CN101386842A

CN101386842A - Deoxyhydroxylputrescinelysine synthase code gene and antisense base sequences thereof

Info

Publication number: CN101386842A
Application number: CNA2008101199939A
Authority: CN
Inventors: 王宏芝; 魏建华; 王彦珍; 李瑞芬; 马荣才
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2008-10-21
Filing date: 2008-10-21
Publication date: 2009-03-18
Anticipated expiration: 2028-10-21
Also published as: CN101386842B

Abstract

The invention discloses an encoding gene of deoxyhypusine synthase and a method for inhibiting expression of the gene and a special antisense nucleotide sequence thereof. The deoxyhypusine synthase of the invention is selected from (a) or (b): (a) a protein which consists of amino acid sequence showed in sequence 4 of the sequence table; (b) a protein which is derived from (a) by substituting and/or deleting and/or adding one or more than one amino acid residue of the amino acid sequence showed in sequence 4 of the sequence table, and encodes the deoxyhypusine synthase. The invention uses antisense nucleotide sequence of DHS to inhibit normal expression of plant endogenous DHS gene, thereby the invention improves the resistance capability of plants against different stress environments, such as drought resistance, low temperature tolerance and salt tolerance. Each resistance is significantly higher than the negative control and the resistances are improved at the same time. Therefore, the encoding gene of the deoxyhypusine synthase and the antisense nucleotide sequence thereof and the method thereof have important significance on improving crop yield.

Description

Deoxidation hydroxyl putrescine Methionin synthasee code gene and antisense base sequences thereof

Technical field

The present invention relates to deoxidation hydroxyl putrescine Methionin synthasee code gene and antisense base sequences thereof, particularly deoxidation hydroxyl putrescine Methionin synthasee code gene and suppress the method and the special antisense nucleotide sequence of this genetic expression.

Background technology

Growth and development of plant is affected by environment very big.Various environment-stress can have a strong impact on the growing state of crop, cause crop failure, cause global grain and ecological crisis.Under the situation of current grain-supply whole world anxiety, improve the resistance of crop, make it overcome various poor environments, as low temperature, high temperature, arid, salt marsh etc., and then improve crop yield, particularly necessary to alleviating grain pressure.

Along with development of molecular biology, people can be familiar with plant anti-(resisting) property mechanism to environment stress on genomic constitution, expression regulation and signal conduction equimolecular level.Through big quantity research, people are obtaining extensive progress aspect morphology, Physiology and biochemistry, biophysics, ecology and the cytogenetics of plant stress-resistance, and some gene (Liang Huimin that in plant stress-resistance reaction, play an important role have been cloned into, the Xiayang, Wang Taiming, plant cold resistance freeze, drought resisting, resistant gene of salt engineering research progress, the grass cultivation journal, 2003,12 (3): 1-7).

Separated already at present and identified a large amount of adversity genes.These genes mainly contain following a few class: a class is an osmotic protection agent genoid, this genoid can increase the synthesis capability of plant perviousness meta-bolites, make plant under water stress, can synthesize more osmoregulation material (as N.F,USP MANNITOL, trimethyl-glycine, trehalose etc.), with the osmotic adjustment ability of raising plant, thereby strengthen the drought resistance of plant; These genes comprise proline(Pro) synthase gene (as proline(Pro) synthetic enzyme P5cs), Polylevulosan synthase gene (as Polylevulosan saccharase gene sac B), the synthetic relative enzyme gene (as betaine-aldehyde dehydrogenase BADH) of trimethyl-glycine, trehalose biosynthesis related genes (as the 6-phosphotrehalose UDP-transglucosylase synthase gene otsA of bacterium, the 6-phosphotrehalose UDP-transglucosylase phosphorylase gene otsB of bacterium) etc.; This genoid is mainly used in the drought resistance of improvement plant.Second class is for removing the relevant enzymes gene of active oxygen; Anti-oxidative defense systematic research in the plant materials under the drought stress is shown, this system is made up of some enzyme system and the antioxidant that can remove active oxygen, as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and xitix (AsA) etc., their collaborative opposing drought stress inductive oxidative damages.The 3rd class is the regulatory gene of the encoding transcription factor; These transcription factors energy regulatory function expression of gene and signal transductions, their overexpressions in transgenic plant can activate the expression of many degeneration-resistant functional genes, therefore, can improve the drought tolerance of plant by transforming regulatory gene.Wherein the DREB transcription factor is the transcription factor of the more resisting abiotic stress of research at present, responds anti-salt and low temperature stress.Owing to be grown in the plant of occurring in nature, usually be subjected to the threat of one or more environmental stresses simultaneously, therefore, seek the various genes that all produce response of coercing, can more effectively improve the adverse-resistant characteristic of farm-forestry crop.

