CN1415754A - Method for producing soybean containg content of high gamma-linolenic acid - Google Patents
Method for producing soybean containg content of high gamma-linolenic acid Download PDFInfo
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Abstract
A process for culturing the soybean with high content of gamma-linolenic acid inclades such steps as separating the nueic acid sequence separated from Mortierella isabellina M6-22 and containing delta 6-fatty acid dehydrogenase gene, configuring expressino carrier, infecting the cotyledonary node and juventile bud of soybean to induce rosette bud, continuous screening with Kan culture medium, cutting off base, immersing in rooting powder, and culturing in rooting culture medium containing no kanamycin for high rooting rate (80%).
Description
Affiliated technical field
The present invention relates to change a kind of host living beings makes it produce Δ
6-fatty acid dehydrogenase and the method that under the effect of this enzyme, produces gamma-linolenic acid.The invention still further relates to and contain the allos adjusting and carry out functional bonded Δ in proper order
6The recombinant expression vector of-fatty acid dehydrogenase coding region.Promotor in the recombinant expression vector is CaMV35S constitutive promoter or soybean β-companion's sphaeroprotein seed promoters.In agriculture bacillus mediated genetic transformation, the bud of growing thickly that induces from cotyledonary node and plumular axis, step sizing in 50mg/L Kan screening culture medium, the survival rate of regeneration bud is 15%, regrowth is changed over to before the root media, earlier its base portion is cut and adopt root-inducing powder to soak into, put into the root media that does not contain kantlex again and cultivate, this method can improve rooting rate and reach 80%.Adopt the inventive method transgenosis in the soybean of defective gamma-linolenic acid, to produce gamma-linolenic acid.
Background technology
The indispensable fatty acid that human body obtains from food is linolic acid (18: 2 Δs
9,12), linolic acid at first is converted into gamma-linolenic acid (18: 3 Δs through ω-6 route
6,9,12), synthetic then arachidonic acid and prostaglandin(PG).Δ
6-fatty acid dehydrogenase is the key enzyme of the synthetic gamma-linolenic acid of linolic acid.Mammiferous cell lacks the desaturase of introducing unsaturated double-bond in the 9th position more than the carbon atom of lipid acid, and self can not synthesize linolic acid, obtains from plant usually.Δ
6The activity of-fatty acid dehydrogenase can be blocked by factors such as the diet of higher fatty acid and Hi CHO, aging, diabetes, hypercholesterolemia blood fat disease, obesity, insobrieties, cause human body gamma-linolenic acid, arachidonic acid synthetic deficiency, thereby suppress the synthetic of human body prostaglandin(PG), cause the remarkable disorder of human endocrine function.Though a large amount of linolic acid is arranged in people's the food, still has a lot of people can not keep the gamma-linolenic acid in the blood and the normal amount of meta-bolites thereof.Document announcement has about 20% to lack gamma-linolenic acid in north American population approximately, yet just can make arachidonic acid content in blood plasma phosphatide increase as long as take in the gamma-linolenic acid of 500mg every day.After adding gamma-linolenic acid, can obviously improve the wet disease patient's of supersensitivity skin, and remove itch, reduce effect the cholesterol drugs requirement; Can obviously alleviate the malaise symptoms that premenstrual syndrome and the preceding pectoralgia patient of menstruation are arranged; To the diabetics, the neurocyte function that can recover to be damaged; To cardiovascular patient, reduce the effect of serum cholesterol and triglyceride, low-density lipoprotein and rising serum high-density LP.It is generally acknowledged that gamma-linolenic acid can increase the drainage of cholesterol, it is synthetic to suppress endogenous cholesterol, changes the composition of lipid acid in the lipoprotein, thereby increases its polarity and flowability.Therefore, directly augment the nutrition trend that gamma-linolenic acid has become high consumption population, and begin in edible tempered oil, to add gamma-linolenic acid, to improve diseases prevention and resistance against diseases in American-European developed country.
From the species of natural generation gamma-linolenic acid, obtain and participate in the biosynthetic genetic material of gamma-linolenic acid, and the genetic material that is separated to is expressed in a kind of allos system that can produce gamma-linolenic acid alone or in combination is very significant.
