CN102140475B - Application of TT1 genes in improving unsaturated fatty acid contents of plants - Google Patents
Application of TT1 genes in improving unsaturated fatty acid contents of plants Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, in particular to an application of TT1 genes in improving unsaturated fatty acid contents of plants, especially oil plants. The technical problem to be solved in the invention is to provide a new effective selection for improving the unsaturated fatty acid contents of the oil plants inr the technical field of transgenes. For solving the technical problem, the invention has the scheme of providing a TT1 gene application in improving the unsaturated fatty acid contents of the oil plants. Experiments prove that the unsaturated fatty acid contents of the oil plants transplanted with the TT1 genes and excessive expressions are obviously improved. The method in the invention for cultivating the oil plants with high unsaturated fatty acid contents is also convenient and effective, and therefore, the invention provides the new effective selection for the field.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the purposes of TT1 gene in improving the plant unsaturated fatty acid content.
Background technology
The oil Lay is one of the world five large oil crops (soybean, cottonseed, Sunflower Receptacle, rape, peanut), is widely distributed.There is China in major country of production, and is Canadian and European.China's oil Lay sown area and gross output all account for the world 1/3rd at present, and Canola oil also is the main food plant oil sources of China, accounts for about 40% of Chinese edible vegetable oil.
Semen Brassicae campestris institute fatty acids is directly determining the quality of rapeseed oil and to people's nutritive value.0), oleic acid (C18: 1), linolic acid (C18: 2), linolenic acid (C18: 3), eicosenoic acid (C20: 1) and erucic acid (C22: 1) seed oil of swede type rape mainly is comprised of 6 kinds of lipid acid, i.e. palmitinic acid (C16:.Wherein, palmitinic acid belongs to saturated fatty acid, and oleic acid, linolic acid, linolenic acid, eicosenoic acid, erucic acid all belong to unsaturated fatty acids, and unsaturated fatty acids is the lipid acid of needed by human.Palmitinic acid is non-digestible, and the mankind are too much edible, can cause obesity and cardiovascular disorder; Oleic acid and linolic acid have nutritive value to the people, and especially linolic acid is that human body can not synthesize, and must replenish from meals; Linolenic acid also is one of human body essential unsaturated fatty acids that can not synthesize, but because its unstable, very easily deterioration by oxidation easily makes grease become bad, reduces nutritive value and the stability of oil; Erucic acid is harmful to human body, and content is higher usually in the living rape variety out of office.
In the double-low rapeseed kind that China promotes at present, the problem that the ubiquity oleic acid content is on the low side, linolenic acid content is higher, owing to lack corresponding germplasm materials, especially low linolenic material, so that the quality breeding of canola fatty acids compositional optimization progress is comparatively slow.Studies show that, be respectively utmost point marked positive correlation and extremely remarkable negative correlation between erucic acid and linolenic acid, the linolic acid.Just can reduce linolenic acid content by selecting to reduce content of erucic acid, and linoleic acid content is increased to some extent.And oleic acid content and linolenic acid content are extremely significantly negative correlation between the two, illustrate that reducing linolenic acid content can make oleic acid content increase to some extent.These contrast indexs are consistent with canola fatty acids quality breeding target.Now, improve the fatty acid component of rape by genetic engineering means and obtained success, cultivated high linoleic acid, high oleic acid and without the rape variety of erucic acid.
In addition, China is second largest energy expenditure big country in the world, and the development substitute energy is very urgent.At present, the production of biodiesel technology take the vegetable seed wet goods as raw material and use are succeeded in developing in succession in China.
Biofuel is a class fatty acid methyl ester, that Vegetable oil lipoprotein takes off a kind of environment-friendly fuel that obtains through esterification behind the glycerine, its raw materials for production are renewable resourcess, and burning biofuel emission greenhouse gas (for example carbonic acid gas) reduces 60% than mineral diesel.The fatty acid carbon chain of low erucic acid rape oil consists of 16～18 carbon, and is close with diesel oil molecules (carbochains about 15) carbon number.Therefore, the biofuel of being produced take low erucic acid rape oil as raw material is the ideal substitute of mineral diesel.
Although biofuel has huge development space, its production cost is high, is a world-famous puzzle that hinders this technology industrialization exploitation always.The bottleneck of development biofuel is raw material, i.e. the amount of raw material and price.Although many woody oleiferous plants can be processed as biofuel, scale is limited.The staple food crops such as the draft such as soybean, peanut oil crops and paddy rice, corn are striven ground, and the potentiality of enlarged-area are little.And as the desirable feedstock of biofuel, rape has its unique advantage:
It is that advantage substitutes biomass energy raw material crop that the states such as Canada, Australia, Korea S, Germany, France, Sweden, Austria establish rape, and wherein the biofuel 80% of at present production of European Union is from rape raw material (Semen Brassicae campestris 60% of production is for the production of biofuel).
Major objective according to current rapeseed breeding and biomass energy exploitation, utilize newest research results, cloned the relevant functional gene of a collection of important character, carried out the systematic study of genetically engineered initiative Semen Brassicae campestris as the biodiesel raw material gordian technique, and proposed further to improve oleaginousness by the metabolic engineering approach, have high oil, high yield, wide suitable, a series of important subject such as " super " rape that resist, adapt to the multiple excellent specific properties such as light cultivation by the cultivation of functional gene pyramiding breeding more, all make substantial progress.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of new effective selection for the raising plant especially field of transgenic technology of the unsaturated fatty acid content of oilseed plant.
