phe?Ile?Asp
275?????????????????280?????????????????285
ttc?ttc?gct?tgg?att?gga?tgg?gct?tat?gat?ctc?aag?aca?gca?tct?aaa????912
Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser?Lys
290?????????????????295?????????????????300
gaa?atg?ata?aag?agt?agg?tcg?gag?aga?act?ggc?gac?ggc?acg?gac?tta????960
Glu?Met?Ile?Lys?Ser?Arg?Ser?Glu?Arg?Thr?Gly?Asp?Gly?Thr?Asp?Leu
305?????????????????310?????????????????315?????????????????320
tgg?ggt?cac?agt?gcc?gat?aaa?cta?aaa?taa????????????????????????????990
Trp?Gly?His?Ser?Ala?Asp?Lys?Leu?Lys
325
<210>6
<211>329
<212>PRT
＜213〉Ostrinia furnacalis
<400>6
Met?Val?Pro?Tyr?Ala?Thr?Thr?Ala?Asp?Gly?His?Pro?Glu?Lys?Asp?Glu
1???????????????5??????????????????10??????????????????15
Cys?Phe?Glu?Asp?Asn?Glu?Ile?Lys?Ser?Asn?Ser?Leu?Pro?Lys?Leu?Glu
20??????????????????25??????????????????30
Ile?Leu?Tyr?Phe?Asn?Val?Met?Thr?Phe?Thr?Phe?Leu?His?Leu?Ser?Ala
35??????????????????40??????????????????45
Leu?Tyr?Gly?Leu?Tyr?Leu?Gly?Phe?Thr?Ser?Val?Lys?Trp?Ala?Thr?Ile
50??????????????????55??????????????????60
Gly?Leu?Gly?Ile?Ile?Phe?Tyr?Phe?Phe?Ala?Glu?Ile?Gly?Ile?Thr?Ala
65??????????????????70??????????????????75??????????????????80
Gly?Ala?His?Arg?Leu?Trp?Ser?His?Arg?Ser?Tyr?Lys?Ala?Lys?Leu?Pro
85??????????????????90??????????????????95
Leu?Glu?Ile?Leu?Leu?Met?Val?Phe?Asn?Ser?Met?Ala?Phe?Gln?Asn?Thr
100?????????????????105?????????????????110
Ala?Leu?Ser?Trp?Ala?Arg?Asp?His?Arg?Val?His?His?Lys?Cys?Pro?Asp
115?????????????????120?????????????????125
Thr?Asn?Gly?Asp?Pro?His?Asn?Ala?Asn?Arg?Gly?Phe?Phe?Tyr?Ser?His
130?????????????????135?????????????????140
Val?Gly?Trp?Leu?Met?Thr?Lys?Lys?Ser?Asp?Glu?Val?Ile?Lys?Gln?Gly
145?????????????????150?????????????????155?????????????????160
Lys?Leu?Cys?Asp?Val?Ala?Asp?Leu?Tyr?Ser?Asn?Pro?Val?Leu?Arg?Phe
165?????????????????170?????????????????175
Gln?Lys?Lys?Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Leu?Cys?Phe?Val?Leu
180?????????????????185?????????????????190
Pro?Thr?Leu?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Thr?Leu?Asn?Asn?Ala
195?????????????????200?????????????????205
Trp?His?Phe?Asn?Met?Phe?Arg?Tyr?Val?Ile?Asn?Leu?Asn?Ala?Thr?Phe
210?????????????????215?????????????????220
Cys?Val?Asn?Ser?Val?Val?His?Lys?Trp?Gly?Tyr?Lys?Pro?Tyr?Asp?Lys
225?????????????????230?????????????????235?????????????????240
Asn?Ile?Cys?Pro?Thr?Gln?Asn?Val?Leu?Leu?Asn?Leu?Ala?Val?Leu?Gly
245?????????????????250?????????????????255
Glu?Ala?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Ala
260?????????????????265?????????????????270
Ala?Glu?Leu?Gly?Asn?Gln?Lys?Met?Asn?Pro?Thr?Thr?Leu?Phe?Ile?Asp
275?????????????????280?????????????????285
Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser?Lys
290?????????????????295?????????????????300
Glu?Met?Ile?Lys?Ser?Arg?Ser?Glu?Arg?Thr?Gly?Asp?Gly?Thr?Asp?Leu
305?????????????????310?????????????????315?????????????????320
Trp?Gly?His?Ser?Ala?Asp?Lys?Leu?Lys
325
<210>7
<211>1017
<212>DNA
＜213〉Heliothis zea (Helicoverpa zea)
<220>
<221>CDS
<222>(1)..(1014)
＜223〉aliphatic alcohol Δ-11 desaturase
<400>7
atg?gcc?caa?agc?tat?caa?tca?act?acg?gtt?ttg?agt?gag?gag?aaa?gaa????48
Met?Ala?Gln?Ser?Tyr?Gln?Ser?Thr?Thr?Val?Leu?Ser?Glu?Glu?Lys?Glu
1???????????????5??????????????????10??????????????????15
cta?aca?ctg?caa?cat?ttg?gtg?ccc?caa?gca?tcg?ccc?agg?aag?tat?caa????96
Leu?Thr?Leu?Gln?His?Leu?Val?Pro?Gln?Ala?Ser?Pro?Arg?Lys?Tyr?Gln
20??????????????????25??????????????????30
ata?gtg?tat?ccg?aac?ctc?att?acg?ttt?ggt?tac?tgg?cac?ata?gcc?gga????144
Ile?Val?Tyr?Pro?Asn?Leu?Ile?Thr?Phe?Gly?Tyr?Trp?His?Ile?Ala?Gly
35??????????????????40??????????????????45
ctt?tat?ggc?ctt?tac?ttg?tgc?ttc?act?tct?gct?aaa?tgg?gct?acg?att????192
Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp?Ala?Thr?Ile
50??????????????????55??????????????????60
tta?ttc?agc?tac?atc?ctc?ttc?gtg?tta?gca?gaa?ata?gga?atc?acg?gct????240
Leu?Phe?Ser?Tyr?Ile?Leu?Phe?Val?Leu?Ala?Glu?Ile?Gly?Ile?Thr?Ala
65??????????????????70??????????????????75??????????????????80
ggc?gct?cac?aga?ctc?tgg?gcc?cac?aaa?act?tac?aaa?gcg?aaa?cta?cca????288
Gly?Ala?His?Arg?Leu?Trp?Ala?His?Lys?Thr?Tyr?Lys?Ala?Lys?Leu?Pro
85??????????????????90??????????????????95
tta?gaa?ata?ctc?tta?atg?gta?ttc?aac?tcc?atc?gct?ttt?caa?aac?tca????336
Leu?Glu?Ile?Leu?Leu?Met?Val?Phe?Asn?Ser?Ile?Ala?Phe?Gln?Asn?Ser
100?????????????????105?????????????????110
gcc?att?gac?tgg?gtg?agg?gac?cac?cga?ctc?cac?cat?aag?tat?agc?gat????384
Ala?Ile?Asp?Trp?Val?Arg?Asp?His?Arg?Leu?His?His?Lys?Tyr?Ser?Asp
115?????????????????120?????????????????125
aca?gat?gct?gat?ccc?cac?aat?gcc?agc?cga?ggg?ttc?ttt?tat?tcc?cat????432
Thr?Asp?Ala?Asp?Pro?His?Asn?Ala?Ser?Arg?Gly?Phe?Phe?Tyr?Ser?His
130?????????????????135?????????????????140
gta?gga?tgg?cta?ctt?gtg?aga?aaa?cat?cct?gaa?gtc?aaa?aag?cga?ggg????480
Val?Gly?Trp?Leu?Leu?Val?Arg?Lys?His?Pro?Glu?Val?Lys?Lys?Arg?Gly
145?????????????????150?????????????????155?????????????????160
aaa?gaa?ctc?aat?atg?tcc?gat?att?tac?aac?aat?cct?gtc?ctg?cgg?ttt????528
Lys?Glu?Leu?Asn?Met?Ser?Asp?Ile?Tyr?Asn?Asn?Pro?Val?Leu?Arg?Phe
165?????????????????170?????????????????175
cag?aaa?aaa?tac?gcc?ata?ccc?ttc?att?ggg?gct?gtt?tgt?ttc?gcc?tta????576
Gln?Lys?Lys?Tyr?Ala?Ile?Pro?Phe?Ile?Gly?Ala?Val?Cys?Phe?Ala?Leu
