CN102277375A - Method for improving alpha-linolenic acid content in transgenic plant seeds - Google Patents
Method for improving alpha-linolenic acid content in transgenic plant seeds Download PDFInfo
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Abstract
The invention discloses a method for improving alpha-linolenic acid content in transgenic plant seeds. The method comprises the steps that: an expression box for specific expression of an omega-3 fatty acid dehydrogenase gene by endosperm is introduced into a plant, and a transgenic plant that has specific expression of the omega-3 fatty acid dehydrogenase gene in endosperm can be obtained through screening; the expression box for specific expression of an omega-3 fatty acid dehydrogenase gene by endosperm comprises a plant endosperm specific promoter and an omega-3 fatty acid dehydrogenase gene connected to the downstream of the promoter. The method of the invention can improve seed quality, and is applicable to large scale production of alpha-linolenic acid and cultivation of transgenic crops that can be inherited stably and have seeds with high alpha-linolenic acid content.
Description
Technical field
The present invention relates to a kind of method that improves alpha-linolenic acid content in the transgenic plant seed, relate to a kind of endosperm specificity promoter that utilizes especially and express the method that the omega-fatty acid dehydrogenase gene improves alpha-linolenic acid content in the transgenic plant seed.
Background technology
Unsaturated fatty acids is the lipid acid of needed by human.Unsaturated fatty acids is divided into two kinds of monounsaturated fatty acids and polyunsaturated fatty acids according to the difference of two key numbers.Position and function according to two keys are divided into polyunsaturated fatty acid ω-3 series and ω-6 series again.Alpha-linolenic acid, EPA, DHA belong to ω-3 series, and linolic acid, gamma-linolenic acid and arachidonic acid belong to ω-6 series.These long chain polyunsaturated fatty acidss are moietys of body tissue microbial film and some vitals (as: pallium, nervous tissue and retina etc.), playing the effect of keeping the cell normal function and increasing body resistance, also is simultaneously the precursor that prostaglandin(PG), prostacyclin and leukotrienes etc. have self instrumentality of strong physiologically active.Omega-fatty acid desaturase (FAD3) is the rate-limiting enzyme of the synthetic serial unsaturated fatty acids of ω-3, can generate alpha-linolenic acid (Fig. 1) (Virginia M.Ursin by the catalysis linolic acid, 2003.J.Nutr.133:4271-4274), alpha-linolenic acid can further be converted into EPA and DHA.Linolic acid also can further be converted into gamma-linolenic acid, arachidonic acid.The mankind can utilize the sugar of absorption and protein to come synthesizing saturated fatty acid and monounsaturated fatty acids, have ω-12, ω-15, ω-18 fatty acid dehydrogenase in the body, can come synthesize polyunsaturated fatty acid by linolic acid and the alpha-linolenic acid that transforms exogenous absorption, but, because there are not ω-3, ω-6 fatty acid dehydrogenases in human body, so these two kinds of lipid acid of linolic acid and alpha-linolenic acid must be from external picked-up.Human in the evolutionary process in 10000000 year, this two classes unsaturated fatty acids ratio maintains (Eaton et al., 1998.World Review ofNutrition and Dietetics.83:12-23) about 1: 1 all the time.And in the agricultural and animal products of current social artificial growth and breed, omega-3 unsaturated fatty acid content is extremely low, the ratio that causes ω-6 and ω-3 in the human body by rose in normal 1: 1 be 15: 1～20: 1 (Simopoulos, 2001.lipids.36suppl:s83-89).Scientific research proves, the lipid acid diet absorption of this serious disproportion can be brought out multiple disease directly or indirectly, as: coronary heart disease, arrhythmia, arteriosclerosis, thrombus, hypertension, allergy, inflammation, senile dementia, dysthymia disorders, visual deterioration, be losing one's memory, osteoporosis, immunity degradation, autism, diabetes, cancer etc. (Stark et al., 2008.Nutr.Rev.66:326-332).Therefore, timely and effective additional omega-3 unsaturated fatty acid is adjusted to proper states with the ratio of ω in the human body-6 and ω-3, is scientific nutrition, returns pressing for of balanced diet structure.
Alpha-linolenic acid is the synthetic precursor of the serial polyunsaturated fatty acids of ω-3 such as EPA, DHA, and the intake of alpha-linolenic acid is directly determining the ratio of ω-6 and the serial unsaturated fatty acids of ω-3.For this reason, the suggestion of United Nations's health organization and world's grain and oil tissue, linolic acid is 5: 1 with the suitable proportion of alpha-linolenic acid in every day meals, the Coming-of-Age Day intake of alpha-linolenic acid be 2.22 restrain (Simopoulos, 2001.lipids.36suppl:s83-89).The existing meals source of alpha-linolenic acid is some abyssal pelagic fishes and some specific spermatophytes, as: rape, soybean, English walnut, flax, purple perilla etc.But, make the intake of alpha-linolenic acid can not satisfy people's dietary needs far away because expensive price of abyssal pelagic fishes and serious day by day heavy metal in sea water pollute, the limited output of spermatophyte.The utilization transgenic method synthesizes and accumulates high-load alpha-linolenic acid in plant seed and can address this problem.Human main food source is food crop, and it belongs to grass mostly, for example paddy rice, wheat, corn etc.Paddy rice is edible in the world most populous, most important food crop.Though it is expressed that the omega-3-aliphatic acid desaturase gene all has in rice paddy seed embryo and endosperm, expression level is consistent and very low relatively.Therefore, alpha-linolenic acid content only accounts for approximately 3/10000ths in the rice paddy seed, and the ratio of linolic acid and alpha-linolenic acid is (Shimada, et al., 2000.Plant Biotechnol.17:43-48) more up to 25: 1.Utilizing transgenic method, improve alpha-linolenic acid content in the food crop seed, is to solve alpha-linolenic acid shortage problem in the meals, and then improves the effective way of health of people.Shimada etc. (2000.Plant Biotechnol.17:43-48) once reported and utilize constitutive promoter overexpression omega-fatty acid dehydrogenase gene in paddy rice can improve alpha-linolenic acid content in the seed.Yet utilize constitutive promoter driving purposes gene whole period of growth and development of plants and plant materials institute in a organized way the high strength expression can bring the potential disadvantageous effect, as: increase transfer-gen plant metabolism burden and unnecessary waste etc., the alpha-linolenic acid content in the Target organ seed is still very low simultaneously.For goal gene is efficiently expressed in plant materials, reduce disadvantageous effect simultaneously again to plant, the research of tissue-specific promoter and application are more and more paid attention to.Recently, applicant clone and evaluation rice endosperm specific expression gluten GluC gene promoter, and the mandate (patent No.: ZL200710098626.0) that patents, the activity of this promotor is active 4.9 times of corn ubiquitin protein promotor ubiqintin-1, and it mainly is expressed in rice paddy seed endosperm centre (Qu et al., 2008.J Exp Bot.59:2417-2424).The rice that common people are eaten is meant the polished rice after the processing, is the endosperm part of rice paddy seed.Therefore, cross expression omega-fatty acid dehydrogenase gene by the rice endosperm specific strong promoter, improve the content of alpha-linolenic acid in the paddy rice polished rice, reduce the ratio of linolic acid and alpha-linolenic acid content simultaneously, thereby the paddy rice that obtains having medical and nourishing function has important and practical meanings.
Summary of the invention
The purpose of this invention is to provide a kind of method that improves alpha-linolenic acid content in the transgenic plant seed.
The method of alpha-linolenic acid content in the raising transgenic plant seed provided by the present invention, be to utilize endosperm specificity promoter to express the method that the omega-fatty acid dehydrogenase gene improves alpha-linolenic acid content in the transgenic plant seed, specifically be that screening obtains the transgenic line of specific expressed described omega-fatty acid dehydrogenase gene in albumen in the expression cassette importing plant with endosperm specific expression omega-fatty acid dehydrogenase gene; The expression cassette of described endosperm specific expression omega-fatty acid dehydrogenase gene comprises the omega-fatty acid dehydrogenase gene that plant endosperm specificity promoter and downstream thereof connect.