Summary of the invention

An object of the present invention is to provide a kind of deoxidation hydroxyl putrescine Methionin synthase and encoding gene thereof.

Deoxidation hydroxyl putrescine Methionin synthase provided by the present invention derives from this uncured tobacco (Nicotianabenthamiana), is following (a) or albumen (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

(b) with the aminoacid sequence of sequence in the sequence table 4 through the replacement of one or several amino-acid residue and/or disappearance and/or the interpolation and the described deoxidation hydroxyl putrescine Methionin synthase of encoding by (a) deutero-protein.

Wherein, sequence 4 is made up of 379 amino acid in the sequence table.

In order to make the deoxidation hydroxyl putrescine Methionin synthase in (a) be convenient to purifying, proteinic N end or C end that can the aminoacid sequence shown in the sequence 4 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1, label

Label	Residue	Sequence
Label	Residue	Sequence	Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH	Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH	FLAG	8	DYKDDDDK
Strep-tag?II	8	WSHPQFEK	FLAG	8	DYKDDDDK
Strep-tag?II	8	WSHPQFEK	c-myc	10	EQKLISEEDL

Above-mentioned (a) but and the deoxidation hydroxyl putrescine Methionin synthase synthetic (b), also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of the deoxidation hydroxyl putrescine Methionin synthase (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna of sequence in the sequence table 3, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The encoding gene of deoxidation hydroxyl putrescine Methionin synthase provided by the present invention is selected from following (1), (2) or (3):

(1) nucleotides sequence is classified the dna molecular shown in the sequence 3 in the sequence table as;

(2) the dna sequence dna hybridization that under stringent condition, limits and the dna molecular of the described deoxidation hydroxyl putrescine Methionin synthase of encoding with (1);

(3) dna sequence dna with (1) qualification has 90% above homology and the dna molecular of the described deoxidation hydroxyl putrescine Methionin synthase of encoding.

Above-mentioned stringent condition is, at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

The recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain above-mentioned arbitrary described encoding gene also belong to protection scope of the present invention.

Another object of the present invention provides a kind of special antisense nucleotide sequence.

Special antisense nucleotide sequence provided by the present invention can be arbitrary section in the antisense base sequences of the above-mentioned encoding gene sequence more than or equal to 25bp; Be specifically as follows the nucleotide sequence shown in the sequence 2 in the sequence table.

The recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain the arbitrary special antisense nucleotide sequence of the present invention also belong to protection scope of the present invention; Can also comprise in the described recombinant vectors being used to the adjusting sequence that realizes that antisense polynucleotides is transcribed, as promotor, terminator.

Wherein, described recombinant vectors can obtain for the multiple clone site of the arbitrary special antisense nucleotide sequence of the present invention being inserted carrier pBI121; Described recombinant vectors is specifically as follows the special antisense nucleotide sequence shown in the sequence in the sequence table 2 inserted and obtains between the XbaI of carrier pBI121 and SacI site.

Last purpose of the present invention provides a kind of method of cultivating the plant of resistance raising.

The method of cultivation resistance plant provided by the present invention is that above-mentioned arbitrary described special antisense nucleotide sequence is imported in the plant, cultivates and obtains the resistance plant.

Generally can adopt following ordinary method that described special antisense nucleotide sequence is imported in the plant, particle bombardment for example, high voltage electric perforation method, liposome method, agriculture bacillus mediated methods for plant transformation or transfection etc.

The present invention utilizes agriculture bacillus mediated method for transformation that described antisense base sequences is imported in the plant, specific as follows: as at first described special antisense nucleotide sequence to be imported in the carrier by recombinant DNA technology, obtain containing the recombinant vectors of described special antisense nucleotide sequence, again described recombinant vectors is imported in the Agrobacterium, obtain containing the reorganization Agrobacterium of described special antisense nucleotide sequence; Utilize agriculture bacillus mediated method for transformation that recombinant DNA molecules is imported in the plant again, thereby the corresponding inherited character of plant materials is changed.

Used recombinant vectors can obtain for the multiple clone site that above-mentioned arbitrary special antisense nucleotide sequence is inserted carrier pBI121 in the inventive method, is specifically as follows the special antisense nucleotide sequence shown in the sequence in the sequence table 2 inserted to obtain between the XbaI of carrier pBI121 and SacI site.

