CN102071203B - Induced promoter - Google Patents

Abstract

The invention discloses an induced promoter comprising the following DNA molecules of (1) or (2) or (3): (1) a DNA molecule shown in a sequence 1 in a sequence table; (2) a DNA molecule which is hybridized with the DNA sequence confined by (1) under the strict condition and has the function of the promoter; and (3) a DNA molecule which has more than 90% homology with the DNA sequence confined by (1) or (2) and has the function of the promoter. The invention discloses the promoter PRO-TaFbox2 sequence of the relevant haloduric gene TaFbox2 of wheat, and the overall length of the nucleotide sequence of the promoter is 3076bp. The 2kb zone of the promoter is analyzed and confirmed to contain a plurality of transcription element binding sites relevant to biological stress induction and non-biological stress induction, which shows that a downstream gene controlled by the promoter is bound to be expressed by the stress induction. The induced promoter has very good application prospect in the biological engineering by serving as the induced expression vector of a plant source.

Description

A kind of inducible promoter

Technical field

The present invention relates to a kind of inducible promoter.

Background technology

In the multilevel hierarchy of gene expression regulation, the regulation and control of transcriptional level are very cost-effective control methods for plant materials, and various regulatory factors are through combining with promotor, open or close expression of gene and reach the purpose of regulation and control.Promotor is topmost a kind of regulative mode in the genetic transcription; Because gene is controlled by different regulatory factors on different levels; This controlling mechanism not only determines the level of genetic expression, also determines the space-time order of genetic expression, therefore; Can think that promotor determines the space-time specificity of genetic expression to a certain extent, occupies crucial status in transcribing link.Promotor is the critical elements of gene engineering expression carrier, and the research promotor is improved the production traits of crop for gene expression regulation mechanism, has crucial meaning like proterties such as pest-resistant, disease-resistant, degeneration-resistant.Obtain the new promotor, particularly specificity promoter of plant origin, can be for the expression of research plant gene, regulate and control and utilize gene engineering method improvement crop to provide the core parts of usefulness.

The promotor of plant gene is divided into three kinds by its expression of gene mode:

1, constitutive promoter.Characteristics are to express to have persistence; Do not receive inducing of extraneous factor, and expression level each etap of organism and different sites do not have notable difference (Li Yikun, Wang Jinfa. higher plant promotor progress BULLETIN OF BOTANY Vol.. Botany Gazette; 1998,15 (supplementary issue) supplementary issue: 1-6.).The constitutive promoter that for example the most often uses in the dicotyledons is cauliflower mosaic virus (CaMV) 35S promoter (Odell J.T.; Nagy F.; Chua N.H..Identification of DNA sequences required for activityof the cauliflower masaic virus 35S promoter.Nature; 1985; 313 (6005): 810-820.), nopaline synthase gene (Nos) and octopine synthase gene (Ocs) promotor, and the rice actin Gene A ct1 promotor (Wang Y., the Zhang W. that from monocotyledons, clone in recent years; Cao.J.; Et al..Characterization of cis-acting elements regulatingtranscription from the promoter of a constitutively active rice actin gene.Mol Cell Biol, 1992,12 (8): 3399-3406.), corn ubiquitin gene Ubi1 promotor (Christensen A.H.; Sharrock R.A..Maize polyubiquitin genes:structure; Thermal perturbation of expression and transcript spl icing and promoteractivity following transfer to protoplasts by electroporation.Plant MolBiol, 1992,18:675-681.).

2, inducible promoter.It promptly is the promotor that under the stimulation of some specific physics or chemical signal, can improve the gene transcription level significantly.The activation of inducible promoter receives inducing of physics or chemical signal, and the molecular structure of promotor all has the sequential structure of enhanser, silencer or similar functions, and experiencing specificity inductive sequence all has tangible specificity, and for example Arabidopis thaliana rd29A gene is under arid, high salinity, low temperature and dormin condition, and transcription factor DREB, ABREB comprises arid response factors DRE with promotor-174～-55 zones and ABA response factors (ABRE) combines, and induces rd29A genetic expression (Seki; M., Narusaka, M., Ishida, J., Nanjo; T., Fuj ita, M., Oono, Y., Kamiya; A., Nakaj ima, M., Enju, A., Sakurai; T., Satou, M., Akiyama, K.; Taji, T., Yamaguchi-Shinozaki, K., Carninci; P., Kawai, J., Hayashizaki, Y.; And Shinozaki, K..Monitoring the expressionprofiles of 7000 Arabidopsis genes under drought, cold and high-salinitystresses using a full-length cDNA microarray.Plant J, 2002,31:279-292.).The part induce the Idiotype promotor have simultaneously tissue specific expression characteristics (Wang Guanlin, Fang Hongjun. plant genetic engineering (second edition). Beijing: Science Press, 2002.).Arabidopis thaliana DREB1A and DREB2A transcription factor be the expression of the regulation and control several genes relevant with arid high-salt stress with low temperature respectively; Simultaneously; The expression of DREB1A and DREB2A also receives (the Liu J. that induces of low temperature and arid high salt; Zhu J.K..A calcium sensor homologrequired for plant salt tolerance.Science, 1998,280:1943-1945.).

