CN102533804B - Artemisia sphaerocephala krasch delta 12 fatty acid dehydrogenase (As flavin adenine dinucleotide 2 (FAD2)) gene and application - Google Patents

Abstract

The invention discloses the sequence of an artemisia sphaerocephala krasch delta 12 fatty acid dehydrogenase (As flavin adenine dinucleotide 2 (FAD2)) gene and a preparation method and application of the gene sequence. Preparation of the artemisia sphaerocephala krasch FAD2 gene includes that ribonucleic acid (RAN) of artemisia sphaerocephala krasch is transcribed into complementary deoxyribonucleic acid (cDNA) in reverse mode, corresponding degenerate primers and nested primers are designed according to FAD2 gene conserved regions of other plants and amplified to obtain the full-length translation region nucleotide sequence of the artemisia sphaerocephala krasch FAD2 gene, functional verification conducted by using transgenic technology illustrates that gene expression enables linoleic acid of tobacco leaves to be improved by 3.57 times, and freezing resistance of transgenic plants is improved remarkably. The artemisia sphaerocephala krasch FAD2 gene can be applied to transgenic operation of oil crops, forge crops and other plants or non-plants.

Description

Roundhead wormwood Δ 12 fatty acid dehydrogenase (As FAD2) gene and purposes

Technical field

The present invention relates to a kind of new gene order, and the preparation method of this gene order and purposes, the present invention relates to Roundhead wormwood Δ 12 fatty acid dehydrogenase (As FAD2) and preparation thereof and purposes exactly.

Background technology

Δ 12 fatty acid dehydrogenase (FAD2) is the key enzyme that plant controls linolic acid synthesis, utilize the FAD2 gene of corresponding plants to carry out gene recombination, beyond the transgenic plant obtained or plant, other genetically modified organism will have new performance by lipid metabolism, cell recognition, immune response, all many-sides such as cold-resistant.Such as: the nucleic acid sequence encoding that relate to the protein that the existence of seed storage compound is correlated with in plant in the nucleic acid molecule of encoding fatty acid desaturase genes from plants disclosed in Chinese invention patent application 200580048074.1 and using method thereof, relates to the purposes in transgenic plant.As: the working method of lipid metabolism related compound, and the method improving oil level and change lipid acid composition in Plants and Seeds; And apply the growth of these new plant polypeptide stimulating plants and/or improve productive rate and/or seed storage, etc. content.Dual-approach gene expression disclosed in Chinese invention patent application 200810186867.5 synchronously suppresses the RNA perturbation technique and the genetic engineering means that describe utilization improvement in the method for acquisition cole with high oleic acid and low erucic acid, it is reticent that interference is carried out in the genetic expression of 1. and 2. going up oleate conversion two pathways metabolisms that are other lipid acid simultaneously, stop oleate conversion to be the lipid acid of other type to greatest extent, realize the method increasing substantially oleic acid content and significantly reduce content of erucic acid.

Nucleotide sequence and the aminoacid sequence of the FAD2 gene of the plants such as peanut, rape, Arabidopis thaliana, parsley, eggplant and cotton has been announced in GenBank.

Summary of the invention

The invention provides a kind of FAD2 gene taking from psammophyte-Roundhead wormwood, and the Preparation method and use of this gene.

Roundhead wormwood FAD2 gene order of the present invention is the SEQ 1 in gene order table.

Roundhead wormwood FAD2 gene preparation method involved in the present invention is: the RNA first extracting Roundhead wormwood, then reverse transcription is cDNA; The nucleotide sequence of the FAD2 gene of other plant of having announced in GenBank and aminoacid sequence is utilized to carry out homology analysis, according to conserved regions design synthesis pair of degenerate primers P1, P2; With the Roundhead wormwood cDNA obtained for template amplification obtains Roundhead wormwood FAD2 core fragment nucleotide sequence; The FAD2 gene core fragment sequence obtained according to checking order designs Roundhead wormwood FAD2 gene specific primer 5 ' end Outside primer P3 and nested primer P4 respectively, respectively with GeneRacer ^tM5 ' P and the 5 ' NP carried in test kit matches, and with Roundhead wormwood cDNA for template, carries out 5 ' outside and nested PCR amplification reaction, obtains 5 ' end nucleotide acid sequence; The Roundhead wormwood FAD2 gene core fragment sequence obtained according to checking order equally designs Roundhead wormwood FAD2 gene specific primer 3 ' end Outside primer P5 and nested primer P6 respectively, respectively with GeneRacer ^tM3 ' P and the 3 ' NP carried in test kit matches, and with Roundhead wormwood cDNA for template, carries out 3 ' outside and nested PCR amplification, obtains 3 ' end nucleotide acid sequence; According to Roundhead wormwood AsFAD25 ' and 3 ' the end nucleotide acid sequence be cloned into, design and primer P7, P8 of this gene coding region two terminal specific, increase and obtain the total length translated region nucleotide sequence of Roundhead wormwood FAD2 gene.Add BamHI and SacI restriction enzyme site sequence respectively at 5 ' end of primer, gene coding region and the plant expression vector of being convenient to the later stage are recombinated.

