CN103525816A - Ginger flower floral specific and damage and pest induced TPS2 (Trehalose-6-Phosphate Synthase 2) promoter and application thereof - Google Patents

Abstract

The invention belongs to the technical field of gene engineering and particularly discloses a ginger flower floral specific and damage and pest induced TPS2 (Trehalose-6-Phosphate Synthase 2) promoter and an application thereof. The promoter has a nucleotide sequence as shown in SEQ ID NO: 2. A target gene controlled by the TPS2 promoter disclosed by the invention is only expressed in a flower tissue of a transgenic tobacco, and the expression is relevant to a flower development process and is induced by damage and pests. In addition, the TPS2 promoter disclosed by the invention can be introduced to a cell of a ginger flower or other plants after being connected to any one plant transformation vector to obtain promoter-containing transgenic fragrance and resistance to be used for production; or a specific molecular marker can also be generated according to the sequence information of the promoter disclosed by the invention and is used for authenticating the fragrance and resistance genotypes of the ginger flower or other plants and realizing the molecular-marker-assisted selective breeding, so that the breeding selection efficiency is increased.

Description

Special TPS2 promotor and the application thereof of inducing with injury, insect pest of a kind of ginger portion

Technical field

The present invention relates to gene engineering technology field, particularly, relate to special TPS2 promotor and the application thereof of inducing with injury, insect pest of a kind of ginger portion.

Background technology

Promotor is to be positioned at a section of structure gene 5 ' end upstream can identify and activate RNA polymerase, makes it to be combined exactly with template DNA, guarantees to transcribe accurately and effectively initial DNA sequence dna.It is the center of plant gene transcription regulation and control, is the key of understanding gene transcription regulation mechanism and expression pattern.

According to the transcriptional profile of promotor, can be divided into: constitutive promoter, tissue or organ specific promoters and inducible promoter.Wherein, organizing specific type promotor is also called organ specific promoter, comprises fruit differential promotor, root-specific promoter, leaf specific promoter and floral organ specific promoter etc.Under the regulation and control of this class promotor, the expression of gene is often only limited to some specific organ or tissue position, and often shows the characteristic of developmental regulation.

Floral specific promoter can make goal gene specific expressed in spending, can effectively control goal gene and spend middle specifically expressing and closely related with flower development process, in addition, also can be subject to the synergy of the controlling elements such as many induction types during floral organ gene action.The controlling element of petunia CHS is found one of them G-box, is the combination territory of associated transcription factor, is light, anoxybiosis and ABA response element, regulated and controled by light and ultraviolet induction, with flower specifically expressing relevant (Hong Yahui etc., 2003).After plant is subject to physical abuse that artificial wound or insect cause, can produce some small-molecule substances and polysaccharide, they are as the expression of semiochemicals induction series of defence gene.Potato lesioned gene protease inhibitor gene Pin II promotor wound induction region is at the proximal end region of promotor,-700～-195 sections and-90CaMV35S promotor are merged, find that chimeric promoters has the activity of wound induction, therefore think that this section controls the abduction delivering of hindering of this gene.

Floral organ specific promoter not only contains outside necessary conservative property cis element, also contains the characteristic sequences of some organizing specific types and inducible promoter.And it is basis that this tissue specificity be take specific histocyte structure and chemical physics signal conventionally, has common decision the such as kind, number and relative position of the element of several control tissue specific expressions in this class promotor simultaneously.

At present the selection of promotor in transgenic technology is mainly adopted to constitutive promoter, under the control of composition type expression promoter, foreign gene all can be expressed at all sites of transgenic plant and different etap, and continue, express efficiently, there is no Space-time speciality, caused the loss of energy, affect the surviving rate of positive plant, cause that plant physiology and aberrant morphogenesis change, thereby affect the normal growth growth of plant.Because organizing specific and inducible promoter can regulate and control the expression of downstream gene effectively, reduce to greatest extent the injury himself being caused due to the accumulation of foreign protein in transgenic plant body.Kasuga etc., by rd29A::DREB1A and 35S::DREB1A transformation of tobacco, compare with rd29A::DREB1A transfer-gen plant, and the hysteresis phenomenon of growing obviously appears in 35S::DREB1A transgene tobacco; The stress tolerance of rd29A::DREB1A transfer-gen plant is also apparently higher than 35S::DREB1A transfer-gen plant.The expression that Pellegrineschi drives DREB1A/CBF3 with stress induced promoter rd29A, has improved the patience of transgenic wheat to drought stress, and has not caused obvious deformity.