The technical scheme that the present invention solves this technical problem has provided the purposes of TT1 gene in improving the plant unsaturated fatty acid content.
Wherein, the nucleotide sequence of above-mentioned TT1 gene:
(1): the nucleotide sequence shown in SEQ ID NO.1 or its degenerate sequence;
Or (2): in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of at least one Nucleotide, and with the same or analogous polypeptide of sequence encoding function of SEQ ID NO.1.Namely also can improve the plant unsaturated fatty acid content.
Further, above-mentioned (2) are: in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of 1 or several (in 10) Nucleotide, and with the same or analogous polypeptide of sequence encoding function of SEQ ID NO.1.
The present invention provides the purposes of polypeptide in improving the plant unsaturated fatty acid content of TT1 genes encoding simultaneously.
TT1 gene in the such use has following nucleotide sequence:
(1): the nucleotide sequence shown in SEQ ID NO.1 or its degenerate sequence;
Or (2): in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of at least one Nucleotide, and can with the same or analogous polypeptide of sequence encoding function of SEQ ID NO.1.
Further, above-mentioned (2) are: in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of 1 or several (in 10) Nucleotide, and with the same or analogous polypeptide of sequence encoding function of SEQ ID NO.1.
The present invention also provides a kind of cultivation to have the method for the plant of high unsaturated fatty acid content.The method may further comprise the steps:
(1) the TT1 gene operationally is connected in expression regulation sequence on the carrier after, form the recombinant vectors of described TT1 gene;
(2) recombinant vectors in the step (1) is changed in the vegetable cell;
(3) obtain transformant through screening, then transformant is cultivated into high unsaturated fatty acid plant or its offspring, the offspring of described plant comprises plant seed and plant tissue.
TT1 gene in the aforesaid method has following nucleotide sequence:
(1): the nucleotide sequence shown in SEQ ID NO.1 or its degenerate sequence;
Or (2): in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of at least one Nucleotide, and can with the same or analogous polypeptide of sequence encoding function of SEQ ID NO.1.
Further, above-mentioned (2) are: in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of 1 or several (in 10) Nucleotide, and with the same or analogous polypeptide of sequence encoding function of SEQ ID NO.1.
Wherein, above-mentioned plant can be the general various plants that need to use the unsaturated fatty acids in its tissue, such as various common oil crops, such as oil crops such as common rape, soybean, cotton, Sunflower Receptacle, sesame, castor-oil plant, Cortex jatrophae, rape or peanuts.
TT1 gene described in the present invention, its basic nucleotide sequence is shown in SEQ ID NO.1 in the sequence table, this gene source mustard in Cruciferae (Brassicaceae, also name Cruciferae) belongs to the plant rape (Brassicanapus) of (Brassica).
The content of above-mentioned raising unsaturated fatty acids can improve greatly for the content of the unsaturated fatty acids in content, the especially seed and fruit of the unsaturated fatty acids that improves each organ such as roots of plants, stem, Ye Hua.
In the present invention, the TT1 gene order of " nucleotide sequence in SEQ ID NO.1 through replace, lack or add at least one Nucleotide derived sequence " nucleotide sequence and the degenerate sequence thereof of polypeptide with the coded protein-active of SEQ ID NO.1 that generally refer to encode.The sequence that this degenerate sequence refers to have in the described sequence one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces.Because the degeneracy of codon, so be low to moderate about 89% the degenerate sequence described sequence of SEQ ID NO.1 of also encoding out with SEQ ID NO.1 homology.In addition, the implication of " nucleotide sequence in SEQ ID NO.1 through replace, lack or add at least one Nucleotide derived sequence " also comprises can be under the rigorous condition of moderate, better under highly rigorous condition with the nucleotide sequence of SEQ ID NO.1 nucleotide sequence hybridization.This term also comprise with SEQID NO.1 in the homology at least 80% of nucleotide sequence, preferably at least 89%, more preferably at least 90%, at least 95% nucleotide sequence best.Identical function in the present invention refers to improve the unsaturated fatty acid content of plant.
This term also comprises encoding to have the variant form of open reading frame sequence among the SEQ ID NO.1 with the albumen of natural SEQ ID NO.1 identical function.These variant forms comprise (but being not limited to): several (are generally 1～90, preferably 1～60, more preferably 1～20,1～10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.Such as the nucleotide sequence shown in the SEQ ID NO:3, the polypeptide of its coding also can improve the unsaturated fatty acid content of plant.
Recombinant vectors of the present invention is the TT1 gene to be inserted in the carrier obtain, and above-mentioned carrier can be selected various carrier, especially carrier for expression of eukaryon known in the art (such as pBI121 or pCAMBIA2301).The present invention transforms host plant cell with above-mentioned recombinant vectors, and screening obtains transformant.Then transformant is cultivated into transgenosis high unsaturated fatty acid plant and offspring thereof, described offspring comprises plant seed and plant tissue.