180?????????????????185?????????????????190
cct?aca?atg?ata?cct?gtt?tac?ttc?tgg?gga?gaa?acc?tgg?tcc?aat?gct????624
Pro?Thr?Met?Ile?Pro?Val?Tyr?Phe?Trp?Gly?Glu?Thr?Trp?Ser?Asn?Ala
195?????????????????200?????????????????205
tgg?cat?atc?acc?atg?ctt?cgc?tac?atc?atg?aac?ctc?aat?gtc?acc?ttt????672
Trp?His?Ile?Thr?Met?Leu?Arg?Tyr?Ile?Met?Asn?Leu?Asn?Val?Thr?Phe
210?????????????????215?????????????????220
ttg?gta?aac?agc?gct?gct?cat?ata?tgg?gga?aac?aag?cct?tat?gac?gca????720
Leu?Val?Asn?Ser?Ala?Ala?His?Ile?Trp?Gly?Asn?Lys?Pro?Tyr?Asp?Ala
225?????????????????230?????????????????235?????????????????240
aaa?ata?tta?cct?gca?caa?aat?gta?gct?gtg?tcg?gtc?gcc?act?ggt?gga????768
Lys?Ile?Leu?Pro?Ala?Gln?Asn?Val?Ala?Val?Ser?Val?Ala?Thr?Gly?Gly
245?????????????????250?????????????????255
gaa?ggt?ttc?cat?aat?tac?cac?cat?gtc?ttc?ccc?tgg?gat?tat?cga?gca????816
Glu?Gly?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Ala
260?????????????????265?????????????????270
gcg?gaa?ctc?ggt?aac?aat?agc?ctc?aat?ctg?acg?act?aaa?ttc?ata?gat????864
Ala?Glu?Leu?Gly?Asn?Asn?Ser?Leu?Asn?Leu?Thr?Thr?Lys?Phe?Ile?Asp
275?????????????????280?????????????????285
tta?ttc?gca?gca?atc?gga?tgg?gca?tat?gat?ctg?aag?acg?gtt?tcg?gag????912
Leu?Phe?Ala?Ala?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Val?Ser?Glu
290?????????????????295?????????????????300
gat?atg?ata?aaa?caa?agg?att?aaa?cgc?act?gga?gat?gga?acg?gat?ctt????960
Asp?Met?Ile?Lys?Gln?Arg?Ile?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Asp?Leu
305?????????????????310?????????????????315?????????????????320
tgg?gga?cac?gaa?caa?aac?tgt?gat?gaa?gtg?tgg?gat?gta?aaa?gat?aaa????1008
Trp?Gly?His?Glu?Gln?Asn?Cys?Asp?Glu?Val?Trp?Asp?Val?Lys?Asp?Lys
325?????????????????330?????????????????335
tca?agt?taa????????????????????????????????????????????????????????1017
Ser?Ser
<210>8
<211>338
<212>PRT
＜213〉Heliothis zea
<400>8
Met?Ala?Gln?Ser?Tyr?Gln?Ser?Thr?Thr?Val?Leu?Ser?Glu?Glu?Lys?Glu
1???????????????5??????????????????10??????????????????15
Leu?Thr?Leu?Gln?His?Leu?Val?Pro?Gln?Ala?Ser?Pro?Arg?Lys?Tyr?Gln
20??????????????????25??????????????????30
Ile?Val?Tyr?Pro?Asn?Leu?Ile?Thr?Phe?Gly?Tyr?Trp?His?Ile?Ala?Gly
35??????????????????40??????????????????45
Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp?Ala?Thr?Ile
50??????????????????55??????????????????60
Leu?Phe?Ser?Tyr?Ile?Leu?Phe?Val?Leu?Ala?Glu?Ile?Gly?Ile?Thr?Ala
65??????????????????70??????????????????75??????????????????80
Gly?Ala?His?Arg?Leu?Trp?Ala?His?Lys?Thr?Tyr?Lys?Ala?Lys?Leu?Pro
85??????????????????90??????????????????95
Leu?Glu?Ile?Leu?Leu?Met?Val?Phe?Asn?Ser?Ile?Ala?Phe?Gln?Asn?Ser
100?????????????????105?????????????????110
Ala?Ile?Asp?Trp?Val?Arg?Asp?His?Arg?Leu?His?His?Lys?Tyr?Ser?Asp
115?????????????????120?????????????????125
Thr?Asp?Ala?Asp?Pro?His?Asn?Ala?Ser?Arg?Gly?Phe?Phe?Tyr?Ser?His
130?????????????????135?????????????????140
Val?Gly?Trp?Leu?Leu?Val?Arg?Lys?His?Pro?Glu?Val?Lys?Lys?Arg?Gly
145?????????????????150?????????????????155?????????????????160
Lys?Glu?Leu?Asn?Met?Ser?Asp?Ile?Tyr?Asn?Asn?Pro?Val?Leu?Arg?Phe
165?????????????????170?????????????????175
Gln?Lys?Lys?Tyr?Ala?Ile?Pro?Phe?Ile?Gly?Ala?Val?Cys?Phe?Ala?Leu
180?????????????????185?????????????????190
Pro?Thr?Met?Ile?Pro?Val?Tyr?Phe?Trp?Gly?Glu?Thr?Trp?Ser?Asn?Ala
195?????????????????200?????????????????205
Trp?His?Ile?Thr?Met?Leu?Arg?Tyr?Ile?Met?Asn?Leu?Asn?Val?Thr?Phe
210?????????????????215?????????????????220
Leu?Val?Asn?Ser?Ala?Ala?His?Ile?Trp?Gly?Asn?Lys?Pro?Tyr?Asp?Ala
225?????????????????230?????????????????235?????????????????240
Lys?Ile?Leu?Pro?Ala?Gln?Asn?Val?Ala?Val?Ser?Val?Ala?Thr?Gly?Gly
245?????????????????250?????????????????255
Glu?Gly?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Ala
260?????????????????265?????????????????270
Ala?Glu?Leu?Gly?Asn?Asn?Ser?Leu?Asn?Leu?Thr?Thr?Lys?Phe?Ile?Asp
275?????????????????280?????????????????285
Leu?Phe?Ala?Ala?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Val?Ser?Glu
290?????????????????295?????????????????300
Asp?Met?Ile?Lys?Gln?Arg?Ile?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Asp?Leu
305?????????????????310?????????????????315?????????????????320
Trp?Gly?His?Glu?Gln?Asn?Cys?Asp?Glu?Val?Trp?Asp?Val?Lys?Asp?Lys
325?????????????????330?????????????????335
Ser?Ser
<210>9
<211>1050
<212>DNA
＜213〉cabbage looper (Trichoplusia ni)
<220>
<221>CDS
<222>(1)..(1047)
＜223〉aliphatic alcohol Δ 11 desaturases
<400>9
atg?gct?gtg?atg?gct?caa?aca?gta?caa?gaa?acg?gct?aca?gtg?ttg?gaa????48
Met?Ala?Val?Met?Ala?Gln?Thr?Val?Gln?Glu?Thr?Ala?Thr?Val?Leu?Glu
1???????????????5??????????????????10??????????????????15
gag?gaa?gct?cgc?aca?gtg?act?ctt?gtg?gct?cca?aag?aca?acg?cca?agg????96
Glu?Glu?Ala?Arg?Thr?Val?Thr?Leu?Val?Ala?Pro?Lys?Thr?Thr?Pro?Arg
20??????????????????25??????????????????30
aaa?tat?aaa?tat?ata?tac?acc?aac?ttt?ctt?aca?ttt?tca?tat?gcg?cat????144
Lys?Tyr?Lys?Tyr?Ile?Tyr?Thr?Asn?Phe?Leu?Thr?Phe?Ser?Tyr?Ala?His
35??????????????????40??????????????????45
tta?gct?gca?tta?tac?gga?ctt?tat?ttg?tgc?ttc?acc?tct?gcg?aaa?tgg????192
Leu?Ala?Ala?Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp
50??????????????????55??????????????????60
gaa?aca?ttg?cta?ttc?tct?ttc?gta?ctc?ttc?cac?atg?tca?aat?ata?ggc????240