Described resistance can be drought resistance and/or cold resistance and/or salt resistance.

Aforesaid method can be applied to specifically can be applicable in the tobacco in the dicotyledons.

The sequence of encoding gene described in the present invention is meant the sequence of coding strand in the encoding gene; The antisense base sequences of described encoding gene (5 ' to 3 ') is meant the sequence of complementary strand from 5 ' end to 3 ' end of above-mentioned coding strand.

The translation of deoxidation hydroxyl putrescine Methionin synthase participation translation initiation factor is postactivated, and translation initiation factor is regulated and control the translation of specific protein by the transportation of controlling specific mRNA.The present invention is the expression by inhibition deoxidation hydroxyl putrescine Methionin synthase, thereby suppresses some proteic expression relevant with stress resistance of plant, and then improves the resistance of plant.

The method of cultivation resistance plant of the present invention is to utilize the antisense base sequences of deoxidation hydroxyl putrescine Methionin synthasee code gene (DHS), the normal expression that has suppressed plant endogenous DHS gene, and then improved plant multiple difference is coerced the resistance of environment, as drought resistance, lower temperature resistance and salt resistance.Experiment shows, the plant that the inventive method obtains, and its drought resistance, lower temperature resistance and salt resistance all are significantly higher than negative control, and these several resistances all are improved simultaneously.In addition, the inventive method is also time saving and energy saving, has reduced cost.Therefore, the method for cultivation resistance plant of the present invention can be used for degeneration-resistant genetic improvement of farm crop and exploitation, and is significant to the output that improves crop, is suitable for applying.

Description of drawings

Fig. 1 is this uncured tobacco NbDHS cDNA clone's a PCR detected result.

Fig. 2 is the detected result that XbaI and SacI enzyme are cut the T-NbDHS-1 plasmid.

Fig. 3 is transgene tobacco growth result in division culture medium.

Fig. 4 is that T0 is for transformed plant Southern qualification result.

Fig. 5 is the Screening and Identification of T1 for the transgene tobacco seed.

Fig. 6 is that T1 identifies for transfer-gen plant PCR.

Fig. 7 handles transgene tobacco growing state down for drought stress.

Fig. 8 handles transgene tobacco growing state down for low temperature stress.

Fig. 9 is a transgene tobacco growing state under the high-salt stress.

Embodiment

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

The separation of embodiment 1, deoxidation hydroxyl putrescine Methionin synthasee code gene DHS cDNA sequence

Deoxidation hydroxyl putrescine Methionin synthasee code gene DHS full length cDNA sequence obtains in conjunction with terminal rapid amplifying (RACE) method of cDNA by reverse transcription PCR (RT-PCR) method.Specifically, be template at first with the total RNA that derives from this uncured tobacco (Nicotiana benthamiana) blade, by reverse transcription, synthetic cDNA first chain; Be template with cDNA first chain again, increase, obtain 868bp DHS cDNA fragment according to the Arabidopis thaliana of having delivered, the homogenic conservative region design of common tobacco (Nicotiana tabaccum) primer.Design forward and reverse primer respectively at this sheet intersegmental part then, by the RACE method, obtain 3 ' and the 5 ' end sequence of cDNA respectively, and then clone full-length gene by the RT-PCR method again.

1, total RNA extracts

Adopt TRIzol (Invitrogen) to extract this uncured tobacco (Nicotiana benthamiana) (national germplasm storehouse) total RNA of old and feeble blade.Get 100mg plant leaf material, after the liquid nitrogen grinding, transfer in the 1.5ml centrifuge tube, add 1ml TRIzol reagent rapidly, 25 ℃ of following incubation 5min; 4 ℃ of centrifugal 10min of following 12000rpm; Get the supernatant water, transfer in the new centrifuge tube, add the 0.2ml chloroform, concuss; 25 ℃ of following incubation 3min; 4 ℃ of centrifugal 15min of following 12000rpm; Get the supernatant water, transfer in the new centrifuge tube; Add equal-volume 5M LiCl, mixing; Static 1hr under-20 ℃, precipitated rna; The centrifugal 45min of 12000rpm, 4 ℃; Abandon supernatant, add 1ml 75% ethanol, the centrifugal 5min of 7500rpm, 2-8 ℃; Repeat 3 times; Abandon ethanol, dry air 10min in the super dressing table; Add the distilled water dissolving 10min that 50 μ l DEPC handle, 55-60 ℃.