3, tissue-specific promoter.Be also referred to as cell or organ specific promoters, under these promoter regulations, expression of gene often only occurs in some specific organ or tissue position, and shows the characteristic of growing adjusting.Tissue-specific promoter is except that the constructional feature that possesses general promotor; The element that also has some control tissue specific expressions; These elements generally are positioned at the upper reaches of TATA-box, and sequence is no more than 30bp usually, and expression of gene is by the common decision of kind, number and the relative position etc. of these elements; And also necessary (the Keller B. of specific gene expression just of these elements; Lamb C.J..Specific location of a plant cellwall glycine-rich protein in protoxylem cells of the vascular system.ProcNatl Acad Sci USA, 1989,86:1529-1533.).

Low temperature, arid and salt are several kinds of the adverse circumstances the most serious that plant faces, and bring about great losses to agriculture prodn, and the resistance of utilizing the biotechnology transgenic technology to improve crop is a cost-effective approach to improve and to utilize salinization soil.In recent years transgenic plant industrialization paces are constantly accelerated.Use the data of International Service Organization (ISAAA) issue according to Agricultural biotechnologies; The country of whole world plantation genetically modified crops in 2008 reaches 25; The genetically modified crops cultivated area increased by 9.4% than 2007, reached 1.25 hundred million hectares (http://www.isaaa.org).China's plantation transgenic cotton against pests area reached 3,800,000 hectares in 2007, accounted for 66% of the Cotton in China total area.At present, a large amount of separated clones of good new gene, but in the plant genetic engineering comparatively widely used promotor CaMV35S promotor from cauliflower mosaic virus is arranged, from promotor (Sanders such as the Act1 of plant itself, Ubi1; P.R., Winter, J.A.; Barnason, A.R., Rogers; S.G., and Fraley, R.T..Comparisonof cauliflower mosaic virus 35S and nopaline synthase promoters intransgenic plants.Nucleic Acids Res; 1987,15:1543-1558.; McElroy, D., Zhang, W., Cao, J., and Wu, R.Isolation of an efficient actin promoter foruse in rice transformation.Plant Cell, 1990,2:163-171.), these all are constitutive promoters.Because the gene that constitutive promoter drives all has expression in various degree in plant different tissues organ, expose some problems in the application gradually.As a rule, people do not hope foreign gene wide expression in the whole plant of transgenic plant, whole growing, because can increase the metabolism burden of plant on the one hand, on the other hand; Some foreign protein is unfavorable for plant normal growth (Kasuga, M., Liu to plant and nonessential even poisonous; Q., Miura, S., Yamaguchi-Shinozaki; K., and Shinozaki, K..Improvingplant drought, salt; And freezing tolerance by gene transfer of a singlestress-inducible transcription factor.Nat Biotechnol, 1999,17:287-291.).Therefore; Research and utilize histoorgan Idiotype promotor and inducible promoter to seem particularly important; Not only can realize when exogenous gene expression implemented, the three-dimensional regulation and control of sky, amount, have many-sided potential value (Venter, M..Synthetic promoters:genetic controlthrough cis engineering.Trends Plant Sci such as economy, environmental protection and Biosafety simultaneously; 2007,12:118-124.).

Summary of the invention

The purpose of this invention is to provide a kind of inducible promoter.

Dna fragmentation provided by the invention (promotor) 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 promoter function;

3) with 1) or 2) dna sequence dna that limits has 90% above homology, and have the dna molecular of promoter function.

The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain said dna fragmentation all belong to protection scope of the present invention.

Said recombinant vectors specifically can be MCS at pCAMBIA-1391Z and inserts said dna fragmentation and obtain recombinant plasmid.Said recombinant vectors specifically can be cuts the recombinant plasmid that obtains between recognition site with SalI and the EcoRI enzyme of the insertion of dna fragmentation shown in the sequence 1 pCAMBIA-1391Z.

Increase total length and any segmental primer thereof of said dna fragmentation to also belonging to protection scope of the present invention.

Said dna fragmentation can be applicable to promote destination gene expression under the environment stress condition.

Said environment stress specifically can be and is drought stress and/or salt stress.

Said goal gene specifically can be gus gene.

Said dna fragmentation can be applicable to promote goal gene specific expressed in plant seed.

Said goal gene specifically can be gus gene.