" operationally being connected in " described in the present invention is expressed as follows situation: namely some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
Beneficial effect of the present invention is: the invention provides the purposes of TT1 gene aspect raising plant unsaturated fatty acid content.The TT1 gene can be used for preparing the transformant of high plant unsaturated fatty acid content, further cultivates the novel oil crops of high-quality edible-type or new forms of energy raw material type.Prove by experiment also that in an embodiment of the present invention having changed the TT1 gene over to also crosses in the oil crops such as rape, soybean, cotton, Sunflower Receptacle, sesame, castor-oil plant, Cortex jatrophae, rape or peanut of expressing, obtain the plant of the above-mentioned oil crops of overexpression TT1 gene, the result shows that unsaturated fatty acid content improves some saturated fatty acid content relative reduces.Show that the TT1 gene can effectively improve the unsaturated fatty acid content of each kind of plant.
The method of novel oil crops that the present invention cultivates edible-type or new forms of energy raw material type is also easy and effective, has good application prospect.
Description of drawings
The PCR electrophoresis result of Fig. 1, TT1 gene overexpression swede type rape.Be followed successively by from left to right Marker (M, clip size is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp) among the figure, plant 1～4 to be detected.
Embodiment
Below by embodiment and by reference to the accompanying drawings, further specify and do not limit the present invention.
Among the following embodiment, all unreceipted concrete experiment conditions, be according to normal condition well known to those skilled in the art, Sambrook for example, the molecular cloning of Russell: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.Among the following embodiment, used cabbage type rape variety is assorted No. nine of another name for Sichuan Province (Sichuan University's Life Science College is preserved); Used Agrobacterium (Agrobacteriumtumefaciensp) is adopted LBA4404 bacterial strain, EHA105 bacterial strain; Intestinal bacteria (E.coli) are adopted DH5 α bacterial strain, and bacterial strain is all available from Qiagen company.Carrier pBI121, pCAMBIA1301 are available from Clontech company.TA clone test kit pUCm-T Vector Kit, Taq Polymerase, restriction enzyme, ligase enzyme, DNA reclaim purification kit and all are purchased from Shanghai living worker's biotechnology company limited.All the other chemical actual commercially available analytical pure that are.Among the following embodiment, " SEQ ID NO:1 " when occurring separately, those skilled in the art can understand that it is the abbreviation of " nucleotide sequence shown in the SEQ ID NO:1 ".
Embodiment one: TT1 gene cloning and obtaining
Atp6 (genebank gi:89279377) gene in the rape is as bait protein, according to yeast two-hybrid method (seeing the disclosed data of Clontech company), screen the est sequence (shown in the SEQ ID NO:2) in the rape, the sequence that screens according to this section again, method by 5 ' RACE (seeing the disclosed data of Takara company) obtains gene of the present invention, and its nucleotide sequence is shown in SEQ ID NO.1 in the sequence table.Design primer according to nucleotide sequence shown in the SEQ ID NO.1:
Upstream primer (SEQ ID NO.3): 5 '-ATGTCGGATGATTTGAGTTTATG-3 ',
Downstream primer (SEQ ID NO.4): 5 '-TCAGACTGGTGTTGGGTTGGATAT-3 '.
Then through the PCR nucleotide sequence shown in the SEQ ID NO.1 that from rape cDNA, increases.To PCR product purification (seeing Qiagen company disclosed PCR product purification data), through sequence verification, obtain the gene order of SEQ ID NO.1.
Embodiment two: turn the preparation of TT1 gene swede type rape
1, goal gene overexpression construction of recombinant plasmid
According to the design of nucleotide sequence shown in SEQ ID NO:1 primer,
Upstream primer (SEQ ID NO.5): 5 '-CGC GGATCCATGTCGGATGATTTGAGTTTATG-3 ',
Downstream primer (SEQ ID NO.6): 5 '-CCGGAGC TCTCAGACTGGTGTTGGGTTGGATAT-3 '.
Through PCR, the nucleotide sequence shown in the complete SEQ ID NO:1 that increases from rape cDNA to PCR product purification (seeing the disclosed data of Qiagen company), carries out the TA clone, and BamH I and Sac I enzyme are cut and identified positive recombinant clone.Positive colony is cut with BamH1 and Sac1 enzyme, and glue reclaims, and is connected (connection site: BamH1 and Sac1) with carrier pBI121, obtains the overexpression recombinant plasmid that contains SEQ ID NO:1.The overexpression recombinant plasmid that will contain SEQ ID NO:1 changes in the Agrobacterium LBA4404, and the method for utilizing hypocotyl to contaminate transforms swede type rape (seeing step 2).
2, the method for plumular axis dip-dye transforms swede type rape
Obtaining of 2-1, aseptic seedling
Choose the swede type rape seed of full seed, 4 ℃ of vernalization of spending the night (the maintenance seed germination is synchronous) are then taken out, with 70% alcohol immersion 30s, 0.1% mercuric chloride (HgCl2) solution soaking 8-10min, aseptic water washing 5 times, filter paper blots, and is inoculated on the MS solid medium.Put in the culturing room 24 ℃, secretly cultivated 2-3 days, then take out illumination 16h/d and continue to sprout.Get 5～7cm (about 7～8 days) aseptic seedling hypocotyl as transformation receptor.