The method that produces gamma-linolenic acid at present is existing many, and Chinese patent 92113085 discloses and " passed through Δ
6-desaturase is produced gamma-linolenic acid ", this patent has mainly been set forth blue algae bacterium Δ
6-desaturase transgenosis algae produces gamma-linolenic acid, but summary of the invention similar to the application's background technology differs greatly.
Summary of the invention
The purpose of this invention is to provide a kind of Mortierella isabellina Δ
6-fatty acid dehydrogenase genetically engineered soybean produces the method for gamma-linolenic acid.Adopt the inventive method transgenosis in the soybean of defective gamma-linolenic acid, to produce gamma-linolenic acid, be applicable to the scale operation of gamma-linolenic acid and be applicable to the change of the fatty acid profile of soybean.
The present invention relates to from Mortierella isabellina M
6-22Isolating Δ
6-fatty acid dehydrogenase gene is the structure gene of total length, and length of nucleotides is 1374bp, 457 amino acid of encoding.
The present invention relates to comprise Δ
6-fatty acid dehydrogenase gene and external source regulatory region are the non-Δ that comes from
6The unit of-fatty acid dehydrogenase gene carries out functional bonded recombinant expression vector.
The invention provides the method that produces the gamma-linolenic acid soybean, by the isolating Δ of the present invention
6The soya cells that-fatty acid dehydrogenase gene transforms and the plant of this cell regeneration.
The present invention also provides and has contained Δ
6The carrier of-fatty acid dehydrogenase cDNA.In different organisms, make up suitable expression vector and instruct Δ
6-fatty acid dehydrogenase coded sequence, this is that those skilled in the art are familiar with.Expression vector as herein described refers to composing type carrier or integrating vector, is used to regulate and control the expression of goal gene.Preferred carrier is a plasmid.This class carrier contains the sequential cell that can effectively carry out genetic expression, and these sequential cell comprise promotor etc.The used soybean of the present invention transforms CaMV35S or the β-companion's sphaeroprotein specific promoter of soybean seeds that contains composing type in the expression vector.
Produce the method for gamma-linolenic acid defective or the organism that lacks gamma-linolenic acid, it comprise with a kind of from fungi isolating Δ
6-fatty acid dehydrogenase gene also is transformed into defective or lacks and express in the organism of gamma-linolenic acid and increase gamma-linolenic acid content, and described fungi is Mortierella isabellina M
6-22(Mortierella isabellina M
6-22), Δ wherein
6-fatty acid dehydrogenase gene structure is SEQ ID NO:1 (Genbank accept number for AF306634).
Described organism is yeast, the tobacco in the higher plant, soybean or the rape of lower eukaryotes.Described yeast is yeast saccharomyces cerevisiae INVSCl (Saccharomyces cereviciae INVScl) and pichia pastoris phaff (Pichia pastoris GS115), and expression vector is respectively pYMICL6 and pPMICL6.
Described tobacco is Nicotiana tabacum cv.xanthi and N.tabacum cv.YN (the big gold dollar in Yunnan), and expression vector is respectively pGAMICL6 and pBMICL6; Described soybean is Jilin 35, Jilin 37, Jilin 43, black farming 37 and Sui Nong 10 kinds, and expression vector is pBMICL6, and the promotor of described expression vector is β-companion's sphaeroprotein soybean seeds promotor or CaMV35S promotor, will have Δ
6The expression vector of the reorganization of-fatty acid dehydrogenase gene induces grow thickly bud and regeneration plant by cotyledonary node and the plumular axis that agrobacterium-mediated transformation infects soybean; Described rape is the recovery system of swede type rape (Brassica napus) 99F045C, 99F044C and the combination of 99F057C triple crossing, is that cotyledon petiole carries out genetic transformation acquisition regeneration plant with agrobacterium-mediated transformation to recovering.
A kind of method that produces the soybean of high gamma-linolenic acid content comprises:
1) with a kind of expression vector soybean transformation cell, described expression vector contains the Δ of SEQ ID NO:1 (Genbank accept number for AF306634)
6-fatty acid dehydrogenase gene, described expression vector are binary vector pBMICL6, and promotor is CaMV35S or specificity soybean β one companion's sphaeroprotein seed promoters of composing type, and selective marker is kantlex or Totomycin;
2) will have Δ
6The expression vector of the reorganization of-fatty acid dehydrogenase gene induces grow thickly bud and regeneration plant by cotyledonary node and the rataria that agrobacterium-mediated transformation infects soybean.