Experiment shows, build the expression vector of AsFAD2, the Transgenic plant leaf Linoleic acid content obtained after transfect tobacco improves 3.57 times; In 12 hours periods that frozen stress (2 DEG C) carries out, transgenic plant frost resistance obviously raises.

It is reported, the increase of unsaturated fatty acids and Genes For Plant Tolerance biology (pathogenic agent, phytopathogen do mutually) and abiotic stress (low temperature, arid, salt, heavy metal etc.) closely related, see: Robert G.Upchurch " Fatty acid unsaturation; mobilization; and regulation in the response of plants to stress " Biotechnol Lett (2008) 30:967-977, therefore, Roundhead wormwood Δ-12 fatty acid dehydrogenase gene likely plays a role in these processes.

The relevant primer that the present invention specifically adopts is by the precious biosynthesizing (precious biotechnology company limited, No. 19, Dongbei 2ed Street ,Economic Development Zone ,Dalian City ,Liaoning Province, postcode 116600) in Dalian, and sequence is as follows:

P1：5’-AARAARGYYATYCCRCCVCAYTG-3’，

P2：5’-TKCCAYCRYIKSATWATRWKG-3’；

P3 is: 5 '-ACATTCATGAGCAATAAGCCAAAGACCC-3 ',

P4 is: 5 '-TGAGGGAGCTGTGGAATATAAGTTGTGG-3 ',

5 ' P is: 5 '-CGACTGGAGCACGAGGACACTGA-3 ',

5 ' NP is: 5 '-GGACACTGACATGGACTGAAGGAGTA-3 ';

P5 is: 5 '-ACATTCATGAGCAATAAGCCAAAGACCC-3 ',

P6 is: 5 '-TGAGGGAGCTGTGGAATATAAGTTGTGG-3 ',

3 ' P is: 5 '-GCTGTCAACGATACGCTACGTAACG-3 ',

3 ' NP is: 5 '-CGCTACGTAACGGCATGACAGTG-3 '.

P7：5’-AGAGGATCCATGGGCGCTGGT-3’

P8：5’-GCGGAGCTCTCAGTCGTACTTGCTTC-3’

Wherein: R=Aor G; Y=C or T; V=A or C or G; K=G orT; S=C or G; W=Aor T.

Roundhead wormwood FAD2 gene involved in the present invention can be applied in the transgenosis work of oil crops, also can apply in the transgenosis work of the crops such as herbage, can do to apply in the transgenosis work of other plant of beyond the region of objective existence except oil plant crop and grass etc., also can apply in except the transgenosis work of other biology exophytic.

Roundhead wormwood (Artemisia sphaerocephala krasch) is a kind of excellent super non-irrigated raw sand binding plant, it is one of important feed of the husky pastoral industry of China, it is the endemic plant in NORTHWEST CHINA, North China, desert and semidesert area, northeast, its anti-blown sand, drought-enduring, cold-resistant, impoverishment tolerant performance is extremely strong.All containing ethyl linoleate in white sand mugwort, seed, linolic acid can be obtained after hydrolysis, Roundhead wormwood seed oil Linoleic acid content can reach more than 80%, total unsaturated fatty acid content can reach 90%, it is the plant that seed oil Linoleic acid content is the highest, see: white Shouning, Yun Xiufang " artemisia oil develops and inquires into " " grain and grease ", 2000 (3): 31-33.Roundhead wormwood seed oil Linoleic acid content is high, and what vegetables oil Linoleic acid content was the highest is exactly artemisia oil (see table 1).

Table 1 artemisia oil and other plant oil linoleic acid content

Oil name	Linoleic acid content (%)	Oil name	Linoleic acid content. (%)
				Artemisia oil	77.4～78.3	Oleum Gossypii semen	40～52
Raisin seed oil	58～78	Sesame oil	35.2～48.4
				Thistle oil	74.5	Peanut oil	33.4～42
Structure offers sacriffices to the gods or the spirits of the dead seed oil	66.6～67.8	Rice pollard oil	28～42
				Sunflower seed oil	66.2	Seabuckthorm Seed Oil	40.3
Wheatgerm oil	44～65	Rice germ oil	38
				Walnut oil	62.2	Sweet oil	3.9～15.0
Barley germs oil	62	Tea-seed oil	11.4
				Fructus Maydis oil	36～62	Palm fibre puts oil	5～11
Soybean oil	49.5～54.5	Oleum Cocois	1.5～2.5

Linolic acid, as a kind of unsaturated fatty acids, have very important nutritive value, and unsaturated fatty acids is one of plant organism essentially consist for human and animal, tool important physiological function.They are the main component of cell membrane lipid, are again important energy substances, or the precursor of some signaling molecules.Such as unsaturated fatty acids is that microbial film function is necessary.Under physiological temp, the polarity glyceride only containing saturated fatty acid can not form bilayer arrangement in microbial film.Introduce the double bond of certain number in the appropriate location of saturated fatty acid, can increase the mobility of film, this is very important to some enzymes be combined on film of activation, has reacted the maintenance ability of biomembrane fluidity under differing temps.We infer because Roundhead wormwood growth is in arid-desert areas, experience the environment stresses such as arid, high temperature, freeze injury for a long time, make himself to evolve out certain defense mechanism, thus improve linolic acid resultant quantity.One of mechanism of action that its linoleic acid content improves is likely that the amino acid of its FAD2 gene nucleotide coding has specific characteristics in catalytic activity.Secondly, the Roundhead wormwood grown under arid, high temperature, freeze injury and the environment stress such as barren has drought resisting, high temperature resistant, resistance toly freezes the quality with impoverishment tolerant.Therefore Roundhead wormwood FAD2 gene is that one can to plant, as oil crops, or pasture crop, or other plant, even other biology of non-plant carries out the best materials of genetic modification.

