CN102127540A - Method for improving soybean quality and DNA (Deoxyribonucleic Acid) molecule special for same - Google Patents

Abstract

The invention discloses a method for improving soybean quality and a DNA (Deoxyribonucleic Acid) molecule special for the same, and provides a DNA molecule which is: (1) a DNA molecule shown as a sequence I in a sequence table or (2) a DNA molecule which is hybridized with the DNA sequence defined in (1) under the strict condition and has the same function as the DNA sequence defined in (1) or (3) a DNA molecule having at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent or at least 99 percent of homology and the same function as the DNA sequence defined in (1). An experiment in the invention indicates that a genetically engineered soybean seed of a transgenic soybean delta12-desaturase gene (FAD2-1) silent expression vector shows a high oleic acid phenotype, and transgenic soybean seed oil remarkably differs from wild type soybean in the aspects of oleic acid, linoleic acid, linolenic acid and palmitic acid.

Description

A kind of method and special DNA molecule thereof that improves soybean quality

Technical field

The present invention relates to plant genetic engineering field, relate in particular to a kind of method and special DNA molecule thereof that improves soybean quality.

Background technology

Soybean [Glycine max (L.) Merr.] belongs to annual leguminous herbaceous plant, in the plantation history in existing more than 4700 year of China.Soybean is important oil crops and food crop, is the important source of edible oil and vegetable-protein in the world.Along with expanding economy and growth in the living standard, people not only increase greatly to the demand of soybean, simultaneously the quality of soybean are also had higher requirement.The quality of soybean quality depends primarily on protein in its seed, the content of fat and the component of lipid acid.

There is multiple lipid acid in nature, and daily edible vegetables oil mainly is made up of 5 kinds of lipid acid, accounts for total fatty acids more than 90%, they are: Palmiticacid (C16:0, palmitic acid), stearic acid (C18:0, stearic acid), oleic acid (C18:1, oleic acid), linolic acid (C18:2, linoleic acid), linolenic acid (C18:3, α-linoleicacid).Saturated degree difference has very big influence to the human cholesterol content in the edible fatty acid, Palmiticacid is saturated fatty acid main in the Vegetable oil lipoprotein, studies show that it can make serum LDL-cholesterol raise, and high-caliber LDL-cholesterol can cause atherosclerosis and cardiovascular disorder in the blood.Different with Palmiticacid, stearic acid can not make LDL-cholesterol levels rising in the blood.Unsaturated fatty acids such as oleic acid, linoleic acid plus linolenic acid can reduce the LDL-cholesterol, therefore can reduce the threat of cardiovascular disorder.But polyunsaturated acid long term high temperature and be exposed under the oxidizing condition very unstablely, the two keys in the consaturated oil are oxidized, produce short chain aldehyde, superoxide and ketone derivatives, can produce bad taste like this, make it to be unsuitable for to explode food very soon.These oxidation productss can rapid absorption be gone into blood, destroy the arterial endothelial cell function, quicken arteriosclerosis and form.Therefore, the edible vegetable oil saturated fatty acid content will be lacked as much as possible, polyunsaturated fatty acid (linoleic (18:2 ω 6) and α-linoleic acid (18:3 ω 3)) must satisfy certain ratio simultaneously, and general ω 6/ ω 3 ratios of recommending are 5-10: 1.Oleic acid is a kind of monounsaturated fatty acids that comprises two keys.From the angle of nutrition, oleic acid to healthy and helpful, because oleic acid double key number order is few, and is not easy oxidized than other polyunsaturated fatty acids.The food easier preservation of food of other polyunsaturated fatty acid processing relatively with oleic acid processing.In thermal treatment and frying process, oleic acid is than other polyunsaturated fatty acids more stable (common polyunsaturated fatty acid is a linoleic acid plus linolenic acid in soybean).Tradition prevents that the method for Fatty Acid Oxidation from being the hydrogen treatment to unsaturated fatty acids.Though hydrogen treatment can reduce the number of two keys, hydrogenation process can produce trans fatty acid.Trans fatty acid is unfavorable to health, the same with saturated fatty acid can the elevating blood cholesterol.Therefore, a major objective to oil crops research is to improve their nutritive value by increasing oleic acid and linoleic ratio.Soybean is important oil crops and food crop, is the main source of edible oil and vegetable-protein in the world.Soybean seeds contains about 40% protein and 20% grease.Soybean oil is except containing a spot of C16 lipid acid, and major part is mainly saturated and undersaturated C18 lipid acid.

In China, soybean is the cultivated area maximum, and output occupy second oil crops.In recent years, along with the raising of China's living standards of the people, the demand of soybean significantly increases.Therefore, the improvement soybean varieties becomes the research focus.

Summary of the invention

An object of the present invention is to provide a kind of special DNA molecule that improves soybean quality.