Described method for transformation is the bud of growing thickly that induces from cotyledonary node and plumular axis, step sizing in 50mg/L Kan screening culture medium, the survival rate of regeneration bud is 15%, and blastogenesis to be grown thickly is grown to 2cm and gone in the root media when high, and to reduce Kan concentration be 25% or remove fully.Described method for transformation is that regrowth is changed over to before the root media, earlier its base portion being cut adopts root-inducing powder to soak into, put into the root media that does not contain kantlex again and cultivate, treat that regrowth grows main root in root media after, change in vermiculite or the soil.
Described soybean is Jilin 35, Jilin 43, Jilin 47, black farming 37 and Sui Nong 10 soybean varieties.
The invention provides the method that in the soya cells of defective gamma-linolenic acid, produces gamma-linolenic acid.These methods have related to functional expression vector in host cell, this expression vector makes Δ
6-fatty acid dehydrogenase is expressed in host cell, makes host cell produce gamma-linolenic acid.The invention still further relates to control is selected in the generation of gamma-linolenic acid in the soybean seeds.On method for transformation, the present invention relates to the condition of resistance screening and regeneration bud root culture.Can improve rooting rate and reach 80%.Adopt the inventive method transgenosis in the soybean of defective gamma-linolenic acid, to produce gamma-linolenic acid, be applicable to the scale operation of gamma-linolenic acid and be applicable to the change of the fatty acid profile of soybean.
Description of drawings
Fig. 1 shows yeast saccharomyces cerevisiae expression vector pYMICL6.This plasmid is the complete Δ that contains by pYES2.0 plasmid (available from Invitrogen company) and this laboratory structure
6The plasmid pTMICL6 structure of-fatty acid dehydrogenase gene (D6D gene) forms.Behind EcoRI and XhoI double digestion, electrophoresis reclaims these two purpose fragments of purifying, through T respectively for plasmid pYES2.0 and pTMICL6
4After DNALigase connects, transformed into escherichia coli DH
5 α, screen and identify recombinant plasmid, called after pYMICL6.
Fig. 2 shows yeast expression vector pPMICL6, and this plasmid is the complete Δ that contains by plasmid pPIC3.5K (available from Invitrogen company) and this laboratory structure
6The plasmid pTMICL6 structure of-fatty acid dehydrogenase gene (D6D gene) forms.Plasmid pPIC3.5K is behind EcoRI and NotI double digestion, and electrophoresis reclaims the big fragment of purifying; With the pTMICL6 plasmid is template, and PCR obtains 5 ' end band EcoRI site, the Δ in 3 ' end band NotI site
6-fatty acid dehydrogenase gene, with EcoRI and NotI double digestion, electrophoresis reclaims purifying D6D gene fragment, with above-mentioned two fragments through T
4After DNALigase connects, transformed into escherichia coli DH
5 α, screen and identify recombinant plasmid, called after pPMICL6.
Fig. 3 shows tobacco expressed carrier pGAMICL6, and this plasmid is the complete Δ that contains of plasmid pGA643 and this laboratory structure
6The plasmid pTMICL6 structure of-fatty acid dehydrogenase gene (D6D gene) forms.Plasmid pGA643 is behind XbaI and BglII double digestion, and electrophoresis reclaims the big fragment of purifying; With the pTMICL6 plasmid is template, and PCR obtains 5 ' end band XbaI site, the Δ in 3 ' end band BglII site
6-fatty acid dehydrogenase gene, with XbaI and BglII double digestion, electrophoresis reclaims purifying D6D gene fragment, with above-mentioned two fragments through T
4After DNALigase connects, transformed into escherichia coli DH
5 α, screen and identify recombinant plasmid, called after pGAMICL6.Plasmid pGA643 is derived from document: GynheungAN, Paul R.Ebert, Amitava Mitra and Sam HA. (1988) Binary rectors.Plant MolecularBiology Manual, A3:1-19.