In the described method, described endosperm specificity promoter can comprise any plant endosperm specificity expression promoter, for example paddy rice glutenin gene promotor, wheat seed storage protein gene promotor, barley seed storage protein gene promotor, zein spirit-soluble gene promotor etc., but be not limited to these promotors.The sequence of described paddy rice glutenin gene promotor is for being 5 ' the 1-2231 position nucleotide sequences (shown in the sequence 7) of EU264107 (GI:166343763) from Genbank Accession Number, the sequence of described wheat seed glutenin gene promotor is for being 5 ' the 1-965 position nucleotide sequences of AJ577815 (GI:42734748) from Genbank Accession Number, the sequence of described barley seed alcohol soluble protein gene promotor is for being 5 ' the 971-1976 position nucleotide sequences of X52572 (GI:18923) from Genbank Accession Number, and the sequence of described zein spirit-soluble gene promotor is for being 5 ' the 752-1449 position nucleotide sequences of X63667 (GI:22551) from Genbank Accession Number.
In the described method, described omega-fatty acid dehydrogenase gene is the encoding gene that catalysis 16:2 (7,10) or 18:2 (9,12) are converted into the key enzyme of 16:3 (7,10,13) or 18:3 (6,9,12) in the unsaturated fatty acids route of synthesis.All omega-fatty acid desaturases all contain the Histidine bunch of three high conservatives, that is: HXXXH, HXXHH, HXXHH.Described omega-fatty acid dehydrogenase gene can comprise any omega-fatty acid dehydrogenase gene sequence of microorganism, plant, animal-origin, be preferably any omega-fatty acid dehydrogenase gene sequence that comprises plant origin, for example soybean, flax, paddy rice, rape etc.Described omega-fatty acid dehydrogenase gene is preferably paddy rice omega-fatty acid dehydrogenase gene or soybean omega-fatty acid dehydrogenase gene; The sequence of described paddy rice omega-fatty acid dehydrogenase gene is for being 5 ' the 223-1380 position nucleotide sequences of AK071185 (GI:32981208) from Genbank Accession Number; The sequence of described soybean omega-fatty acid dehydrogenase gene is for being 5 ' the 56-1186 position nucleotide sequences of AY204710 (GI:27902572) from Genbank AccessionNumber.
In the described method, described transgenic plant can comprise conventional biological method transformed plant cells or tissue or organs such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity are led, agriculture bacillus mediated or particle gun by using, and obtain transgenic plant cells or tissue or organ and differentiation, regenerated whole plant and clone or its offspring thus.The expression cassette of described endosperm specific expression omega-fatty acid dehydrogenase gene imports in the plant by the plant expression vector that contains this expression cassette.The recombinant expression vector of described plant expression vector for obtaining between the Sma I that the expression cassette of described omega-fatty acid dehydrogenase gene is inserted into the GluC-pGPTV carrier and the Sac I restriction enzyme site.
In the described method, described plant is a grass.Described grass is paddy rice, corn, wheat, Chinese sorghum, oat, barley, rye etc.
Described method for screening comprises: the method screening by Molecular Identification obtains the T0 of the transgenic line of specific expressed described omega-fatty acid dehydrogenase gene in endosperm for plant, with T0 for the plant selfing, resulting T1 is for hereditary segregating population, detect transfer-gen plant T2 for the alpha-linolenic acid content in the seed endosperm by gas chromatographic analysis, filter out the high transgenic line of alpha-linolenic acid content in the seed endosperm.
Described method for screening also is included in the transgenic line that the screening acquisition is isozygotied in the transgenic line that alpha-linolenic acid content is high in the described seed endosperm.
The method of the transfer-gen plant of the genetic stability of high alpha-linolenic acid content in the acquisition seed of the present invention specifically can may further comprise the steps:
1) utilizes the RT-PCR method, separate obtaining the omega-fatty acid dehydrogenase gene.
2) be connected in endosperm specificity promoter with separating the omega-fatty acid dehydrogenase gene that obtains in the step 1), make up plant expression vector.
3) with step 2) in the plant expression vector that builds transform plant receptor tissue, cultivate and obtain T
0For transfer-gen plant.
4) T that obtains in PCR, Southern, Northern, the Western hybridization analysis step 3)
0For transformed plant, screening is contained the T of endosperm specificity promoter and omega-fatty acid dehydrogenase gene simultaneously
0For transformed plant.
5) incubation step 4) the middle T that obtains that screens
0For the resulting T of transformed plant selfing
1For hereditary segregating population, detect transfer-gen plant T by gas chromatographic analysis
2For the alpha-linolenic acid content in the seed endosperm, filter out the high transgenic line of alpha-linolenic acid content in the seed endosperm.
6) filter out the transfer-gen plant of high alpha-linolenic acid content in the seed endosperm that isozygotys in the transgenic line that alpha-linolenic acid content is high from the seed endosperm that step 5) obtains.Wherein, separation that can be by the pcr analysis goal gene can detect alpha-linolenic acid content in the seed endosperm by gas chromatographic analysis.
The present invention creatively utilizes endosperm specificity promoter to drive the omega-fatty acid dehydrogenase gene and expresses specifically in the transgenic plant seed endosperm, the content of alpha-linolenic acid in the transgenic plant seed can be improved, and the transfer-gen plant of alpha-linolenic acid content height in the seed and genetic stability can be obtained.According to the inventive method, from soybean (Glycine max) and paddy rice (Oryza sativa cv.kitaake), separate respectively by RT-PCR and to obtain omega-fatty acid dehydrogenase gene (GmFAD3 and OsFAD3), connect after paddy rice glutenin gene GluC promotor, make up plant expression vector and carry out genetic transformation, obtain transfer-gen plant.Show by gas chromatographic analysis detected result, at the T of genetic stability alpha-linolenic acid content in the seed endosperm
3In transgenic paddy rice, alpha-linolenic acid content reaches as high as 4.86 grams/500 grams in the polished rice of OsFAD3 transgenic paddy rice strain system, and alpha-linolenic acid and linoleic ratio were up to 6.9: 1.Every day, edible 200 gram polished rice just can reach the alpha-linolenic acid Coming-of-Age Day intake standard of United Nations's health organization suggestion.Illustrate that the inventive method can utilize seed to make bio-reactor and produce the necessary omega-3 polyunsaturated fatty acids of human body, obtain the plant seed that alpha-linolenic acid content improves and linoleic acid content reduces, but the genetically modified crops of high alpha-linolenic acid content in the seed of cultivation genetic stability.Method of the present invention and being applied as utilizes researchs such as bio-technology improvement seed quality, molecule medicine farm to lay a good foundation, and has great application prospect.
Further illustrate the present invention below in conjunction with embodiment, and do not constitute the restriction to claim scope of the present invention, used in an embodiment term and abbreviation are general term of those skilled in the art and abbreviation.
Description of drawings
Fig. 1 is ω in Mammals and the plant-6 and ω-3 series fatty acid pathways metabolism synoptic diagram.
Fig. 2 A is a paddy rice omega-fatty acid desaturase structure gene nucleotide sequence.
Fig. 2 B is a paddy rice omega-fatty acid desaturase structure gene aminoacid sequence.
Fig. 3 A is a soybean omega-fatty acid desaturase structure gene nucleotide sequence.
Fig. 3 B is a soybean omega-fatty acid desaturase structure gene aminoacid sequence.
Fig. 4 A is the electrophoretogram of pcr amplification omega-fatty acid desaturase from rice cDNA.
Fig. 4 B is the electrophoretogram of pcr amplification omega-fatty acid desaturase from soybean cDNA.
Fig. 5 merges the structural representation of paddy rice omega-fatty acid dehydrogenase gene carrier GluC-OsFAD3-35S-HPT for the GluC promotor.
Fig. 6 merges the structural representation of soybean omega-fatty acid dehydrogenase gene carrier GluC-GmFAD3-35S-HPT for the GluC promotor.
Fig. 7 is for changeing GluC::OsFAD3 carrier T
0Electrophoretogram for the rice plant pcr analysis.
Fig. 8 is for changeing GluC::GmFAD3 carrier T
0Electrophoretogram for the rice plant pcr analysis.
Fig. 9 A is for changeing the T of GluC::OsFAD3 carrier
0For rice plant is the Southern results of hybridization of probe with the hygromycin gene.