2-2, hypocotylar preculture
Rape hypocotyls is cut into 7mm left and right sides segment, and being uniformly dispersed places the preculture (visible hypocotyl chap) of carrying out in the pre-culture medium (MS+2mg/L 6-BA, 1mg/L 2,4-D, 2.5mg/L AgNO3,19.62mg/L Syringylethanone) 2～3 days.
2-3, hypocotylar dip-dye and altogether cultivation
Picking contains the Agrobacterium of the overexpression recombinant plasmid of SEQ ID NO:1, be inoculated in and contain the 20mg/L Streptomycin sulphate, the 50mg/L kantlex, in the LB liquid nutrient medium of 40mg/L Rifampin, 28 ℃ are shaken the bacterium rear collection thalline that spends the night, be resuspended in the MS liquid nutrient medium that contains the 100mg/L Syringylethanone to OD600=0.4～0.6,28 and ℃ shake bacterium 1～2h.
To immerse respectively through the rape hypocotyls of pre-incubated stalwartness 30s～1min in the Agrobacterium bacterium liquid of the overexpression recombinant plasmid that contains SEQ ID NO:1, during this constantly vibration bacterium liquid is fully contacted with rape hypocotyls.Blot rapidly unnecessary bacterium liquid with aseptic filter paper, rape hypocotyls is lain against on the common substratum (MS+2mg/L 6-BA, 1mg/L 2,4-D, 2.5mg/L AgNO3,19.62mg/L Syringylethanone), cultivate altogether 2d.
2-4, screening and culturing and spore induction
Two kinds of rape hypocotyls after the common cultivation are accessed respectively continuation cultivation in the division culture medium (MS+2mg/L 6-BA, 1mg/L 2,4-D, 2.5mg/L AgNO3,19.62mg/L Syringylethanone).Per 2 weeks are upgraded substratum once, cultivate for 4 weeks, obtain the callus bud.
2-5, take root
At screening culture medium (MS+2mg/L 6-BA, 2.5mg/L AgNO3, the 500mg/L Pyocianil, the 10mg/L kantlex) treats on that two kinds of callus buds grow to when 4-6 sheet true leaf is arranged, bud is downcut from callus, move in the root media (1/2MS, 0.15mg/L NAA, 250mg/L cephamycin).When treating that the regrowth root growth is flourishing, culture tank is moved to outdoor 2～3d, then will cultivate cover and open, hardening 2～3d in culturing room.
2-6, potted plant cultivation
The overexpression transfer-gen plant that will contain SEQ ID NO:1 grows complete root system at root media respectively, it is changed over to potted plant.
3, the PCR of transgene rape detects
After regeneration plant is grown up in soil, respectively get blade and use on a small quantity the total DNA of CTAB method extracting, do template with the DNA that extracts, carry out respectively PCR and detect.Goal gene overexpression swede type rape transgenic line detects:
Upstream primer (SEQ ID NO.7): 5 '-ATTTCATTTGGAGAGAACACGG-3 ';
Downstream primer (SEQ ID NO.8): 5 '-TCAGACTGGTGTTGGGTTGGATAT-3 '.
Then agarose electrophoresis detects, and detected result is seen Fig. 1, has target stripe to occur then representing goal gene and has changed swede type rape over to.The size of detected purpose band and expection SEQ ID NO:1 in the same size are about 860bp.It is for subsequent use to make SEQ ID NO:1 nucleotide sequence overexpression swede type rape plant and seed.
Embodiment three: the mensuration that turns fatty acid content in the TT1 gene swede type rape seed
Concrete measuring method referring to " gas chromatographic analysis of fatty acid content in the Semen Brassicae campestris " (" the Qinghai agriculture and forestry science and technology, the 4th phase in 2006, Wang Ninghui).
Instrument: the U.S. 6890N of Agilent company gas chromatograph comprises flame ionization ditector; The full-automatic hydrogen generator of Hewlett-Packard's analytical technology SPA-300A of institute type in Beijing; The TGA-2000A of Shanghai Huaai Chromatographic Analysis Co., Ltd. type low-noise air pump.
Chromatographic column: the U.S. INNOWAX30m of Agilent company capillary column.Post case temperature: 175 ℃ of initial temperatures, temperature programming behind the maintenance 15min, 5 ℃ to 210 ℃ of per minutes keep 17min.250 ℃ of carrier gas nitrogen of 240 ℃ of detector temperatures of temperature of vaporization chamber, sample size 1 μ l, splitting ratio 8: 1.
Reagent: sherwood oil, anhydrous diethyl ether or benzene, potassium hydroxide, dehydrated alcohol.Be mixed with 0.4N potassium hydroxide-methanol solution; 1: 1 sherwood oil-diethyl ether solution.