Fig. 4 shows soybean expression vector pBMICL6, and the building process of this plasmid is seen embodiment 2.Plasmid pBI121 is derived from document: Jefferson RA.Tony AK, Michael WB. (1987a) .Gus fusions: β-glucuronidase as asensitive and versatile gene fusion marker in higher plants.EMBO J., 6 (13): 1301-1307.
Embodiment
Following examples further specify the present invention.Embodiment 1 is from Mortierella isabellina M
6-22The middle Δ that separates
6-fatty acid dehydrogenase nucleotide sequence
From cultivating the Mortierella isabellina M of 48h
6-22Mycelium extracts total RNA, and (20 (1): 44～50,2001), by reverse transcription test kit (Promega company) specification sheets, be template with the total RNA of 1ug, the first chain cDNA is synthesized in reverse transcription, is that template is carried out pcr amplification with this cDNA for Li Mingchun etc., fungus system.
According to delivering different plant species Δ in the document
6-fatty acid dehydrogenase nucleotide sequence relatively mostly has the conserved sequence in the cytochrome b5 district (Cytb5) of electron transfer function and Histidine (His) conserved regions I, II, III district.We are according to the aminoacid sequence design clone Mortierella isabellina Δ of Cytb5 district (HPGG) and His conserved regions III (QIEHHLFP)
6The part primer of-fatty acid dehydrogenase nucleotide sequence and total length primer:
Part primer (according to two conserved regions: the Cytb5 and the HisIII of structure gene)
Upstream primer: 5 ,-TTTGTCCCTGATCATCCCGGTGG-3,
Downstream primer: 5 ,-AGGGAACAAGTGGTGGTCAATCTG-3,
Total length primer: (according to the initial sum terminator nucleotide sequence of structure gene)
Upstream primer: 5 '〉TAGGCTGAATTCATGGCTGCTGCTCCCAGTGTGAGGACG＜3 '
Downstream primer: 5 '〉AACTGCCTCGAGTTACTGCGCCTTACCCATCTTGGAGGC＜3 ' carry out pcr amplification reaction in following reaction system:
PCR reaction system (25ul), in the 0.2ml thin-walled tube, carry out:
| Composition | Volume (μ l) | Final concentration |
| H
2Primer downstream, O Taq | 17.25 2.5 1 1 1 2 0.25 | ----1 * Buffer 0.4mmol/L 0.4mmol/L 0.4mmol/L 1.25U/ reaction |
Undertaken by following PCR response procedures:
94 ℃ of 2min of program 1 (cycle1)
94 ℃ of 1min of program 2 (cycle2～36), 46 ℃ of 100s, 72 ℃ of 100s
72 ℃ of 15min of program 3 (cycle37)
The PCR that uses the part primer to carry out on cDNA produces the special band of an about 1.0kb of size, is 1071bp through this length of nucleotides that checks order.Through finding that relatively this nucleotide sequence contains Cytb5 and HisI, II, III district, prove that clone's cDNA fragment is a Δ
6The part of-fatty acid dehydrogenase nucleotide sequence.