Fig. 9 B is for changeing the T of GluC::GmFAD3 carrier
0For rice plant is the Southern results of hybridization of probe with GmFAD3.
Figure 10 A is for changeing the T of GluC::OsFAD3 carrier
0For rice plant is the Northern results of hybridization of probe with OsFAD3.
Figure 10 B is for changeing the T of GluC::GmFAD3 carrier
0For rice plant is the Northern results of hybridization of probe with GmFAD3.
Figure 11 A is for changeing the T of GluC::OsFAD3 carrier
0Is antigenic Western results of hybridization for rice plant with OsFAD3.
Figure 11 B is for changeing the T of GluC::GmFAD3 carrier
0Is antigenic Western results of hybridization for rice plant with GmFAD3.
Figure 12 A is for changeing GluC::OsFAD3 carrier T
1Electrophoretogram for the rice plant pcr analysis.
Figure 12 B is for changeing GluC::OsFAD3 carrier T
2Electrophoretogram for the rice plant pcr analysis.
Figure 13 A is for changeing GluC::GmFAD3 carrier T
1Electrophoretogram for the rice plant pcr analysis.
Figure 13 B is for changeing GluC::GmFAD3 carrier T
2Electrophoretogram for the rice plant pcr analysis.
Figure 14 A is non-transgenic rice paddy seed gas chromatographic analysis result.
Figure 14 B is T
2For OsFAD3 transgenic paddy rice seed gas chromatographic analysis result.
Figure 15 is T
1, T
2, T
3Gas chromatographic analysis result for alpha-linolenic acid content in transfer-gen plant and the non-transgenic plant.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
One, the acquisition of paddy rice omega-fatty acid dehydrogenase gene
From GenBank, search paddy rice omega-fatty acid desaturase nucleotide sequence (GenBank number is AK071185), design primer amplification paddy rice omega-fatty acid dehydrogenase gene (OsFAD3).For ease of vector construction, on every pair of primer, add two restriction enzyme sites (shown in the underscore) successively.
Paddy rice omega-fatty acid dehydrogenase gene amplimer: forward primer is F2:5 '-AA
CCCGGGATGGTTAAAGACACAAAG-3 ' (
SmaI) (sequence 1), reverse primer is R2:5 '-AA
GAGCTCTCAGTCTCGTTGCGAGTGGA-3 ' (
Sac I) (sequence 2).
With Trizol test kit (available from Invitrogen company), from paddy rice Kitaake (the Oryza sativaL.cv.Kitaake of blooming back 12 days, Qu and Takaiwa, Plant Biotech J 2004,2:113-125, Institute of Botany, Chinese Academy of Sciences preserves) extract total RNA in the blade, according to reverse transcription test kit specification sheets (available from Promega company), with the total RNA of 2 μ g is template, and the first chain cDNA is synthesized in reverse transcription.With the rice cDNA is template, is primer with F2 and R2, carries out pcr amplification paddy rice omega-fatty acid dehydrogenase gene sequence.Reaction system is 25 μ l:cDNA, 1 μ l, forward primer (10pM) 1 μ l, reverse primer (10pM) 1 μ l, dNTP mixture (10mM) 1 μ l, ExTaqDNA polysaccharase 10 * buffer 2.5 μ l, water 18.3 μ l, ExTaqDNA polysaccharase (5U/ μ l) 0.2 μ l.Response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1min, 36 circulations, last 72 ℃, 10min.
Pcr amplification obtains the purpose band (Fig. 4 A, among Fig. 4 A, the 1st swimming lane is a molecular weight standard, the 2nd swimming lane is the omega-fatty acid dehydrogenase gene of paddy rice) of about 1.2kb.Reclaim amplified production, be directly connected on the pMD18-T carrier (available from TaKaRa company), check order, the result shows, the paddy rice omega-fatty acid dehydrogenase gene sequence size that amplification obtains is 1158bp, as shown in Figure 2, this extension increasing sequence be that 5 of AK071185 ' end 223-1380 position nucleotide sequence is in full accord from Genbank Accession Number, amino acid sequence coded is for being the aminoterminal 1-385 amino acids residue (Fig. 2 A, Fig. 2 B) of BAG92361 from Genbank Accession Number.Wherein, square frame indicates among Fig. 2 B is three conservative Histidines of omega-fatty acid desaturase bunch.To check order to detect and show and contain the segmental recombinant vectors called after of paddy rice omega-fatty acid dehydrogenase gene pMD-OSFAD3.
Two, the plant expression vector construction of omega-fatty acid dehydrogenase gene and genetic transformation
1, the GluC promotor merges the plant expression vector construction of paddy rice omega-fatty acid dehydrogenase gene
With Sma I and Sac I double digestion pMD-OsFAD3 plasmid and GluC-pGPTV binary expression vector (Qu etal., 2008.J Exp Bot.59:2417-2424), recovery contains the endonuclease bamhi of the 1158bp of paddy rice omega-fatty acid dehydrogenase gene, and the fragment that recovery is obtained is inserted into the GluC-pGPTV carrier.Contain the GluC promotor among the GluC-pGPTV, it is 5 of EU264107 (GI:166343763) ' end 1-2331 position nucleotide sequence (shown in the sequence 7) that the nucleotides sequence of this GluC promotor is classified as from Genbank Accession Number, and the GluC promotor is a specific expressing promoter in the albumen; Institute of Botany, Chinese Academy of Sciences has preserved the GluC-pGPTV carrier) SmaI and Sac I enzyme recognition site between.Contain paddy rice omega-fatty acid dehydrogenase gene OsFAD3 plant expression vector called after pGPTV-GluC-OsFAD3 (Fig. 5) with what obtain, among the pGPTV-GluC-OsFAD3, the downstream of GluC promotor is connected with paddy rice omega-fatty acid dehydrogenase gene, and the GluC promotor starts the expression of paddy rice omega-fatty acid dehydrogenase gene.
2, change the acquisition of pGPTV-GluC-OsFAD3 and pGPTV-GluC-GmFAD3 plant expression vector paddy rice.
With freeze-thaw method pGPTV-GluC-OsFAD3 is imported Agrobacterium EHA105 (Hood et al., Transgenic Res.1993.2:208-218, Institute of Botany, Chinese Academy of Sciences preserves) in, then by agrobacterium-mediated transformation rice transformation Kitaake embryo callus.Utilize hygromycin selection method (carrying out), obtain 24 strain T0 for transfer-gen plant according to the J.6:271-282 described method of document Hiei et al.1994.Plant.
Utilize PCR method that the commentaries on classics pGPTV-GluC-OsFAD3 paddy rice that above-mentioned screening obtains is carried out the PCR Molecular Detection.Being used to detect the primer that changes pGPTV-GluC-OsFAD3 plant expression vector regeneration plant is: forward primer is F4:5 '-TGTGAATTTAGTGTAGTTTG-3 ' (sequence 3), and reverse primer is R4:5 '-TCAGTCTCGTTGCGAGTGGA-3 ' (sequence 4).The PCR reaction system is 25 μ l:cDNA, 1 μ l, forward primer (10pM) 1 μ l, reverse primer (10pM) 1 μ l, dNTP mixture (10mM) 1 μ l, rTaqDNA polysaccharase 10 * buffer 2.5 μ l, water 18.3 μ l, rTaqDNA polysaccharase (5U/ μ l) 0.2 μ l.Response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1.5min, 36 circulations, last 72 ℃, 10min.The purpose band that pcr amplification obtains 1.6kb is the detection positive.The result shows: obtain 22 strain PCR and detect male commentaries on classics pGPTV-GluC-OsFAD3 paddy rice T0 for plant (Fig. 7).M is a molecular weight standard among Fig. 7, and the 2nd～24 swimming lane is respectively the regeneration plant that changes pGPTV-GluC-OsFAD3 paddy rice expression vector.
PCR detects male T0 for changeing seed that the pGPTV-GluC-OsFAD3 paddy rice tied and being T1 generation by the plant that this seed grows up to, and the rest may be inferred, and T2, T3 represent the 2nd generation of transfer-gen plant and the 3rd generation respectively.