A kind of dna molecular provided by the invention is following 1) or 2) or 3) dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) under stringent condition with 1) dna sequence dna hybridization that limits and dna molecular with identical function;

3) with 1) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have the dna molecular of 99% homology and concrete identical function at least at least.

Sequence 1 in the sequence table is made up of 1818 deoxyribonucleotides, from 5 ' the 1st to 538 terminal deoxyribonucleotide is 538 base forward sequences between soybean Δ 12-delta 8 desaturase genes (FAD2-1) coding region sequence 315～852, the the 539th to 1280 deoxyribonucleotide from 5 ' end is intron (PDK), is 538 base inverted repeats between soybean Δ 12-delta 8 desaturase genes (FAD2-1) coding region sequence 315～852 from 5 ' the 1281st to 1818 deoxyribonucleotide of holding.

Described stringent condition is at 6 * SSC, in the solution of 0.5%SDS, 68 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain described dna molecular also are the scope of protection of the invention.

Described recombinant vectors is that described dna molecular is inserted the recombinant vectors that the SmaI site of pBI121-Lec carrier obtains.

Another object of the present invention provides a kind of method that changes soybean quality.

Method provided by the invention, the FAD2-1 genetic expression in the soybean that comprises the steps: to suppress to set out obtains comparing with the soybean that sets out the purpose soybean that quality improves;

Described quality improves the content that the content that is embodied in unsaturated fatty acids in the described purpose soybean is higher than unsaturated fatty acids in the described soybean that sets out.

The content of described unsaturated fatty acids is the ratio that described unsaturated fatty acids accounts for total fatty acids.

Described unsaturated fatty acids is an oleic acid.

The set out method of the FAD2-1 genetic expression in the soybean of described inhibition comprises the steps: described dna molecular is imported in the described soybean that sets out by described recombinant vectors.

The kind of the described soybean that sets out is black farming 44; Described FAD2-1 gene is in the sequence table shown in the sequence 2.

The application of described dna molecular in reticent soybean FAD2-1 gene and/or inhibition FAD2-1 genetic expression;

Or the application of described recombinant vectors in reticent soybean FAD2-1 gene and/or inhibition FAD2-1 genetic expression; More than using all is the scope of protection of the invention.

Of the present invention experiment showed, FAD2-1 gene process vector construction obtains a carrier that contains FAD2-1 gene, intron and FAD2-1 gene reverse sequence, obtains one and can carry out RNA interferential carrier.This carrier is imported in the soybean, obtain changeing the gmFAD2-1 soybean, compare with the wild-type soybean, this commentaries on classics gmFAD2-1 soybean seeds shows high oleic acid phenotype, the genetically engineered soybean seed is at oleic acid, linolic acid, and linolenic acid and palmitinic acid aspect all significantly are different from the wild-type soybean.

Description of drawings

Fig. 1 is the T-DNA district synoptic diagram of reticent expression vector

Fig. 2 is that agriculture bacillus mediated hypocotyl transforms the PCR detected result that obtains the reticent expression vector genetically engineered soybean of gmFAD2-1

Fig. 3 is that agriculture bacillus mediated hypocotyl transforms the Southern hybridization detected result that obtains the reticent expression vector genetically engineered soybean of gmFAD2-1

Fig. 4 is the lipid acid proximate analysis diagrammatic sketch of wild-type soybean and the reticent expression vector genetically engineered soybean of gmFAD2-1

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Restriction enzyme EcoRV, BamHI, ClaI, KpnI, XhoI and connection, the flat used enzyme of benefit, TA VectorI carrier are all available from TaKaRa company.

Plant expression vector pKANNIBAL is documented in Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants.Plant J 27 (6): among the 581-590, the public can obtain from Institute of Botany, Chinese Academy of Sciences.

Plant expression vector pBI121-Lec is documented in Liu Q, Singh SP, among Green AG (2002) the High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing.Plant Physiology 129:1732-1743, the public can obtain from Institute of Botany, Chinese Academy of Sciences.

One, the structure of the reticent expression vector of soybean Δ 12-delta 8 desaturase genes (FAD2-1)