The promotor PRO-TaFbox2 sequence of wheat salt tolerance genes involved TaFbox2 disclosed by the invention, this promotor nucleotide sequence total length is 3076bp, and is high near the position conservative property of gene coding region, difference mainly concentrates on-500bp zone in addition.Promotor 2kb zone is analyzed, contained a plurality of and biological coercing and induce the relevant combination of elements site of transcribing, show that its downstream gene of controlling should receive the expression of stress-inducing with abiotic stress.Promotor of the present invention has tissue specificity, and can receive the environment stress abduction delivering, can in biotechnology, have good application prospects as the inducible expression vector of plant-sourced.

Description of drawings

Fig. 1 transcribes component analysis for MITE.

Fig. 2 detects (arrow indication position is the purpose fragment) for transfer-gen plant PCR; P: positive control; N: non-transgenic plant; H:H ₂O; M:marker.

Fig. 3 detects for transfer-gen plant southern hybridization; The A:HindIII enzyme is cut the result; B: results of hybridization.

Fig. 4 is transfer-gen plant Gus dyeing; A: seedling leaf; B: germination period seedling; C: ripening stage seed.

Fig. 5 is for changeing empty carrier plant Gus dyeing; A: seedling leaf; B: germination period seedling; C: ripening stage seed.

Gus dyeing behind Fig. 6 stress-inducing in seedling stage; A: change the empty carrier plant; B: transfer-gen plant; C12: deepfreeze 12H; C2: deepfreeze 2H; P12:PEG handles 12H; P2:PEG handles 2H; S12: salt is handled 12H; S2: salt is handled 2H; N: be untreated.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.

The preparation of substratum and solution:

The compound method of NB minimum medium (1L) is following: KNO ₃2830mg, (NH ₄) ₂SO ₄463mg, KH ₂PO ₄400mg, MgSO ₄7H ₂O 185mg, CaCl ₂2H ₂O 166mg, FeSO ₄7H ₂O 27.85mg, Na ₂-EDTA 37.25mg, MnSO ₄4H ₂O 10mg, H ₃BO ₄3mg, ZnSO ₄7H ₂O 2mg, Na ₂MoO ₄2H ₂O 0.25mg, CuSO ₄5H ₂O 0.025mg, CoCl ₂6H ₂O 0.025mg, KI0.75mg, vitamin (VB1) 10mg, pyridoxine hydrochloride (VB6) 1mg, nicotinic acid (VB5) 1mg; Inositol 100mg, caseinhydrolysate 300mg, Stimulina 500mg, proline(Pro) 500mg; Sucrose 30g, CEH (hydrolysis network albumen) 300mg, vegetable jelly 2.6g, constant volume is to 1L; PH is adjusted into 5.8,121 ℃ of sterilization 20min.

NBco is substratum altogether: the NB minimum medium adds 2, the Syringylethanone (AS) of 4-D 2mg/L and 100 μ M, adjustment pH 5.5.

Prescreen substratum NBps:NB minimum medium adds 2,4-D 2mg/L and cephamycin (cef) 500mg/L, and adjustment pH is 5.8.

Screening culture medium NBs:NB minimum medium adds 2,4-D 2mg/L, and cephamycin (cef) 500mg/L, Hyg 30mg/L, adjustment pH is 5.8.

MS minimum medium: get 50mL MS macroelement (20 *), 5mL MS trace element (200 *), 5mL molysite (200 *), 5mL MS organic composition (200 *); Mixing; With sucrose 30g, with the NaOH adjust pH to 5.8 of 1mol/L, solid MS adds the 7g agar powder again; Be settled to 1000mL, 121 ℃ of sterilization 20min;

MS macroelement (20 *) is (1000mL): NH ₄NO ₃33000mg, KNO ₃38000mg, CaCl ₂2H ₂O 8800mg, MgSO ₄7H ₂O 7400mg, KH ₂PO ₄3400mg;

MS trace element (200 *) is (1000mL): KI 166mg, H ₃BO ₃1240mg, MnSO ₄7H ₂O 4460mg, ZnSO ₄7H ₂O 1720mg, Na ₂MoO ₄2H ₂O 50mg, CuSO ₄2H ₂O 5mg, CoCl ₂2H ₂O 5mg;

Molysite (200 *) is (1000mL): FeSO47H ₂O 5560mg, Na ₂EDTA2H ₂O 7460mg;

MS organic composition (200 *) is (1000mL): inositol (Inositol) 20000mg, glycocoll (Glycine) 400mg, nicotinic acid (Nicotinic acid) 100mg, vitamin (Vitamin B ₁) 100mg, pyridoxine hydrochloride (Vitamin B ₆) 100mg.

Regeneration culture medium MSpr:MS minimum medium adds NAA 1mg/L in advance, BAP 2mg/L, cephamycin (cef) 500mg/L, Hyg 30mg/L, adjustment pH 5.8.

Regeneration culture medium MSr:MS minimum medium adds NAA 0.5mg/L, BAP 3mg/L, cephamycin (cef) 500mg/L, Hyg 30mg/L, adjustment pH 5.8.