According to the common practice, generally mostly be to adopt the seed kind of plant to extract RNA.The present invention shows through related experiment, adopting the blade of Roundhead wormwood to extract its RNA comparatively adopts other tissue extraction RNA of Roundhead wormwood more convenient, and the FAD2 gene that the present invention extracts is expressed in blade, therefore keeps membrane lipids fluidity in plant stress-resistance process, plays prior effect; The maximum position that utilizes of herbage is its blade, improves its blade Linoleic acid content, is conducive to domestic animal and searches for food, absorb.Show through repetition test, adopt the blade of Roundhead wormwood, particularly extract with the tender leaf in 3 week age and have fabulous effect.

The total length translated region of Roundhead wormwood FAD2 gene obtained is built carrier for expression of eukaryon, in model plant tobacco, verifies its function, found that its expression improves tobacco linoleic acid content significantly, and significantly increase its resistance to low temperature.

Accompanying drawing illustrates:

Fig. 1: Fig. 3-1.AsFAD2 gene open reading frame amplification agarose gel electrophoresis figure

Note: M:DNA molecular weight standard; 1:AsFAD2 gene open reading frame pcr amplification product 2: positive colony PCR primer

With primer P7 and P8 through RT-PCR method amplification containing the AsFAD2 gene ORF of BamHI and SacI restriction enzyme site, obtain the single bright band of an about 1200bp clearly, consistent with the 1175bp inferred.After this fragment being connected with pSIMPLE-18EcoR V/BAP cloning vector, carry out bacterium colony PCR qualification, the clip size that result obtains and RT-PCR increase consistent (Fig. 1) of obtaining.By this positive colony called after pSI-AsFad2.

Fig. 2: Fig. 3-2.pSI-AsFad2 and PBI121 double digestion figure

Note: M:DNA molecular weight standard; 1:SacI and BamHI double digestion plasmid PBI121; 2:SacI and BamHI double digestion recombinant plasmid pSI-AsFad2;

Double digestion is carried out with SacI and BamHI respectively after positive colony pSI-AsFad2 and plant expression vector pBI121 is extracted plasmid, agarose gel electrophoresis detected result as shown in Figure 2, pSI-AsFad2 has two fragments after double digestion, large fragment is sticky end pSIMPLE-18EcoR V/BAP carrier, and small segment is AsFad2 gene ORF; PBl121 also has two fragments after double digestion, and large fragment is sticky end pBl121 carrier, and small segment is Gus gene fragment, illustrates that plasmid pSI-AsFad2 and pBI121 has cut completely.

Fig. 3: Roundhead wormwood PBI121-AsFad2 plant expression vector construction schematic diagram

Sticky end pBl121 carrier is carried out being connected (Fig. 3) with AsFad2 gene ORF, transformation of E. coli reacts, then positive colony plasmid is carried out specific PCR amplification and restriction enzyme digestion, result as shown in Figure 3-4, the Restriction fragment length of the fragment that PCR amplifies and plasmid is all at about 1200bp, in the same size, tentatively think that AsFad2 gene ORF is correctly connected on PBI121 expression vector.Positive colony bacterium liquid delivers to the raw work order-checking in Shanghai, and sequencing result display goal gene sequence is correct, and direction of insertion is accurate, shows to obtain plant expression vector PBI121-AsFad2.

Fig. 4 recombinant plasmid PBI121-AsFad2 double digestion and bacterium colony PCR identify

Note: M:DNA molecular weight standard; 1:SacI and BamHI double digestion plasmid PBI121-AsFad2; 2: positive colony PCR primer;

Fig. 5 recombinant plasmid PBI121-AsFad2 Agrobacterium GV3101 transforms PCR and detects figure

Note: M:DNA molecular weight standard; 1,2,3,4: positive colony PCR primer

Utilize electroporated method, carrier PBI121-AsFad2 plasmid DNA is imported Agrobacterium GV3101 competent cell, through pcr amplification, detected through gel electrophoresis, the size of the fragment obtained that increases and AsFAD2 gene ORF clip size consistent (Fig. 5).Show that goal gene has been recombinated Agrobacterium, obtain Agrobacterium engineering bacteria.

Fig. 6: the conversion of transgene tobacco and plant regeneration process

Note: A tobacco and Agrobacterium Dual culture; B, C callus induction; D, E Bud polarization; F Furcation defects;

Fig. 7: the linolic acid structural formula that mass spectrum records.

Fig. 8: the winter resistance of transgenic plant is than contrast photo.