Glu?Thr?Leu?Leu?Phe?Ser?Phe?Val?Leu?Phe?His?Met?Ser?Asn?Ile?Gly
65??????????????????70??????????????????75??????????????????80
atc?acc?gca?ggg?gct?cac?cga?ctc?tgg?act?cac?aag?act?ttc?aaa?gcc????288
Ile?Thr?Ala?Gly?Ala?His?Arg?Leu?Trp?Thr?His?Lys?Thr?Phe?Lys?Ala
85??????????????????90??????????????????95
aaa?ttg?cct?ttg?gaa?att?gtc?ctc?atg?ata?ttc?aac?tct?tta?gcc?ttt????336
Lys?Leu?Pro?Leu?Glu?Ile?Val?Leu?Met?Ile?Phe?Asn?Ser?Leu?Ala?Phe
100?????????????????105?????????????????110
caa?aac?acg?gct?att?aca?tgg?gct?aga?gaa?cat?cgg?cta?cat?cac?aaa????384
Gln?Asn?Thr?Ala?Ile?Thr?Trp?Ala?Arg?Glu?His?Arg?Leu?His?His?Lys
115?????????????????120?????????????????125
tac?agc?gat?act?gat?gct?gat?ccc?cac?aat?gcg?tca?aga?ggg?ttc?ttc????432
Tyr?Ser?Asp?Thr?Asp?Ala?Asp?Pro?His?Asn?Ala?Ser?Arg?Gly?Phe?Phe
130?????????????????135?????????????????140
tac?tcg?cat?gtt?ggc?tgg?cta?tta?gta?aaa?aaa?cat?ccc?gat?gtc?ctg????480
Tyr?Ser?His?Val?Gly?Trp?Leu?Leu?Val?Lys?Lys?His?Pro?Asp?Val?Leu
145?????????????????150?????????????????155?????????????????160
aaa?tat?gga?aaa?act?ata?gac?atg?tcg?gat?gta?tac?aat?aat?cct?gtg????528
Lys?Tyr?Gly?Lys?Thr?Ile?Asp?Met?Ser?Asp?Val?Tyr?Asn?Asn?Pro?Val
165?????????????????170?????????????????175
tta?aaa?ttt?cag?aaa?aag?tac?gca?gta?ccc?tta?att?gga?aca?gtt?tgt????576
Leu?Lys?Phe?Gln?Lys?Lys?Tyr?Ala?Val?Pro?Leu?Ile?Gly?Thr?Val?Cys
180?????????????????185?????????????????190
ttt?gct?ctt?cca?act?ttg?att?cca?gtc?tac?tgt?tgg?ggc?gaa?tcg?tgg????624
Phe?Ala?Leu?Pro?Thr?Leu?Ile?Pro?Val?Tyr?Cys?Trp?Gly?Glu?Ser?Trp
195?????????????????200?????????????????205
aac?aac?gct?tgg?cac?ata?gcc?tta?ttt?cga?tac?ata?ttc?aat?ctt?aac????672
Asn?Asn?Ala?Trp?His?Ile?Ala?Leu?Phe?Arg?Tyr?Ile?Phe?Asn?Leu?Asn
210?????????????????215?????????????????220
gtg?act?ttc?cta?gtc?aac?agt?gct?gcg?cat?atc?tgg?ggg?aat?aag?cct????720
Val?Thr?Phe?Leu?Val?Asn?Ser?Ala?Ala?His?Ile?Trp?Gly?Asn?Lys?Pro
225?????????????????230?????????????????235?????????????????240
tat?gat?aaa?agc?atc?ttg?ccc?gct?caa?aac?ctg?ctg?gtt?tcc?ttc?cta????768
Tyr?Asp?Lys?Ser?Ile?Leu?Pro?Ala?Gln?Asn?Leu?Leu?Val?Ser?Phe?Leu
245?????????????????250?????????????????255
gca?agt?gga?gaa?ggc?ttc?cat?aat?tac?cat?cac?gtc?ttt?cca?tgg?gat????816
Ala?Ser?Gly?Glu?Gly?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp
260?????????????????265?????????????????270
tac?cgc?aca?gca?gaa?tta?ggg?aat?aac?ttc?ctg?aat?ttg?acg?acg?ctg????864
Tyr?Arg?Thr?Ala?Glu?Leu?Gly?Asn?Asn?Phe?Leu?Asn?Leu?Thr?Thr?Leu
275?????????????????280?????????????????285
ttc?att?gat?ttt?tgt?gcc?tgg?ttt?gga?tgg?gct?tat?gac?ttg?aag?tct????912
Phe?Ile?Asp?Phe?Cys?Ala?Trp?Phe?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Ser
290?????????????????295?????????????????300
gta?tca?gag?gat?att?ata?aaa?cag?aga?gct?aaa?cga?aca?ggt?gac?ggt????960
Val?Ser?Glu?Asp?Ile?Ile?Lys?Gln?Arg?Ala?Lys?Arg?Thr?Gly?Asp?Gly
305?????????????????310?????????????????315?????????????????320
tct?tca?ggg?gtc?att?tgg?gga?tgg?gac?gac?aaa?gac?atg?gac?cgc?gat????1008
Ser?Ser?Gly?Val?Ile?Trp?Gly?Trp?Asp?Asp?Lys?Asp?Met?Asp?Arg?Asp
325?????????????????330?????????????????335
ata?aaa?tct?aaa?gct?aac?att?ttt?tat?gct?aaa?aag?gaa?tga????????????1050
Ile?Lys?Ser?Lys?Ala?Asn?Ile?Phe?Tyr?Ala?Lys?Lys?Glu
340?????????????????345
<210>10
<211>349
<212>PRT
＜213〉cabbage looper
<400>10
Met?Ala?Val?Met?Ala?Gln?Thr?Val?Gln?Glu?Thr?Ala?Thr?Val?Leu?Glu
1???????????????5??????????????????10??????????????????15
Glu?Glu?Ala?Arg?Thr?Val?Thr?Leu?Val?Ala?Pro?Lys?Thr?Thr?Pro?Arg
20??????????????????25??????????????????30
Lys?Tyr?Lys?Tyr?Ile?Tyr?Thr?Asn?Phe?Leu?Thr?Phe?Ser?Tyr?Ala?His
35??????????????????40??????????????????45
Leu?Ala?Ala?Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp
50??????????????????55??????????????????60
Glu?Thr?Leu?Leu?Phe?Ser?Phe?Val?Leu?Phe?His?Met?Ser?Asn?Ile?Gly
65??????????????????70??????????????????75??????????????????80
Ile?Thr?Ala?Gly?Ala?His?Arg?Leu?Trp?Thr?His?Lys?Thr?Phe?Lys?Ala
85??????????????????90??????????????????95
Lys?Leu?Pro?Leu?Glu?Ile?Val?Leu?Met?Ile?Phe?Asn?Ser?Leu?Ala?Phe
100?????????????????105?????????????????110
Gln?Asn?Thr?Ala?Ile?Thr?Trp?Ala?Arg?Glu?His?Arg?Leu?His?His?Lys
115?????????????????120?????????????????125
Tyr?Ser?Asp?Thr?Asp?Ala?Asp?Pro?His?Asn?Ala?Ser?Arg?Gly?Phe?Phe
130?????????????????135?????????????????140
Tyr?Ser?His?Val?Gly?Trp?Leu?Leu?Val?Lys?Lys?His?Pro?Asp?Val?Leu
145?????????????????150?????????????????155?????????????????160
Lys?Tyr?Gly?Lys?Thr?Ile?Asp?Met?Ser?Asp?Val?Tyr?Asn?Asn?Pro?Val
165?????????????????170?????????????????175
Leu?Lys?Phe?Gln?Lys?Lys?Tyr?Ala?Val?Pro?Leu?Ile?Gly?Thr?Val?Cys
180?????????????????185?????????????????190
Phe?Ala?Leu?Pro?Thr?Leu?Ile?Pro?Val?Tyr?Cys?Trp?G1y?Glu?Ser?Trp
195?????????????????200?????????????????205
Asn?Asn?Ala?Trp?His?Ile?Ala?Leu?Phe?Arg?Tyr?Ile?Phe?Asn?Leu?Asn
210?????????????????215?????????????????220
Val?Thr?Phe?Leu?Val?Asn?Ser?Ala?Ala?His?Ile?Trp?Gly?Asn?Lys?Pro
225?????????????????230?????????????????235?????????????????240
Tyr?Asp?Lys?Ser?Ile?Leu?Pro?Ala?Gln?Asn?Leu?Leu?Val?Ser?Phe?Leu
245?????????????????250?????????????????255
Ala?Ser?Gly?Glu?Gly?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp
260?????????????????265?????????????????270
Tyr?Arg?Thr?Ala?Glu?Leu?Gly?Asn?Asn?Phe?Leu?Asn?Leu?Thr?Thr?Leu