1. the acquisition of the total RNA of soybean

(the black farming 44 of soybean (Glycine max) is documented in full be group, Du Weiguang, Chen Yi, Luan Xiaoyan, Liu Xinlei, Wang Fengli the soybean of the black farming 44 of kind in greenhouse to get cultivation, the seed selection of the black farming 44 of new soybean varieties and Different Ways of Planting are to the influence of its yield and quality, the Heilungkiang agricultural sciences, 2004,5:1-3, in, the public can obtain from Institute of Botany, Chinese Academy of Sciences.) developmental seed, add the quick grinding powder of liquid nitrogen, place the centrifuge tube of 1.5ml; The Trizol solution that adds 1mL Invitrogen company, whirlpool mixes, and 25 ℃ left standstill 5 minutes; Add 200 μ L chloroforms, build, concuss centrifuge tube 15 seconds, 25 ℃ left standstill 2～3 minutes; 2～8 ℃, centrifugal 15 minutes of 12,000 * g; The careful absorption in the new centrifuge tube of upper strata water to adds 500 μ l isopropanol precipitating RNA, and-20 ℃ left standstill 10 minutes behind the mixing; 2～8 ℃, under 12,000 * g centrifugal 10 minutes, RNA formed a gelatinous precipitate; Remove supernatant liquor, add the aqueous ethanolic solution washing and precipitating of 1mL 75%, vortex suspension RNA precipitation; 2～8 ℃, centrifugal 5 minutes of 7,500 * g; Remove supernatant liquor, dry RNA on super clean bench; Add the aseptic DEPC water of 30～50 μ L, 55～60 ℃ of incubations 10 minutes are with abundant dissolving RNA; Preserve standby down for-20 ℃.

2.gmFAD2-1 the clone of partial sequence

(Gen bank, L43920), design also entrusts Shanghai to give birth to the synthetic following primer of worker, is cloned in 538bp gene segment between the 315th base and the 852nd base according to the soybean FAD2-1 gene order of having reported.Primer sequence is: 315-F:5 '-TTCCACCTCCTTCCTCAA-3 ', 852-R:5 '-ATAGCAGCCAAACCAACC-3 '.

Total RNA being diluted to 0.1 μ g/ μ L, getting 2 μ L, is primer with Oligo dT (18), uses the M-MLV enzyme (catalog number (Cat.No.): M1701) carry out reverse transcription reaction available from Promega (U.S.) company.Concrete operations are undertaken by the specification sheets of the M-MLV enzyme of Promega (U.S.) company, and it is synthetic that Oligo dT (18) primer entrusts Shanghai to give birth to the worker.The sequence of Oligo dT (18) is 5 '-TTTTTTTTTTTTTTTTT-3 '.With the reverse transcription product is that template is carried out the PCR reaction by following system: add in 0.2mL PCR pipe (50 μ L system): 1.95 μ L ddH ₂O, 10 * PCR buffer II, 1.0 μ L, 25mM MgCl ₂1.5 μ L, 250 μ M dNTPs, 2.5 μ L 315-F (10 μ M), 2.5 μ L 852-R (10 μ M), reverse transcription product 1.0 μ L are available from the Pyrobest archaeal dna polymerase (catalog number (Cat.No.): DR005A) (5U/ μ L) 0.3 μ L of TaKaRa company.The pcr amplification condition is: 94 ℃ of pre-sex change 5min; 94 ℃ of 30s; 57 ℃ of 30s; 72 ℃ of 1min; 30 circulations; 72 ℃ are extended 7min.The last 4 ℃ of preservations of the PCR product that obtains.

Above-mentioned PCR product is sent to order-checking, and the result has sequence 1 in the sequence table from 5 ' terminal 1-538 position Nucleotide for this PCR product; With the sequence and the Gen bank of this PCR product, L43920 (sequence 2) comparison, sequence is just the same, and this PCR product is soybean FAD2-1 gene.

3.RNAi the structure of silent carrier

The PCR product two ends that step 2 is obtained add A, and are specific as follows:

Get 7 μ L behind the PCR product purification that step 2 obtains, add 1 μ L Taq archaeal dna polymerase, 10 * reaction buffer and (contain MgCl ₂), add dATP to final concentration 0.2mM, add the Taq archaeal dna polymerase of 5U, adding deionized water to end reaction volume is 10 μ L, hatches 15-30 minute for 70 ℃, promptly obtains adding the PCR product of A;

To add the PCR product of A and pUCm-T vector carrier (available from the green skies, Shanghai Bioisystech Co., Ltd, catalog number (Cat.No.): D2006) connect, obtain carrier, through order-checking, the result is that the sequence in the sequence table 1 is connected the carrier that obtains from 5 ' terminal 1-538 position Nucleotide with the pUCm-T carrier for this carrier, with this carrier called after pUCm-T (FAD2-1).

Obtain the big fragment of carrier with BamH I/Cla I double digestion pKANNIBAL carrier, and the big fragment of this carrier is carried out cohesive end mend that flat (Klenow mends flat, TaKaRa), obtains mending the flat terminal big fragment of carrier;

Use EcoR V and BamH I double digestion pUCm-T (FAD2-1) carrier again, obtain soybean FAD2-1 gene fragment; Be connected with soybean FAD2-1 gene fragment mending the flat terminal big fragment of carrier, obtain carrier, this carrier is checked order, the result is for the carrier of this carrier for sequence in the sequence table 1 is obtained BamH I to the pKANNIBAL carrier and Cla I restriction enzyme site from the Nucleotide shown in the 1281-1818 position of 5 ' end, with this carrier called after pKANNIBAL+538;

Then, use KpnI/Xho I double digestion pUCm-T (FAD2-1) carrier again, reclaim the goal gene small segment, the big fragment of carrier that this small segment is connected to the pKANNIBAL+538 carrier of the same double digestion of process connects, and obtains pKANNIBAL 538+538; Through order-checking, pKANNIBAL 538+538 is for inserting the carrier that obtains among the pKANNIBAL carrier KpnI/Xho I with sequence in the sequence table 1 from the Nucleotide shown in the 1-538 position of 5 ' end.