Three, the Molecular Detection of transgenic rice plant
1, PCR detects male T
0The Southern hybridization that generation is changeed the pGPTV-GluC-OsFAD3 rice plant detects
Extract PCR with the CTAB method and identify the T that is positive
0In generation, changeed the genomic dna of pGPTV-GluC-OsFAD3 rice plant blade, about 40 μ g DNA add after the 50 unit abundant enzymes of EcoR I cut, after 0.8% agarose gel electrophoresis separates, DNA is transferred on the nylon membrane (available from Amershampharmacia company) that positively charged examines with the NaOH of 0.4N.Film after the transfer 65 ℃ of prehybridizations 5 hours in the 0.5M sodium phosphate buffer that contains 7% (g/ml) SDS, with (α-
32P) the dCTP random priming carries out probe mark (available from Chinese Fu Rui company).The hybridization probe sequence that is used to change paddy rice omega-fatty acid dehydrogenase gene plant is: 5 '-AACTGCAGGAATTCGCAAGGAATCGGTCAATACA-3 ' (sequence 5); 5 '-AAGGTACCGTCGACTTCTACACAGCCATCGGTC-3 ' (sequence 6).65 ℃ of hybridization were washed film twice in 65 ℃ after 20 hours in containing 2 * SSC of 0.1%SDS, each 15 minutes, wash film once, 15 minutes in 65 ℃ again in 1 * SSC of 0.1%SDS.Press the X-ray sheet after 24～48 hours, radioactive automatic developing is contrast with non-transgenic paddy rice Kitaake.The result is shown in Fig. 9 A.The Southern results of hybridization shows, can both detect the hybridization band in each transgenic line, and in different strain systems, the number of hybridization band and the position of appearance are incomplete same, this omega-fatty acid dehydrogenase gene that shows paddy rice successfully is incorporated in the rice genome, and is separate conversion individuality between each transgenic line.Among Fig. 9 A, the 1st swimming lane is the contrast of non-transgenic plant, and 2～11 swimming lanes are T
0In generation, changeed paddy rice omega-fatty acid dehydrogenase gene plant.
2, PCR detects male T
0The Northern hybridization that generation is changeed the seed of pGPTV-GluC-OsFAD3 paddy rice detects
Extract PCR with the Trizol test kit and detect male T
0In generation, changeed bloom total RNA of back 12 days rice paddy seed of pGPTV-GluC-OsFAD3 rice plant, the total RNA of about 20 μ g transfers to RNA on the nylon membrane (available from Amersham pharmacia company) of positively charged nuclear with 20 * SSC after 1.2% agarose gel electrophoresis separates.With (α-
32P) the dCTP random priming carries out probe mark (available from Chinese Fu Rui company).The hybridization probe sequence that is used to change paddy rice omega-fatty acid dehydrogenase gene plant is: 5 '-AACTGCAGGAATTCGCAAGGAATCGGTCAATACA-3 '; 5 '-AAGGTACCGTCGACTTCTACACAGCCATCGGTC-3 '.65 ℃ of hybridization were washed film twice in 65 ℃ after 20 hours in containing 2 * SSC of 0.1%SDS, each 15 minutes, wash film once, 15 minutes in 65 ℃ again in 1 * SSC of 0.1%SDS.Press the X-ray sheet after 24～48 hours, radioactive automatic developing is contrast with non-transgenic paddy rice Kitaake.The result is shown in Figure 10 A.The Northern results of hybridization shows, in changeing the pGPTV-GluC-OsFAD3 rice paddy seed, compares with the non-transgenic adjoining tree, and this gene presents in various degree raising at transcriptional level.This shows that in the transfer-gen plant seed endosperm GluC promotor can drive the transcription initiation of paddy rice omega-fatty acid dehydrogenase gene effectively, specifically.Among Figure 10 A, the 1st swimming lane is the contrast of non-transgenic plant, and 2～11 swimming lanes are T
0In generation, changeed paddy rice omega-fatty acid dehydrogenase gene plant.
3, transgenic paddy rice T
0Western hybridization for seed detects
From the mature seed that changes the pGPTV-GluC-OsFAD3 paddy rice, extract total protein, after seed fully grinds, add extraction buffer: 0.05M Tris-Hcl, 10% glycerol, 10mM PMSF, pH7.0, at 13000 * g centrifugal 10 minutes, collect supernatant, add sample-loading buffer (12.5mM Tris, pH6.8,20% glycerol, 2% (g/ml) SDS, 0.001% (g/ml) bromophenol blue, 2% (V/V) 0.3M 2-ME), in boiling water, heated 2 minutes.Behind 12% (g/ml) SDS-PAGE gel electrophoresis, albumen is transferred to (available from Millipore company) on the pvdf membrane with semidrying.Film seals successively, rinsing, one anti-, rinsing, two anti-, rinsing, ECL super quick luminescent solution reaction (available from Amersham Biosciences company), compressing tablet, development, photographic fixing, scanning, is contrast with non-transgenic paddy rice Kitaake.The result is shown in Figure 11 A.The Western results of hybridization shows, compares with the contrast of non-transgenic plant, and the protein expression level of the omega-fatty acid dehydrogenase gene of paddy rice has increase in various degree in the transgenic line.Among Figure 11 A, the 1st swimming lane is the contrast of non-transgenic plant, and 2～11 swimming lanes are T
0In generation, changeed paddy rice omega-fatty acid dehydrogenase gene plant.
4, the evaluation of transgenic paddy rice homozygous lines
The T that only contains a copy that Molecular Detection is positive
0Obtain T for transgenic line by selfing
1Generation, T
1Obtain T for transgenic line by selfing
2Generation, detect and screening by PCR method, finally obtain the paddy rice homozygous lines that alpha-linolenic acid content improves.In theory, T
1For homozygous lines proportion in the transgenic line is 1/4, and the heterozygosis strain is that proportion is 2/4.With T
1Corresponding T of generation
2In generation isolating strain not taking place is homozygous lines, and and T
1Corresponding T of generation
2Need only separative strain in generation and be this T
1For strain is heterozygosis strain system.In the self progeny that pure and mild strain is, the proterties that goal gene is controlled can not separated.Proterties neat and consistent between the plant, heredity that can be stable and expression.
Being used to detect the primer that changes pGPTV-GluC-OsFAD3 plant expression vector strain system is: forward primer is F4:5 '-TGTGAATTTAGTGTAGTTTG-3 ', and reverse primer is R4:5 '-TCAGTCTCGTTGCGAGTGGA-3 '.The PCR reaction system is 25 μ l:cDNA, 1 μ l, forward primer (10pM) 1 μ l, reverse primer (10pM) 1 μ l, dNTP mixture (10mM) 1 μ l, rTaqDNA polysaccharase 10 * buffer 2.5 μ l, water 18.3 μ l, rTaqDNA polysaccharase (5U/ μ l) 0.2 μ l.Response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1.5min, 36 circulations, last 72 ℃, 10min.The purpose band that pcr amplification obtains 1.6kb is the detection positive.The result shows: obtain 20 strain PCR and detect male commentaries on classics pGPTV-GluC-OsFAD3 paddy rice T
1For plant (Figure 12 A).M is a molecular weight standard among Figure 12 A, and the 2nd～23 swimming lane is respectively the plant that changes pGPTV-GluC-OsFAD3 paddy rice expression vector.Choose T
1For identifying in the plant that the plant selfing that is positive obtains T
2For plant, obtain unseparated homozygous lines (Figure 12 B) by the PCR screening.M is a molecular weight standard among Figure 12 B, and the 2nd～24 swimming lane is respectively the T that changes pGPTV-GluC-OsFAD3 paddy rice expression vector
2Plant.
Four, the gas chromatographic analysis of transgenic paddy rice seed
1, T
2In generation, changeed the gas chromatographic analysis of pGPTV-GluC-OsFAD3 rice paddy seed alpha-linolenic acid
To change pGPTV-GluC-OsFAD3 paddy rice T
2Grind to form fine powder for mature seed, pack into and be equipped with in advance in the interior target band plug glass test tube of 10 μ l (6 μ g) 17:0 lipid acid, in test tube, seal behind adding 1.5ml sulfuric acid methanol solution (5% sulfuric acid+95% methyl alcohol) inflated with nitrogen.Test tube is placed on 85 ℃ of water-baths 1 hour, the cooling back adds the 1.5ml Skellysolve A and 1ml water stops methylation reaction, and centrifugal 10 minutes of vibration back 2500rpm collects supernatant liquor, nitrogen dries up the back and adds the normal hexane that 20 μ l heavily steam, and gets 1 μ l sample and carries out gas chromatographic analysis.Gas chromatograph device model is the HP6890GC of Hewlett-Packard, and chromatographic column is HPINNOWAX, column length 30m, internal diameter 0.25mm, coat-thickness 0.25 μ m, post oven temperature, degree be set to gradient increased temperature (170～210 ℃, 5 ℃/min), be contrast with non-transgenic paddy rice Kitaake.