275?????????????????280?????????????????285
Phe?Ile?Asp?Phe?Cys?Ala?Trp?Phe?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Ser
290?????????????????295?????????????????300
Val?Ser?Glu?Asp?Ile?Ile?Lys?Gln?Arg?Ala?Lys?Arg?Thr?Gly?Asp?Gly
305?????????????????310?????????????????315?????????????????320
Ser?Ser?Gly?Val?Ile?Trp?Gly?Trp?Asp?Asp?Lys?Asp?Met?Asp?Arg?Asp
325?????????????????330?????????????????335
Ile?Lys?Ser?Lys?Ala?Asn?Ile?Phe?Tyr?Ala?Lys?Lys?Glu
340?????????????????345
<210>11
<211>990
<212>DNA
＜213〉argyrotaenia velutinana (Argyrotaenia velutinana)
<220>
<221>CDS
<222>(1)..(987)
＜223〉aliphatic alcohol Δ 11 desaturases
<400>11
atg?gct?cca?aat?gcg?gaa?gat?att?gaa?acg?aat?atg?cca?gaa?act?gaa????48
Met?Ala?Pro?Asn?Ala?Glu?Asp?Ile?Glu?Thr?Asn?Met?Pro?Glu?Thr?Glu
1???????????????5??????????????????10??????????????????15
gag?aac?tgg?gaa?aca?tta?gta?gca?cct?caa?gca?gcg?cct?aga?aaa?tat????96
Glu?Asn?Trp?Glu?Thr?Leu?Val?Ala?Pro?Gln?Ala?Ala?Pro?Arg?Lys?Tyr
20??????????????????25??????????????????30
caa?att?gtg?tat?aaa?agc?ctc?tta?act?ttt?ggc?tac?gga?cac?ctc?gct????144
Gln?Ile?Val?Tyr?Lys?Ser?Leu?Leu?Thr?Phe?Gly?Tyr?Gly?His?Leu?Ala
35??????????????????40??????????????????45
ggt?cta?tat?ggt?tta?tat?ttg?tgc?ttt?act?tcc?gct?aaa?tgg?caa?act????192
Gly?Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp?Gln?Thr
50??????????????????55??????????????????60
att?gga?ctt?gct?atc?atc?ctc?cac?gcg?atg?gca?atc?ttg?ggc?atc?aca????240
Ile?Gly?Leu?Ala?Ile?Ile?Leu?His?Ala?Met?Ala?Ile?Leu?Gly?Ile?Thr
65??????????????????70??????????????????75??????????????????80
gca?ggc?gct?cac?cga?ctc?tgg?aca?cac?aga?gca?tac?aaa?gcg?acg?gtg????288
Ala?Gly?Ala?His?Arg?Leu?Trp?Thr?His?Arg?Ala?Tyr?Lys?Ala?Thr?Val
85??????????????????90??????????????????95
ccc?ctc?caa?atc?atc?ctc?ata?atc?ttc?aac?tcc?ctg?tcg?ttc?caa?aac????336
Pro?Leu?Gln?Ile?Ile?Leu?Ile?Ile?Phe?Asn?Ser?Leu?Ser?Phe?Gln?Asn
100?????????????????105?????????????????110
agc?gcc?ttt?act?tgg?atc?aga?gac?cac?aga?ctc?cac?cac?aag?tat?agt????384
Ser?Ala?Phe?Thr?Trp?Ile?Arg?Asp?His?Arg?Leu?His?His?Lys?Tyr?Ser
115?????????????????120?????????????????125
gac?aca?gac?ggg?gat?ccc?cac?aat?gca?acc?aga?ggg?ttc?ttt?tac?tct????432
Asp?Thr?Asp?Gly?Asp?Pro?His?Asn?Ala?Thr?Arg?Gly?Phe?Phe?Tyr?Ser
130?????????????????135?????????????????140
cat?atc?gga?tgg?ctg?ttg?gtg?agg?aaa?cac?cct?gaa?gtc?atg?aag?agg????480
His?Ile?Gly?Trp?Leu?Leu?Val?Arg?Lys?His?Pro?Glu?Val?Met?Lys?Arg
145?????????????????150?????????????????155?????????????????160
gga?aga?atg?acc?gag?atg?tcg?gat?att?tac?agc?aat?cct?atc?ata?atg????528
Gly?Arg?Met?Thr?Glu?Met?Ser?Asp?Ile?Tyr?Ser?Asn?Pro?Ile?Ile?Met
165?????????????????170?????????????????175
ttt?caa?aaa?aac?tac?gct?ata?cct?ttc?ata?ggc?acg?gtg?tgt?ttc?gta????576
Phe?Gln?Lys?Asn?Tyr?Ala?Ile?Pro?Phe?Ile?Gly?Thr?Val?Cys?Phe?Val
180?????????????????185?????????????????190
ctt?ccc?aca?ata?ata?ccc?atg?tac?ttc?tgg?gga?gag?acg?ttg?aac?aac????624
Leu?Pro?Thr?Ile?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Thr?Leu?Asn?Asn
195?????????????????200?????????????????205
gct?tgg?cat?ata?acg?gtg?ctg?cgc?tac?att?ttt?agc?ctc?aac?tgc?ata????672
Ala?Trp?His?Ile?Thr?Val?Leu?Arg?Tyr?Ile?Phe?Ser?Leu?Asn?Cys?Ile
210?????????????????215?????????????????220
ttc?ctc?gtg?aac?agc?gca?gcc?cat?tta?tac?ggc?tac?aag?cca?tac?gac????720
Phe?Leu?Val?Asn?Ser?Ala?Ala?His?Leu?Tyr?Gly?Tyr?Lys?Pro?Tyr?Asp
225?????????????????230?????????????????235?????????????????240
aag?aac?att?ttg?cca?gcg?gaa?aac?aaa?gca?gct?tca?atc?gca?tct?ttt????768
Lys?Asn?Ile?Leu?Pro?Ala?Glu?Asn?Lys?Ala?Ala?Ser?Ile?Ala?Ser?Phe
245?????????????????250?????????????????255
gga?gaa?gcc?ttc?cat?aac?tat?cat?cat?gtg?ttt?cct?tgg?gac?tac?aga????816
Gly?Glu?Ala?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg
260?????????????????265?????????????????270
gct?tct?gaa?cta?ggt?aat?ata?aca?atg?aat?tgg?aca?ata?tat?ttc?att????864
Ala?Ser?Glu?Leu?Gly?Asn?Ile?Thr?Met?Asn?Trp?Thr?Ile?Tyr?Phe?Ile
275?????????????????280?????????????????285
gat?ttc?ttc?gct?tgg?atc?ggc?tgg?gct?tac?gac?ttg?aaa?act?gca?tcg????912
Asp?Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser
290?????????????????295?????????????????300
gat?gag?act?att?aaa?agc?aga?ata?aaa?aga?act?ggc?gat?ggt?act?gac????960
Asp?Glu?Thr?Ile?Lys?Ser?Arg?Ile?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Asp
305?????????????????310?????????????????315?????????????????320
ttc?tcg?ggc?cag?caa?ata?tac?gca?aga?tga????????????????????????????990
Phe?Ser?Gly?Gln?Gln?Ile?Tyr?Ala?Arg
325
<210>12
<211>329
<212>PRT
＜213〉argyrotaenia velutinana
<400>12
Met?Ala?Pro?Asn?Ala?Glu?Asp?Ile?Glu?Thr?Asn?Met?Pro?Glu?Thr?Glu
1???????????????5??????????????????10??????????????????15
Glu?Asn?Trp?Glu?Thr?Leu?Val?Ala?Pro?Gln?Ala?Ala?Pro?Arg?Lys?Tyr
20??????????????????25??????????????????30
Gln?Ile?Val?Tyr?Lys?Ser?Leu?Leu?Thr?Phe?Gly?Tyr?Gly?His?Leu?Ala
35??????????????????40??????????????????45
Gly?Leu?Tyr?Gly?Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp?Gln?Thr
50??????????????????55??????????????????60
Ile?Gly?Leu?Ala?Ile?Ile?Leu?His?Ala?Met?Ala?Ile?Leu?Gly?Ile?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Gly?Ala?His?Arg?Leu?Trp?Thr?His?Arg?Ala?Tyr?Lys?Ala?Thr?Val
85??????????????????90??????????????????95