Cut pKANNIBAL 538+538 with XbaI and XhoI enzyme then, the purpose fragment that obtains is carried out cohesive end and is mended flat, to mend flat purpose fragment through cohesive end is connected with the pBI121-Lec plant expression vector fragment of cutting through the SmaI enzyme, obtain connecting product, transformed into escherichia coli obtains transformant, extracts the plasmid of transformant and sends to order-checking, the result is for the carrier of this plasmid for obtaining between the SmaI site with the 1 insertion pBI121-Lec of the sequence in the sequence table, with this carrier called after pBI121-Lec-FAD2-1; This carrier is reticent expression vector.Fig. 1 is the T-DNA district synoptic diagram of reticent expression vector.

Dna molecular called after gmFAD2-1 in the sequence table shown in the sequence 1, sequence 1 in the sequence table is made up of 1818 deoxyribonucleotides, from 5 ' the 1st to 538 terminal deoxyribonucleotide is 538 base forward sequences between soybean Δ 12-delta 8 desaturase genes (FAD2-1) coding region sequence 315～852, the the 539th to 1280 deoxyribonucleotide from 5 ' end is intron (PDK), is the inverted repeats of 538 bases between soybean Δ 12-delta 8 desaturase genes (FAD2-1) coding region sequence 315～852 from 5 ' the 1281st to 1818 deoxyribonucleotide of holding.

Artificial synthesized sequence 1 is inserted between the SmaI site of pBI121-Lec, and the carrier that obtains is pBI121-Lec-FAD2-1.

Two, change the acquisition of gmFAD2-1 soybean plant strain

1. the acquisition of acceptor soybean hypocotyl

1) test materials: soybean (Glycine max) kind: black farming 44 is designated hereinafter simply as the wild-type soybean.

2) soybean seeds surface sterilization: select anosis, full soybean seeds and be tiled in the uncovered culture dish, put into built-in one moisture eliminator that fills 100mL clorox stoste beaker, slowly add the 4mL concentrated hydrochloric acid along beaker then, the close drying device places the stink cupboard 18h that sterilizes.Seed after the sterilization is placed in the germination medium (500mg/L agar, pH 5.8 for 1/2MS minimum medium+15,000mg/L sucrose+7), places under 25 ℃, the condition of 120 μ Em-2S-1 light intensity (periodicity of illumination 18/6h) and cultivate 5d.

3) preparation of explant:

When cultivating 5d, sprout soybean peel this moment and break, primary leaf (primary leaves) does not also expose cotyledon.In Bechtop, take out soybean, carefully remove kind of a skin, cotyledon is peeled off.With scalpel terminal bud is excised then, the hypocotyl that cuts the 1cm length with apical meristem is as the acceptor soybean hypocotyl.

2. Agrobacterium preparation and conversion

1) Agrobacterium preparation

The competent preparation of Agrobacterium: single bacterium colony (the Hood EE of picking soil agrobacterium tumefaciens EHA105, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants.Transgenic Res 2:208-218, the public can obtain from Institute of Botany, Chinese Academy of Sciences.) be inoculated in 5mL and contain the 50mg/L Rifampin, the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate (contain 10, the 000mg/L Tryptones, 10, the 000mg/L yeast extract, 5,000mg/L sodium-chlor, pH7.0) in, 28 ℃, 250rpm shaking culture spend the night; Be inoculated in 40mL by 1: 100 and contain the 50mg/L Rifampin, continue to cultivate 4-6h in the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate; 4 ℃, 5, the centrifugal 10min of 000rpm removes supernatant; CaCL with 600 μ L ice precooling 0.05mol/L ₂The solution washing thalline; 4 ℃, 5, the centrifugal 10min of 000rpm removes supernatant; CaCL with 200 μ L ice precooling 0.05mol/L ₂Resuspended thalline is in 4 ℃ of preservations.