2, the segmental clone of DHS 868 bp cDNA

(1) cDNA first chain is synthetic

Use ThermoScript ^TMRT-PCR test kit (Invitrogen), cDNA first chain is synthesized in explanation to specifications.With oligo (dT) ₂₀Be primer, 42 ℃ of insulation 1hr add 1 μ l RNase H (2U/ μ l) afterwards, and 37 ℃ are incubated 10 minutes.

(2) the segmental pcr amplification of cDNA

With 1 μ l cDNA, the first chain reaction mixture is template, polymerase chain reaction (PCR) amplification DHS cDNA fragment, upstream primer is DHSf1:5 '-TTCACTTCAAACCTTGTTTCTTC, and downstream primer is DHSr1:5 '-CCATGATACAGCTTCATCAGG.Reaction system is 25 μ l, wherein comprises: 2.5 μ l 10X PCR Buffer, 1.5 μ l MgCl ₂(25mM), the upstream primer DHSf1 of 0.5 μ l dNTP mix (10mM each), each 1 μ l and downstream primer DHSr1 (10 μ M), 1 μ lcDNA, the first chain reaction mixture, 0.15 μ l Taq archaeal dna polymerase (5U/ μ l) and 17.35 μ l H ₂O.PCR reaction operational conditions is as follows: 95 ℃, and 5min; 94 ℃, 30s, 62 ℃, 30s ,-1 ℃/cycle, 72 ℃, 40s, 12cycles; 94 ℃, 30s, 50 ℃, 30s, 72 ℃, 40s, 30cycles; 72 ℃, 5min.After reaction finishes, get 20 μ l PCR products electrophoresis detection on 1% sepharose, the result as shown in Figure 1, wherein M is marker, swimming lane 1 is a pcr amplification product.

(3) the electrophoresis purifying of dna fragmentation reclaims

Adopt the Vitagene-DNA sepharose to reclaim test kit, purifying DHS cDNA fragment: electrophoresis finishes and downcut the purpose band under ultraviolet lamp, is placed in the 1.5ml EP pipe; The gel fusing agent that adds 3 times of volumes mixes back 75 ℃ of temperature and bathed 8 minutes, during be interrupted and mix, gel is thoroughly melted; The height that adds 0.5 times of gel fusing agent volume mixes from liquid sequence solution; Get a new centrifugal adsorption column, place the 2ml centrifuge tube, the solution of previous step gained is added in the centrifugal adsorption column, 3600rpm, centrifugal 1min abandons filtrate; Centrifugal adsorption column is put back in the centrifuge tube, added 0.5ml washings W1,3600rpm, centrifugal 30s abandons filtrate; Centrifugal adsorption column is put back in the centrifuge tube, added 0.7ml washings W2,3600rpm, centrifugal 30s abandons filtrate, repeats to drill and does once; Centrifugal adsorption column is put back in the centrifuge tube, and the centrifugal 1min of 12000rpm removes rinsing liquid as far as possible; Centrifugal adsorption column is placed clean 1.5ml centrifuge tube, prepare the film centre at DNA and add 25 μ l water, room temperature leaves standstill 1min, and the centrifugal 1min eluted dna of 12000rpm obtains the DHS cDNA fragment of purifying.

(4) DHS cDNA fragment cloning and order-checking

Use pGEM-T Easy (promega) carrier cloning DHS cDNA fragment: get the cDNA fragment of 4 μ l purifying, add 5 μ l 2xT4 ligase enzyme damping fluids successively, 0.5 μ l pGEM-T Easy vector (50ng/ μ l), 0.5 μ l T ₄Dna ligase (3U/ μ l), 4 ℃ of temperature are bathed and are spent the night.Obtain the product that is connected of DHS cDNA fragment and pGEM-T Easy carrier.

(article No.: 18265017), in the super clean bench, will connect product and join in the 50 μ l DH5 α competent cells, put 30 minutes on ice by mixing available from invitrogen for the bacillus coli DH 5 alpha competent cell; 42 ℃, heat shock 90s; Ice bath 2min; 250 μ l LB liquid nutrient mediums are cultivated 1hr for 37 ℃; 150 μ l cell suspension mixings are coated on contain 100 μ g/ml Amp, X-gal (40mg/L), on the LB solid medium of IPTG (40mg/L), 37 ℃ of lucifuges are inverted overnight incubation.