The PCR that uses the total length primer to carry out on cDNA produces the fragment of an about 1.4kb of size, through order-checking and this nucleotide sequence relatively, finds to contain Cytb5 and HisI, II, III district, proves that the cDNA fragment of cloning is a Δ
6-fatty acid dehydrogenase nucleotide sequence.Sequencing result shows the Mortierella isabellina Δ
6-fatty acid dehydrogenase structure gene total length is 1374 Nucleotide, 457 amino acid of encoding altogether, three Histidine conserved regions that the film desaturase is common, respectively at 172～176 of aminoacid sequence, 209～213,395～402 places, aminoacid sequence of deriving and known cyanobacteria (Reddy A S.et al., Plant Mol.Biol.1993,27:293～300), plant Borrago officinalis (Sayanova O.etal.Proc.Natl.Acad Sci.USA1997,94:4211～4216), the Δ of nematode (Napier J A.et al.Biochem.J.1998,330:611～614)
6-fatty acid dehydrogenase is similar, with the Mortierella alpina Δ
6The maximum homology of the nucleotide sequence of-fatty acid dehydrogenase is 94%, with the plant Borrago officinalis be 53%, with nematode be 55%.As other species Δ
6-fatty acid dehydrogenase is the same, contains equally at the N-terminal of aminoacid sequence to be similar to cytochrome b
5The protein sequence HPGG in zone.This sequence is Δ
6-fatty acid dehydrogenase structure gene SEQ ID NO:1 (Genbank accept number be AF306634).Embodiment 2 Mortierella isabellina Δs
6The structure of-fatty acid dehydrogenase gene expression vector in soybean transforms
This example is only limited to the structure of pBMICL6
Go up PCR from plasmid pTMICL6 and obtain 5 ' end band XbaI site, the Δ in 3 ' end band SacI site
6-fatty acid dehydrogenase gene is cut with XbaI and SacI enzyme, with the Δ in the PCR product
6-fatty acid dehydrogenase gene fragment is replaced gus gene and is cloned in the pBI221 plasmid.Form new plasmid pBI221MICL6, this gene is under the control of CaMV35S promotor (or β-companion's sphaeroprotein seed promoters), will contain the Δ of CaMV35S promotor then by HindIII and EcoRI restriction site
6The dna fragmentation of-fatty acid dehydrogenase gene clones into that another one contains in the carrier pBI121 plasmid of kantlex selected marker, constructs to contain Δ
6The plasmid pBMICL6 (see figure 4) of-fatty acid dehydrogenase gene.By electric transformation technology, this expression vector plasmid is introduced in the agrobacterium tumefaciens lba4404, with this bacterium plant is carried out conversion test.Embodiment 3 agriculture bacillus mediated soybean heredities transform
(1) cultivation of Agrobacterium and preparation
The single bacterium colony of Agrobacterium that picking contains expression vector from the flat board of 4 ℃ of preservations inserts and contains 50mg/LKan, 25mg/L Rif, the YEP substratum of 25mg/L Str, 28 ℃, to logarithmic phase, use MS (.Mtuashige T then about 160r/min shaking culture 18-20h, Skoog F.Physiol Plant, 1962,21:473-479) liquid nutrient medium is with 5-10 times of bacterium liquid dilution, as the engineering bacteria liquid of Agrobacterium-mediated Transformation.
(2) cultivation of aseptic seedling
Picking does not have scab, plants the intact soybean seeds of skin, after the clear water soaked overnight, and 70% ethanol disinfection 1min, 10-15% Losantin sterilization 15min, aseptic water washing 3-4 time removes kind of a skin, place the 1/2MS solid medium, 25-30 ℃ of cultivation obtains aseptic seedling after several days.
(3) agriculture bacillus mediated soybean heredity transforms
Directly regeneration induction plant and callus induces the MS substratum of the different tethelin 6BA+IBA of used various interpolations (6-benzylaminopurine+indolebutyric acid).
Cut cotyledon, cotyledonary node, plumular axis, the true leaf of aseptic seedling, adopt the method for multi-point injection and immersion to infect respectively.Be inoculated in the corresponding M S substratum, cultivate 2-3d altogether, change in the MS substratum that contains 50mg/L Kan and 500mg//L Cef (saitomycin), two all subcultures once, at the wound director bud that goes out to grow thickly, the bud of the growing thickly formation regeneration plant of growing up gradually.Since the difference at soybean genotype, the position of drawing materials, bud ratio and transformation efficiency variant (seeing Table 1).
The influence that different soybean varieties of table 1 and different explants are sprouted to induced bundle
Table?1?The?affection?of?adventitious?bud?on?soybean?genetype?and?explant
Kind explant inoculation number that resistance transformation efficiency of several bud ratio cards that sprouts
Cultivars????Explant????Infection????Number?of????Rate?of???Kan?resistance????Rate?of
number???????adventitious?adventitious????????????????transformation
bud??????????bud
Ji is educated 43 cotyledons 86 0000
Jiyu43 cotyledonary node 84 179 213 15 17.9
Plumular axis 86 162 188 11 12.8
True leaf 86 0000
Black agricultural 36 cotyledons 20 0000
Heinong36 cotyledonary node 20 38 190 2 10.0
Plumular axis 19 31 163 1 5.3
True leaf 20 0000
Black agricultural 37 cotyledons 119 0000
Heinong37 cotyledonary node 120 230 192 16 13.3
Plumular axis 120 207 173 9 7.5
True leaf 120 0000
As can be seen from Table 1, cotyledonary node is easy to induced bundle and sprouts, and the bud number of growing thickly is up to 7 more than, and plumular axis also is easy to induced bundle and sprouts, the bud difficulty but cotyledon and true leaf differentiation are grown thickly.The time that induces the bud of growing thickly from cotyledonary node and plumular axis is roughly similar, and the former is higher than the latter number of the bud of growing thickly, and how tiny latter's regeneration plant is.From table 1, it can also be seen that, the soybean induced bundle of different genotype several obvious differences of sprouting, it is higher that Ji is educated 43 bud ratios.Therefore, adopting cotyledonary node and plumular axis induced bundle to sprout is optimal selection.