1mol/L phosphate buffered saline buffer (pH7.2) is (1000mL): NaH ₂PO ₄38.9g, Na ₂HPO ₄258.12g; Adjust pH to 7.2, ddH ₂O is settled to 1000mL, 121 ℃ of sterilization 20min.

0.2mol/L NaH ₂PO ₄(pH7.0) (100mL): 3.12g NaH ₂PO ₄2H ₂O is dissolved in sterilization ddH ₂Among the O, be settled to 100mL.

0.2mol/L Na ₂HPO ₄(pH7.0) (100mL): 7.17g Na ₂HPO ₄12H ₂O is dissolved in sterilization ddH ₂Among the O, be settled to 100mL.

GUS histochemical stain reaction solution (20mL): 0.2mol/L Na ₂HPO ₄(pH7.0) 5mL, EDTA (0.5mol/L, pH8.0) 5mL, Tween-2080 μ L, K ₃[Fe (CN) ₆] (170mmol/L) 118 μ L, K ₄[Fe (CN) ₆] (170mmol/L) 118 μ L, X-Gluc (20mg/mL) 1mL; Add ddH ₂O is settled to 20mL ,-20 ℃ of storages after the packing.

Biomaterial:

Agrobacterium GV3101: Liu Fuxiu, Zhao Qin, Ruan Xiaolei, He Yunwei, Li Huaping. the reticent son that suppresses of RNA of paddy rice knurl dwarf virus genome the 11st segment encoding. Science Bulletin, 2008,53 (1): 96-103; Institute of Crop Science, Chinese Academy of Agricultural Science.

Rice paddy seed (Kitaake, the precocious japonica rice variety of Hokkaido, Japan): Yuan Limin, Chou Ming, Wang Peng, Wang Zhiqin, Yang Jianchang .C4 transgenic paddy rice rice shoot blade pore and leaf sheath vascular bundle constitutional features. Scientia Agricultura Sinica, 2006,39 (5): 902-909; Institute of Crop Science, Chinese Academy of Agricultural Science.

PCAMBIA-1391Z:Enrico Scarpella, Erik J.Simons and Annemarie H.Meijer.Multiple Regulatory Elements Contribute to the Vascular-specific Expressionof the Rice HD-Zip Gene Oshox1in Arabidopsis.Plant and Cell Physiology 200546 (8): 1400-1410; Institute of Crop Science, Chinese Academy of Agricultural Science.

Chinese spring: Chen Feng, He Zhonghu, Chen Dongsheng, Zhang Chunli, Xia Xianchun. the variation of Chinese spring grain hardness puroindoline allele detects. Scientia Agricultura Sinica, 2007,40 (2): 217-224; Institute of Crop Science, Chinese Academy of Agricultural Science; Primary source is unclear.

The discovery of embodiment 1, PRO-TaFbox2 (promotor of TaFbox2) sequence

One, the discovery of PRO-TaFbox2 sequence

1, the screening of BAC mixing pit

(1) getting 5ul behind the bacterial classification mixing that the BAC mixing pit is preserved is inoculated in the LB substratum; Shaking speed 220rpm, 37 ℃ of overnight cultures; Alkaline lysis method of extracting plasmid DNA increases according to TaFbox gene ORFs design primer, positive mixing pit is carried out next step screening.

(2) after the preservation bacterium liquid dilution with positive BAC mixing pit, be coated on LB solid culture primary surface, 37 ℃ of incubated overnight, the mono-clonal of 5 times of clone's numbers in mixing pit of picking is inoculated in 384 orifice plates 37 ℃ of overnight cultures, the freezing preservation of substratum then.

(3) be prepared into mixing pit with all mono-clonals in every 384 orifice plate, be inoculated in overnight cultures in the LB substratum, alkaline lysis method of extracting plasmid DNA, PCR detects.

(4) mixing pit to the clone that was positive in the last step carries out the positive monoclonal screening.16 clones that walk crosswise in (A-P) and 24 files (1-24) in 384 orifice plates are mixed respectively, obtain 40 littler clone's mixing pits altogether.Be template with its bacterium liquid respectively, use the allele specific PCR primer amplification, detect and whether contain positive colony.The point of crossing of walking crosswise with file of containing positive colony is exactly possible positive monoclonal; Mono-clonal to corresponding position in 384 orifice plates is verified with the PCR primer.

2, the extraction of BAC DNA

Qiagen Large-Construct Kit extracts BAC DNA.

3, order-checking

With check order PCR reaction of BigDyeR Terminator v3.1Cycle Sequencing Kit (ABI), ABI3730 XL DNA Analyzer is used in order-checking.

Utilize China spring BAC cloning and sequencing, obtained the promotor PRO-TaFbox2 of wheat TaFbox2 gene, total length 3076bp (seeing the sequence 1 of sequence table).