3 of white clones on the picking LB flat board place 3ml LB substratum respectively, 37 ℃ of concussion overnight incubation; Plasmid DNA is subsequently extracted and the EcoRI restriction enzyme reaction carries out with reference to " molecular cloning " (ColdSpring Harbor Laboratory Press, edition1,1989).The size of electrophoresis detection endonuclease bamhi.The plasmid that contains recombinant clone is through the order-checking of dioxygen method, and the segmental nucleotide sequence of DHS cDNA of acquisition is shown in sequence in the sequence table 1, and length is 868bp.The plasmid vector called after T-NbDHS that will contain nucleotide sequence shown in the sequence 1 in the sequence table.

3, DHS gene cDNA 3 ' and 5 ' terminal amplification

By RACE technology amplification DHS cDNA 3 ' and 5 ' stub area.According to DHS 868bp cDNA fragments sequence design shell type Auele Specific Primer DHSf2:5 '-GAGGCTGTCCATGCTGGTTCGAG, DHSf3:5 '-GCCTAAGCATCATGTTTGCAATG and DHSr2:5 '-GGCGTCACCAAGATTAGCAGCTTGG, the DHSr3:5 '-CAATCTTCTATGGGCTGCTCATGTG that obtains above, use 5 '/3 ' RACE reagent Kit (Luo Shi Applied Science Fiction Co., catalog number (Cat.No.): 03353621001), the scheme that provides to specifications, by multiplex PCR, amplify DHS cDNA3 ' and 5 ' stub area respectively.The amplification PCR fragment reclaims, clones, checks order through the electrophoresis purifying, obtains DHS cDNA3 ' and 5 ' end sequence.After the sequence of gained and the 868 bp cDNA fragments sequence that obtain are previously spliced, obtain the full length coding region nucleotide sequence (being open reading frame) of this gene, shown in sequence in the sequence table 3.Infer that by DNAMAN software this open reading frame encoded protein matter sequence is shown in sequence in the sequence table 4.

Embodiment 2, tobacco resistance improve experiment

One, the structure of expression of plants recombinant vectors pAND

With the T-NbDHS plasmid is template, utilizes primer DHS5 ': GAGCTCCTCATAAAATGCCTTGCACC (introducing the SacI site) and DHS3 ': TCTAGAACCATTTCGCATCATATTG (introducing the XbaI site) carries out pcr amplification, obtains the DHS cDNA fragment of 516bp, and the PCR fragment of gained is connected acquisition T-NbDHS-1 carrier with pGEM-T Easy carrier, import in the intestinal bacteria competence, and plasmid is extracted in the evaluation back.Plasmid is behind XbaI and SacI double digestion, and electrophoresis detection, result are as shown in Figure 2, show the band that cuts out 3.0kb and 516bp, reclaim the fragment of 516bp, check order again, its sequence from shown in the Nucleotide of 5 ' terminal 268-783 position, shows that fragment sequence is correct as sequence in the sequence table 1.

Contain cauliflower mosaic virus 35S promoter (CaMV35S promotor) and Agrobacterium rouge alkali synthetase gene 3 ' terminator (NOS terminator) among the plant expression vector pBI121, it can regulate transcribing of antisense base sequences.With restriction enzyme XbaI and the SacI enzyme expression vector pB1121 (Clontech, the U.S.) that cuts plant, reclaim the fragment of 13kb;

Get the 516bp fragment of 1ul and the 13bk fragment of 10ul, the connection damping fluid I that adds 10ul, 16 ℃ of connections are spent the night, transformed into escherichia coli, extract plasmid, carry out that PCR detects, enzyme is cut and detected and order-checking, acquisition contains the recombinant plasmid vector of 516bpDHS antisense base sequences, 516bp DHS antisense base sequences (from 5 ' end to 3 ' end) is shown in sequence in the sequence table 2,516bp DHS antisense base sequences is arranged in carrier CaMV35S ' promotor downstream, the recombinant vectors called after pAND carrier that structure is correct.

Two, expression of plants recombinant vectors pAND transformation of tobacco

1, preparation Agrobacterium competent cell: the single bacterium colony of picking Agrobacterium C58 is seeded in the 3ml LB substratum (contain the tsiklomitsin that final concentration is 5mg/L, final concentration is the Rifampin of 50mg/L) 28 ℃ of concussion overnight incubation; Get the 1ml culture and be seeded in the 100ml LB substratum, 28 ℃ of concussions are cultured to OD ₆₀₀Be 0.5 (about 5-8hr); Collect bacterium liquid, the centrifugal 10min of 4000rpm, 4 ℃ with 50ml polypropylene pipe; Abandon supernatant liquor, with 15ml precooling pure water (18 Ω) suspension cell precipitation, the centrifugal 10min of 4000rpm, 4 ℃; Repeat the step once; Abandon supernatant liquor, with 10% glycerine (preparation of 18 Ω pure water) the suspension cell precipitation of 10ml precooling, the centrifugal 10min of 4000rpm, 4 ℃; Abandon supernatant liquor, with the 10% glycerine suspension cell precipitation of 0.75ml (every 100ml bacterium liquid) precooling; Be divided in the 1.5ml EP pipe, every pipe 40 μ l behind the liquid nitrogen freezing, preserve in-80 ℃ of refrigerators.