Pro?Leu?Gln?Ile?Ile?Leu?Ile?Ile?Phe?Asn?Ser?Leu?Ser?Phe?Gln?Asn
100?????????????????105?????????????????110
Ser?Ala?Phe?Thr?Trp?Ile?Arg?Asp?His?Arg?Leu?His?His?Lys?Tyr?Ser
115?????????????????120?????????????????125
Asp?Thr?Asp?Gly?Asp?Pro?His?Asn?Ala?Thr?Arg?Gly?Phe?Phe?Tyr?Ser
130?????????????????135?????????????????140
His?Ile?Gly?Trp?Leu?Leu?Val?Arg?Lys?His?Pro?Glu?Val?Met?Lys?Arg
145?????????????????150?????????????????155?????????????????160
Gly?Arg?Met?Thr?Glu?Met?Ser?Asp?Ile?Tyr?Ser?Asn?Pro?Ile?Ile?Met
165?????????????????170?????????????????175
Phe?Gln?Lys?Asn?Tyr?Ala?Ile?Pro?Phe?Ile?Gly?Thr?Val?Cys?Phe?Val
180?????????????????185?????????????????190
Leu?Pro?Thr?Ile?Ile?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Thr?Leu?Asn?Asn
195?????????????????200?????????????????205
Ala?Trp?His?Ile?Thr?Val?Leu?Arg?Tyr?Ile?Phe?Ser?Leu?Asn?Cys?Ile
210?????????????????215?????????????????220
Phe?Leu?Val?Asn?Ser?Ala?Ala?His?Leu?Tyr?Gly?Tyr?Lys?Pro?Tyr?Asp
225?????????????????230?????????????????235?????????????????240
Lys?Asn?Ile?Leu?Pro?Ala?Glu?Asn?Lys?Ala?Ala?Ser?Ile?Ala?Ser?Phe
245?????????????????250?????????????????255
Gly?Glu?Ala?Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg
260?????????????????265?????????????????270
Ala?Ser?Glu?Leu?Gly?Asn?Ile?Thr?Met?Asn?Trp?Thr?Ile?Tyr?Phe?Ile
275?????????????????280?????????????????285
Asp?Phe?Phe?Ala?Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser
290?????????????????295?????????????????300
Asp?Glu?Thr?Ile?Lys?Ser?Arg?Ile?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Asp
305?????????????????310?????????????????315?????????????????320
Phe?Ser?Gly?Gln?Gln?Ile?Tyr?Ala?Arg
325
<210>13
<211>1071
<212>DNA
＜213〉green head volume moth (Planotortrix octo)
<220>
<221>CDS
<222>(1)..(1068)
＜223〉aliphatic alcohol Z10 desaturase
<400>13
atg?cca?cca?aat?tca?gag?gaa?aca?gtg?cta?tgt?gaa?aag?gaa?gac?cac????48
Met?Pro?Pro?Asn?Ser?Glu?Glu?Thr?Val?Leu?Cys?Glu?Lys?Glu?Asp?His
1???????????????5??????????????????10??????????????????15
gag?aag?ctg?gtg?gcg?cca?caa?gcg?gct?acc?agg?aaa?cat?gag?ctg?gca????96
Glu?Lys?Leu?Val?Ala?Pro?Gln?Ala?Ala?Thr?Arg?Lys?His?Glu?Leu?Ala
20??????????????????25??????????????????30
ata?gtg?ccc?atc?tca?ctc?ttc?act?tac?tgg?cac?gtc?gct?ggc?ttg?tac????144
Ile?Val?Pro?Ile?Ser?Leu?Phe?Thr?Tyr?Trp?His?Val?Ala?Gly?Leu?Tyr
35??????????????????40??????????????????45
ggg?ctg?tat?ctc?atc?ttt?gct?gaa?gcg?aaa?tgg?cag?acc?gta?gtg?ttc????192
Gly?Leu?Tyr?Leu?Ile?Phe?Ala?Glu?Ala?Lys?Trp?Gln?Thr?Val?Val?Phe
50??????????????????55??????????????????60
act?ctc?ttc?acc?tac?aac?gcc?ggc?att?ctg?ggc?atc?act?gca?ggg?tcc????240
Thr?Leu?Phe?Thr?Tyr?Asn?Ala?Gly?Ile?Leu?Gly?Ile?Thr?Ala?Gly?Ser
65??????????????????70??????????????????75??????????????????80
cac?cgc?ctc?tgg?gcc?cac?aag?aca?tac?aag?gcc?aag?aga?ccc?cta?gaa????288
His?Arg?Leu?Trp?Ala?His?Lys?Thr?Tyr?Lys?Ala?Lys?Arg?Pro?Leu?Glu
85??????????????????90??????????????????95
acc?ctg?ctc?atg?gta?ttc?cat?agt?ctg?acg?agc?cag?aac?acc?gtg?cgg????336
Thr?Leu?Leu?Met?Val?Phe?His?Ser?Leu?Thr?Ser?Gln?Asn?Thr?Val?Arg
100?????????????????105?????????????????110
cac?tgg?gca?agg?gac?cat?cgg?ttc?cat?cac?aag?tac?agc?gac?aca?gac????384
His?Trp?Ala?Arg?Asp?His?Arg?Phe?His?His?Lys?Tyr?Ser?Asp?Thr?Asp
115?????????????????120?????????????????125
gcc?gac?ccg?cac?aat?gcg?act?cga?ggt?ttc?ttc?tac?tcc?cac?gta?ggc????432
Ala?Asp?Pro?His?Asn?Ala?Thr?Arg?Gly?Phe?Phe?Tyr?Ser?His?Val?Gly
130?????????????????135?????????????????140
tgg?ctg?ctg?gtc?aag?aaa?cac?ccc?gag?gtc?ctc?aga?cgg?tcg?aag?acc????480
Trp?Leu?Leu?Val?Lys?Lys?His?Pro?Glu?Val?Leu?Arg?Arg?Ser?Lys?Thr
145?????????????????150?????????????????155?????????????????160
atc?gac?atg?tcc?gac?att?tac?aac?aat?cca?gtg?ttg?cgg?ttc?cag?aaa????528
Ile?Asp?Met?Ser?Asp?Ile?Tyr?Asn?Asn?Pro?Val?Leu?Arg?Phe?Gln?Lys
165?????????????????170?????????????????175
aac?tac?ggc?ctc?cca?gtg?ata?aca?tta?ttc?gcc?tac?gtc?ctc?cca?gct????576
Asn?Tyr?Gly?Leu?Pro?Val?Ile?Thr?Leu?Phe?Ala?Tyr?Val?Leu?Pro?Ala
180?????????????????185?????????????????190
ctc?ata?cca?atg?tac?tgc?tgg?gaa?gaa?acc?ctg?aac?aac?gcc?tgg?cat????624
Leu?Ile?Pro?Met?Tyr?Cys?Trp?Glu?Glu?Thr?Leu?Asn?Asn?Ala?Trp?His
195?????????????????200?????????????????205
ata?aac?cta?ctg?cga?atc?ata?gcc?aac?ctc?cac?gct?tcc?tgt?ctt?gtc????672
Ile?Asn?Leu?Leu?Arg?Ile?Ile?Ala?Asn?Leu?His?Ala?Ser?Cys?Leu?Val
210?????????????????215?????????????????220
aac?agc?gca?gca?cac?gcc?ttc?ggt?aac?aaa?ccg?tac?gac?aag?cac?ata????720
Asn?Ser?Ala?Ala?His?Ala?Phe?Gly?Asn?Lys?Pro?Tyr?Asp?Lys?His?Ile
225?????????????????230?????????????????235?????????????????240
gca?gcc?acg?caa?atc?tcc?acc?cta?tcc?ttc?ata?act?tta?ggg?gag?tgt????768
Ala?Ala?Thr?Gln?Ile?Ser?Thr?Leu?Ser?Phe?Ile?Thr?Leu?Gly?Glu?Cys
245?????????????????250?????????????????255
ttc?cat?aac?tac?cac?cac?gtc?ttc?ccc?tgg?gat?tat?agg?acg?gcg?gag????816
Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Thr?Ala?Glu
260?????????????????265?????????????????270
ctg?ggg?aat?aat?tgg?ttg?aac?atg?acg?acg?ctg?ttc?att?gat?ttt?ttc????864
Leu?Gly?Asn?Asn?Trp?Leu?Asn?Met?Thr?Thr?Leu?Phe?Ile?Asp?Phe?Phe
275?????????????????280?????????????????285
gcg?tgg?gtc?ggc?tgg?gcg?tat?gat?ttg?aag?act?gct?tct?gat?ggg?atg????912
Ala?Trp?Val?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser?Asp?Gly?Met