Express in the transgenic line of paddy rice omega-fatty acid dehydrogenase gene 10 mistakes, the content Schwellenwert of alpha-linolenic acid is 1.13 ± 0.26g in per 500 gram rice, and maximum is 4.87 ± 0.16g, and mean value is 3.13 ± 0.15g.Compare with the non-transgenic adjoining tree, the content of alpha-linolenic acid has improved 5.5～24.5 times respectively, and mean value is 15.5 times (table 1).In the table 1, contrast and be the basic plant of non-commentaries on classics, 1～10 for changeing the rice plant of GluC::OsFAD3 carrier.
To a T
2In generation, crosses expression paddy rice omega-fatty acid dehydrogenase gene transgenic line and carries out gas chromatographic analysis, and the result shows that compare with the non-transgenic adjoining tree, the peak value of alpha-linolenic acid presents nearly 8 times transition (Figure 14).Wherein, Figure 14 A is the non-transgenic plant, and Figure 14 B is for changeing the rice plant of GluC::OsFAD3 carrier, and what red arrow was indicated is that alpha-linolenic acid goes out the peak position accordingly.
Table 1 T2 is for the gas chromatographic analysis knot that changes alpha-linolenic acid content in GluC::OsFAD3 vector gene and the non-transgenic rice
To changeing the rice plant T of GluC::OsFAD3 carrier
2Carry out statistical analysis for linolenic acid in the transgenic paddy rice seed and linoleic ratio, the result shows that in the non-transgenic adjoining tree, the ratio of alpha-linolenic acid and linoleic acid content is 0.044 ± 0.003; Express in the transgenic line of OsFAD3 gene 10 mistakes, the Schwellenwert of the ratio of alpha-linolenic acid and linoleic acid content is 0.36 ± 0.10, and maximum is 6.85 ± 1.78, and mean value is 2.28 ± 0.38.Compare with the non-transgenic adjoining tree, the ratio of the two has improved 8.2～155.7 times, and mean value is 51.8 times (table 1).In the table 1, contrast and be the basic plant of non-commentaries on classics, 1～10 for changeing the rice plant of GluC::OsFAD3 carrier;
2, T
1, T
2, T
3Gas chromatographic analysis for the transgenic paddy rice seed alpha-linolenic acid
Choose the rice strain of the highest commentaries on classics GluC::OsFAD3 carrier of alpha-linolenic acid content, will change the rice plant T of GluC::OsFAD3 carrier
1, T
2, T
3For going up sample after the step process described in each 10 seeds process step 1, the result shows: at T
1, T
2, T
3In generation, crosses and expresses in the transgenic line of soybean and paddy rice omega-fatty acid dehydrogenase gene, the alpha-linolenic acid content of per 500 gram rice is followed successively by 4.79 grams, 4.90 grams, 4.92 grams (Figure 15), this shows under the GluC promoters driven, cross expression paddy rice omega-fatty acid dehydrogenase gene can genetic stability to T
3Generation.Among Figure 13,1 is the contrast of non-transgenic plant, and 2 for changeing the rice plant of GluC::GmFAD3 carrier, and 3 for changeing the rice plant of GluC::OsFAD3 carrier.
3, T
3Gas chromatographic analysis for alpha-linolenic acid in the transgenic paddy rice polished rice
Choosing the highest preceding 7 strains of rice plant alpha-linolenic acid content of changeing the GluC::OsFAD3 carrier is, each strain system selects a seed, a ripe transgenic paddy rice seed is cut in half, wherein half uses rice polisher (KettElectrical Laboratory, Tokyo, Japan) ground for 30 seconds, carry out the gas chromatographic analysis of alpha-linolenic acid after the processing in half polished rice and corresponding second half brown rice process step 1.
Express in the transgenic line of paddy rice omega-fatty acid dehydrogenase gene 7 mistakes, the content Schwellenwert of alpha-linolenic acid is 1.46 ± 0.10g in per 500 gram rice, and maximum is 4.86 ± 0.17g, and mean value is 3.25 ± 0.20g.Compare with corresponding brown rice, the content of alpha-linolenic acid has improved 1.1 times (table 2) respectively.In the table 2, C1～C7 is for changeing the paddy rice brown rice of GluC::OsFAD3 carrier; P1～P7 is for changeing the paddy rice polished rice of GluC::OsFAD3 carrier.
Table 2.T
3The gas chromatographic analysis result of alpha-linolenic acid content in the paddy rice brown rice of generation commentaries on classics GluC::OsFAD3 carrier and the polished rice
One, the acquisition of soybean omega-fatty acid dehydrogenase gene
From GenBank, search soybean omega-fatty acid desaturase nucleotide sequence (GenBank number is AY204710), design primer amplification soybean omega-fatty acid dehydrogenase gene (GmFAD3).For ease of vector construction, on every pair of primer, add two restriction enzyme sites (shown in the underscore) successively.
Soybean omega-fatty acid dehydrogenase gene amplimer: forward primer is F3:5 '-AACCCGGGATGGCGGCGTCGGCGACCCAG-3 ' (Sma I), and reverse primer is R3:5 '-AAGAGCTCTCACTTGTGCTTAGCATCTTC-3 ' (Sac I).
With Trizol test kit (available from Invitrogen company), (Shen Nong 25104 from the back 12 days soybean that blooms, professor Yu Jianzhang of Agricultural University Of Shenyang is so kind as to give, this kind also can be available from East Asia, Liaoning seed company limited) extract total RNA in the blade, according to reverse transcription test kit specification sheets (available from Promega company), with the total RNA of 2 μ g is template, and the first chain cDNA is synthesized in reverse transcription.With soybean cDNA is template, is primer with F3 and R3, carries out pcr amplification soybean omega-fatty acid dehydrogenase gene sequence.Reaction system is 25 μ l:cDNA, 1 μ l, forward primer (10pM) 1 μ l, reverse primer (10pM) 1 μ l, dNTP mixture (10mM) 1 μ l, ExTaqDNA polysaccharase 10 * buffer 2.5 μ l, water 18.3 μ l, ExTaqDNA polysaccharase (5U/ μ l) 0.2 μ l.Response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1min, 36 circulations, last 72 ℃, 10min.
Pcr amplification all obtains the purpose band of about 1.2kb, and (among Fig. 4 B, the 1st swimming lane is a molecular weight standard, and the 2nd swimming lane is respectively the omega-fatty acid dehydrogenase gene of soybean.)。Reclaim amplified production, be directly connected on the pMD18-T carrier (available from TaKaRa company), check order, the result shows, the soybean omega-fatty acid dehydrogenase gene sequence size that amplification obtains is 1131bp, as shown in Figure 3, this extension increasing sequence be that 5 of AY204710 ' end 56-1186 position nucleotide sequence is in full accord from Genbank Accession Number, amino acid sequence coded is for being aminoterminal 1-376 amino acids residue (Fig. 3 A of AAO24263 from GenbankAccession Number, Fig. 3 B), wherein, square frame indicates among Fig. 3 B is three conservative Histidines of omega-fatty acid desaturase bunch.To check order to detect and show and contain the segmental recombinant vectors called after of soybean omega-fatty acid dehydrogenase gene pMD-GmFAD3.