When inducing the bud length of growing thickly to 2cm from cotyledonary node and plumular axis, the bud of will growing thickly downcuts its base portion and adopts root-inducing powder to soak into, and changes root induction substratum (MS+VB over to
6(1mg/L)+nicotinic acid (1mg/L)+VitB1 (10mg/L)+inositol (100mg/L)+Cef (500mg/L)+IBA (1.5mg/L) pH5.8).Bud to be grown thickly changes regrowth in the root media (blended eutrophy soil) cultivation after growing main root.Embodiment 4 kantlex are to the test of soybean selective pressure
This tests used soybean varieties is Jilin 43, black farming 36 and Hei Nong 37, expression vector is pBMICL6, plant screening mark gene is a kalamycin resistance gene, compound concentration is respectively 0mg/L, 5mg/L, 25mg/L, 50mg/L, 75mg/L, the kantlex of 100mg/L carries out the selective pressure test of soybean callus tissue growth and differentiation regeneration bud in genetic transformation.Inoculate the 10/ware of embryo callus subculture (establishing 5 repetitions) of the basically identical that grows, around cultured continuously on the division culture medium that is added with different concns Kan.Inoculation differentiation 3/bottle of regeneration buds (establishing 10 repetitions) are around cultured continuously on the division culture medium that is added with different concns Kan.25-30 ℃ of cultivation, 12h illumination cultivation, the changing conditions (see figure 5) of observing callus growth and differentiation regeneration bud.
Fig. 5 shows that the Kan concentration of 25-50mg/L all reaches 50% restraining effect to soybean embryo callus and differentiation regeneration bud, and particularly when Kan concentration was 50mg/L, the survival rate of regeneration bud was lower than 15%.So in this Study on Transformation, beginning used Kan screening concentration is 50mg/L, treat that regeneration bud grows to 2cm when high, removes Kan fully in root media.The molecular Biological Detection of embodiment 5 genetically engineered soybeans
(1) PCR of genetically engineered soybean detects
Garbled kanamycin-resistant callus tissue of that mycin of card taking and regeneration plant, usefulness CTAB method (. Fu Rongzhao, Sun Yongru, Jia Shirong chief editor, plant genetic transformation technology handbook, China Science Tech Publishing House, 1994) extract total DNA, be used for round pcr amplification Δ
6The gene of-fatty acid dehydrogenase.
Two primer sequences are 5 '-GGCTTCTAGAATGGCTGCTGCTCCCAGT-3 ' and 5 '-CTGCGAGCTCTTACTGCGCCTTACC-3 '.The PCR reaction conditions is: 95 ℃ of sex change 5min, and then at 94 ℃ of sex change 1min, 53 ℃ of annealing 1min carry out 35 circulations under the condition of 72 ℃ of extension 2min, and last 72 ℃ are extended 10min again.Electrophoresis result is seen Fig. 6.
(2) southern of genetically engineered soybean plant detects
A large amount of total DNA that extract PCR reacting positive soybean transgene seedling are after the abundant enzyme of HindIII is cut, through steps such as electrophoresis, commentaries on classics films, with the Δ of digoxigenin labeled
6The gene probe hybridization of-fatty acid dehydrogenase, concrete testing sequence is seen the test kit working instructions.Results of hybridization is seen Fig. 7 and 8.