Embodiment

The specific embodiment of the present invention and relevant experimental data are below provided:

One, take Roundhead wormwood as material extraction RNA, reverse transcription is cDNA

Roundhead wormwood (Artemisia sphaerocephala) seed picks up from Alxa Zuoqi, Nei Mongol, sowing is in nutrition pot, be placed in greenhouse, cultivate according to a conventional method, during 3 week age, getting the tender blade of children is material, by the raw work (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd in Shanghai, No.698, Xiangmin Road, Chedun Industry District, Songjiang District, Shanghai City, postcode 201611) UNIQ-10 pillar Trizol total serum IgE extraction agent box specification sheets carries out RNA extraction.According to pressing precious biological (precious biotechnology company limited, No. 19, Dongbei 2ed Street ,Economic Development Zone ,Dalian City ,Liaoning Province, postcode 116600) PrimeScript ^tM1st Strand cDNA Synthesis Kit specification sheets carries out reverse transcription, obtains cDNA.

According to the common practice, generally mostly be to adopt the seed kind of plant to extract RNA.But experiment shows, adopt the seed kind of Roundhead wormwood to extract RNA difficulty very, often cannot realize operation, show through repetition test, adopt the blade of Roundhead wormwood, particularly extract with the tender leaf in 3 week age and have fabulous effect.

Two, core fragment RT-PCR increases

Peanut, sesame, rape, Sunflower Receptacle, Arabidopis thaliana, parsley, the nucleotide sequence of the plant FAD2 gene such as eggplant and cotton and the aminoacid sequence announced in GenBank is utilized to carry out homology analysis, according to conserved regions design pair of degenerate primers P1, P2.

Following component configuration PCR reaction solution is added successively in 200 μ l PCR pipe:

Mix gently, PCR reaction conditions is:

The goal gene product that glue purification and recovery obtain, is connected to pGM-T Vector, is converted into E.coliDH5 α competent cell, chooses positive colony and checked order by the raw work in Shanghai after bacterium colony PCR identifies.Obtain length and be approximately 880bp Roundhead wormwood FAD2 core fragment.

Three, the clone (5 '-RACE and 3 '-RACE) of Roundhead wormwood FAD2 gene 5 ' and 3 ' end

Place's amount (carrying out according to Invitrogen RACE test kit operational guidance) before 3.1 Roundhead wormwood total serum IgE reverse transcriptions

3.1.1 Roundhead wormwood total serum IgE dephosphorylation group

Dephosphorylation radical reaction:

(1) getting 1.5ml centrifuge tube is placed on ice, adds following reagent successively:

(2) mix gently, of short duration collected by centrifugation liquid.

(3) 50 DEG C of incubation 1h, to be of short durationly centrifugally placed on ice.

RNA precipitin reaction:

(1) 90 μ l DEPC water, 100 μ l phenol are added: chloroform (25: 24), whirlpool concussion 30s.

The centrifugal 5min of 12000rpm at (2) 20 DEG C, draws supernatant (about 100 μ l) and is transferred in new centrifuge tube.

(3) 2 μ l 10mgml are added successively ^-1mussel Glycogen, 10 μ l 3molL ^-1naAc (pH5.2) and 220 μ l 95% ethanol, put upside down mixing, ice bath 10min.

(4) 4 DEG C, the centrifugal 20min of 12000rpm, abandon supernatant, carefully do not discard precipitation, adds 500 μ l 70% ethanol, put upside down mixing.

(5) 4 DEG C, the centrifugal 20min of 12000rpm, carefully suck ethanol, recentrifuge discards residual ethanol.

Drying precipitated 1-2min at (6) 20 DEG C, adds 7 μ l DEPC water dissolution, for lower step goes cap to react for subsequent use.

3.1.2 Roundhead wormwood mRNA removes cap sequence

Cap is gone to react:

(1) getting 1.5ml centrifuge tube is placed on ice, adds following reagent successively:

(2) mix gently, of short duration collected by centrifugation liquid.

(3) 37 incubation 1h, to be of short durationly centrifugally placed on ice.

RNA precipitin reaction: the same 3.1.1 of method.

3.1.3 the Roundhead wormwood mRNA after cap is gone to be connected with RNA oligo

Ligation:

(1) 0.25 μ g GeneRacer is being contained ^tMadd the above-mentioned reaction solution of 7 μ l in the centrifuge tube of RNA Oligo, mix gently, of short duration collected by centrifugation liquid.

(2) 65 DEG C of incubation 5min, eliminate RNA secondary structure.

(3) ice bath 2min is of short duration centrifugal.

(4) following reagent is added successively:

(5) mix gently, of short duration centrifugal.

(6) 37 DEG C of incubation 1h, to be of short durationly centrifugally placed on ice.

RNA precipitin reaction: the same 3.1.1 of method.Precipitate by 10 μ l DEPC water dissolution.

The synthesis of 3.2 first chain cDNA

(1) in 10 μ l ligated RNA of above-mentioned acquisition, following reagent is added:

(2) mix gently, of short duration collected by centrifugation liquid.

(3) 65 DEG C of incubation 5min to remove RNA secondary structure, ice bath 1min, of short duration collected by centrifugation liquid.

(4) in adding following reagent successively on ice:

(5) mix gently, of short duration collected by centrifugation liquid.