Flower is as the important reproductive organ of plant, utilizing plant hybrid vigour manual creation sterile line, study some genes with the Expression element of floral organ and particularly play an important role in the genetically engineered researchs such as function of transcription factor.Wherein anther specific expression promoter and pollen promotor have played very important effect.Utilize anther tapetum specific promoter TA29, A9 etc. to build male sterility gene, create male infertility plant and tobacco, rape etc., succeed.Chang little Li (2009) proceeds to the special CHS gene promoter of the colored portion being cloned into from lily in Arabidopis thaliana and finds to have specific expressed at the inflorescence position of transformed plant.The discoveries such as Kobayashi, the CHS gene promoter that clone obtains from three flower rough gentian (Getiana triflora) can be specific expressed at the petal lip of petunia.Lewinsohn etc. adopt E8 promotor, and by the LIS Gene into Tomato of fairy maiden's fan, mellow fruit synthesizes and discharges the obvious S-phantol of fragrance and 8-hydroxyl phantol.Davidovich etc. (2007) adopt PG promotor, and lemon sieve alkene Geraniol synthetic enzyme (GES) gene is proceeded to tomato, and this gene overexpression in Transgenic tomato fruit causes the accumulation volume of Geraniol to increase, and successfully changes tamato fruit local flavor.

Development along with plant gene engineering technology, organizing specific and inducible promoter can be used for manual control target gene accurate expression under specific developmental stage, particular tissues organ and the relevant adverse environmental factor of plant, at the resistance of Crop Improvement, the aspects such as local flavor of viewing and admiring shape, improving fruit and vegetables that improve flowers, have important application prospect.

Summary of the invention

Object of the present invention is in order to overcome in prior art the defect of the rarely seen report of research of and injury special to ginger portion, insect pest evoked promoter, and the TPS2 promotor of the special and injury of a kind of ginger portion, insect pest induction is provided.

Another object of the present invention is to provide the application that a kind of ginger portion TPS2 promotor special and that injury, insect pest are induced changes the peculiar proterties of flower portion such as pattern, the fragrance of a flower and fertility and improves the new variety of plant of insect resistance capacity in cultivation.

The present invention's above-mentioned purpose that is achieved by the following technical programs:

The TPS2 promotor that portion is special and injury, insect pest are induced, its nucleotide sequence is as shown in SEQIDNO:2.This promotor total length is 1922bp, and section length is respectively 782bp and 321bp.TPS2 promotor is given ginger floral base because of the special and injury of HcTPS2 flower portion, insect pest abduction delivering feature.After TPS2 promoter sequence of the present invention and gus gene amalgamation and expression transformation of tobacco, target gene is only spent tissue expression tobacco, and its expression is relevant with flower development process, and in blade, express be hurt, insect pest and methyl jasmonate induction.

Through software analysis, obtain object fragment p-320, p-780 and the p-1928 that described TPS2 promotor contains 35 ' end series disappearance, the sequence of p-320, p-780 and p-1928 is as shown in SEQIDNO:2～4.

, described recombinant vectors is for to insert TPS2 promoter sequence (SEQIDNO:2) as mentioned above in the multiple clone site of the carrier that sets out.The described carrier that sets out can be all types of plant expression vectors of often using in the art, and preferably, the present invention's plant expression vector used is pCAMBIA1300G plant expression vector.

, described recombinant vectors is for inserting object fragment p-320, p-780 or the p-1928 sequence of 5 ' end series disappearance of TPS2 promotor as mentioned above in the multiple clone site of the carrier that sets out.The described carrier that sets out can be all types of plant expression vectors of often using in the art, and preferably, the present invention's plant expression vector used is pCAMBIA1300G plant expression vector.