Plasmid is to the conversion of Agrobacterium: add the gmFAD2-1 silent carrier plasmid DNA of about 0.5 μ g in the competence Agrobacterium, mixing is placed 5min on ice gently; Place liquid nitrogen 3min then; Be transferred to 37 ℃ of incubation 5min rapidly; Add 500 μ L YEP liquid nutrient mediums, 28 ℃ of shaking culture 5-6h; 5, the centrifugal 3min of 000rpm removes most of supernatant liquor, surplus 200 μ L, suspension thalline; Evenly coat the YEP that contains 50mg/L Rifampin, 50mg/L Streptomycin sulphate and 50mg/L kantlex and select on the flat board, be inverted for 28 ℃ and cultivated two days, obtain single bacterium colony.Picking list colony inoculation contains the 50mg/L Rifampin in 5mL, in the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate, 28 ℃, 250rpm shaking culture spend the night, get 1 μ L and carry out the PCR evaluation, primer is 315-F:5 '-TTCCACCTCCTTCCTCAA-3 ', 852-R:5 '-ATAGCAGCCAAACCAACC-3 ', the fragment that the result obtains 538bp is positive bacteria, this positive bacteria is extracted plasmid send to order-checking, the result is pBI121-Lec-FAD2-1 for this plasmid, the positive plasmid of this plasmid contains the positive bacterial strain of bacterial strain of this plasmid, called after EHA105/gmFAD2-1.

2) preparation of carrier-containing reorganization Agrobacterium solution

The above-mentioned reorganization bacterium EHA105/gmFAD2-1 that obtains is seeded in 28 ℃ of shaking culture 24h in the YEP substratum that contains 50mg/L Rifampin, 50mg/L kantlex and 50mg/L Streptomycin sulphate, to increased logarithmic phase (the about 0.6-0.8 of OD600), the bacterium liquid of the bacterium EHA105/gmFAD2-1 that recombinates this moment is orange can be used for transforming.

The bacterium liquid of the reorganization bacterium EHA105/gmFAD2-1 of OD600 0.8 is put into the 30mL centrifuge tube, the centrifugal 10min of 3725rpm, abandon supernatant, thalline at the bottom of the collection tube is total to culture medium (1/2MS minimum medium, 3.9g/L2-morphine acid-ethyl sulfonic acid with liquid, 0.2g/L Sulfothiorine, 400mg/L L-halfcystine, 0.154g/L dithiothreitol (DTT), 200 μ mol/L Syringylethanones, the 4mg/L 6-benzyladenine, the 5mg/L Silver Nitrate, 30,000mg/L sucrose, pH 5.6) resuspended thalline, bacterial concentration is transferred to the 0D value be 0.5,25 ℃ and leave standstill behind the 1h standbyly, obtain containing the reorganization Agrobacterium solution of foreign gene.

2) infect

Will 324 immerse in the reorganization Agrobacterium solution that contain foreign gene of the above-mentioned acquisition of 30mL and infect by the 1 acceptor soybean hypocotyl that obtains, during constantly vibration, the rotating speed of described shaking culture is 130g/min, time of infection is 4h;

3) cultivate altogether

To take out through the soybean hypocotyl after infecting, on aseptic filter paper, blot bacterium liquid, be placed on the common culture medium (add 6, the liquid of 000mg/L agar is culture medium altogether) that is covered with filter paper, 3d under 25 ℃ of shading conditions obtains common cultivation back soybean hypocotyl.

After cultivating altogether soybean hypocotyl is used distilled water flushing 3 times successively, be total to culture medium flushing 4 times with containing Pyocianil and Reflin liquid.

4) bud inducing culture

Soybean hypocotyl after will washing is then transferred to bud inducing culture (1/2MS minimum medium, 4mg/L 6-benzyladenine, 0.2mg/L indolebutyric acid, 30,000mg/L sucrose, 300mg/L Pyocianil, the 300mg/L Reflin, 8,000mg/L agar, the 5mg/L Silver Nitrate, pH 5.8) go up and recover to cultivate, culture temperature is 27 ℃, intensity of illumination is 70 μ Em-2S-1, photoperiod control 18/6h cultivates 6d, obtains inducing the back hypocotyl.

5) screening and culturing

After above-mentioned recovery cultivated induce the back hypocotyl transfer to screening culture medium (the bud inducing culture adds the 100mg/L kantlex, pH5.8) on, subculture once obtains indefinite bud (this moment, indefinite bud length was 2.5cm) after cultivating for 4 weeks weekly.27 ℃ of the temperature of described screening and culturing, intensity of illumination are 70 μ Em ^-2S ^-1, photoperiod control 18/6h light/dark.

6) regeneration plant takes root and transplanting

When treating the indefinite bud elongation for 2.5cm, the base portion of being close to indefinite bud downcuts it, the base portion (being otch) of the indefinite bud that downcuts immersed in the 2mg/L indolebutyric acid aqueous solution soak 5min, change root media (1/2MS minimum medium over to after blotting with aseptic filter paper then, 20,000mg/L sucrose, the 150mg/L Reflin, the 10mg/L kantlex, 0.8% agar, the pH value is 5.6,27 ℃, cultivated 15 days, and obtained having the seedling of the adventive root of 2cm, seedling is taken out, water washes away the substratum that root adheres to, be transplanted to then (turfy soil and vermiculite mass ratio 1: 1) in the composite soil, change the greenhouse over to after 1 week of cultivation of preserving moisture and carry out normal cultivation management, obtain 76 strain T0 for the regeneration soybean plant strain that changes gmFAD2-1.