Two, PRO-TaFbox2 sequential analysis

1, transcribes the combination of elements site

Promotor 3KB zone is analyzed; At the translation initiation codon upper reaches except core parts; Contain a plurality of and biological coercing and induce the relevant combination of elements site (seeing table 1) of transcribing with abiotic stress; Show that its downstream gene of controlling should receive the expression of stress-inducing, wherein (9 of methyl jasmonate class binding sites infer that this promotor possibly receive inducing of jasmonate acid signal to MeJA.

The site statistical study of table 1 upstream region of gene promotor induced reaction associated retroviral combination of elements

2, MITE element

PRO-TaFbox2 has a Stowaway type MITE original paper to insert in the zone of upstream of coding region-1kb; This MITE element is transcribed component analysis; Discovery contains TATA-box, CAAT-box and the transcription factor binding site point (see figure 1) relevant with auxin response in more than 100 short base.Therefore, the insertion of MITE also might produce certain influence to the expression pattern of its downstream gene.

The compliance test result of embodiment 2, promotor

One, the acquisition of promotor (PRO-TaFbox2) DNA

It is following to design a pair of primer:

Nucleotide sequence design primer according to PRO-TaFbox2 adds restriction enzyme site SalI and EcoRI, and primer sequence is following:

Primer?F：5’- GTCGACGACTGCGAGTTGCTG-3’；

Primer?R：5’- GAATTCGACAAGTTGAGAAGGAA-3’。

The gDNA that extracts Chinese spring obtains goal gene with above primer to carrying out pcr amplification as template.

PCR reaction system such as table 2.

Table 2PCR reaction system

Response procedures: 94 ℃ of 5min, (94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 1min) * 32,72 ℃ of 10min.Flat end adds A reaction system such as table 3.

Table 3 adds the A reaction system

Composition	Volume (μ l)
		10×Buffer	2
Mg 2+	1.8
		Taq(5U/μl)	0.16
dNTP(25mM)	0.15
		PCR?product	15
TV	20

Response procedures: 72 ℃ of 30min.

The PCR product digs glue and reclaims behind electrophoresis on 1% sepharose.Gel recovery test kit with hundred Tyke Time Inc.s carries out purifying.

Two, the structure of recombinant expression vector

(CAMBIA, Canberra Australia), cut product with enzyme behind the purifying of the skeleton carrier that obtains and step 1 and are connected, and connect more than 2 hours under the room temperature to cut pCAMBIA-1391Z with restriction enzyme SalI and EcoRI enzyme.Linked system is a table 4.

Table 4pGEM-Teasy Vector linked system

Composition	Volume (μ l)
		T4?DNA?ligase	1μl
2×T4?DNA?ligase	2.5μl
		Skeleton carrier	1μl
The PCR product	0.5μl
		TV	5μl

Connect product thermal shock method transformed into escherichia coli (Trans-T1; Article No.: CD501; Available from full Shi Jin biotech firm Beijing Quanshijin Biotechnology Co., Ltd).Sequencing result shows, obtains driving with PRO-TaFbox2 the plant binary expression vector (promotor shown in the sequence 1 is inserted the SalI of pCAMBIA-1391Z and the recombinant expression vector between the EcoRI site) of Cus gene.

Three, the Agrobacterium competent preparation of shocking by electricity

1) picking Agrobacterium GV3101 mono-clonal from the streak plate is inoculated in the 5ml YEB substratum 28 ℃, 250rpm overnight cultures;

2) be inoculated in the 500ml YEP liquid nutrient medium (containing 50mg/L kantlex, 50mg/L Rifampin) in 1: 200 ratio, being cultured to OD600 is about 0.6-0.8;

3) bacterium liquid branch is installed in the 50ml centrifuge tube of precooling and sterilization, 4 ℃, the centrifugal 10min of 4000rpm collect thalline;

4) abandon supernatant, add 50ml 10% (volumn concentration) glycerine suspension thalline (glycerine is prepared with ultrapure water, and sterilization), 4 ℃, the centrifugal 10min of 4000rpm;

5) abandon supernatant, add 25ml 10% (volumn concentration) glycerine suspension thalline, 4 ℃, the centrifugal 10min of 4000rpm;

6) abandon supernatant, add 5ml 10% (volumn concentration) glycerine suspension thalline, 4 ℃, the centrifugal 10min of 4000rpm;

7) abandon supernatant, add 1ml 10% (volumn concentration) glycerine suspension thalline, be packed as 20 μ l/ pipe, be stored in behind the liquid nitrogen flash freezer-70 ℃ subsequent use.

Four, the electric shock of Agrobacterium transforms

On the cell of Gibico company Porter electric shock appearance, carry out.