A kind of recombinant bacterium of recombinant vectors as mentioned above that comprises.

A kind of clone of recombinant vectors as mentioned above that comprises.

TPS2 promotor is in the application of cultivating the peculiar proterties of flower portion such as changing pattern, the fragrance of a flower and fertility and improving the transgenic plant new variety of insect resistance capacity.

Cultivate a method for the transgenosis fragrance of a flower or resistance plant, concrete steps are: will contain recombinant vectors as above and import the vegetable cell that sets out, thereby obtain the transgenosis fragrance of a flower or resistance plant that TPS2 promotor starts destination gene expression.

Preferably, described goal gene is gus gene.

The present invention comprises that part connects the upper suitable formed mosaic gene of objective function gene effectively using the promoter fragment of three kinds of length as primary structure equally, and in genome, comprises the plant of this promoter fragment and the seed of this kind of plant.

According to promoter DNA sequence information provided by the invention, those skilled in the art can easily obtain the promoter DNA sequence being equal to by the following method: (1) obtains by database retrieval; (2) take promoter dna fragment is the genomic library acquisition of probe screening ginger or other plant; (3) according to promoter DNA sequence information design oligonucleotides primer, by the method for pcr amplification, from the genome of ginger or other plant, obtain; (4) on the basis of promoter DNA sequence, with gene engineering method transformation, obtain; (5) by the method for chemosynthesis, obtain.

Promoter DNA sequence provided by the invention has important using value.One of application is that described promoter DNA sequence is connected to any plant conversion carrier, with any method for transformation, promoter DNA sequence is imported to vegetable cell, can obtain and express the special and injury of described colored portion, insect pest abduction delivering feature, thereby be applied to produce.Promoter DNA sequence construct of the present invention, in plant conversion carrier, can regulate and control the downstream gene of amalgamation and expression, thereby strengthen plant, produces the fragrance of a flower and strengthens the abilities such as resistance.

The Another application of promoter DNA sequence provided by the invention is to produce specific molecule marker according to described gene order information, includes but not limited to SNP(mononucleotide polymorphic), SSR(simple sequence repeats polymorphic), RFLP(restriction enzyme length is polymorphic), CAP(cutting amplified fragments is polymorphic).With these marks, can identify the fragrance of a flower genotype of ginger or other plant, for molecular marker assisted selection breeding, thereby improve the efficiency of selection of breeding.

Compared with prior art, the present invention has following beneficial effect:

The present invention is combined clone's TPS2 promoter DNA sequence with control pattern, the fragrance of a flower and resistance objective function gene, contribute to produce new pattern, the fragrance of a flower and resistance plant, and can not produce the chain problem of bad gene in the genome of following appearance in traditional breeding technology, and can shorten breeding time.In addition, the present invention can further provide or apply transfer-gen plant and the corresponding seed of new pattern, the fragrance of a flower and resistance that above-mentioned TPS2 promoter dna fragment obtains, and the plant transforming with recombinant chou of the present invention or by the seed of this class plant acquisition.Can promotor of the present invention be proceeded to other plant by the mode of sexual hybridization.

Figure of description

The transgene tobacco GUS of Fig. 1 .TPS2 promotor and GUS amalgamation and expression is at different tissues expression specificity; A: the different tissues GUS situation that dyes; B: different tissues GUS enzyme activity situation.

The transgene tobacco GUS of Fig. 2 .TPS2 promotor and GUS amalgamation and expression is in the expression of floral organ different development stage; A: the different development stage GUS situation that dyes, wherein illustrates 1 for unopened petal; 2 is the petal of half-open position; 3 is the petal under state in full bloom; 4 is old and feeble petal; B: different development stage GUS enzyme activity situation, wherein illustrates 1～4 same A.

Fig. 3. transgene tobacco GUS methyl jasmonate treatment in petal of different lengths fragment TPS2 promotor and GUS amalgamation and expression, the expression after injury is processed; A: GUS dyeing situation in petal after different treatment; B: GUS enzyme activity situation in petal after different treatment; Diagram 1 be untreated petal, 2 petals that be methyl jasmonate treatment, and 3 for injuring the petals of processing.