2, plant expression vector pAND electric shock changes among the Agrobacterium C58: take out the C58 electric shock and use competent cell in refrigerator, put on ice and slowly melt; 2 μ l plasmids are joined in the competent cell, and mixing is put on ice; Competent cell and plasmid pAND DNA mixture are joined in the electric shock cup of precooling; 2.5kv 400 Ω shock by electricity under the 25 μ F conditions; Add 1ml LB substratum to the electric shock cup rapidly; Complete soln in the electric shock cup is transferred in the 1.5ml EP pipe 28 ℃ of incubation 1hr; Centrifugal 2 minutes of 4000rpm abandons 800 μ l supernatants, surpluss 200 μ l; Re-suspended cell is coated on the cell suspension mixing on the LB solid medium that contains 5mg/L tsiklomitsin, 50mg/L Rifampin and 50mg/L kantlex; After treating that liquid absorbs fully, 28 ℃ of lucifuges are inverted and are cultivated 3 days visible bacterium colonies.Detect positive Agrobacterium-mediated Transformation through PCR, obtain to contain the agrobacterium strains of plant expression vector pAND.

3, the conversion of tobacco

Single bacterium colony of the fresh Agrobacterium that contains plant expression vector pAND of picking is inoculated into (containing final concentration is the 5mg/L tsiklomitsin, and final concentration is the 50mg/L Rifampin, and final concentration is the 50mg/L kantlex) in the LB liquid nutrient medium, and 28 ℃ of shaking culture are spent the night.Get the LB liquid nutrient medium that 1ml bacterium liquid adds fresh 40ml, 28 ℃ 220 rev/mins shaking culture 2-3h (OD ₆₀₀=0.4-0.8), stand-by.

Adopt leaf dish method transformation of tobacco: cut eugonic tobacco tissue cultured seedling blade, be cut into about 1cm with blade ²Immerse in the Agrobacterium of dilution, infected 5 minutes; Blot unnecessary bacterium liquid with sterilization filter paper, place on the common substratum, cultivate altogether in the dark; Cultivation is transferred to illumination cultivation on the division culture medium two days later altogether; Grow (about about 2 weeks) behind the green bud, go to subculture medium (being division culture medium) and go up succeeding transfer culture, every through 2 all succeeding transfer culture once; (Fig. 3) went to root media when the budlet that differentiates grew to this 2-3cm; After taking root, transplant to flowerpot hot-house culture.Required substratum is as follows: 1) be total to culture medium: MS+6-BA 1mg/L+NAA 0.1mg/L; 2) differentiation screening culture medium: MS+6-BA 1mg/L+NAA 0.1mg/L+Kan 200mg/L+cef 500mg/L; Root media: MS+NAA 0.1mg/L+Kan 100mg/L+cef 200mg/L.The composition of MS substratum is as shown in table 2.With the tobacco W38 that changes empty carrier pBI121 over to as blank.

The composition of table 2, MS substratum

	Title	Content (mg/L)
	Title	Content (mg/L)	Macroelement	NH ₄NO ₄	1650
	KNO ₃	1900	Macroelement	NH ₄NO ₄	1650
	KNO ₃	1900		CaCl ₂·2H ₂O	440
	MgSO ₄·7H ₂O	370		CaCl ₂·2H ₂O	440
	MgSO ₄·7H ₂O	370		KH ₂PO ₄	170
Trace element	KI	0.83		KH ₂PO ₄	170
Trace element	KI	0.83		H ₃BO ₃	6.2
	MnSO ₄·4H ₂O	22.3		H ₃BO ₃	6.2
	MnSO ₄·4H ₂O	22.3		ZnSO ₄·7H ₂O	8.6
	Na ₂MoO ₄·2H ₂O	0.25		ZnSO ₄·7H ₂O	8.6
	Na ₂MoO ₄·2H ₂O	0.25		CuSO ₄·5H ₂O	0.025
	CoCl ₂·6H ₂O	0.025		CuSO ₄·5H ₂O	0.025
	CoCl ₂·6H ₂O	0.025	Molysite	Na ₂-EDTA·2H ₂O	37.3
	FeSO ₄·7H ₂O	27.8	Molysite	Na ₂-EDTA·2H ₂O	37.3
	FeSO ₄·7H ₂O	27.8	Organic composition	Nicotinic acid	0.5
	Pyridoxine hydrochloride (VB ₆)	0.5	Organic composition	Nicotinic acid	0.5
	Pyridoxine hydrochloride (VB ₆)	0.5		Nicotinic acid VitB1 (VB ₁)	0.1
	Glycine	2.0		Nicotinic acid VitB1 (VB ₁)	0.1
	Glycine	2.0		Inositol	100.0