The recombinant expression vector that 1) Agrobacterium competent cell, electric shock cup and step 2 is prepared (or pCAMBIA-1391Z, as contrast) be put into together on ice and melt until competent cell;

2) open the switch of electric shock appearance, and in electric shock tank, put into mixture of ice and water and carry out precooling;

3) recombinant expression vector with the preparation of 1 μ l step 2 adds filling in the competent centrifuge tube of Agrobacterium of having melted, flicks pipe end mixing;

4) plasmid of mixing and Agrobacterium competence are transferred in the electric shock cup together, note that bubble is not arranged, and the cup that will shock by electricity is put in the electric shock tank;

5) electric shock tank and electric shock appearance are connected, switch is twisted to CHARGE, by MP voltage is risen to more than 390 volts, switch is turned to ARM, when treating that voltage drops to 390 volts, (shock parameters: electric capacity is 330 μ F by the trigger key; Electric shock speed is fast);

6) take out conversion fluid, add 500 μ l YEP liquid nutrient mediums, 28 ℃, 220rpm recovery cultivation 3h;

7) get 3 μ l nutrient solutions and add an amount of YEP liquid nutrient medium and be uniformly coated on the YEP solid plate that contains kantlex (50mg/L) and Rifampin, be inverted for 28 ℃ and cultivated 2 days.

Five, conversion is with the preparation of Agrobacterium bacterium liquid

Get PCR and detect correct Agrobacterium bacterium liquid 5 μ l and be inoculated in the 5ml YEB liquid nutrient medium (containing the 50mg/L kantlex, the 50mg/L Rifampin), 28 ℃, 250rpm are cultivated 30h.Bacterium liquid was transferred in the 300mlYEB liquid nutrient medium by 1: 400, and 28 ℃, 250rpm are cultivated 14h to OD6001.5-3.0.7000rpm, 4 ℃ of centrifugal 15min collect bacterium liquid, and resuspended thalline is in the conversion penetrating fluid (1/2MS+2%Sucrose, 6-BA:0.01mg/L, VB1:10mg/L, VB6:1mg/L, Silwet L-77:0.02%) of 2 times of volumes.

Six, rice conversion

1, after ripe paddy rice (Kitaake) seed carries out surface sterilization 1min with 70% (volumn concentration) ethanol; With 20% (volumn concentration) Youxiaolin (dripping a Tween-20) sterilization 20min; Aseptic water washing 4-5 time blots on filter paper, and the embryo of seed places on the NB solid medium up; 26 ℃, the cultivation down of 16h/8h photoperiod, evoked callus.

2, behind the 10-15d, peel the callus that mature embryo scultellum director goes out, change new NB solid medium succeeding transfer culture under identical condition over to.The callus of 3-4cm size promptly can be used for infecting conversion.

3, the Agrobacterium bacterium liquid with step 5 is evenly coated in dull and stereotyped (containing 50mg/L kantlex and 50mg/L Rifampin) the last 28 ℃ of dark culturing 2-3d of solid YEP; Scrape bacterium with aseptic spoon from flat board and (contain 100 μ M Syringylethanones in the common culture medium AAM of liquid; Be called for short AS) in, the adjustment bacterial concentration to OD600 be 0.2-0.5.

4, the embryo callus of step 2 is put into the agrobacterium suspension submergence of step 3, shaken 15min frequently gently.The bacteria-removing liquid that inclines blots the unnecessary bacterium liquid in surface with the rice callus tissue with aseptic filter paper, places NBco altogether on the substratum, and 28 ℃, dark are cultivated 2-3d altogether.

5, the callus of cultivating 2-3d is altogether given a baby a bath on the third day after its birth time with sterilized water, cleans 15min with the water of the cephamycin (cef) that contains 500mg/L, and the water flushing once dries up moisture with callus again, forwards on the prescreen substratum NBps that contains Totomycin 26 ℃ of dark cultivations to.

6, change the screening culture medium NBs that contains Totomycin over to, induction of resistance callus 2-3 time behind the cultivation 7d.Treat that resistant calli is long after a certain size, select golden yellow color, resistant calli that quality is compact changes preparatory regeneration culture medium MSpr over to, the identical resistant calli in source is moved to the same area of substratum, as same strain system, 26 ℃ of illumination cultivation.

7, change regeneration culture medium MSr behind the 7d over to,, differentiation green up to going out sprouted.The seedling that grows moves in the test tube of root media 1/2MS root culture behind 3-4cm.In root culture 2-3 week, treat to can be moved to hot-house culture after seedling grows sturdy root system.Can transplant land for growing field crops results seed after cultivating for some time, obtain T1 for seed.

The plant that T1 is grown up to for seed carries out PCR evaluation and Southern evaluation (the part qualification result is seen Fig. 2 and Fig. 3), identifies that the male plant is transfer-gen plant.Identify that male T1 obtains T2 for seed for the plant selfing.T2 is identified for seed.

Six, Gus histochemical stain

1, tissue specificity

Respectively with T2 for transfer-gen plant seed, T2 for changeing the sowing of empty carrier plant seed, get respectively seedling leaf,, germination period seedling, ripening stage seed detect.The detection step is following: at room temperature use 1mol/L phosphate buffered saline buffer (pH7.2) to wash twice rice tissue, each 15min adds GUS histochemical stain reaction solution, 37 ℃ of lucifuge incubation 24h afterwards.Outwell reaction solution, 70% ethanol is fixed several hours for 4 ℃, and 95% ethanol decolorization is taken a picture.