Get 20 of Semen Brassicae campestris, put into mortar and grind.Add 1: 1 sherwood oil-diethyl ether solution 3.5ml, ground and mixed is abundant.Dissolve things inside is poured in the little centrifuge tube of 5ml into the centrifugal 2min of 10000rpm.Move the 2ml supernatant to the little centrifuge tube of another 5ml, add 1ml0.4N potassium hydroxide-methanol solution mixing, room temperature is placed 30min, adds 2ml distilled water and shakes up, the centrifugal 2min of 10000rpm.It is stand-by that the absorption supernatant places the gas-chromatography sample bottle that sample bottle is put into automatic sampler in order.The seed oil of swede type rape mainly is comprised of 6 kinds of lipid acid, be palmitinic acid (C16: 0), oleic acid (C18: 1), linolic acid (C18: 2), linolenic acid (C18: 3), eicosenoic acid (C20: 1) and erucic acid (C22: 1), statistics is as shown in table 1:
Table 1 swede type rape seed fatty acid content cartogram (unit: %)
Can be found out by fatty acid content result statistics, transgenosis type rape (TT1-1, TT1-2, TT1-3) than non-transgenic type rape (WT, i.e. wild-type) 1), linolic acid (C18: 2) on average exceed 18.97%, 33.72% aspect the content at oleic acid (C18:; Palmitinic acid (C16: 0) reduction by 71.65%, linolenic acid (C18: 3) reduce by 40.18%.All in all, the unsaturated fatty acid content of transgenosis type rape (oleic acid, linolic acid, linolenic acid, eicosenoic acid, erucic acid) has improved 15.65% than non-transgenic type.
Illustrate that the TT1 gene has improved unsaturated fatty acid content in the Semen Brassicae campestris, especially favourable to HUMAN HEALTH oleic acid and linolic acid have reduced saturated fatty acid content simultaneously; Therefore turn TT1 genotype rape also because its higher unsaturated fatty acid content, lower saturated fatty acid content, more meet the standard of health plant oil, also become the desirable feedstock of preparing biological diesel oil.
Wherein, above-mentioned plant can be the general various plants that need to use the unsaturated fatty acids in its tissue, such as various common oil crops, such as oil crops such as common soybean, cotton, Sunflower Receptacle, sesame, castor-oil plant, Cortex jatrophae, rape or peanuts.
Embodiment four: turn the preparation of TT1 gene soybean and the mensuration of fatty acid content
1, goal gene overexpression construction of recombinant plasmid
According to the design of nucleotide sequence shown in SEQ ID NO:1 primer,
Upstream primer (SEQ ID NO.5): 5 '-CGC GGATCCATGTCGGATGATTTGAGTTTATG-3 ',
Downstream primer (SEQ ID NO.6): 5 '-CCGGAGC TCTCAGACTGGTGTTGGGTTGGATAT-3 '.
Through PCR, the nucleotide sequence shown in the complete SEQ ID NO:1 that increases from rape cDNA to PCR product purification (seeing the disclosed data of Qiagen company), carries out the TA clone, and BamH I and Sac I enzyme are cut and identified positive recombinant clone.Positive colony is cut with BamH1 and Sac1 enzyme, and glue reclaims, and is connected (connection site: BamH1 and Sac1) with carrier pBI121, obtains the overexpression recombinant plasmid that contains SEQ ID NO:1.The overexpression recombinant plasmid that will contain SEQ ID NO:1 changes among the Agrobacterium EHA105, screening positive clone on the LB flat board that contains Rifampin (rif) 50mg/L, kantlex (kan) 50mg/L, bacterium colony PCR detection validation.
2, the genetic transformation of soybean
The preparation of 2-1, Agrobacterium bacterium liquid
Picking contains the Agrobacterium of overexpression recombinant plasmid of SEQ ID NO:1 in containing corresponding antibiotic LB liquid nutrient medium, 28 ℃ are shaken the bacterium rear collection thalline that spends the night, be resuspended in the MS liquid nutrient medium that contains the 100mg/L Syringylethanone to OD600=0.4～0.6,28 and ℃ shake bacterium 1～2h.
The acquisition of 2-2, explant
It is intact to get kind of skin, without the soybean seeds of scab, and 75% ethanol disinfection 30s, 0.1% mercuric chloride sterilization 8min, aseptic water washing 3～5 times.
Seed after the sterilization is inoculated in the MS solid medium, behind the 26 ℃ of dark cultivation of cultivation 2d, changes illumination cultivation over to, behind 5～7d, get aseptic seedling and vertically cut from cotyledonary node, keep 1～2mm hypocotyl, cut terminal bud and the lateral bud of sprouting, put into the preculture substratum.
The acquisition of 2-3, gene transformation and resistant plant
The explant of preculture 1d is put into OD600=0.5～0.6 Agrobacterium bacterium liquid contaminate 15min, be seeded in afterwards on the common culture medium, cultivate altogether 3d under 26 ℃ of dark or the low light level.Explant after cultivating altogether changes over to except in the bacterium culture medium.Cultivate 5～7d and change screening culture medium over to, per 10～12d subculture 1 time.When the long 2cm of Multiple Buds, change its cutting-out over to root media.Culture temperature is 26 ℃, photoperiod 18/6h.After regeneration plant is taken root and grown 5 above compound leaves, open culturing bottle, in growth cabinet, behind hardening 4～5d, clean substratum, it is transplanted in the little basin that fills the bacterium matrix of going out, continue to cultivate.