(6) 25 DEG C of incubation 5min, 50 DEG C of incubation 1h, 70 DEG C of incubation 5min, ice bath 2min, of short duration collected by centrifugation liquid.

(7) 1 μ l RNase H (2U μ l is added ^-1), 37 DEG C of incubation 20min, of short duration collected by centrifugation liquid.

(8)-20 DEG C save backup and maybe can carry out 5 ' and 3 ' outside P CR amplified reaction.

3.35 ' end pcr amplification

The FAD2 gene core fragment sequence obtained according to checking order designs 5 ' end Outside primer P3 and nested primer P4 respectively, respectively with GeneRacer ^tM5 ' P and the 5 ' NP carried in test kit matches.With the Roundhead wormwood cDNA obtained described in 3.2 for template carries out 5 ' outside and nested PCR amplification reaction.

5 ' outside P CR amplification

Following component configuration PCR reaction solution is pressed in 200 μ l PCR pipe:

Mix gently, of short duration centrifugal, PCR reaction system is as follows:

Get 5 μ l PCR primer and carry out 1.0% agarose gel electrophoresis, testing goal fragment.

5 ' nested PCR amplification

Following component configuration PCR reaction solution is pressed in 200 μ l PCR pipe:

Mix gently, of short duration centrifugal, PCR reaction system is as follows:

Get 5 μ l PCR primer and carry out 1.0% agarose gel electrophoresis, testing goal fragment.

The target gene 5 ' end PCR primer that glue purification and recovery obtain, is connected to pGM-T Vector, is converted into E.coli DH5 α competent cell, chooses positive colony and checked order by the raw work in Shanghai after bacterium colony PCR identifies.

3.43 ' end pcr amplification

The Roundhead wormwood FAD2 gene core fragment sequence obtained according to checking order designs 3 ' end Outside primer P5 and nested primer P6 respectively, respectively with GeneRacer ^tM3 ' P and the 3 ' NP carried in test kit matches.With the Roundhead wormwood cDNA obtained in 3.2 for template carries out 3 ' outside and nested PCR amplification.

3 ' outside P CR amplification

Following PCR reaction solution is configured successively in 200 μ l PCR pipe:

Of short duration centrifugal after mixing gently, PCR reaction conditions is as follows:

Get 5 μ l PCR primer and carry out 1.0% agarose gel electrophoresis, testing goal fragment.

3 ' nested PCR amplification

Following PCR reaction solution is configured successively in 200 μ l PCR pipe:

Of short duration centrifugal after mixing gently, PCR reaction conditions is as follows:

Get 5 μ l PCR products and carry out 1.0% agarose gel electrophoresis, testing goal fragment.

The target gene 5 ' end PCR primer that glue purification and recovery obtain, is connected to pGM-T Vector, is converted into E.coli DH5 α competent cell, chooses positive colony and checked order by the raw work in Shanghai after bacterium colony PCR identifies.

Through order-checking, 5 ' and 3 ' RACE nested PCR amplification product be respectively 344bp and 512bp, there is partial nucleotide overlapping with aforementioned obtained Roundhead wormwood Δ 12-fatty acid dehydrogenase core fragment, and finding that the fragment that increases out and other plant Δ 12-fatty acid dehydrogenase gene 5 ' and 3 ' are held and had homology with comparison in GeneBank.Therefore, increase obtain 5 ' and 3 ' RACE-PCR product be respectively same cDNA 5 ' and 3 ' hold.

Four, pcr amplification translated region full length sequence

According to Roundhead wormwood AsFAD25 ' and 3 ' the end nucleotide acid sequence be cloned into, design and primer P7, P8 of this gene coding region two terminal specific, increase and obtain the total length translated region nucleotide sequence (Fig. 1) of Roundhead wormwood FAD2 gene.Add BamHI and SacI restriction enzyme site sequence respectively at 5 ' end of primer, gene coding region and the plant expression vector of being convenient to the later stage are recombinated.

Following PCR reaction solution is configured successively in 200 μ l PCR pipe:

Of short duration centrifugal after mixing gently, PCR reaction conditions is as follows:

Get 5 μ l PCR primer and carry out 1.0% agarose gel electrophoresis, testing goal fragment.

The goal gene product that glue purification and recovery obtain, is connected to pGM-T Vector, is converted into E.coliDH5 α competent cell, chooses positive colony and checked order by the raw work in Shanghai after bacterium colony PCR identifies.Obtain the Roundhead wormwood FAD2 full length gene translated region cDNA sequence that total length is 1158bp, see SEQ1 in gene order table.

Five, Roundhead wormwood Δ 12-fatty acid dehydrogenase gene high efficiency plant expression vector structure and the conversion in tobacco

5.1 material

5.1.1 vegetable material: tobacco NC89.

5.1.2 bacterial strain and carrier

Intestinal bacteria TOP10 competent cell, PGM-T carrier cloning test kit is purchased from sky, Beijing root.

Agrobacterium strains GV1301, plant expression vector PBI121 are this room and preserve.