(3) the black agricultural 37T of genetically engineered soybean
0Gas-chromatography (GC) for seed is analyzed
The genetically engineered soybean seed is through deionized water wash three times, 50 ℃ of oven dry.After the grinding, add 5% KOH-CH
3OH solution, 70 ℃ of reaction 3h add 14%BF again
3-CH
30H, 70 ℃ of reaction 1.5h form fatty acid methyl ester.Chloroform with 1: 4: hexane extracting twice, united extraction liquid.Add an amount of anhydrous Na
2SO
4Dry extraction liquid leaves standstill 1h, removes Na
2SO
4, dry up with nitrogen.With a little normal hexane Hui Rong, standby.The GLA methyl esters of producing with Sigma company is standard substance, and normal hexane is the solvent working sample.Instrument is Tianjin, island GC-7A, pillar: DEGS 0.25mm * 25m, splitting ratio: 100: 1, column temperature: 180 ℃, tail blew: 50ml/min, the vaporizer temperature: 250 ℃, detector: hydrogen flameionization detects.
Measurement result is seen accompanying drawing 9.Calculate black agricultural 37 soybean T according to sectional area
0For one linolenic acid content of r in the seed is 18.2% of polyunsaturated fatty acid.
Claims (9)
1, a kind ofly produce the method for gamma-linolenic acid defective or the organism that lacks gamma-linolenic acid, it is characterized in that it comprise with a kind of from fungi isolating Δ
6-fatty acid dehydrogenase gene also is transformed into defective or lacks and express in the organism of gamma-linolenic acid and increase gamma-linolenic acid content, and described fungi is Mortierella isabellina M
6-22(Mortierella isabellina M
6-22), Δ wherein
6-fatty acid dehydrogenase gene structure is SEQ ID NO:1 (Genbank accept number for AF306634).
2, the organism at defective or shortage gamma-linolenic acid according to claim 1 produces the method for gamma-linolenic acid, it is characterized in that described organism is yeast, the tobacco in the higher plant, soybean and the rape of lower eukaryotes.
3, the organism at defective or shortage gamma-linolenic acid according to claim 2 produces the method for gamma-linolenic acid, it is characterized in that described yeast is yeast saccharomyces cerevisiae INVSCl (Saccharomycescereviciae INVScl) and pichia pastoris phaff (Pichia pastoris GS115), expression vector is respectively pYMICL6 and pPMICL6 (seeing Fig. 1,2).
4, the organism at defective or shortage gamma-linolenic acid according to claim 2 produces the method for gamma-linolenic acid, it is characterized in that described tobacco is Nicotiana tabacum cv.xanthi and N.tabacumcv.YN (the big gold dollar in Yunnan), expression vector is respectively pGAMICL6 and pBMICL6 (seeing Fig. 3,4); Described soybean is Jilin 35, Jilin 37 and Jilin 43 soybean, black farming 37 and Sui Nong 10, and expression vector is pBMICL6, and the promotor of described expression vector is CaMV35S or β-companion's sphaeroprotein soybean seeds promotor, will have Δ
6The expression vector of the reorganization of-fatty acid dehydrogenase gene induces grow thickly bud and regeneration plant by cotyledonary node and the plumular axis that agrobacterium-mediated transformation infects soybean; Described rape is the recovery system of swede type rape (Brassicanapus) 99F045C, 99F044C and the combination of 99F057C triple crossing, is that cotyledon petiole carries out genetic transformation acquisition regeneration plant with agrobacterium-mediated transformation to recovering.
5, a kind of method that produces the soybean of high gamma-linolenic acid content is characterized in that:
1) with a kind of expression vector soybean transformation cell, described expression vector contains the Δ of SEQ ID NO:1 (Genbank accept number for AF306634)
6-fatty acid dehydrogenase structure gene, described expression vector are binary vector pBMICL6 (see figure 4), and promotor is CaMV35S or specificity soybean β one companion's sphaeroprotein seed promoters of composing type, and selective marker is kantlex or Totomycin;
2) will have Δ
6The expression vector of the reorganization of-fatty acid dehydrogenase gene induces grow thickly bud and regeneration plant by cotyledonary node and the rataria that agrobacterium-mediated transformation infects soybean.