From dip-dye to the differentiation state that screens 50 days explant of differentiation as shown in Figure 3, A is the tobacco W38 negative control that changes empty carrier pBI121 over to, and its explant breaks up hardly or breaks up slowly, and under the screening of kan resistance, jaundice is die gradually; The explant of B for contaminating through the Agrobacterium bacterium liquid that contains the pAND plasmid differentiated several strain independence seedlings, just can cut this moment to change in the screening root media to cultivate.

Three, the evaluation of transformed plant

1, Southern Analysis and Identification T0 is for transformed plant

Southern hybridization is carried out according to the explanation of " molecular cloning " (Cold Spring Harbor Laboratory Press, edition 1,1989).Use HindIII and EcoRI double digestion Plant Genome, the restriction enzyme digestion and electrophoresis result is shown in Fig. 4 A, and swimming lane 1 is a pAND plasmid positive control, and swimming lane 4 and 12 is for changing the tobacco W38 negative control of empty carrier pBI121 over to, swimming lane 15 is Marker, the tobacco of all the other swimming lanes for transforming; The result shows that the genomic dna of transfer-gen plant obtains the special band of a 1.5kb after enzyme is cut.

The Southern results of hybridization is shown in Fig. 4 B.The result shows that except the transformation plant of the 7th swimming lane, other transformation of tobacco seedling all can hybridize the object tape of 1.5kb, shows thus in the genomes of these plant to have inserted tobacco antisense DHS gene fragment.2 negative control W38 (the 4th and the 12nd swimming lane) all do not hybridize object tape, because the existence of DHS native gene is arranged, so all Plant Genome sample lane 2 bands greater than 2kb of all having mixed out.

2, T1 is for the Screening and Identification of transgene tobacco

With part transgenosis T0 for plantlet of transplant to physical environment, growth and maturity under field conditions (factors), accept seed, at the enterprising row filter of the MS of kan200-800mg/l substratum, sow together with the tobacco W38 seed that changes empty carrier pBI121 over to simultaneously and do contrast after the seed disinfection.Cultivate and observe, the W38 seed still can normal growth during Kan concentration≤400mg/l, and when Kan concentration reaches 600mg/L, the growing state of transgenic seed and contrast seed has obvious difference, and as shown in Figure 5: all negative control W38 seeds all can not germinate in the MS of kan 600mg/l substratum or germinating growth jaundice dead (Fig. 5 B) gradually after about 10 days; And transgenic seed, because F1 separated for the generation, a part of seed is germinating growth normally, in addition a part and negative control W38 seed performance the same (Fig. 5 A).

Randomly draw the resistance seedling that the MS substratum of a part of Kan 600mg/L filters out, extract genomic dna, be PCR and identify, carry out PCR with following primer and identify: DHS5 ': GSGCTCCTCATAAAATGCCTTGCACC and DHS3 ': TCTAGAACCATTTCGCATCATATTG, result show that the seedling DNA sample that screens through Kan can both detect the existence of foreign gene as shown in Figure 6, and the seedling that can grow out from the MS substratum of kan 600mg/L all is transfer-gen plants.Can be PCR the plant of the MS of kan 600mg/l substratum normal growth and detect positive seedling, and the seed of W38 can't be grown on this proof strength (Fig. 5 and Fig. 6).

3, the phenotypic evaluation of transgene tobacco

(1) drought resistance is identified

The little transplantation of seedlings of positive transgenosis that above-mentioned screening obtains was cultivated 2 months to the nutrition soil of vermiculite and fertilizer composition, and the seedling growth is stable, stops then watering 15 days, to carry out environment stress, observes DHS and reduces the transfer-gen plant phenotypic character of expressing.The result is (the A group is the W38 negative control, and the B group is transfer-gen plant) as shown in Figure 7.Show and stop to water 15 days the transfer-gen plant and the phenotypic character of W38 contrast.The result shows that 2 W38 contrasts are all seriously wilted, and the whole shrivelled shrinkages of blade stop growing impending death; And in the transfer-gen plant, the performance of each strain system is different, and the strain that has is that growing way is fine, almost do not wilt, and wilting shape in various degree appears in the strain that has system, and is fairly good but blade is held green property, and leaf surface is smooth, does not occur the phenomenon of shrivelled shrinkage as yet.The drought-resistant ability that shows the strain system that changes antisense DHS gene over to is significantly improved.