Culture medium prescription is as follows:
Pre-culture medium: MS+3.5mg/L 6-BA, pH 5.8;
Dip-dyeing solution: MS+6.0mg/L 6-BA+200 μ mol/L Syringylethanone (AS), pH 5.4;
Be total to substratum: MS+6.0mg/L 6-BA+200 μ mol/L AS, pH5.8;
Remove bacterium culture medium: MS+0.2mg/L 6-BA+150mg/L cephamycin (cef)+150mg/L Pyocianil (cab), pH 5.8;
Screening culture medium: MS+0.2mg/L 6-BA+150mg/L cef+150mg/L cab+100mg/L kan, pH 5.8;
Root media: MS+1.0mg/L IBA+100mg/L cef+100mg/L cab, pH 5.8.
3, the PCR of transfer-gen plant detects
After regeneration plant is grown up in soil, respectively get blade and use on a small quantity the total DNA of CTAB method extracting, do template with the DNA that extracts, carry out respectively PCR and detect:
Upstream primer (SEQ ID NO.7): 5 '-ATTTCATTTGGAGAGAACACGG-3 ';
Downstream primer (SEQ ID NO.8): 5 '-TCAGACTGGTGTTGGGTTGGATAT-3 '.
Then 1.0% agarose electrophoresis detects, and detected result is seen Fig. 1, has target stripe to occur then representing goal gene and has changed soybean over to.The size of detected purpose band and expection SEQ ID NO:1 in the same size are about 860bp.It is for subsequent use to make SEQ ID NO:1 nucleotide sequence overexpression soybean plant strain.
4, turn the mensuration of TT1 gene soybean fat acid content
Determination of fatty acid adopts the vapor-phase chromatography of fatty acid methyl vinegar, referring to embodiment three.
Five lipid acid indexs that selected soybean oil is measured are usually measured, and the result is as follows:
Table 2 soybean fat acid content cartogram (unit: %)
Can be found out by fatty acid content result statistics, transgenosis type soybean (TT1-1, TT1-2, TT1-3) than non-transgenic type soybean (WT, i.e. wild-type) 2) and linolenic acid (C18: 3) on average exceed 10.21%, 1.09% aspect the content at linolic acid (C18:.All in all, the unsaturated fatty acid content of transgenosis type soybean (oleic acid, linolic acid, linolenic acid) has improved 6.59% than non-transgenic type.
Illustrate that the TT1 gene has improved unsaturated fatty acid content in the soybean, has reduced saturated fatty acid content simultaneously; Therefore turn the TT1 Soybean Genotypes also because its higher unsaturated fatty acid content, lower saturated fatty acid content, more meet the standard of health plant oil, also become the desirable feedstock of preparing biological diesel oil.
Embodiment five: turn the preparation of TT1 gene Sunflower Receptacle
1, goal gene overexpression construction of recombinant plasmid
According to the design of nucleotide sequence shown in SEQ ID NO:1 primer,
Upstream primer (SEQ ID NO.5): 5 '-CGC GGATCCATGTCGGATGATTTGAGTTTATG-3 ',
Downstream primer (SEQ ID NO.6): 5 '-CCGGAGC TCTCAGACTGGTGTTGGGTTGGATAT-3 '.
Through PCR, the nucleotide sequence shown in the complete SEQ ID NO:1 that increases from rape cDNA to PCR product purification (seeing the disclosed data of Qiagen company), carries out the TA clone, and BamH I and Sac I enzyme are cut and identified positive recombinant clone.Positive colony is cut with BamH1 and Sac1 enzyme, and glue reclaims, and is connected (connection site: BamH1 and Sac1) with carrier pBI121, obtains the overexpression recombinant plasmid that contains SEQ ID NO:1.The overexpression recombinant plasmid that will contain SEQ ID NO:1 changes among the Agrobacterium EHA105, screening positive clone on the LB flat board that contains Rifampin (rif) 50mg/L, kantlex (kan) 50mg/L, bacterium colony PCR detection validation.
2, the genetic transformation of Sunflower Receptacle
The preparation of 2-1, Agrobacterium bacterium liquid
Picking contains the Agrobacterium of overexpression recombinant plasmid of SEQ ID NO:1 in containing corresponding antibiotic LB liquid nutrient medium, 28 ℃ are shaken the bacterium rear collection thalline that spends the night, be resuspended in the MS liquid nutrient medium that contains the 100mg/L Syringylethanone to OD600=0.4～0.6,28 and ℃ shake bacterium 1～2h.
The preparation of 2-2, explant
Select full, big or small all seeds of even anosis insect pest, peel off, 70% ethanol infiltrates 1min, aseptic water washing 2 times; The 1%AgNO3 3min that sterilizes, aseptic water washing 3 times; Planting seed on the MS0 solid medium, is germinateed in 28 ℃ of dark, obtain aseptic seedling behind cultivation 36～48h; The root, cotyledon and the leaf primordium that cut aseptic seedling are exposed stem apex, then vertically cut, and the explant that obtains contains shoot apical meristem and 2 half the cotyledon axillalry buds of half.
The acquisition of 2-3, gene transformation and resistant plant
Place bacterium liquid fully to infiltrate 10min the Shoot tip explants of preparation, fully blot the unnecessary bacterium liquid in explant surface with aseptic filter paper after taking out, place on the common culture medium, under 28 ℃ of dark conditions, cultivate altogether 3d, and establish contrast.
Explant behind the common cultivation 3d is moved to 2 weeks of cultivation on the screening culture medium, and then take turns screening (the every wheel for 2 weeks) through 2～3.The resistant buds of selecting is moved on on the root media root induction.