5.1.3 enzyme and molecular biology reagents

UNIQ-10 pillar Trizol total serum IgE extraction agent box is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.PrimeScriptTM 1st Strand cDNA Synthesis Kit, high-fidelity DNA polymerase, TaKaRa DNA Kination Kit are all purchased from TaKaRa.The little extraction reagent kit of plasmid is purchased from TIANGEN Biotech (Beijing) Co., Ltd..DNA fast purifying/recovery test kit, 6-BA, NAA are all purchased from Beijing Ding Guo Bioisystech Co., Ltd.Restriction enzyme, T4DNA ligase enzyme are purchased from Fermentas.Kantlex (Kan), cephamycin (Cefo) and Rifampin (Rif) are purchased from Sigma.Other reagent is domestic analytical pure.Primer synthesis and sequencing are completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

5.1.4 key instrument

The single Bechtop of SW-CJ-1G type, the intelligent growth cabinet of RXZ, ultraviolet spectrophotometer ( nD-1000), constant-temperature shaking incubator (SUKUN SKY-2102C), electric conversion instrument (BLO-RAD Micropulserim).

5.1.5 substratum

YEB substratum; MS substratum

Division culture medium:

MS+Kan(100mg/L)+Cefo(200mg/L)+NAA(0.1mg/L)+6-BA(1.5mg/L)

Root media:

1/2MS+Kan(100mg/L)+Cefo(200mg/L)+NAA(0.02mg/L)

5.2 method

5.2.1 plant expression vector construction (Fig. 2)

5.2.1.1 design of primers and synthesis

According to the Roundhead wormwood fad2 full-length gene nucleotide sequence be cloned into, design and primer P7, P8 of this gene coding region two terminal specific, add BamHI and SacI restriction enzyme site sequence respectively at 5 ' end of primer.

4.2.1.2PCR amplification

Following PCR reaction solution is configured successively in 200 μ l PCR pipe:

Get 5 μ l PCR primer and carry out 1.0% agarose gel electrophoresis, testing goal fragment.

5.2.1.3PCR in reaction solution, 5 ' terminal phosphateization of DNA fragmentation is reacted

With reference to TaKaRa DNA KinationKit test kit specification sheets, method is as follows:

(1) in 200 μ l centrifuge tubes, prepare following reaction solution, full dose is 20 μ l.

(2) 37 DEG C of reaction 30min.

(3) 70 DEG C of heating 5-10min make enzyme deactivation.

(4) get above-mentioned solution electrophoresis on the sepharose of 1.0% of 5 μ l, the DNA in above-mentioned solution is carried out quantitatively thick.

5.2.1.45 ' DNA fragmentation of terminal phosphate and the connection of pSIMPLE-18EcoR V/BAP Vector and conversion

With reference to TaKaRa DNA Kination Kit test kit specification sheets, method is as follows:

(1) in 200 μ l centrifuge tubes, prepare following DNA solution, full dose is 5 μ l.

(2) the Solution I of 5 μ l is added.

(3) 16 DEG C of reaction 1h.

(4) full dose is converted in DH5 α competent cell.

5.2.1.5 the screening of positive colony, qualification and sequential analysis

Choose the recombinant plasmid bacterium liquid called after pSI-AsFad2 that the correct activity of sequential analysis is higher, carry out subsequent experimental.

5.2.1.6pSI-AsFad2 and the plasmid extraction of PBI 121

Carry out with reference to the large extraction reagent kit specification sheets of sky, Beijing root plasmid.

5.2.1.7pSI-AsFad2 cut back to close (Fig. 3) with the enzyme of PBI 121 plasmid

Plasmid pSI-Fad2 containing object fragment and PBI121 plasmid are carried out BamHI and SacI double digestion respectively, and the two method is identical, as follows:

(1) in 200 μ l centrifuge tubes, each reagent is added by the following enzyme system of cutting:

(2) mix, slight of short duration centrifugal, at the bottom of liquid collecting to pipe.

(3) 37 DEG C of incubation 2h.

(4) 1% agarose gel electrophoresis detection enzymes cut effect, recovery enzyme cuts object fragment.Of short duration centrifugal after mixing.

5.2.1.8 endonuclease bamhi connects

(1) in 200 μ l centrifuge tubes, each reagent is added by the following enzyme system of cutting:

(2) mix, slight of short duration centrifugal 3-5 second.

(3) 22 DEG C of incubation 18h.

(4) 65 DEG C, 10min, makes T4DNA Ligase inactivation.

(5) draw 10 μ l to be converted in DH5 α competent cell.

5.2.1.9 the screening of recombinant plasmid, qualification and sequential analysis (Fig. 4)

Choose the recombinant plasmid bacterium liquid bacterium liquid called after PBI121-AsFad2 that the correct activity of a pipe sequential analysis is higher, extract plasmid, carry out subsequent experimental.

5.2.2 the genetic transformation of tobacco

5.2.2.1 the preparation (sorbyl alcohol method) of Agrobacterium GV3101 competent cell

(1) picking list colony inoculation is in 10mL YEB liquid nutrient medium, 28 DEG C of concussion cultivation 14 ~ 16h that spend the night.

(2) 4 DEG C, 6000rpm, 5min, abandon supernatant, collects thalline, aseptic filter paper is controlled dry being placed on ice.