(2) winter hardiness is identified

The little transplantation of seedlings of positive transgenosis that above-mentioned screening obtains was cultivated 2 months to the nutrition soil of vermiculite and fertilizer composition, the seedling growth is stable, then seedling being changed over to 4 ℃ from the natural light greenhouse damages to plants caused by sudden drop in temperature and coerces 3 days, placed 3 days under the room temperature afterwards, observe the seedling upgrowth situation, the result is (A1 and A2 are the W38 negative control, and B1 and B2 are transgenic line) as shown in Figure 8.The result shows that the strain of W38 negative control shows as the plant lodging, and blade is sagging to rot, and is subjected to tangible injury from low temperature; And two transgenic lines are still stood upright, and most of blade does not have sagging rotting yet, and keep green, continued growth, the phenomenon that is not significantly damaged to plants caused by sudden drop in temperature.The cold tolerance that shows the strain system that changes antisense DHS gene over to is significantly improved.

(3) anti-salt is identified

The little transplantation of seedlings of positive transgenosis that above-mentioned screening obtains was cultivated 2 months to the nutrition soil of vermiculite and fertilizer composition, the seedling growth is stable, from nutrition soil, dig out seedling (the careful excavation then, avoid injuring its root system), clean the soil that is attached on the root system with clear water, place then on the floating plate of perforation, its root system is inserted contain in the MS liquid nutrient medium of 3%NaCl, cultivated 6 days, and took out and take a picture.The result as shown in Figure 9, wherein left side two strains are the W38 negative control, right side three strains are No. 10 strains of transgenosis systems.Show that growth is after 6 days in the MS substratum of the NaCl 3%, all blades of 2 on left side contrast strain are wilted, surface staining, and the whole plant very weak and limp weakness that becomes, the part leaf cell is damaged simultaneously, causes the blade browning to rot; And the blade of the transgenosis strain of 3 the DHS downward modulations in right side still keeps natural gloss, and it is healthy and strong upright that whole plant still keeps, and blade does not show the septic phenomenon of brownization yet, shows good anti-salt feature.The significantly anti-high salt ability that has that shows the strain system that changes antisense DHS gene over to.

Sequence table

<160>4

<210>1

<211>868

<212>DNA

＜213〉this uncured tobacco of Nicotiana (Nicotiana benthamiana)

<400>1

<210>2

<211>516

<212>DNA

＜213〉this uncured tobacco of Nicotiana (Nicotiana benthamiana)

<400>2

<210>3

<211>1140

<212>DNA

＜213〉this uncured tobacco of Nicotiana (Nicotiana benthamiana)

<400>3

<210>4

<211>379

<212>Pro

＜213〉this uncured tobacco of Nicotiana (Nicotiana benthamiana)

<400>4

Claims

1, deoxidation hydroxyl putrescine Methionin synthase is selected from following (a) or (b):

2, the encoding gene of the described deoxidation hydroxyl of claim 1 putrescine Methionin synthase.

3, encoding gene according to claim 2 is characterized in that: described encoding gene is selected from following (1), (2) or (3):

4, the recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain claim 2 or 3 described encoding genes.

5, a kind of special antisense nucleotide sequence is the arbitrary section sequence more than or equal to 25bp in the antisense base sequences of arbitrary described encoding gene in claim 2 or 3.

6, special antisense nucleotide sequence according to claim 5, it is characterized in that: the nucleotide sequence of described special antisense nucleotide sequence is shown in sequence in the sequence table 2.

7, the recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain arbitrary described special antisense nucleotide sequence in claim 5 or 6.

8, a kind of method of cultivating the resistance plant is that arbitrary described special antisense nucleotide sequence in claim 5 or 6 is imported in the plant, cultivates and obtains the resistance plant.

9, method according to claim 8 is characterized in that: described special antisense nucleotide sequence imports in the plant by recombinant vectors.

10, method according to claim 9 is characterized in that: described resistance is drought resistance and/or cold resistance and/or salt resistance; Described plant is a dicotyledons.