Adopting uses the same method changes empty carrier pBI121-Lec in the soybean (Glycine max) over to, obtains changeing the empty carrier soybean.Identify through PCR, primer is PDK-89F:5 '-TATAAATATATTGTTTACATAAACA-3 ' and PDK-581R:5 '-ATACTATATAAAATGATAGATCTTG-3 ', the result does not have the intron fragment (PDK) of 493bp, proves not change purpose fragment gmFAD2-1 over to.

Three, change the Molecular Detection of gmFAD2-1 soybean plant strain

1. the PCR of genetically engineered soybean plant screening

1) preparation of transgenic soybean gene group DNA (CTAB method)

Take by weighing 2 gram T0 and grind fast down in liquid nitrogen for the regeneration soybean leaves that changes gmFAD2-1, with powder transfer to the 30mL centrifuge tube; The extraction damping fluid that adds 60 ℃ of preheatings of 6mL (extracts damping fluid: 100mM TrisCl (pH8.0); 20mM EDTA (pH 8.0); 1.4M NaCl; 2%CTAB; 0.3% beta-mercaptoethanol; 2%PVP) change gently mix rearmounted 60 ℃ of water-baths 30 minutes; Take out cool isopyknic chloroform that adds after 25 ℃: primary isoamyl alcohol (24: 1), behind the reversing mixing gently 25 ℃ down 12, centrifugal 10 minutes of 000rpm; Draw supernatant, repeat extracting once; (protecting upward not muddy mutually); The 5M NaCl that adds 1/2 volume in the supernatant liquor adds the 95% cold ethanol of two volumes again behind the mixing, placed 30 minutes for 4 ℃ behind the mixing; 4 ℃ 5, centrifugal 6 minutes of 000rpm; Abandon supernatant, and with 70% cold ethanol washing and precipitating, 37 ℃ drying precipitated; Be dissolved in the 500 μ L TE solution and go in the centrifuge tube of 1.5ml; Add 5 μ L RNaseA, in 37 ℃ of insulations 1 hour; Add isopyknic phenol: chloroform (1: 1), behind the reversing mixing gently, in 12, centrifugal 10 minutes of 000rpm; Use isopyknic phenol: chloroform: extracting is once again for primary isoamyl alcohol (25: 24: 1); Use isopyknic chloroform again: primary isoamyl alcohol (24: 1) extracting once; The 2M sodium acetate that adds 1/10 volume in the supernatant liquor adds the dehydrated alcohol of two volumes again behind the mixing, 4 ℃ of placements are spent the night behind the mixing; 4 ℃ 12, centrifugal 10 minutes of 000rpm; After the 70% ethanol washing and precipitating, 37 ℃ drying precipitated; The distilled water dissolving of 100 μ L obtains DNA, places 4 ℃ of preservations.

2) PCR screening

DNA with said extracted is a template, is primer with PDK-89F:5 '-TATAAATATATTGTTTACATAAACA-3 ' and PDK-581R:5 '-ATACTATATAAAATGATAGATCTTG-3 ', carries out pcr amplification, and the soybean transformant is carried out preliminary screening.It is synthetic that primer PDK-89F and PDK-581R entrust Shanghai to give birth to the worker.The negative contrast of DNA with wild-type soybean and the extraction of commentaries on classics empty carrier soybean.

The result as shown in Figure 2, what show among Fig. 2 is the PCR detected results of wherein 6 strains system, the numbering of swimming lane is followed successively by 1 from left to right among the figure, 2,3,4,5,6,7,8,9, wherein 1 represents the molecular weight marker thing, and 2 expressions are the negative control of template with the DNA that changes the extraction of empty carrier soybean, 3 expressions are the negative control of template with the wild-type soy bean DNA, the different transgenic line of 4 to 9 expressions.

The result is the positive plant that obtains 493bp, obtains the positive T0 of 30 strains altogether for the raw soybean again that changes gmFAD2-1.Soybean and wild-type soybean with commentaries on classics empty carrier pBI121-Lec are that template does not all have the segmental amplification of purpose.