Culture medium prescription is as follows:
The MS0 substratum: MS+2% sucrose+0.8% agar, pH 5.8;
GBA:MS0+0.5mg/L BAP+0.25mg/L IAA+0.1mg/L GA3+30g/L sucrose+0.8% agar, pH 5.8;
Be total to culture medium: GBA+100 μ mol/L Syringylethanone (AS)+30g/L sucrose+0.8% agar, pH 5.8;
Screening culture medium: GBA+400mg/L Pyocianil (cab)+10mg/L Totomycin+30g/L sucrose+0.8% agar, pH 5.8;
Root media: 1/2MS0+0.2mg/L NAA+250mg/L cab+5mg/L Totomycin+30g/L sucrose+0.8% agar, pH5.8.
3, the PCR of transfer-gen plant detects
After regeneration plant is grown up in soil, respectively get blade and use on a small quantity the total DNA of CTAB method extracting, do template with the DNA that extracts, carry out respectively PCR and detect:
Upstream primer (SEQ ID NO.7): 5 '-ATTTCATTTGGAGAGAACACGG-3 ';
Downstream primer (SEQ ID NO.8): 5 '-TCAGACTGGTGTTGGGTTGGATAT-3 '.
Then 1.0% agarose electrophoresis detects, and detected result is seen Fig. 1, has target stripe to occur then representing goal gene and has changed Sunflower Receptacle over to.The size of detected purpose band and expection SEQ ID NO:1 in the same size are about 860bp.It is for subsequent use to make SEQ ID NO:1 nucleotide sequence overexpression Sunflower Receptacle plant.
4, turn the mensuration of TT1 gene Sunflower Receptacle fatty acid content
Determination of fatty acid adopts the vapor-phase chromatography of fatty acid methyl vinegar, referring to embodiment three.
Five lipid acid indexs that selected sunflower seed oil is measured are usually measured, and the result is as follows:
Table 3 sunflower seed oil fatty acid content cartogram (unit: %)
Can be found out by fatty acid content result statistics, transgenosis type Sunflower Receptacle (TT1-1, TT1-2, TT1-3) than non-transgenic type Sunflower Receptacle (WT, i.e. wild-type) 1), linolic acid (C18: 2) on average exceed 2.77%, 7.11% aspect the content at oleic acid (C18:.All in all, the unsaturated fatty acid content of transgenosis type Sunflower Receptacle (oleic acid, linolic acid, linolenic acid) has improved 9.91% than non-transgenic type.
Illustrate that the TT1 gene has improved unsaturated fatty acid content in the sunflower seeds, has reduced saturated fatty acid content simultaneously; Therefore turn TT1 genotype Sunflower Receptacle also because its higher unsaturated fatty acid content, lower saturated fatty acid content, more meet the standard of health plant oil, also become the desirable feedstock of preparing biological diesel oil.
Embodiment six: turn the preparation of TT1 gene cotton
1, goal gene overexpression construction of recombinant plasmid
According to the design of nucleotide sequence shown in SEQ ID NO:1 primer,
Upstream primer (SEQ ID NO.5): 5 '-CGC GGATCCATGTCGGATGATTTGAGTTTATG-3 ',
Downstream primer (SEQ ID NO.6): 5 '-CCGGAGC TCTCAGACTGGTGTTGGGTTGGATAT-3 '.
Through PCR, the nucleotide sequence shown in the complete SEQ ID NO:1 that increases from rape cDNA to PCR product purification (seeing the disclosed data of Qiagen company), carries out the TA clone, and BamH I and Sac I enzyme are cut and identified positive recombinant clone.Positive colony is cut with BamH1 and Sac1 enzyme, and glue reclaims, and is connected (connection site: BamH1 and Sac1) with carrier pBI121, obtains the overexpression recombinant plasmid that contains SEQ ID NO:1.The overexpression recombinant plasmid that will contain SEQ ID NO:1 changes in the Agrobacterium LBA4404.
2, the genetic transformation of cotton
The preparation of 2-1, Agrobacterium bacterium liquid
Picking contains the Agrobacterium of the overexpression recombinant plasmid of SEQ ID NO:1, be inoculated in and contain the 20mg/L Streptomycin sulphate, the 50mg/L kantlex, in the LB liquid nutrient medium of 40mg/L Rifampin, 28 ℃ are shaken the bacterium rear collection thalline that spends the night, be resuspended in the MS liquid nutrient medium that contains the 100mg/L Syringylethanone to OD600=0.6～1,28 and ℃ shake bacterium 1～2h.
The preparation of 2-2, explant
Cotton seeds soaks with 95% vitriol oil sloughs surperficial short flannel, and flushing with clean water is dried in the place, cool place after falling surperficial sulfuric acid.The seed that obtains first with 75% alcohol immersion sterilization 30s, is removed ethanol and with aseptic water washing 1～2 time, soaks 15min with 0.1% mercuric chloride, then wash 5～6 times with sterilized water.Seed after the sterilization put into be lined with double-deck filter paper and by the aseptic bottle of moistening mistake, under 28 ℃～30 ℃ conditions, cultivate.When cultivating hypocotyl length to 1 behind the 2～3d～2cm left and right sides, germinating seed is inserted in the MS solid medium, continue to cultivate.Treating to can be used for when seedling grows to 8～12cm stem apex behind about 5～7d transforms.