290?????????????????295?????????????????300
gtc?gaa?gct?agg?gct?aaa?agg?acg?ggg?gat?ggc?acg?aat?ctg?tgg?ggg????960
Val?Glu?Ala?Arg?Ala?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Asn?Leu?Trp?Gly
305?????????????????310?????????????????315?????????????????320
tgg?ggg?gat?gag?gat?ctg?ggg?agg?gag?gag?ggg?ggt?gag?gaa?gtg?ttt????1008
Trp?Gly?Asp?Glu?Asp?Leu?Gly?Arg?Glu?Glu?Gly?Gly?Glu?Glu?Val?Phe
325?????????????????330?????????????????335
tac?ggg?tgg?gga?gat?aga?gat?atg?aag?gat?acc?agt?ggg?gtt?aga?gtt????1056
Tyr?Gly?Trp?Gly?Asp?Arg?Asp?Met?Lys?Asp?Thr?Ser?Gly?Val?Arg?Val
340?????????????????345?????????????????350
tat?tca?caa?gag?taa????????????????????????????????????????????????1071
Tyr?Ser?Gln?Glu
355
<210>14
<211>356
<212>PRT
＜213〉green head volume moth
<400>14
Met?Pro?Pro?Asn?Ser?Glu?Glu?Thr?Val?Leu?Cys?Glu?Lys?Glu?Asp?His
1???????????????5??????????????????10??????????????????15
Glu?Lys?Leu?Val?Ala?Pro?Gln?Ala?Ala?Thr?Arg?Lys?His?Glu?Leu?Ala
20??????????????????25??????????????????30
Ile?Val?Pro?Ile?Ser?Leu?Phe?Thr?Tyr?Trp?His?Val?Ala?Gly?Leu?Tyr
35??????????????????40??????????????????45
Gly?Leu?Tyr?Leu?Ile?Phe?Ala?Glu?Ala?Lys?Trp?Gln?Thr?Val?Val?Phe
50??????????????????55??????????????????60
Thr?Leu?Phe?Thr?Tyr?Asn?Ala?Gly?Ile?Leu?Gly?Ile?Thr?Ala?Gly?Ser
65??????????????????70??????????????????75??????????????????80
His?Arg?Leu?Trp?Ala?His?Lys?Thr?Tyr?Lys?Ala?Lys?Arg?Pro?Leu?Glu
85??????????????????90??????????????????95
Thr?Leu?Leu?Met?Val?Phe?His?Ser?Leu?Thr?Ser?Gln?Asn?Thr?Val?Arg
100?????????????????105?????????????????110
His?Trp?Ala?Arg?Asp?His?Arg?Phe?His?His?Lys?Tyr?Ser?Asp?Thr?Asp
115?????????????????120?????????????????125
Ala?Asp?Pro?His?Asn?Ala?Thr?Arg?Gly?Phe?Phe?Tyr?Ser?His?Val?Gly
130?????????????????135?????????????????140
Trp?Leu?Leu?Val?Lys?Lys?His?Pro?Glu?Val?Leu?Arg?Arg?Ser?Lys?Thr
145?????????????????150?????????????????155?????????????????160
Ile?Asp?Met?Ser?Asp?Ile?Tyr?Asn?Asn?Pro?Val?Leu?Arg?Phe?Gln?Lys
165?????????????????170?????????????????175
Asn?Tyr?Gly?Leu?Pro?Val?Ile?Thr?Leu?Phe?Ala?Tyr?Val?Leu?Pro?Ala
180?????????????????185?????????????????190
Leu?Ile?Pro?Met?Tyr?Cys?Trp?Glu?Glu?Thr?Leu?Asn?Asn?Ala?Trp?His
195?????????????????200?????????????????205
Ile?Asn?Leu?Leu?Arg?Ile?Ile?Ala?Asn?Leu?His?Ala?Ser?Cys?Leu?Val
210?????????????????215?????????????????220
Asn?Ser?Ala?Ala?His?Ala?Phe?Gly?Asn?Lys?Pro?Tyr?Asp?Lys?His?Ile
225?????????????????230?????????????????235?????????????????240
Ala?Ala?Thr?Gln?Ile?Ser?Thr?Leu?Ser?Phe?Ile?Thr?Leu?Gly?Glu?Cys
245?????????????????250?????????????????255
Phe?His?Asn?Tyr?His?His?Val?Phe?Pro?Trp?Asp?Tyr?Arg?Thr?Ala?Glu
260?????????????????265?????????????????270
Leu?Gly?Asn?Asn?Trp?Leu?Asn?Met?Thr?Thr?Leu?Phe?Ile?Asp?Phe?Phe
275?????????????????280?????????????????285
Ala?Trp?Val?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Ala?Ser?Asp?Gly?Met
290?????????????????295?????????????????300
Val?Glu?Ala?Arg?Ala?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Asn?Leu?Trp?Gly
305?????????????????310?????????????????315?????????????????320
Trp?Gly?Asp?Glu?Asp?Leu?Gly?Arg?Glu?Glu?Gly?Gly?Glu?Glu?Val?Phe
325?????????????????330?????????????????335
Tyr?Gly?Trp?Gly?Asp?Arg?Asp?Met?Lys?Asp?Thr?Ser?Gly?Val?Arg?Val
340?????????????????345?????????????????350
Tyr?Ser?Gln?Glu
355
<210>15
<211>20
<212>DNA
＜213〉composition sequence
<220>
＜223〉description of composition sequence: Oligonucleolide primers
<400>15
atyachgccg?gkkmycaymg??????????????????????????????????????????????20
<210>16
<211>20
<212>DNA
＜213〉composition sequence
<220>
＜223〉description of composition sequence: Oligonucleolide primers
<400>16
ggraabdygt?grtggwagtt??????????????????????????????????????????????20
<210>17
<211>20
<212>DNA
＜213〉composition sequence
<220>
＜223〉description of composition sequence: Oligonucleolide primers
<400>17
ccccaycrnc?tstggwcnca??????????????????????????????????????????????20
<210>18
<211>25
<212>DNA
＜213〉composition sequence
<220>
＜223〉description of composition sequence: Oligonucleolide primers
<400>18
ccctctagar?tgrrwarttr?tgrwa???????????????????????????????????????25
<210>19
<211>18
<212>DNA
＜213〉composition sequence
<220>
＜223〉description of composition sequence: Oligonucleolide primers
<400>19
taatacgact?cactatag??????????????????????????????????????????????18
<210>20
<211>19
<212>DNA
＜213〉composition sequence
<220>
＜223〉description of composition sequence: Oligonucleolide primers
<400>20
acataactaa?ttacatgat????????????????????????????????????????????19
<210>21
<211>996
<212>DNA
＜213〉cotton pink bollworm (Pectinophora gossypiella)
<220>
<221>CDS
<222>(1)..(993)
＜223〉coded delta-11-desaturase
<400>21
atg?gcc?cct?tac?tta?aag?gag?ggt?aac?gca?att?ttg?gaa?aaa?tac?gaa????48
Met?Ala?Pro?Tyr?Leu?Lys?Glu?Gly?Asn?Ala?Ile?Leu?Glu?Lys?Tyr?Glu
1???????????????5??????????????????10??????????????????15
acc?cta?aaa?gcg?cct?cag?gcg?gga?cca?aga?aaa?tat?caa?ata?att?tat????96
Thr?Leu?Lys?Ala?Pro?Gln?Ala?Gly?Pro?Arg?Lys?Tyr?Gln?Ile?Ile?Tyr
20??????????????????25??????????????????30
ccg?aat?tta?ata?acg?ttt?gga?tat?gga?cac?atc?gcg?gca?ttg?tat?gga????144
Pro?Asn?Leu?Ile?Thr?Phe?Gly?Tyr?Gly?His?Ile?Ala?Ala?Leu?Tyr?Gly
35??????????????????40??????????????????45