2. the Southern hybridization analysis of genetically engineered soybean

Get above-mentioned PCR and detect the DNA 20 μ gs of male T0, cut with the HindIII enzyme for the regeneration soybean leaves that changes gmFAD2-1.Then through 0.8% agarose gel electrophoresis separate and be transferred to Hybond N+ nylon membrane (Amersham, UK).Use digoxin (DIG) mark with PDK-89F:5 '-TATAAATATATTGTTTACATAAACA-3 ' then, PDK-581R:5 '-ATACTATATAAAATGATAGATCTTG-3 ' makees probe for the PDK fragment (intron) of primer amplification and carries out hybridization analysis.To change empty carrier soy bean DNA and the negative contrast of wild-type soybean, with the positive contrast of plasmid pBI121-Lec-FAD2-1.

Adopt asymmetric PCR method label probe.PCR DIG probe synthetic agent box according to Boehringer Mannheim company carries out following operation: add 5 μ L, 10 * PCR buffer in the 50 μ L reaction systems, 5 μ L, 10 * PCRDIG mix, the 50pmol upstream primer, the 5pmol downstream primer, 0.75 μ L enzyme mixture, the 100pg template DNA, the dNTP with unmarked DIG compares reaction simultaneously.Reaction conditions has change slightly according to different templates.Reaction finishes rear electrophoresis certification mark efficient, and the probe behind the mark should be bigger than the molecular weight of product that does not have mark, and-20 ℃ frozen standby.

The result as shown in Figure 3, what Fig. 3 showed is the southern hybridization detected results of wherein 5 strain systems, the numbering of swimming lane is followed successively by 1 from left to right among the figure, 2,3,4,5,6,7,8, wherein 1 represents the molecular weight marker thing, as positive control, the different PCR of 3 to 7 expressions detects male T0 for the regeneration soybean strain system that changes gmFAD2-1 with plasmid pBI121-Lec-FAD2-1 in 2 expressions, 8 expressions with the DNA that changes the empty carrier soybean as negative control.

From Fig. 3 we as can be seen, PCR detects male T0 and exists the reverse repeating structure that comprises the segmental gmFAD2-1 of PDK for the regeneration soybean plant strain that changes gmFAD2-1, do not hybridize band and change the empty carrier soybean, illustrate that comprising the fragment that gmFAD2-1 oppositely repeats to suppress in the gmFAD2-1 silent carrier has been incorporated in the soybean gene group.Change the empty carrier soybean and do not hybridize band, plasmid pBI121-Lec-FAD2-1 has the hybridization band.Proof obtains the positive T0 of 30 strains altogether for the raw soybean again that changes gmFAD2-1 like this.Wild-type soybean and commentaries on classics empty carrier soybean result do not have significant difference.

Four, change the fatty acid analysis of gmFAD2-1 genetically engineered soybean seed

1. the extraction of total fat

Results are planted and are numbered 1,2,3,4,5 positive T0 for the regeneration soybean mature seed that changes gmFAD2-1 (being T1 for the regeneration soybean seeds that changes gmFAD2-1) in the greenhouse.Mature seed extracts total fat with the nondestructive cotyledon fritter away from the soybean plumular axis end (account for seed 1/10) that cuts of clean operation cutter.The soybean cotyledon fritter last 0.5g of taking by weighing that pulverizes rapidly in liquid nitrogen is moved in the 10ml tool plug glass test tube, in test tube, add the 3mL chloroform immediately: the mixed solution of methyl alcohol (volume ratio is 1: 2), mixing vibrates on eddy mixer, 4 ℃ leave standstill the chloroform of adding 1mL after 10～20 minutes, add the 1M KCl aqueous solution of 1.8mL behind the mixing again, make chloroform in the final extracting solution: methyl alcohol: the volume ratio of KCl is 1: 1: 0.9.Through abundant vibration the last in 2500rpm centrifugal 10 minutes, absorption contained lower floor's chloroform phase of film fat.After concentrating with nitrogen, in-20 ℃ of stored for future use.With wild-type soybean and commentaries on classics empty carrier soybean is contrast.

2. the separation of monoester

The employing thin layer chromatography carries out, and chromatoplate (the G type, 10cm * 10cm) available from Qingdao marine chemical industry factory.Thin-layer chromatography first to the exhibition liquid be chloroform: methyl alcohol: water (volume ratio: 65: 25: 4), second to the exhibition liquid be chloroform: methyl alcohol: strong aqua (volume ratio: 65: 35: 5).After finishing, on chromatoplate, evenly sprays chromatography 0.01% Primulin aqueous acetone solution (acetone: water=60: 40), dry up the back in the down definite position of different film fat in thin plate of ultraviolet lamp (360nm).

3. monoester fatty acid compositional analysis

With blade fixed film fat composition on the silica-gel plate is scraped to pack into and be equipped with in advance in 10 μ L (6 μ g) the 17:0 lipid acid in the target test tube with ground stopper, in test tube, add 1.5mL sulfuric acid methanol solution (5% sulfuric acid+95% methyl alcohol), seal behind the 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 is ended 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.The gas chromatograph 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).By with the known standard product in the retention time of lipid acid (37-component FAME mix, Supelco 47885-U) compare, determine the kind of the lipid acid that methylates in the sample.Heptacanoic acid (17:0 is available from Sigma company) calculates the absolute content of triglyceride as interior mark.With wild-type soybean and commentaries on classics empty carrier soybean is contrast.