The acquisition of 2-3, gene transformation and resistant plant
To grow to the aseptic seedling of 8～12cm, peel off a slice cotyledon with tweezers, after blade scratches apical meristem gently, the absorbent cotton that is moistened with Agrobacterium will be put into seedling stem apex place, behind the dip-dye 20min unnecessary bacterium liquid be sucked.Place on the common substratum, in 22 ℃ of lower cultivations; Behind the 3d, a large amount of aqua sterilisa flushing stem apexs with containing 300mg/L Cef suck excessive moisture with sterilization filter paper and are placed on the recovery media, cultivate 7d under 28 ℃～30 ℃ illumination conditions; After transfer on the screening culture medium, cultivate under the same conditions 20～25d, then every 20d subculture is 1 time, the stem apex or the indefinite bud that survive after the screening are transferred on the root induction substratum, cultivate under the same conditions, 20d follow-up generation 1 time, transfer on the root induction substratum that contains IBA and carry out root induction, adventive root appears behind about 20d.
Culture medium prescription is as follows:
The MSB substratum: MS+B5 is organic;
Be total to substratum: MSB+3% glucose+0.1mg/L 6-BA+0.1mg/L NAA+40mg/L AS+0.2%Phytogel, pH 5.0;
Screening culture medium: MSB+3% glucose+0.1mg/L 6-BA+0.1mg/L NAA+400mg/L Cef+0.2%Phytogel, pH 5.8;
Recovery media: MSB+3% glucose+0.1mg/L 6-BA+0.1mg/L NAA+400mg/L Cef+0.2%Phytogel, pH 5.8;
Root induction substratum: 1/2MS+2% glucose+0.4mg/L IBA+400mg/L Cef+0.2%Phytogel, pH5.8.
3, the PCR of transfer-gen plant detects
After regeneration plant is grown up in soil, respectively get blade and use on a small quantity the total DNA of CTAB method extracting, do template with the DNA that extracts, carry out respectively PCR and detect:
Upstream primer (SEQ ID NO.7): 5 '-ATTTCATTTGGAGAGAACACGG-3 ';
Downstream primer (SEQ ID NO.8): 5 '-TCAGACTGGTGTTGGGTTGGATAT-3 '.
Then 1.0% agarose electrophoresis detects, and detected result is seen Fig. 1, has target stripe to occur then representing goal gene and has changed cotton over to.The size of detected purpose band and expection SEQ ID NO:1 in the same size are about 860bp.It is for subsequent use to make SEQ ID NO:1 nucleotide sequence overexpression cotton plants.
4, turn the mensuration of TT1 gene cotton fatty acid content
Determination of fatty acid adopts the vapor-phase chromatography of fatty acid methyl vinegar, referring to embodiment three.
Five lipid acid indexs that selected Oleum Gossypii semen is measured are usually measured, and the result is as follows:
Table 4 cottonseed oil fatty acid content cartogram (unit: %)
Can be found out by fatty acid content result statistics, transgenosis type cottonseed (TT1-1, TT1-2, TT1-3) than non-transgenic type cottonseed (WT, i.e. wild-type) 1), linolic acid (C18: 2) on average exceed 5.85%, 5.36% aspect the content at oleic acid (C18:.All in all, the unsaturated fatty acid content of transgenosis type cottonseed (oleic acid, linolic acid, linolenic acid) has improved 11.26% than non-transgenic type.
Illustrate that the TT1 gene has improved unsaturated fatty acid content in the cottonseed, has reduced saturated fatty acid content simultaneously; Therefore turn TT1 genotype cotton also because its higher unsaturated fatty acid content, lower saturated fatty acid content, more meet the standard of health plant oil, also become the desirable feedstock of preparing biological diesel oil.
Claims (3)
1.TT1 the purposes of gene in improving the plant unsaturated fatty acid content, the nucleotides sequence that it is characterized in that described TT1 gene are classified as shown in the SEQ ID NO.1 or be the degenerate sequence of sequence shown in the SEQ ID NO.1; Described plant is swede type rape, soybean, cotton or Sunflower Receptacle.
2.TT1 the purposes of the polypeptide of genes encoding in improving the plant unsaturated fatty acid content; The nucleotides sequence of described TT1 gene is classified as shown in the SEQ ID NO.1 or is the degenerate sequence of sequence shown in the SEQ ID NO.1; Described plant is swede type rape, soybean, cotton or Sunflower Receptacle.
3. cultivate the high unsaturated fatty acid plant method, it is characterized in that may further comprise the steps:
(1), the TT1 gene operationally is connected in expression regulation sequence on the carrier after, form the recombinant vectors of described TT1 gene;
(2), the recombinant vectors in the step (1) is changed in the vegetable cell;
(3), obtain transformant through screening, then transformant is cultivated into high unsaturated fatty acid transfer-gen plant or its offspring, the offspring of described plant comprises plant seed and plant tissue:
The nucleotides sequence of described TT1 gene is classified as shown in the SEQ ID NO.1 or is the degenerate sequence of its SEQ ID NO.1;
Described plant is swede type rape, soybean, cotton or Sunflower Receptacle.
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