Transgene tobacco GUS methyl jasmonate in blade of Fig. 4 .TPS2 promotor and GUS amalgamation and expression, the abduction delivering of injury and insect pest; A: GUS dyeing situation in different treatment rear blade; B: GUS enzyme activity situation in different treatment rear blade, illustrate 1 for untreated, 2 is methyl jasmonate treatment, and 3 process for injury, and 4 is insect pest processing.

Fig. 5. the PCR of the transgenic tobacco plant of different lengths fragment TPS2 promotor and GUS amalgamation and expression detects figure; A:P-1928 transfer-gen plant PCR detects; B:P-780 transfer-gen plant PCR detects; C:P-320 transfer-gen plant PCR detects.

Fig. 6 .TPS2 promotor p-780 fragment and floral base are because of the PCR detection figure of the transgenic tobacco plant of HcTPS1 amalgamation and expression; A: HcTPS2 promotor special primer amplification for transgene tobacco; B: Totomycin primer specific primer amplification for transgene tobacco; C: HcTPS1 gene specific primer amplification for transgene tobacco.

Embodiment

Below in conjunction with the drawings and specific embodiments, further describe the present invention.Unless stated otherwise, the reagent adopting in embodiment and method are conventional reagent and the method for using in this area.

Embodiment 1

The acquisition of TPS2 promoter sequence

S1. the extraction of the total DNA of ginger blade: by the 0.1～0.2g ginger flowering plant blade powder transfer through liquid nitrogen grinding to 1.5mL centrifuge tube.Add 1mLCTAB solution, 65 ℃ of water-baths 30 minutes; Every 10 minutes, put upside down once.Then, to adding 1mL phenol in centrifuge tube: chloroform (1: 1), put upside down several times, centrifugal 10 minutes of 10000rpm, shifts the new centrifuge tube of supernatant to, adds isopyknic chloroform: primary isoamyl alcohol (24: 1), mixes centrifugal 10 minutes of 10000rpm.Shift the new centrifuge tube of supernatant to.Add isopyknic Virahol, put upside down and mix, centrifugal 10 minutes of 10000rpm, removes supernatant, and 70% ethanol is washed once, and vacuum is drained, and is dissolved in the sterilized water of 30 μ L, for pcr amplification.Consisting of of described CTAB solution: Tris100mM, NaCl1.4M, 20mMEDTA, CTAB2%, mercaptoethanol 0.1%.

S2. adopt 5 ' end upstream regulatory sequence of hiTAIL-PCR technology clone HcTPS2 gene: according to the sequence of the ginger HcTPS2 gene obtaining before contriver, (see Li and Fan, 2011, Molecular Cloning and Expression Analysis of a Terpene Synthase Gene, HcTPS2, in Hedychium coronarium.Plant Molecular Biology Reporter29 (1): 35-42), at 3 outside specificity nested primer sp1 of its 5 ' end design, sp2, sp3, random degenerated primer design is with reference to 5 random degenerated primers that design in (2007) documents such as Liu (Liu, 2007, BioTechniques, 43(5): 649-656).Concrete sequence is as follows:

sp1：5'-CGGATATCATGGGAGCACAGATAGTAGTGC-3'（SEQID NO:6）

sp2：5'-CTACACGATCACTACTTTGGAGCCTTGTCCTTC-3'（SEQID NO:7）

sp3：5'-ATGAGGTCAGAGTTGTCGTGTGGATTTGGGCGTAG-3'（SEQIDNO:8）

LAD-1：5'-ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA-3'（SEQIDNO:9）

LAD-2：5'-ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT-3'（SEQIDNO:10）

LAD-3：5'-ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA-3'（SEQIDNO:11）

LAD-4：5'-ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT-3'（SEQIDNO:12）

AC1：5'-ACGATGGACTCCAGAG-3'（SEQIDNO:13）

The system of amplification and program are with reference to the carrying out in (2007) documents such as Liu.Amplifying DNA fragmentation, from agarose gel, reclaim and be connected to pMD-19Tvector, Transformed E .coli.DH5 α, enzyme checks order after cutting and identifying.Sequencing sequence is as shown in SEQIDNO:1; Sequence shown in SEQIDNO:1 comprises the part ORF sequence of the gene after promotor TPS2 sequence and initiator codon.Sequence before initiator codon is exactly the nucleotide sequence of promotor TPS2, and the long 1922bp of sequence, as shown in SEQIDNO:2.