6, the method for the soybean of generation high gamma-linolenic acid content according to claim 5, it is characterized in that described method for transformation is the bud of growing thickly that induces from cotyledonary node and plumular axis, step sizing in 50mg/L Kan screening culture medium, the survival rate of regeneration bud is 15%, blastogenesis to be grown thickly is grown to 2cm and is gone in the root media when high, and to reduce Kan concentration be 25% or remove fully.
7, the method for the soybean of generation high gamma-linolenic acid content according to claim 6, it is characterized in that described method for transformation is that regrowth is changed over to before the root media, earlier its base portion being cut adopts root-inducing powder to soak into, putting into the root media that does not contain kantlex again cultivates, after treating that regrowth grows main root in root media, change in vermiculite or the soil.
8, the method for the soybean of generation high gamma-linolenic acid content according to claim 5 is characterized in that described soybean is Jilin 35, Jilin 43, Jilin 47, black farming 37 and Sui Nong 10 soybean varieties.
9, the method for the soybean of the described generation high gamma-linolenic acid of claim 5 content is characterized in that described Δ
6-fatty acid dehydrogenase gene is the total length structure gene that obtains by the RT-PCR method, and its length of nucleotides is 1374bp, 457 amino acid of encoding.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948759A (en) * | 2010-09-14 | 2011-01-19 | 南京工业大学 | Mortierella isabellina and application thereof |
| CN101979624A (en) * | 2010-10-11 | 2011-02-23 | 南京工业大学 | Method for preparing microbial oil containing rich gamma-linolenic acid |
| CN102399832A (en) * | 2011-11-08 | 2012-04-04 | 四川农业大学 | Forage grease producing method by solid state fermentation of maize starch and wheat bran by mortierella isabellina |
| CN102839134A (en) * | 2012-09-24 | 2012-12-26 | 山东大学 | Yeast strain for producing alpha-linolenic acid, culture method and application thereof |
| CN104250651A (en) * | 2014-07-28 | 2014-12-31 | 昆明理工大学 | Delta-6 fatty acid dehydrogenase gene and application thereof |
| CN104250650A (en) * | 2014-07-28 | 2014-12-31 | 昆明理工大学 | Delta-6 fatty acid dehydrogenase gene and application thereof |
| WO2023273420A1 (en) * | 2021-07-02 | 2023-01-05 | 河南大学 | Application of soybean gene promoters peif1 and peif1-i in soybeans, arabidopsis thaliana and tobacco |
-
2002
- 2002-12-13 CN CNB021555958A patent/CN1176215C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948759A (en) * | 2010-09-14 | 2011-01-19 | 南京工业大学 | Mortierella isabellina and application thereof |
| CN101948759B (en) * | 2010-09-14 | 2012-02-01 | 南京工业大学 | Mortierella isabellina and application thereof |
| CN101979624A (en) * | 2010-10-11 | 2011-02-23 | 南京工业大学 | Method for preparing microbial oil containing rich gamma-linolenic acid |
| CN102399832A (en) * | 2011-11-08 | 2012-04-04 | 四川农业大学 | Forage grease producing method by solid state fermentation of maize starch and wheat bran by mortierella isabellina |
| CN102399832B (en) * | 2011-11-08 | 2013-12-18 | 四川农业大学 | Forage grease producing method by solid state fermentation of maize starch and wheat bran by mortierella isabellina |
| CN102839134A (en) * | 2012-09-24 | 2012-12-26 | 山东大学 | Yeast strain for producing alpha-linolenic acid, culture method and application thereof |
| CN102839134B (en) * | 2012-09-24 | 2014-04-16 | 山东大学 | Yeast strain for producing alpha-linolenic acid, culture method and application thereof |
| CN104250651A (en) * | 2014-07-28 | 2014-12-31 | 昆明理工大学 | Delta-6 fatty acid dehydrogenase gene and application thereof |
| CN104250650A (en) * | 2014-07-28 | 2014-12-31 | 昆明理工大学 | Delta-6 fatty acid dehydrogenase gene and application thereof |
| WO2023273420A1 (en) * | 2021-07-02 | 2023-01-05 | 河南大学 | Application of soybean gene promoters peif1 and peif1-i in soybeans, arabidopsis thaliana and tobacco |
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| CN1176215C (en) | 2004-11-17 |
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