Two, the plant expression vector construction of omega-fatty acid dehydrogenase gene and genetic transformation
1, the GluC promotor merges the plant expression vector construction of soybean omega-fatty acid dehydrogenase gene
With Sma I and Sac I difference double digestion pMD-GmFAD3 plasmid, reclaim the endonuclease bamhi of the 1131bp that contains soybean omega-fatty acid dehydrogenase gene.The fragment that recovery is obtained is inserted into GluC-pGPTV carrier (Quet al. respectively, 2008.J Exp Bot.59:2417-2424, contain the GluC promotor among the GluC-pGPTV, it is 5 of EU264107 ' end 1-2331 position nucleotide sequence that the nucleotides sequence of this GluC promotor is classified as from Genbank Accession Number, and the GluC promotor is a specific expressing promoter in the albumen) Sma I and Sac I enzyme recognition site between.The plant expression vector called after pGPTV-GluC-GmFAD3 (Fig. 6) that will contain soybean omega-fatty acid dehydrogenase gene GmFAD3.Among the pGPTV-GluC-GmFAD3, the downstream of GluC promotor is connected with soybean omega-fatty acid dehydrogenase gene, and the GluC promotor starts the expression of soybean omega-fatty acid dehydrogenase gene.
2, change the acquisition of pGPTV-GluC-GmFAD3 plant expression vector paddy rice
With freeze-thaw method pGPTV-GluC-GmFAD3 is imported among the Agrobacterium EHA105 (Hood et al., Transgenic Res.1993.2:208-218) jointly, then by agrobacterium-mediated transformation rice transformation Kitaake embryo callus.Utilize hygromycin selection method (carrying out), obtain 24 strain T according to the J.6:271-282 described method of document Hiei et al.1994.Plant
0For transfer-gen plant.
Utilize PCR method that the commentaries on classics pGPTV-GluC-GmFAD3 paddy rice that above-mentioned screening obtains is carried out the PCR Molecular Detection.Being used to detect the primer that changes pGPTV-GluC-GmFAD3 plant expression vector regeneration plant is: forward primer is F5:5 '-TGTGAATTTAGTGTAGTTTG-3 ', and reverse primer is R5:5 '-TCACTTGTGCTTAGCATCTTC-3 '.The PCR reaction system is 25 μ l:cDNA, 1 μ l, forward primer (10pM) 1 μ l, reverse primer (10pM) 1 μ l, dNTP mixture (10mM) 1 μ l, rTaqDNA polysaccharase 10 * buffer 2.5 μ l, water 18.3 μ l, rTaqDNA polysaccharase (5U/ μ l) 0.2 μ l.Response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1.5min, 36 circulations, last 72 ℃, 10min.The purpose band that pcr amplification obtains 1.6kb is the detection positive.The result shows: obtain 22 strain PCR and detect male commentaries on classics pGPTV-GluC-GmFAD3 paddy rice T
0For plant (Fig. 8).M is a molecular weight standard among Fig. 8, and the 2nd～24 swimming lane is respectively the regeneration plant that changes the pGPTV-GluC-GmFAD3 plant expression vector.
PCR detects male T0 for changeing seed that the pGPTV-GluC-GmFAD3 paddy rice tied and being T by the plant that this seed grows up to
1In generation, the rest may be inferred, T
2, T
3Represent the 2nd generation of transfer-gen plant and the 3rd generation respectively.
Three, the Molecular Detection of transgenic rice plant
1, PCR detects male T0 for the Southern hybridization detection of changeing the pGPTV-GluC-GmFAD3 rice plant
Extract PCR with the CTAB method and identify the T that is positive
0In generation, changeed the genomic dna of pGPTV-GluC-GmFAD3 rice plant blade, about 40 μ gDNA add after the 50 unit abundant enzymes of EcoR I cut, after 0.8% agarose gel electrophoresis separates, DNA is transferred on the nylon membrane (available from Amershampharmacia company) that positively charged examines with the NaOH of 0.4N.Film after the transfer 65 ℃ of prehybridizations 5 hours in the 0.5M sodium phosphate buffer that contains 7% (g/ml) SDS, with (α-
32P) the dCTP random priming carries out probe mark (available from Chinese Fu Rui company).The hybridization probe sequence that is used to change soybean omega-fatty acid dehydrogenase gene plant is: 5 '-ATGGTTAAAGACACAAAG-3 '; 5 '-TCAGTCTCGTTGCGAGTGGA-3 '.65 ℃ of hybridization were washed film twice in 65 ℃ after 20 hours in containing 2 * SSC of 0.1%SDS, each 15 minutes, wash film once, 15 minutes in 65 ℃ again in 1 * SSC of 0.1%SDS.Press the X-ray sheet after 24～48 hours, radioactive automatic developing is contrast with non-transgenic paddy rice Kitaake.The result is shown in 9B.The Southern results of hybridization shows, can both detect the hybridization band in each transgenic line, and in different strain systems, the number of hybridization band and the position of appearance are incomplete same, this omega-fatty acid dehydrogenase gene that shows soybean successfully is incorporated in the rice genome, and is separate conversion individuality between each transgenic line.Among Fig. 9 B, the 1st swimming lane is the contrast of non-transgenic plant, and 2～11 swimming lanes are T
0In generation, changeed soybean omega-fatty acid dehydrogenase gene plant.
2, PCR detects male T
0The Northern hybridization that generation is changeed the seed of pGPTV-GluC-GmFAD3 paddy rice detects
Extract T with the Trizol test kit
0In generation, changeed bloom total RNA of back 12 days rice paddy seed of pGPTV-GluC-GmFAD3 rice plant, the total RNA of about 20 μ g transfers to RNA on the nylon membrane (available from Amersham pharmacia company) of positively charged nuclear with 20 * SSC after 1.2% agarose gel electrophoresis separates.With (α-
32P) the dCTP random priming carries out probe mark (available from Chinese Fu Rui company).The hybridization probe sequence that is used to change soybean omega-fatty acid dehydrogenase gene plant is: 5 '-ATGGTTAAAGACACAAAG-3 '; 5 '-TCAGTCTCGTTGCGAGTGGA-3 '.65 ℃ of hybridization were washed film twice in 65 ℃ after 20 hours in containing 2 * SSC of 0.1%SDS, each 15 minutes, wash film once, 15 minutes in 65 ℃ again in 1 * SSC of 0.1%SDS.Press the X-ray sheet after 24～48 hours, radioactive automatic developing is contrast with non-transgenic paddy rice Kitaake.The result is shown in Figure 10 B.The Northern results of hybridization shows that in the rice paddy seed that changes soybean omega-fatty acid dehydrogenase gene, this gene presents different expression degree at transcriptional level, and does not detect this expression of gene in the non-transgenic adjoining tree; This shows that in the transfer-gen plant seed endosperm GluC promotor can drive the transcription initiation of the omega-fatty acid dehydrogenase gene of paddy rice and soybean effectively, specifically.Among Figure 10 B, the 1st swimming lane is the contrast of non-transgenic plant, and 2～11 swimming lanes are T
0In generation, changeed soybean omega-fatty acid dehydrogenase gene plant.
3, PCR detects male T
0The Western hybridization that generation is changeed the seed of pGPTV-GluC-GmFAD3 paddy rice detects
Detect male T from PCR
0Generation change the pGPTV-GluC-GmFAD3 paddy rice mature seed in extract total protein, after seed fully grinds, add extraction buffer: 0.05M Tris-Hcl, 10% glycerol, 10mM PMSF, pH7.0, at 13000 * g centrifugal 10 minutes, collect supernatant, add sample-loading buffer (12.5mM Tris, pH6.8,20% glycerol, 2% (g/ml) SDS, 0.001% (g/ml) bromophenol bule, 2% (V/V) 0.3M 2-ME), in boiling water, heated 2 minutes.Behind 12% (g/ml) SDS-PAGE gel electrophoresis, albumen is transferred to (available from Millipore company) on the pvdf membrane with semidrying.Film seals successively, rinsing, one anti-, rinsing, two anti-, rinsing, ECL super quick luminescent solution reaction (available from Amersham Biosciences company), compressing tablet, development, photographic fixing, scanning, is contrast with non-transgenic paddy rice Kitaake.The result is shown in Figure 11 B.The Western results of hybridization shows that the omega-fatty acid dehydrogenase gene of soybean all has protein expression in various degree in different transgenic lines, and does not detect this expression of gene in the non-transgenic adjoining tree.Among Figure 11 B, the 1st swimming lane is the contrast of non-transgenic plant, and 2～11 swimming lanes are T
0In generation, changeed soybean omega-fatty acid dehydrogenase gene plant.