Submit the nucleotide sequence of promotor TPS2 (shown in SEQIDNO:2) to online promoter Analysis software, the cis-regulating element comprising in prediction promotor, found that: the nucleotide sequence of promotor TPS2 is divided into the object fragment of 35 ' end series disappearance; 35 ' end deletion sequence is respectively p-320, p-780 and p-1928.P-320 length is about shown in 320bp(SEQIDNO:3); Shown in the about 781bp(SEQIDNO:4 of p-780); Shown in the about 1921bp(SEQIDNO:5 of p-1928).

TPS2 promoter regulation component analysis

Embodiment 2

TPS2 promotor and GUS amalgamation and expression transformation of tobacco

The sequence of the TPS2 promotor S1. obtaining according to embodiment 1, design packet is containing the primer for increase 35 ' end deletion sequence p-320, p-780 and p-19283 of Hind III and BamHI restriction enzyme site.Primer sequence is as follows:

The primer of amplification p-320:

p-320-F：5'-GGCCAAGCTTATACGTGCATGTTACAAGTATC-3'（SEQIDNO:14）

The primer of amplification p-780:

p-780-F：5'-GGCCAAGCTTCTGTTTCAAAACATCCAATTCCTC-3'（SEQIDNO:15）

The primer of amplification p-1928

p-1928-F：5'-GGCCAAGCTTACTGATAAGATAAGTTGC-3'（SEQID NO:16）

Downstream primer is:

R：5'-GGCGGATCCAATTTCAACTTCAAGTCCTGAC-3'（SEQID NO:17）

S2.pCAMBIA1300G plant expression vector carries out double digestion with Hind III and BamHI restriction enzyme, and 1% sepharose reclaims large fragment.Utilize T4DNA ligase enzyme that object fragment orientation is inserted into plant expression vector.To connect product and transform intestinal bacteria (E.coli) DH5 α competent cell, and through enzyme, cut and check order after evaluation, obtain respectively three kinds of recombinant plant expression vectors.Preparation agrobacterium tumefaciens EHA105 competent cell, proceeds to Agrobacterium by above-mentioned recombinant plant expression vector.

S3. the aseptic blade of tobacco is cut into the fritter of 0.5cm * 0.5cm, is seeded in and on inducing culture, carries out preculture, illumination cultivation 2～3d.Get the material after preculture 2～3d, add the Agrobacterium suspension bacteria liquid preparing, infect 5～10min, outwell bacterium liquid, taking-up explant is placed on aseptic thieving paper and sucks the bacterium liquid adhering to, and is again placed on former pre-culture medium 28 ℃ of dark 3d that cultivate.Explant after common cultivation is transferred in antibacterial screening culture medium and selects to cultivate after 20d, and the transformant of explant will produce resistant calli, and these materials are proceeded to and on corresponding selective differentiation substratum, differentiate bud cultivation.In screening culture medium, induce after approximately 4 weeks, more than indefinite bud grows to 2cm, the young shoot that leaf plate edge is grown cuts from base portion and leaf dish, inserts to contain on the root media of selecting to press and carries out root culture.Plant to be planted is long-living while going out a large amount of root, and tissue cultured seedling is taken out from group training chamber, unscrews bottle cap, at indoor placement 5～7d, shines 1～2h sun every day; Then take out young plant, clean the substratum on root system, be transplanted in sterilized compost, transplant the transparent plastics being with holes on back cover, to keep 75～85% humidity, 25 ℃, illumination 1500～2500Lux, the photoperiod is with 14～16h/8～10h hour dark of illumination every day.Cultivate 10～15d left and right, move on to the outdoor Adaptable growth that carries out.