cta?tac?ctg?tgc?ttc?acc?tcg?gct?aaa?tgg?gct?act?att?ctt?ctt?gga????192
Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp?Ala?Thr?Ile?Leu?Leu?Gly
50??????????????????55??????????????????60
tac?atg?ttg?ttc?atc?gta?agt?gaa?ctc?ggc?atc?aca?gcg?ggc?gct?cac????240
Tyr?Met?Leu?Phe?Ile?Val?Ser?Glu?Leu?Gly?Ile?Thr?Ala?Gly?Ala?His
65??????????????????70??????????????????75??????????????????80
aga?ctg?tgg?gca?cac?aag?act?tac?aaa?gcc?aag?ctg?cct?ctg?gaa?att????288
Arg?Leu?Trp?Ala?His?Lys?Thr?Tyr?Lys?Ala?Lys?Leu?Pro?Leu?Glu?Ile
85??????????????????90??????????????????95
ctc?cta?atg?gtg?ttc?aac?tca?att?gcc?ttt?caa?aac?act?gcc?gtc?acc????336
Leu?Leu?Met?Val?Phe?Asn?Ser?Ile?Ala?Phe?Gln?Asn?Thr?Ala?Val?Thr
100?????????????????105?????????????????110
tgg?gtg?aag?gac?cac?cgc?gct?cac?cac?aag?tac?agc?gac?acc?gat?gca????384
Trp?Val?Lys?Asp?His?Arg?Ala?His?His?Lys?Tyr?Ser?Asp?Thr?Asp?Ala
115?????????????????120?????????????????125
gac?cct?cac?aac?gcc?acg?aga?gga?ctc?ttc?tac?tca?cac?atc?ggg?tgg????432
Asp?Pro?His?Asn?Ala?Thr?Arg?Gly?Leu?Phe?Tyr?Ser?His?Ile?Gly?Trp
130?????????????????135?????????????????140
cta?cta?gtc?aag?aaa?cac?ccc?gaa?gtc?att?aaa?cgc?gga?aaa?caa?atc????480
Leu?Leu?Val?Lys?Lys?His?Pro?Glu?Val?Ile?Lys?Arg?Gly?Lys?Gln?Ile
145?????????????????150?????????????????155?????????????????160
gat?tac?agc?gat?ata?tgt?aac?aat?cct?gta?ctg?agg?ttc?cag?aaa?aag????528
Asp?Tyr?Ser?Asp?Ile?Cys?Asn?Asn?Pro?Val?Leu?Arg?Phe?Gln?Lys?Lys
165?????????????????170?????????????????175
tat?gcg?gtc?ccg?ttc?atc?ggg?acc?atg?tgc?ttc?gtg?ctt?ccg?act?gtg????576
Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Met?Cys?Phe?Val?Leu?Pro?Thr?Val
180?????????????????185?????????????????190
gta?cca?atg?tac?ttt?tgg?ggg?gag?agc?ctg?agg?aac?gcg?tgg?cat?gtc????624
Val?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Ser?Leu?Arg?Asn?Ala?Trp?His?Val
195?????????????????200?????????????????205
aac?ctg?ctt?cgg?tac?gcg?ctc?agc?ctc?cat?gcc?acg?ttc?cta?gtt?aac????672
Asn?Leu?Leu?Arg?Tyr?Ala?Leu?Ser?Leu?His?Ala?Thr?Phe?Leu?Val?Asn
210?????????????????215?????????????????220
agc?gcc?gcg?cac?tac?tgg?ggc?acc?aaa?cct?tac?gat?aag?aac?cta?gtt????720
Ser?Ala?Ala?His?Tyr?Trp?Gly?Thr?Lys?Pro?Tyr?Asp?Lys?Asn?Leu?Val
225?????????????????230?????????????????235?????????????????240
gcg?tca?caa?aac?gtg?tcc?gtc?tct?ctg?ttg?acg?agc?ggc?gaa?gga?tac????768
Ala?Ser?Gln?Asn?Val?Ser?Val?Ser?Leu?Leu?Thr?Ser?Gly?Glu?Gly?Tyr
245?????????????????250?????????????????255
cac?aac?tac?cac?cat?gcg?ttc?cca?tgg?gac?tac?cgc?gcc?gcc?gaa?tta????816
His?Asn?Tyr?His?His?Ala?Phe?Pro?Trp?Asp?Tyr?Arg?Ala?Ala?Glu?Leu
260?????????????????265?????????????????270
ggc?aac?aac?ttc?gtc?aac?ttg?acg?acg?aag?ttt?ata?gat?ttc?ttc?gcc????864
Gly?Asn?Asn?Phe?Val?Asn?Leu?Thr?Thr?Lys?Phe?Ile?Asp?Phe?Phe?Ala
275?????????????????280?????????????????285
tgg?att?ggc?tgg?gct?tac?gac?ctt?aag?acg?gtc?cct?aag?gac?ttg?gtt????912
Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Val?Pro?Lys?Asp?Leu?Val
290?????????????????295?????????????????300
ata?agc?cgc?atg?aaa?aga?acc?ggc?gac?ggt?acc?tcc?cta?tgg?ggt?tgg????960
Ile?Ser?Arg?Met?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Ser?Leu?Trp?Gly?Trp
305?????????????????310?????????????????315?????????????????320
ggt?gag?aaa?tat?gac?gga?aaa?cga?atg?aaa?cgc?taa????????????????????996
Gly?Glu?Lys?Tyr?Asp?Gly?Lys?Arg?Met?Lys?Arg
325?????????????????330
<210>22
<211>331
<212>PRT
＜213〉cotton pink bollworm
<400>22
Met?Ala?Pro?Tyr?Leu?Lys?Glu?Gly?Asn?Ala?Ile?Leu?Glu?Lys?Tyr?Glu
1???????????????5??????????????????10??????????????????15
Thr?Leu?Lys?Ala?Pro?Gln?Ala?Gly?Pro?Arg?Lys?Tyr?Gln?Ile?Ile?Tyr
20??????????????????25??????????????????30
Pro?Asn?Leu?Ile?Thr?Phe?Gly?Tyr?Gly?His?Ile?Ala?Ala?Leu?Tyr?Gly
35??????????????????40??????????????????45
Leu?Tyr?Leu?Cys?Phe?Thr?Ser?Ala?Lys?Trp?Ala?Thr?Ile?Leu?Leu?Gly
50??????????????????55??????????????????60
Tyr?Met?Leu?Phe?Ile?Val?Ser?Glu?Leu?Gly?Ile?Thr?Ala?Gly?Ala?His
65??????????????????70??????????????????75??????????????????80
Arg?Leu?Trp?Ala?His?Lys?Thr?Tyr?Lys?Ala?Lys?Leu?Pro?Leu?Glu?Ile
85??????????????????90??????????????????95
Leu?Leu?Met?Val?Phe?Asn?Ser?Ile?Ala?Phe?Gln?Asn?Thr?Ala?Val?Thr
100?????????????????105?????????????????110
Trp?Val?Lys?Asp?His?Arg?Ala?His?His?Lys?Tyr?Ser?Asp?Thr?Asp?Ala
115?????????????????120?????????????????125
Asp?Pro?His?Asn?Ala?Thr?Arg?Gly?Leu?Phe?Tyr?Ser?His?Ile?Gly?Trp
130?????????????????135?????????????????140
Leu?Leu?Val?Lys?Lys?His?Pro?Glu?Val?Ile?Lys?Arg?Gly?Lys?Gln?Ile
145?????????????????150?????????????????155?????????????????160
Asp?Tyr?Ser?Asp?Ile?Cys?Asn?Asn?Pro?Val?Leu?Arg?Phe?Gln?Lys?Lys
165?????????????????170?????????????????175
Tyr?Ala?Val?Pro?Phe?Ile?Gly?Thr?Met?Cys?Phe?Val?Leu?Pro?Thr?Val
180?????????????????185?????????????????190
Val?Pro?Met?Tyr?Phe?Trp?Gly?Glu?Ser?Leu?Arg?Asn?Ala?Trp?His?Val
195?????????????????200?????????????????205
Asn?Leu?Leu?Arg?Tyr?Ala?Leu?Ser?Leu?His?Ala?Thr?Phe?Leu?Val?Asn
210?????????????????215?????????????????220
Ser?Ala?Ala?His?Tyr?Trp?Gly?Thr?Lys?Pro?Tyr?Asp?Lys?Asn?Leu?Val
225?????????????????230?????????????????235?????????????????240
Ala?Ser?Gln?Asn?Val?Ser?Val?Ser?Leu?Leu?Thr?Ser?Gly?Glu?Gly?Tyr
245?????????????????250?????????????????255