4, the evaluation of transgenic paddy rice homozygous lines
Being used to detect the primer that changes pGPTV-GluC-GmFAD3 plant expression vector strain system is: forward primer is F5:5 '-TGTGAATTTAGTGTAGTTTG-3 ', and reverse primer is R5:5 '-TCACTTGTGCTTAGCATCTTC-3 '.The PCR reaction system is 25 μ l:cDNA, 1 μ l, forward primer (10pM) 1 μ l, reverse primer (10pM) 1 μ l, dNTP mixture (10mM) 1 μ l, rTaqDNA polysaccharase 10 * buffer 2.5 μ l, water 18.3 μ l, rTaqDNA polysaccharase (5U/ μ l) 0.2 μ l.Response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1.5min, 36 circulations, last 72 ℃, 10min.The purpose band that pcr amplification obtains 1.6kb is the detection positive.The result shows: obtain 19 strain PCR and detect male commentaries on classics pGPTV-GluC-GmFAD3 paddy rice T
1For plant (Figure 13 A).M is a molecular weight standard among Figure 13 A, and the 2nd～24 swimming lane is respectively the plant that changes the pGPTV-GluC-GmFAD3 plant expression vector.Choose T
1For identifying in the plant that the strain that is positive is that selfing obtains T
2For plant, obtain unseparated homozygous lines (Figure 13 B) by the PCR screening.M is a molecular weight standard among Figure 13 B, and the 2nd～24 swimming lane is respectively the T that changes pGPTV-GluC-GmFAD3 paddy rice expression vector
2Plant.
Four, the gas chromatographic analysis of transgenic paddy rice seed
1, T
2In generation, changeed the gas chromatographic analysis of pGPTV-GluC-GmFAD3 rice paddy seed alpha-linolenic acid
To change pGPTV-GluC-GmFAD3 paddy rice T
2Grind to form fine powder for mature seed, pack into and be equipped with in advance in the interior target band plug glass test tube of 10 μ l (6 μ g) 17:0 lipid acid, in test tube, seal behind adding 1.5ml sulfuric acid methanol solution (5% sulfuric acid+95% methyl alcohol) inflated with nitrogen.Test tube is placed on 85 ℃ of water-baths 1 hour, the cooling back adds the 1.5ml Skellysolve A and 1ml water stops methylation reaction, and centrifugal 10 minutes of vibration back 2500rpm collects supernatant liquor, nitrogen dries up the back and adds the normal hexane that 20 μ l heavily steam, and gets 1 μ l sample and carries out gas chromatographic analysis.Gas chromatograph device model is the HP6890GC of Hewlett-Packard, and chromatographic column is HPINNOWAX, column length 30m, internal diameter 0.25mm, coat-thickness 0.25 μ m, post oven temperature, degree be set to gradient increased temperature (170～210 ℃, 5 ℃/min), be contrast with non-transgenic paddy rice Kitaake.
Change in the pGPTV-GluC-GmFAD3 rice strain at 10, the content Schwellenwert of alpha-linolenic acid is 0.78 ± 0.07g in per 500 gram rice, and maximum is 4.06 ± 0.21g, and mean value is 2.48 ± 0.13g; Compare with the non-transgenic adjoining tree, the content of alpha-linolenic acid has improved 4.0～20.5 times, and mean value is 12.5 times (table 3).In the table 3, contrast and be the basic plant of non-commentaries on classics, 1～10 for changeing the rice plant of GluC::GmFAD3 carrier.
To T
2Carry out statistical analysis for linolenic acid in the transgenic paddy rice seed and linoleic ratio, the result shows that in the non-transgenic adjoining tree, the ratio of alpha-linolenic acid and linoleic acid content is 0.044 ± 0.003; Express in the transgenic line of GmFAD3 gene 10 mistakes, the Schwellenwert of the ratio of alpha-linolenic acid and linoleic acid content is 0.21 ± 0.02, and maximum is 2.88 ± 0.41, and mean value is 1.32 ± 0.15; Compare with the non-transgenic adjoining tree, the ratio of the two has improved 4.8～65.5 times, and mean value is 30.0 times (table 3).
Table 3.T
2In generation, changeed the gas chromatographic analysis result of alpha-linolenic acid content in GluC::GmFAD3 vector gene and the non-transgenic rice
2, T
1, T
2, T
3Gas chromatographic analysis for the transgenic paddy rice seed alpha-linolenic acid
Choose the highest commentaries on classics pGPTV-GluC-GmFAD3 rice strain of alpha-linolenic acid content, will change the T of pGPTV-GluC-GmFAD3 rice strain
1, T
2, T
3For going up sample after the step process described in 10 seeds process steps 1, the result shows: at T
1, T
2, T
3In generation, crosses and expresses in the transgenic line of soybean and paddy rice omega-fatty acid dehydrogenase gene, the alpha-linolenic acid content of per 500 gram rice is followed successively by 3.97 grams, 4.06 grams, 4.03 grams (Figure 15), this shows under the GluC promoters driven, cross the soybean omega-fatty acid dehydrogenase gene of expressing can genetic stability to T
3Generation.Among Figure 15,1 is the contrast of non-transgenic plant, and 2 for changeing the rice plant of GluC::GmFAD3 carrier, and 3 for changeing the rice plant of GluC::OsFAD3 carrier.
Sequence table
<160>7
<210>1
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
aacccgggat?ggttaaagac?acaaag 26
<210>2
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
aagagctctc?agtctcgttg?cgagtgga 28
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
tgtgaattta?gtgtagtttg 20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
tcagtctcgt?tgcgagtgga 20
<210>5
<211>34
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
aactgcagga?attcgcaagg?aatcggtcaa?taca 34
<210>6
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
aaggtaccgt?cgacttctac?acagccatcg?gtc 33
<210>7
<211>2331
<212>DNA
＜213〉paddy rice (Oryza sativa Japonica Group)
<400>7
gttcaagatt?tatttttggt?atttaattta?cttgcttaag?tcagatatat?tcccatcgtt 60
gcaggtttgt?cacttagtat?tattattaag?cgctctagca?ctaggactct?ggataaataa 120
gaaagtttat?tcacgaggct?agagtagtaa?tcaataacat?aagcgtggtg?tctaggtcag 180
cggttatctt?catatgtagt?gtgctccatg?gaaagtgagg?taggaggaag?gtggtgacag 240
tcccgtccgt?cctttgtatc?cctccatgtt?cgggtatatc?atagagctac?aggctagact 300
tagcttggca?gactagggga?gagccggtgc?tcgaagcaat?ccatgaggct?ttacatttaa 360
cataagttag?taaattaacc?cataggaatc?atctctagac?tgaacctacc?agtagttgtg 420
cttggatata?attatattcc?tacatataca?tacacgttcc?ctgcgattag?atacccttgg 480
aatactctaa?ggtgaagtgc?tacagcggta?tccgtgcgct?tgcggattta?tctgtgaccg 540
tatcaaatac?caacaggtag?atacaaggaa?tcatctctcc?tatccattgg?tttatcatct 600
tttaaaatta?tctcttgctc?tcctattgcc?tctgcaactg?cggataggtg?tttctcaaca 660
atgaaggttg?tgaagaatgc?tttgtgcaac?aagatggatg?acaagtatct?cagccatagc 720
ctcatttgct?ttgtagaaaa?ggatatgtcg?gacacaatca?ctaagtatca?ccgtggaaag 780
gatgcactgt?atgccctatc?tatatttacc?atttagtaat?atttatatgg?cttgtgctaa 840
ctttatgttg?tctttacagg?caataacatt?atttggaagg?catatctata?tattactatt 900
taagataatg?taatatctca?aagtttttat?aagctgcaat?gaggtgagtt?tcacttagct 960
ttctaacttg?ttatgagtta?tagatgcatg?ccaccagtca?ttttttatct?tgcatcagcc 1020
cctgcctgtt?agaatatgtt?tctttgtctg?ggagtccatg?tcaactagcc?aatttccaaa 1080
tatatgaaca?aaactatgtg?gcctttgtaa?cccaaatgag?ataaagacta?ctctccatag 1140
aaatttagca?aacatggcac?tcaaagaaaa?tgtgttggat?agtttcatca?tgcatacaaa 1200
agcaacactt?ttgaactacc?attccaaatc?ctttttgtaa?attatctttg?cttaacacta 1260
cccctttgag?caaatgtggc?tttgtgcgga?aaaaactcaa?acttggtagg?gtagacatcc 1320
atttatataa?ttggatccat?gtacataagt?tgttgagtac?ttcaagtact?tacccttgtg 1380
atatacatct?caaatatatt?gaagaagaga?agttcttttt?ttgagagagg?ttgaagaaga 1440
gaagtttgtc?catagctgaa?gaggagtttt?atagtgtcta?gcttaccttg?ctgctgattg 1500
catgtctaaa?atgtcgttta?atttgggcta?taatgaaata?ttcaccaata?tttctgctgg 1560
tctattaaag?tttaatagtt?actcgtaact?catttatttt?gggctataat?ttaatattca 1620