S4.CTAB method is extracted resistant plant leaves genomic DNA, with the HcTPS2 promotor special primer that comprises Hind III and BamHI restriction enzyme site of design in advance, carries out pcr amplification, can amplify the positive plant of band.

Through PCR, detect positive transfer-gen plant, with not genetically modified plant as negative control, getting the positions such as root, stem, leaf, petal, calyx, column cap and flower pesticide dyes, decolouring, observes gus protein in the colour developing situation of Different Organs tissue and different development stage and adopts chemoluminescence method to detect GUS enzymic activity.During different tissues GUS dyeing, flower pesticide, calyx, column cap (Figure 1A), presents blueness in petal (Fig. 2 B); At leaf (Fig. 3 B), in stem and root (Figure 1A), without obvious blue, show that TPS2 promotor presents colored specifically expressing characteristic; Different tissues GUS enzyme assay result shows, GUS enzymic activity is expressed the highest in flower pesticide, is secondly petal, calyx and column cap, and in stem and leaf, enzyme activity is very low, sees Figure 1B, consistent with coloration result.The GUS coloration result of different development stage shows: unopened petal does not have blueness, and half-open, the petal under in full bloom and aging state presents blueness, and petal blueness in full bloom is the most obvious.(Fig. 2 A) GUS enzyme assay simultaneously result also shows that unopened petal enzyme activity is very low, and along with petal is open, enzyme activity improves.Under state in full bloom, it is the highest that enzymic activity reaches, and declines senescence phase (Fig. 2 B).

Transfer-gen plant and the pocket knife scuffing that transfer-gen plant use was not disinfected are carried out to damage experiment, after 20min, GUS staining examine and GUS enzyme assay.The larva of insect prodenia litura is placed on the blade of transfer-gen plant and transfer-gen plant not, Continuous Observation, after insect bite after 20min, GUS staining examine and GUS enzyme assay.The methyl jasmonate of 0.2mM is applied on the blade and petal of transfer-gen plant and transfer-gen plant not, after 20min, GUS staining examine and GUS enzyme assay.GUS dyeing shows: after methyl jasmonate and injury are processed, the blueness of petal is obviously than the blue color of untreated petal dark (Fig. 3 A), three fragment comparisons simultaneously, and promoter fragment is longer, and blueness is darker.Blade after methyl jasmonate and injury are processed all presents obvious blueness.GUS enzyme activity determination shows, after petal methyl jasmonate and injury are processed, GUS activity ratio is untreated to be significantly improved (Fig. 3 B).Untreated normal activities of enzymes in leaf is extremely low, but methyl jasmonate, after injury and insect bite processing, GUS enzymic activity be significantly increased (Fig. 4 B).

Embodiment 3

TPS2 promotor and floral base are because of HcTPS1 amalgamation and expression transformation of tobacco

With HcTPS1 clone recombinant plasmid, do template, with the special primer of introducing SpeI and BamHI restriction enzyme site, carry out pcr amplification.PCR product reclaims test kit with TaKaRa and reclaims, and reclaims product and directly with SpeI and BamHI restriction enzyme, carries out double digestion, and 1% sepharose reclaims object fragment.Adopt the above-mentioned p-780 plant expression vector containing HcTPS2 promotor building to carry out double digestion with SacI and BamHI restriction enzyme, 1% sepharose reclaims large fragment.Enzyme is cut product and is connected, and transforms bacillus coli DH 5 alpha.Through enzyme, cut and check order after evaluation, obtaining recombinant plant expression vector.Preparation agrobacterium tumefaciens EHA105 competent cell, proceeds to Agrobacterium by above-mentioned recombinant plant expression vector.