(3) the 1mL 10% glycerine Eddy diffusion thalline of precooling is added, ice-water bath 5min.

(4) 4 DEG C, 6000rpm, 5min, abandon supernatant, collected by centrifugation thalline, and control is dry.

(5) repeating step (4) twice.

(6) with the 1M sorbyl alcohol Eddy diffusion thalline of 0.5mL precooling.

(7) often pipe 40 μ l packing, put into after liquid nitrogen flash freezer-70 DEG C preserve or 4 DEG C save backup.

5.2.2.2 Agrobacterium GV3101 transforms (electric shocking method)

(1) get the GV3101 competent cell that 40 μ l have just dissolved on ice, the PBI121-Fad2 recombinant plasmid adding 10 μ l precoolings on ice flicks mixing.

(2) " A.tumefaciens " in electroporated instrument choosing " bacterial protocol screen ".

(3) sample that step (1) mixes is added the electric shock cup that aseptic, dry precooling treatment is crossed, electric shock cup is put into ShockPod, cover lid.

(4) press after " plus " key and take out electric shock cup, add 1mL YEB liquid nutrient medium immediately, be transferred in 1.5mL centrifuge tube, record and check that whether electric shock time, voltage is correct.

(5) 30 DEG C, 3h is cultivated in 200rpm concussion.

(6) get on YEB solid medium flat board that 500 μ l bacterium liquid are applied to containing kantlex (final concentration 100 μ g/mL) and Rifampin (final concentration 50 μ g/mL), 28 DEG C of light culture 2 ~ 3d, observe transformation efficiency.5.2.2.3 the Agrobacterium PCR containing recombinant plasmid PBI121-Fad2 identifies (Fig. 5)

Single bacterium colony on picking YEB flat board, be inoculated in 2mLYEB liquid nutrient medium (containing kantlex 100 μ g/mL and Rifampin 50 μ g/mL), 28 DEG C of 200rpm light culture 1 ~ 2d, compare with unconverted Agrobacterium, carry out bacterium colony PCR qualification.

5.2.2.4 genetic transformation (leaf disk method) (Fig. 6) of tobacco

(1) the Agrobacterium bacterium liquid containing plant expression vector is evenly coated with and YEB flat board (containing kantlex 100 μ g/mL and Rifampin 50 μ g/mL), 28 DEG C of overnight incubation.

(2) with rifle head scraping thalline gently, proceed in MS liquid nutrient medium, make suspension OD600 ≈ 0.5.

(3) tobacco tests for sterility is cut off leaf margin and main lobe arteries and veins, be cut into 0.5cm size box.

(4) explant cut is soaked 5-10 minute in the Agrobacterium bacterium liquid (OD600 ≈ 0.5) suspended.

(5) blotted by blade surface bacterium liquid on aseptic filter paper, upper surface proceeds to surface down and is covered with on the MS solid medium of one deck filter paper, 28 DEG C of light culture 3 days.

(6) be forwarded on division culture medium by blade from Dual culture base, every two weeks subcultures once.

(7) when resistant buds grows to 2-3cm height, bud is forwarded on root media and carries out root culture.

Six, turn Roundhead wormwood Δ 12-fatty acid dehydrogenase gene tobacco linoleic acid content to measure

6.1 linolic acid pre-treating process:

After tobacco leaf is ground to form powdery in liquid nitrogen, get 1g (being accurate to 0.1g) sample and be placed in centrifuge tube with a lid, add 12ml chloroform/methanol/formic acid (volume ratio is 10: 10: 1), in-20 DEG C of refrigerator overnight.Centrifugal, get supernatant liquor, then it is centrifugal afterwards to add 4.4ml chloroform/methanol/water (volume ratio is 5: 5: 1), merges twice supernatant liquor.Add 6ml containing concentration 0.2M H ₃pO ₄with 1M KCl solution, cleaning organic phase.After organic phase N2 is dried up, add 0.5ml chloroform to dissolve, be transferred in round-bottomed flask the methanolic solution adding 1ml2.5%, back flow reaction 90 minutes under 80 DEG C of water-baths, be cooled to room temperature, add 1.5ml0.9% sodium-chlor and 1ml normal hexane, after jolting, get supernatant liquor with glue head dropper, cross in 4 DEG C of preservations after the filter membrane of 0.45 micron, to be measured.

The detection of 6.2 lipid acid:

Use U.S. Agilent company 6890N type gas chromatograph and 5975C mass spectrograph, adopt automatic sampling, each sample continuous sample introduction six times.

Chromatographic condition: adopt quartz capillary column (Agilent DB-FFAP, 30.0m × 250.0 μm × 0.5 μm, top temperature 250 DEG C) sample size to be 0.2 μ l; Splitting ratio is 10: 1; Injector temperature is 200 DEG C; Temperature programming: from 150 DEG C, keeps 3min, rises to 210 DEG C, keep 12 minutes with 15 DEG C/min; Column flow rate is 1.1ml/min; Carrier gas is high-purity helium (99.999%).

Mass Spectrometry Conditions: ionizer is 70eV; Ion source temperature 230 DEG C; Level Four bar temperature 150 DEG C; Interface temperature is 250 DEG C; Solvent delay is 1.5min.