The result of transfer-gen plant sees Fig. 4.The result who changes the empty carrier plant sees Fig. 5.The result shows: promotor of the present invention (PRO-TaFbox2) can drive the Gus protein expression, a little less than leaf tissue is expressed, and expresses stronger at seed.Empty carrier adopts 35S promoter, thus in the contrast Gus protein expression is arranged also, but close in each tissue.

2, inducibility

Respectively with T2 for transfer-gen plant seed, T2 for changeing empty carrier plant seed soaked overnight, change over to then in the petridish that is covered with filter paper, when treating that seedling grows to 5cm, change in the plastics casing that fills the Hoagland nutritive medium and cultivate.When seedling grows to two leaves wholeheartedly the time, carry out respectively that salt stress is handled, drought stress is handled and low temperature stress is handled, the treatment time is 0h, 2h, 12h.

Salt stress is handled: the seedling root is immersed in the aqueous solution that contains 250mM NaCl 25 ℃ of cultivations.

Drought stress is handled: the seedling root is immersed in the aqueous solution that contains 16% (quality percentage composition) PEG 25 ℃ of cultivations.

Low temperature stress is handled: seedling is placed on the middle mutually cultivation of 4 ℃ of artificial climates.

The result sees Fig. 6.Transfer-gen plant is under PEG and salt processing, and the Gus dyeing of root strengthens, but variation is very little under cold coercing, and shows that this promotor can be expressed by the stress-inducing of arid and salt and strengthen.Commentaries on classics empty carrier adjoining tree is organized at each all has expression, and is coercing under the processing, and expression amount does not have obvious gap.

Sequence table

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<210>1

<211>3076

<212>DNA

< 213>Triticum wheat (Triticum aestivum L.)