The aseptic blade of tobacco is cut into the fritter of 0.5cm * 0.5cm, is seeded in and on inducing culture, carries out preculture, illumination cultivation 2～3d.Get the material after preculture 2～3d, add the Agrobacterium suspension bacteria liquid preparing, infect 5～10min, outwell bacterium liquid, taking-up explant is placed on aseptic thieving paper and sucks the bacterium liquid adhering to, and is again placed on former pre-culture medium 28 ℃ of dark 3d that cultivate.Explant after common cultivation is transferred in antibacterial screening culture medium and selects to cultivate after 20d, and the transformant of explant will produce resistant calli, and these materials are proceeded to and on corresponding selective differentiation substratum, differentiate bud cultivation.In screening culture medium, induce after approximately 4 weeks, more than indefinite bud grows to 2cm, the young shoot that leaf plate edge is grown cuts from base portion and leaf dish, inserts to contain on the root media of selecting to press and carries out root culture.Plant to be planted is long-living while going out a large amount of root, and tissue cultured seedling is taken out from group training chamber, unscrews bottle cap, at indoor placement 5～7d, shines 1～2h sun every day; Then take out young plant, clean the substratum on root system, be transplanted in sterilized compost, transplant the transparent plastics being with holes on back cover, to keep 75～85% humidity, 25 ℃, illumination 1500～2500Lux, the photoperiod is with 14～16h/8-10h hour dark of illumination every day.Cultivate 10～15d left and right, move on to the outdoor Adaptable growth that carries out.

CTAB method is extracted resistant plant leaves genomic DNA, with HcTPS2 promotor special primer, hygromycin phosphotransferase gene primer hpt II (hygromycin phosphotransferase gene sequence is shown in SEQIDNO:24) and HcTPS1 gene specific primer (HcTPS1 gene order is shown in the national patent ZL201110223418.5 that Agricultural University Of South China obtains) carry out pcr amplification, can amplify the positive plant of band.

HcTPS2 promotor special primer:

P1：5'-GGCGAGCTCCTGTTTCAAAACATCCAATTCCTC-3'（SEQID NO:18）

P2：5'-GGCCAAGCTTAATTTCAACTTCAAGTCCTGAC-3'（SEQID NO:19）

Totomycin primer hpt II:

P1：5'-CTTCTACACAGCCATCGGTCCAGA-3'（SEQIDNO:20）

P2：5'-GATGTAGGAGGGCGTGGATATGTC-3'（SEQIDNO:21）

HcTPS1 special primer:

P1：5'-GCAACTAGTATGTCTCTCTTCCAACCTGC-3'（SEQIDNO:22）

P2：5'-GGCGGATCCTTACAATAGGATAGGGTTGATC-3'（SEQID NO:23）

Transgenic tobacco plant volatile matter SPME-GC/MS analyzes: transgene tobacco and contrast are carried out to volatile matter employing solid-phase microextraction-gaschromatographic mass spectrometry methods analyst with the flower that injury and insect pest gene are bloomed respectively.Adopt the fresh tobacco leaf of 1.0g or petal, be placed in 100mL triangular pyramidal bottle, add the ethyl decylate 5 μ L of 31.6ng/ μ L as internal standard substance, with masking foil, seal, insert 100 μ m polydimethyl oxygen alkane (PMDS) extracting fiber heads, in 25 ℃ of head space sampling 30min.Adopt Agilent7890/5975C-GC/MSD gas chromatograph-mass spectrometer to analyze.Chromatographic condition is HP-5 quartz capillary tubule column length 30m, internal diameter 0.25mm, and the thick 0.25 μ m of liquid film, carrier gas He, flow velocity is 1mL/min, column head pressure 7.1Psi, mean flow rate is 36.262cm/sec, the residence time is 1.3789min.Temperature programming: 40 ℃, keep 2min; Heat-up rate with 10 ℃/min rises to 250 ℃, keeps 5min.250 ℃ of program shunting/Splitless injecting samples mouth temperature, injection port is set as Splitless injecting samples mode, and diffluence pass purge flow rate is that 60mL/min is at 0.75min.The mass spectrum collecting is analyzed by Agilent5975 data analysis software, according to standard sample retention time and relative retention value and check with reference to related documents, determine the chemical composition of the volatile matter of plant, and quantitative with the ratio of interior mark ethyl decylate peak area according to each component peaks area, determine the chemical composition of tobacco volatile matter.Result shows: in the transgene tobacco blade after injury is processed, occurred the unexistent monoterpene ocimene of wild-type tobacco; Tobacco primary pest prodenia litura, take food after tobacco leaf, the monoterpene firpene content in transgene tobacco has raise 4～6 times, newly-generated monoterpene ocimene; The flower aroma substance Linaool volatile quantity of transgene tobacco has improved 10～15 times.