6.3 sample tests (Fig. 7)

By NIST spectrogram library searching, and coordinate the comparison of Fatty acid standards material, it is qualitative to carry out lipid acid, is undertaken quantitatively by typical curve.

6.4 contrasts detect with transgenic plant linoleic acid content

Table 1 contrasts and detects with transgenic plant linoleic acid content

Detect through GC-MS, find to turn the linoleic acid content that Roundhead wormwood Δ 12 fatty acid dehydrogenase gene significantly increases its blade.Transgenic plant leaf linoleic acid content is 3.57 times of non-transgenic plant.

6.5 contrasts detect with transgenic plant frost resistance

Result shows that the winter resistance of transgenic plant is significantly increased than contrast: 2 DEG C process 12 hours, and contrast there occurs obvious wilting, and transgenic plant phenotype normal (Fig. 8).

Table 2 contrasts and transgenic plant proline(Pro) and mda content

After subzero treatment, there is not considerable change as a kind of osmotic protection material content in transgenic plant in proline(Pro), and in wild-type tobacco, its content only has 42% under normal temps.Lipid peroxidation metabolism product mda significantly raises after wild-type tobacco freezing treatment, is 2.86 times of contrast; And transgene tobacco mda content does not have considerable change.These all absolutely prove that the expression of Roundhead wormwood Δ 12 fatty acid dehydrogenase in tobacco improves its frost resistance.

Claims (7)

1. a preparation method for Roundhead wormwood Δ 12 fatty acid dehydrogenase gene, is characterized in that the RNA first extracting Roundhead wormwood, then reverse transcription is cDNA; The nucleotide sequence of the FAD2 gene of other plant of having announced in GenBank and aminoacid sequence is utilized to carry out homology analysis, according to conserved regions design synthesis pair of degenerate primers P1, P2; With the Roundhead wormwood cDNA obtained for template amplification obtains Roundhead wormwood FAD2 core fragment nucleotide sequence; The FAD2 gene core fragment sequence obtained according to checking order designs Roundhead wormwood FAD2 gene specific primer 5 ' end Outside primer P3 and nested primer P4 respectively, respectively with GeneRacer ^tM5 ' P and the 5 ' NP carried in test kit matches, and with Roundhead wormwood cDNA for template, carries out 5 ' outside and nested PCR amplification reaction, obtains 5 ' end nucleotide acid sequence; The Roundhead wormwood FAD2 gene core fragment sequence obtained according to checking order equally designs Roundhead wormwood FAD2 gene specific primer 3 ' end Outside primer P5 and nested primer P6 respectively, respectively with GeneRacer ^tM3 ' P and the 3 ' NP carried in test kit matches, and with Roundhead wormwood cDNA for template, carries out 3 ' outside and nested PCR amplification, obtains 3 ' end nucleotide acid sequence; By obtained three sections of sequences: Roundhead wormwood FAD2 core fragment nucleotide sequence, 5 ' end nucleotide acid sequence and 3 ' end nucleotide acid sequence splice, and obtain target gene, the primer used is:

Upstream primer P1 is:

5’-AARAARGYYATYCCRCCVCAYTG-3’，

Downstream primer P2 is:

5’-TKCCAYCRYIYSATWATRW KG-3’，

P3 is: 5 '-ACATTCATGAGCAATAAGCCAAAGACCC-3 ',

P4 is: 5 '-TGAGGGAGCTGTGGAATATAAGTTGTGG-3 ',

5 ' P is: 5 '-CGACTGGAGCACGAGGACACTGA-3 ',

5 ' NP is: 5 '-GGACACTGACATGGACTGAAGGAGTA-3 ',

P5 is: 5 '-ACATTCATGAGCAATAAGCCAAAGACCC-3 ',

P6 is: 5 '-TGAGGGAGCTGTGGAATATAAGTTGTGG-3 ',

3 ' P is: 5 '-GCTGTCAACGATACGCTACGTAACG-3 ',

3 ' NP is: 5 '-CGCTACGTAACGGCATGACAGTG-3 ',

Wherein: R=A or G; Y=C or T; V=A or C or G; K=G or T; S=C or G; W=A or T.

2. the preparation method of Roundhead wormwood Δ 12 fatty acid dehydrogenase gene according to claim 1, is characterized in that the blade adopting Roundhead wormwood extracts RNA.

3. the preparation method of Roundhead wormwood Δ 12 fatty acid dehydrogenase gene according to claim 2, is characterized in that the Roundhead wormwood Δ 12 fatty acid dehydrogenase gene sequence prepared is SEQ ID No1.

4. the application of Roundhead wormwood Δ 12 fatty acid dehydrogenase gene according to claim 3 in the transgenosis work of oil crops.

5. the application of Roundhead wormwood Δ 12 fatty acid dehydrogenase gene according to claim 3 in the transgenosis work of pasture crop.

6. Roundhead wormwood Δ 12 fatty acid dehydrogenase gene according to claim 3 is removing the application in the transgenosis work in the exophytic plant described in claim 4 or 5.

7. the application of Roundhead wormwood Δ 12 fatty acid dehydrogenase gene according to claim 3 in the transgenosis work removing other biology exophytic.