His?Asn?Tyr?His?His?Ala?Phe?Pro?Trp?Asp?Tyr?Arg?Ala?Ala?Glu?Leu
260?????????????????265?????????????????270
Gly?Asn?Asn?Phe?Val?Asn?Leu?Thr?Thr?Lys?Phe?Ile?Asp?Phe?Phe?Ala
275?????????????????280?????????????????285
Trp?Ile?Gly?Trp?Ala?Tyr?Asp?Leu?Lys?Thr?Val?Pro?Lys?Asp?Leu?Val
290?????????????????295?????????????????300
Ile?Ser?Arg?Met?Lys?Arg?Thr?Gly?Asp?Gly?Thr?Ser?Leu?Trp?Gly?Trp
305?????????????????310?????????????????315?????????????????320
Gly?Glu?Lys?Tyr?Asp?Gly?Lys?Arg?Met?Lys?Arg
325?????????????????330

Claims (20)

1. be used to produce the method for the triglyceride level that comprises unsaturated fatty acids, it is included in the organism that is selected from plant, yeast, fungi and algae, recombinant expressed at least a fatty acid desaturase from lepidopterous insects.
2. the method for claim 1, wherein fatty acid desaturase can produce two keys in C8, C9, C10, C11 or the C12 position of lipid acid, fatty acid coa A ester or other derivative of fatty acid.
3. method as claimed in claim 1 or 2, wherein fatty acid desaturase produces cis or trans double bond specifically in lipid acid, fatty acid coa A ester or other derivative of fatty acid of the fatty acid chain length with 16 or 18 carbon atoms.
4. as any described method in the claim 1 to 3, wherein fatty acid desaturase and SEQ ID NO:2, one of 4,6,8,10,12,14 or 22 described fatty acid desaturases have at least 65% homology.
5. as any described method, the wherein nucleotide sequence of fatty acid desaturase in the claim 1 to 4
A) in its sense strand, comprise SEQ ID NO:15 or 17 described sequence motifs, or
B) in its antisense strand, comprise SEQ ID NO:16 or 18 described sequence motifs.
6. as any described method in the claim 1 to 5, wherein unsaturated fatty acids comprises conjugated linolic acid.
7. as any described method in the claim 1 to 6, wherein organism is selected from: volume branch Mucor (Mucor circinelloides), Mortierella alpina (Mortierella alpina), fantastic corruption mould (Pythium insidiosum), crooked Cryptococcus (Cryptococcus curvatus), Semen Lini, soybean, Semen Brassicae campestris, coconut, oil palm, safflower, Viscotrol C plant, peanut, cocoa tree and Sunflower Receptacle.
8. the triglyceride level by producing as method as described in the claim 1 to 7 any.
9. the purposes of triglyceride level as claimed in claim 8 is used to prepare food, feed, makeup or fine chemicals.
10. have the active polypeptide of E11-desaturase, described polypeptide preferentially transforms C16 and/or C18 aliphatic alcohol lipid acid and comprises at least one and is selected from following sequence:
A) aminoacid sequence shown in SEQ ID NO:2,4,6 and 22 and
B) with SEQ ID NO:2,4,6 or 22 shown in wherein a kind of aminoacid sequence have at least 65% homology aminoacid sequence and
C) comprise the aminoacid sequence of at least 20 continuous amino acid residue segments in the sequence shown in SEQ ID NO:2,4,6 or 22.
11. polypeptide as claimed in claim 10, it is described with the aminoacid sequence shown in the SEQ ID NO:22.
12. coding is as the nucleotide sequence of polypeptide as described in the claim 11.
13. recombinant expression cassettes, it is included at least one coding under the promoter regulation that works in plant, plant biological body, algae, yeast or the fungi from the nucleotide sequence of the fatty acid desaturase of lepidopterous insects.
14. recombinant expression cassettes as claimed in claim 13, wherein promotor works at least a organism that is selected from volume branch Mucor, Mortierella alpina, mould, the crooked Cryptococcus of fantastic corruption, soybean, Semen Lini, Semen Brassicae campestris, coconut, oil palm, safflower, Viscotrol C plant, peanut, cocoa beans and Sunflower Receptacle.
15. as claim 13 or 14 described recombinant expression cassettes, wherein fatty acid desaturase is selected from following polypeptide of sequence description to comprise at least one:
A) aminoacid sequence as shown in SEQ ID NO:2,4,6,8,10,12,14 or 22 and
B) with one of the aminoacid sequence shown in the SEQ ID NO:2,4,6,8,10,12,14 or 22 have at least 65% homology aminoacid sequence and
C) comprise the aminoacid sequence of at least 20 continuous amino acid residue segments of sequence as shown in SEQ ID NO:2,4,6,8,10,12,14 or 22.
16. as any described recombinant expression cassettes in the claim 13 to 15, wherein fatty acid desaturase is described with the aminoacid sequence shown in the SEQ ID NO:22.
17. recombinant expression vector, it comprises at least one as any described expression cassette in the claim 13 to 16.
18. transgenic organism, it comprises at least one as any described recombinant expression cassettes or the recombinant expression vector described in claim 17 in the claim 13 to 16.
19. transgenic organism as claimed in claim 18, it is the plant biological body.
20. as claim 18 or 19 described transgenic organisms, it is selected from: volume branch Mucor, Mortierella alpina, mould, the crooked Cryptococcus of fantastic corruption, soybean, Semen Lini, Semen Brassicae campestris, coconut, oil palm, safflower, Viscotrol C plant, peanut, cocoa tree and Sunflower Receptacle.
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