<400>1

gactgcgagt?tgctgcattc?ggctgcagtt?ttcttgcggt?aggatcaggc?gagccagtta 60

aaaaagtcaa?acatgcagcg?catgaaccgg?atcatatcat?ggtttttttc?tctgatcggg 120

gcccctacga?tttgcggggc?cctgtgcgat?tgcaccgctt?gcacaccctc?ctcgacgggc 180

ctggttgtag?cacgtatgcg?tcgagttaaa?gtatatgcat?taatggatct?ctctacagtt 240

tatccacaac?ccatcaaggc?cgcaatgtga?tttgtggttt?atgggagaca?gtgtgatgcc 300

tttatgaaat?gtgcttcttg?tacgtgaatc?ctgaatcctg?gacgcaattg?ccaggtccgt 360

atttcccata?tatgttgacc?gctacttata?tcaatcttgg?aaggaacaac?ttgaagttgt 420

gttacctttg?ggagtttttg?cctgacctta?atggtgtcct?tgcaaggcca?ggagcgtgta 480

tgcctccgtt?tttaaagatc?acaggaaatg?gagtgggttg?agtaaagtct?attttaaacc 540

ttgaagttgt?attgtatgtg?tggtcgaaat?taaactggga?tcactcactt?ctctcactta 600

ctatgtgggc?ctacatgtca?tattcatttc?ttcctactga?actttttaaa?tatatgatga 660

acttttttta?atatacaaat?tttttataaa?aaacatactg?aacaattttc?taaagaatat 720

tattaaaatt?aataggtttt?caaatatgta?gctcctaaga?aataacaacc?attttaattt 780

taaaaaataa?caactatttg?ggaggggggg?gggtgagaaa?caacctaaaa?gactgaagat 840

tttttttata?tcgctcacgg?gcaaattgtg?ccaccaacct?gctcttgcta?tcagccaagc 900

ttgcgggtgg?taaataagag?ctcttaagaa?tggggtcaac?taaccaacgg?tatccccggg 960

aggagttcac?agcgggtacg?catgggtgaa?ccgcgtgata?tggccaaccg?ccgtcacatg 1020

tcatgctttg?ggcacatctg?tgggagcaca?attttttgtc?tccatgcgtt?tttttaagat 1080

tttgtttgtt?atttcataaa?ttttctcgac?aaaaaagatc?catacttttt?ttgtgtgttt 1140

tcctcgcgtg?aagcacacta?tgtgtttttt?ctgattttta?tttttatttc?catttttcca 1200

aaagaattag?tttctgattt?ttttgttttc?tgcttttttc?agaaaaaggt?ttgtccgaat 1260

ctattaacat?gagatctaat?tttgaagatg?tcaacgcaag?aaacgcaagg?ataataatgg 1320

ttcatggttt?gggcgcaaga?tttaatagat?aaaaagttta?ggaagaatga?atatacaaaa 1380

aaaaacctag?gctgtgacaa?gtgtcgtaca?tacagtacac?catttgtcat?cacaaaaaga 1440

tgtgaggatg?acatttgcaa?agagtatcct?tgaccaatga?tttcacaaca?attactacag 1500

tttctttttg?acaatctcgc?taaagctttt?ttttttttga?gaaaccgcta?aagctttatt 1560

atgtcagtca?caagatttta?gggccgagtt?ttctgcgggc?cggtctaatt?tgggccgaag 1620

cccgtagagc?gtcttctcct?tgcgtgacgc?actccccaca?aaatcacaaa?cggcctcata 1680

cacacaggag?acaggacgaa?caagaaccct?aatccccaaa?tcctccagcc?ctctctcaaa 1740

tcttgagacc?cgatccctaa?atcggaccat?ggattgacta?tgcggcagca?tggattgagc 1800

tcctgccgat?cccggatgtg?gcgggcagcg?gcggcggtct?ggaagctcgc?cggcaggaca 1860

ggacaagcgg?aggttgagca?gcggcgtctg?acttaatagg?tacgacatca?aaactactcc 1920

ctccgttcct?aaatacaagt?gtttctacag?atttcaatat?agactacata?cggagcaaaa 1980

tgagtgaatc?taccctctaa?actatgtcta?tatacatcct?tatgtggtcc?atattaaaaa 2040

ctctagaaga?ctttatattt?aggaatggag?gaagtactat?ttaggttaac?tgctatgggc 2100

ggtctcattt?ctttgcagct?aatactcctc?cagcgttagg?ttctgcgtcg?aagtaacaga 2160

tgtatagtca?cagtcacaga?aatgcaagtc?aagtgcctca?ataaaaattc?tgaaaggatt 2220

ctcaaaaaca?aaaacaaaaa?agttgtaaaa?ggagcctgta?aaattctaac?accgtgtatc 2280

catgttaata?gttagtcggc?tagactacgt?ttttgaactt?gtacttaatg?gagtggttgt 2340

agaacaaaga?tgtcacattg?ttaaccatgt?tgggctagct?tggttgctgc?actggagcga 2400

tttgcttgag?actctacgtt?tcgtgtttgg?ttgccacact?gagatggcgc?taatttggaa 2460

ataaaccatt?tattcgtaat?tacattcccg?gaagggttcc?tccttgaaca?aatgtctcat 2520

ttatggatac?actgggagca?aaattgaaaa?ccctagctag?tatttattct?ggaaagataa 2580

ctgatcaaag?agacgtagct?tcactcaggc?acgtccgtca?gactcaggcg?tccgacttgc 2640

agcccaccgt?gcctggcact?ccatcctcga?ggcggtcgtt?ggtgagaatg?aaggaaccga 2700

tgaaaacaaa?agactacctt?tacatttagc?tttcacaaag?tgtatttaag?tcttgacgtt 2760

ttgtcctcag?gcgttgtttg?caaccagtaa?gtttcaaata?gtattagcaa?acttgttaca 2820

cgccgctatc?aatagcttgt?actattttga?tttattcttt?gttgtttggc?aattaattca 2880

actatctgag?ccactgtaac?ttttgttccc?agccgtcagc?cgcagaaccg?ccgttgatag 2940

ccacttccat?tgttgccgct?ccgtgcttca?gtagactggt?atgcctgttc?tgcctccgtg 3000

ctctttcttc?catcctttca?ttctgtcctg?cactgatgtt?tgatctggtt?attttcgttt 3060

tccttctcaa?cttgtc 3076

Claims (9)

1. a dna fragmentation is the dna molecular shown in the sequence in the sequence table 1.

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

3. recombinant vectors as claimed in claim 2 is characterized in that: said recombinant vectors obtains recombinant plasmid for the MCS at pCAMBIA-1391Z inserts the said dna fragmentation of claim 1.

4. recombinant vectors as claimed in claim 3 is characterized in that: said recombinant vectors is cut the recombinant plasmid that obtains between recognition site for the SalI and the EcoRI enzyme that dna fragmentation shown in the sequence 1 are inserted pCAMBIA-1391Z.

5. the primer of the said dna fragmentation total length of amplification claim 1 is right; The right sequence of said primer is following:

5’- GTCGACGACTGCGAGTTGCTG-3’；

5’- GAATTCGACAAGTTGAGAAGGAA-3’。

6. the said dna fragmentation of claim 1 is promoting paddy rice application in the destination gene expression under the environment stress condition;

Said environment stress is drought stress and/or salt stress.

7. application as claimed in claim 6 is characterized in that: said goal gene is a gus gene.

8. the application of the said dna fragmentation of claim 1 in the promotion goal gene is specific expressed in plant seed; Said plant is a paddy rice.

9. application as claimed in claim 8 is characterized in that: said goal gene is a gus gene.

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