The result is as follows: total fatty acids comprises in the wild-type soybean of mensuration and the commentaries on classics gmFAD2-1 soybean seeds: oleic acid, linolic acid, linolenic acid and palmitinic acid, and wherein oleic acid, linolic acid, linolenic acid are unsaturated fatty acidss, palmitinic acid is a saturated fatty acid.The height that unsaturated fatty acids accounts for the ratio of total fatty acids is an important indicator of estimating soybean oil quality quality, and the quality of the high more soybean oil of ratio that unsaturated fatty acids accounts for total fatty acids is good more.

The wild-type soybean is obviously different for the regeneration soybean seeds lipid acid existence of changeing gmFAD2-1 with T1, is specially:

The ratio that the oleic acid of wild-type soybean (C18:1) accounts for total fatty acids is 18.1% (per-cent that accounts for total fatty acids just is equivalent to the numerical value of absolute yield with regard to the soybean oil quality); 5 strain T1 are respectively for the ratio that the regeneration soybean seeds oleic acid that changes gmFAD2-1 accounts for total fatty acids: 81.9%, 79.1%, 73.6%, 71.5%, 80.8%;

The ratio that the linolic acid of wild-type soybean accounts for total fatty acids is 46.4%; 5 strain T1 are respectively for the ratio that the regeneration soybean seeds linolic acid that changes gmFAD2-1 accounts for total fatty acids: 3.4%, 5.8%, 10.4%, 11.7%, 2.3%;

The ratio that the linolenic acid of wild-type soybean accounts for total fatty acids is 11.9%; T1 is 3.5%, 2.9%, 3.9%, 5.4%, 4.1% for the regeneration soybean seeds linolenic acid content that changes gmFAD2-1;

From as can be seen above-mentioned, the ratio of saturated fatty acid descends: the ratio that wild-type soybean palmitinic acid accounts for total fatty acids is 12.3%; 5 strain T1 are respectively for the ratio that the regeneration soybean seeds palmitinic acid that changes gmFAD2-1 accounts for total fatty acids: 7.3%, 8.7%, 7.8%, 7.1%, 8.1%;

With above result's mapping as shown in Figure 4, the result that the lipid acid that wherein last figure is the wild-type soybean is measured; Figure below is to be numbered the result that 1 T1 measures for the lipid acid of the raw soybean again that changes gmFAD2-1.Change gmFAD2-1 soybean seeds oil as can be seen at oleic acid, linolic acid, linolenic acid and palmitinic acid aspect significantly are different from the wild-type soybean.Wild-type soybean and commentaries on classics empty carrier soybean result do not have significant difference.

Claims (10)

1. a dna molecular is following 1) or 2) or 3) dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) under stringent condition with 1) dna sequence dna hybridization that limits and dna molecular with identical function;

3) with 1) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have the dna molecular of 99% homology and concrete identical function at least at least.

2. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain the described dna molecular of claim 1.

3. recombinant vectors according to claim 2 is characterized in that: described recombinant vectors is that the described dna molecular of claim 1 is inserted the recombinant vectors that the multiple clone site of pBI121-Lec carrier obtains.

4. method that changes soybean quality, the FAD2-1 genetic expression in the soybean that comprises the steps: to suppress to set out obtains comparing with the soybean that sets out the purpose soybean that quality improves;

Described quality improves the content that the content that is embodied in unsaturated fatty acids in the described purpose soybean is higher than the described soybean unsaturated fatty acids that sets out.

5. method according to claim 4 is characterized in that:

The content of described unsaturated fatty acids is the ratio that described unsaturated fatty acids accounts for total fatty acids.

6. according to claim 4 or 5 described methods, it is characterized in that:

Described unsaturated fatty acids is an oleic acid.

7. according to arbitrary described method among the claim 4-6, it is characterized in that:

The set out method of the FAD2-1 genetic expression in the soybean of described inhibition comprises the steps: the described dna molecular of claim 1 is imported in the described soybean that sets out by claim 2 or 3 described recombinant vectorss.

8. require arbitrary described method among the 4-7 according to profit, it is characterized in that: the kind of the described soybean that sets out is black farming 44; Described FAD2-1 gene is in the sequence table shown in the sequence 2.

9. the application of the described dna molecular of claim 1 in reticent soybean FAD2-1 gene and/or inhibition FAD2-1 genetic expression.

10. claim 2 or the 3 described recombinant vectorss application in reticent soybean FAD2-1 gene and/or inhibition FAD2-1 genetic expression.

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