cctatgtttt?tgttagtcta?ttttatttcc?ctagtgtgca?ctagcttaac?cccaaattag 1680
ttttgaacac?ttaacctaaa?tgtgtctatt?atggtcagac?actctctcac?ggcactctaa 1740
caaaaagtga?attttgttgt?tatgtttttg?tcatgatctc?acaagcaatg?tacatgtacg 1800
tttctagagt?gcaatcttat?gctagcctga?ttgtgaattt?agtgtagttt?gttttctctt 1860
tttgtagcta?cactaccaat?aacctattgt?cctctagtca?taccacgtaa?tcacaaggca 1920
aatccctaac?tctcaccttt?aaaagcatgt?ctttattttc?ttgggtggca?ctaatacaaa 1980
atctttttca?gcattcctat?gtgcgatagc?aagaaaacat?ggcataactc?ttgcttcact 2040
ctaacaaaaa?aaacactttt?ccaactttaa?aacaatggta?tctatgtgtt?taatgatcaa 2100
tcaagcatat?aatgacttac?aagtttttac?ctatgccctt?tttgcatcat?cttgtttgca 2160
acagacaaac?tagatattcc?tttaggctat?aaacacatca?gcatgataaa?gagattaggt 2220
aagtttgtta?tccctttttg?catatattct?cgtctactcc?gtgtatataa?gcccctctcc 2280
tccaactcgt?ccatccatca?ccaagagcag?tgggaaacta?agtgaataac?t 2331
Claims (10)
1. method that improves alpha-linolenic acid content in the transgenic plant seed, be that screening obtains the transgenic line of specific expressed described omega-fatty acid dehydrogenase gene in albumen in the expression cassette importing purpose plant with endosperm specific expression omega-fatty acid dehydrogenase gene; The expression cassette of described endosperm specific expression omega-fatty acid dehydrogenase gene comprises the omega-fatty acid dehydrogenase gene that plant endosperm specificity promoter and downstream thereof connect.
2. method according to claim 1 is characterized in that: described endosperm specificity promoter is paddy rice glutenin gene promotor, wheat seed storage protein gene promotor, barley seed storage protein gene promotor or zein spirit-soluble gene promotor; The sequence of described paddy rice glutenin gene promotor is for being 5 ' the 1-2331 position nucleotide sequences of EU264107 from Genbank AccessionNumber, the sequence of described wheat seed storage protein gene promotor is for being 5 ' the 1-965 position nucleotide sequences of AJ577815 from Genbank Accession Number, the sequence of described barley seed storage protein gene promotor is for being 5 ' the 971-1976 position nucleotide sequences of X52572 from Genbank Accession Number, and the sequence of described zein spirit-soluble gene promotor is for being 5 ' the 752-1449 position nucleotide sequences of X63667 from GenbankAccession Number.
3. method according to claim 1 and 2, it is characterized in that: described omega-fatty acid dehydrogenase gene is the encoding gene that derives from the omega-fatty acid desaturase of plant, is preferably the encoding gene of the omega-fatty acid desaturase that derives from soybean, flax, paddy rice or rape.
4. according to any described method among the claim 1-3, it is characterized in that: described omega-fatty acid dehydrogenase gene is paddy rice omega-fatty acid dehydrogenase gene or soybean omega-fatty acid dehydrogenase gene; The sequence of described paddy rice omega-fatty acid dehydrogenase gene is for being 5 ' the 223-1380 position nucleotide sequences of AK071185 from Genbank Accession Number; The sequence of described soybean omega-fatty acid dehydrogenase gene is for being 5 ' the 56-1186 position nucleotide sequences of AY204710 from GenbankAccession Number.
5. according to any described method among the claim 1-4, it is characterized in that: the expression cassette of described endosperm specific expression omega-fatty acid dehydrogenase gene imports in the plant by the plant expression vector that contains this expression cassette.
6. method according to claim 5 is characterized in that: the recombinant expression vector of described plant expression vector for obtaining between the Sma I that the expression cassette of described omega-fatty acid dehydrogenase gene is inserted into the GluC-pGPTV carrier and the Sac I restriction enzyme site.
7. according to any described method among the claim 1-6, it is characterized in that: described method for screening comprises: the T that obtains the transgenic line of specific expressed described omega-fatty acid dehydrogenase gene in endosperm by the method screening of Molecular Identification
0For plant, with T
0For the plant selfing, resulting T
1For hereditary segregating population, detect transfer-gen plant T by gas chromatographic analysis
2For the alpha-linolenic acid content in the seed endosperm, filter out the transgenic line that alpha-linolenic acid content in the seed endosperm is higher than described purpose plant.
8. method according to claim 7 is characterized in that: described method for screening also is included in alpha-linolenic acid content in the described seed endosperm and is higher than screening in the transgenic line of described purpose plant and obtains the transgenic line that isozygotys.
9. method according to claim 7 is characterized in that: the method for described Molecular Identification comprises that PCR identifies and/or Southern hybridization is identified and/or Northern hybridization is identified and/or Western hybridization is identified.
10. according to any described method among the claim 1-9, it is characterized in that: described plant is monocotyledons or endosperm type dicotyledons, is preferably grass, most preferably is paddy rice, corn, wheat, Chinese sorghum, oat, barley or rye.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776213A (en) * | 2012-08-28 | 2012-11-14 | 江南大学 | Desaturase omega 3 Des for biosynthesis of polyunsaturated fatty acids |
| CN104480110A (en) * | 2013-08-30 | 2015-04-01 | 中国农业科学院生物技术研究所 | Corn tissue specific promoter and application thereof |
| CN106032539A (en) * | 2015-03-17 | 2016-10-19 | 中国科学院植物研究所 | Method for culturing safe transgenic plant having increased alpha-linolenic acid content in seeds |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101120091A (en) * | 2004-12-20 | 2008-02-06 | 巴斯福植物科学有限公司 | Nucleic acid molecules encoding fatty acid desaturase genes from plants and methods of use |
-
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101120091A (en) * | 2004-12-20 | 2008-02-06 | 巴斯福植物科学有限公司 | Nucleic acid molecules encoding fatty acid desaturase genes from plants and methods of use |
Non-Patent Citations (3)
| Title |
|---|
| ANAI, T.等: "Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene", 《PLANT CELL REP》 * |
| MARTA VASCONCELOS等: "Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene", 《PLANT SCIENCE》 * |
| QU,LQ.等: "Expression pattern and activity of six glutelin gene promoters in transgenic rice", 《 JOURNAL OF EXPERIMENTAL BOTANY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776213A (en) * | 2012-08-28 | 2012-11-14 | 江南大学 | Desaturase omega 3 Des for biosynthesis of polyunsaturated fatty acids |
| CN102776213B (en) * | 2012-08-28 | 2014-07-30 | 江南大学 | Desaturase omega 3 Des for biosynthesis of polyunsaturated fatty acids |
| CN104480110A (en) * | 2013-08-30 | 2015-04-01 | 中国农业科学院生物技术研究所 | Corn tissue specific promoter and application thereof |
| CN106032539A (en) * | 2015-03-17 | 2016-10-19 | 中国科学院植物研究所 | Method for culturing safe transgenic plant having increased alpha-linolenic acid content in seeds |
| CN106032539B (en) * | 2015-03-17 | 2019-07-26 | 中国科学院植物研究所 | The method for cultivating the safe transgenic plant that alpha-linolenic acid content improves in seed |
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