SEQUENCE LISTING

<110> Agricultural University Of South China

<120>special TPS2 promotor and the application thereof of inducing with injury, insect pest of a kind of ginger portion

<130>

<160> 24

<170> PatentIn version 3.3

<210> 1

<211> 1962

<212> DNA

<213>artificial sequence

<400> 1

tactgataag ataagttgcg ctttccctgc atatgaaata gtggccttcg gccacgccgt 60

gaccctctgg gtaagcgagt ctagaagtgt gctgtagtta gagattttga gtttctggga 120

ggcaatggga atgcccaggt atctgaaagg catgtcccct atctggaatc cagtgatggt 180

caaaagctgt tcccgtgtct gagtatctat ccctgtcatg tagatgttgg acttagctat 240

attggccatg aggcttgcta tatgagtaaa ctgtgataag cagtcaacta gctgagtcac 300

cgaagatatg tcctctctgg atagcaagag caggtcatct gcatagatca agtacactat 360

cctaagatgc caaaagctac tgtcgaacag atgaaaggag gaggaattgc gaatagtctt 420

cttgttctag ttgtaagatg tctgaactct catttacttg taattttttc tttcaaattt 480

tttcttgtag cataactcat gcacgacttg ctccaaatgc tggcgttatc ttgaactgga 540

ttgctattga tgttttttcc tgttcttttt ttcaggagct tgtgagttca gtcaaaaatg 600

tgcttccttg gttacaagcg gttctgtcgt tggaaagacc gacggtcaca ctattcatcc 660

gcgctgtgat tttgattgtc aaatacaagt aagttgtacc ctgaaatgat acggttgtta 720

cctttggtta tataaccttt tatgctcaag ctatttagta ctcggttcta aataaaaaaa 780

aatcacacta ttttatttag gatcagctac attgaactta tttcaaatta caatcaaagt 840

gtattcgtcc attgctatca tgctagggca ataatcacct tgtcattcaa cagggaaatg 900

gttggatttg caattgcaat gattgcacta ttactagttg cagcgatact ttgggccaga 960

gtgaatggaa ttggagaaag atgccgagag attgttgtgg acaccacctt tgatcaaaca 1020

atgatggaaa gcatcgttgc agcccagcac agtctgaata acttgcatga gctcgttaag 1080

gaaactaata tcacgatttt gagaatattc tcgataataa tcggaaaggc tccaaaggta 1140

ctgtttcaaa acatccaatt cctccacgac tctctcccaa catttaacag agctattgtc 1200

atatctacag caagctaatg atgtgatgtg ggcaatgatt gcagtagcag tcctgttggt 1260

ggtaatcccg ttcaagtata ttgttatggg gctaactttg tacttgttca tagctaattc 1320

aaggattgcg aagcgcatgt tgaaccaaca gggaaatcga cggctgaaag agtggtggga 1380

gtcgatccct gtcattccag ttcgaacagt gagcagtagt ccttgataga aatggtaaga 1440

ctgatttggg aaaaggtgaa acgatagtct gtttatgaaa cttactgcct catcagcttt 1520

tcttttcaca attgaatttt gcttcatcat ggttttctgt tccttcaatt ctaagttctc 1580

tgtagttttt ttttctttgt gtgcgctcat gtaatcgtct catacgtgca tgttacaagt 1640

atccttgcaa ttttggtcac ccctcgagta aaacttacat aaatcaatat gtagcttgtt 1700

gtaccttgta gagtggcctt tttttttttt cataatttaa aagaagacgg tgttcaattt 1760

gaatcgaatg gtgtactgta atcatcctta taatgacatc tacgcccaaa tccacacgac 1820

aactctgacc tcatcgggta cgattacgaa ctatgaagga caaggctcga aagtagtgat 1880

cgtgtaggtt tattgcaaaa tgaaagaaga agattcaagg tcaggacttg aagttgaaat 1920

tgatggcaaa atgcactact gtctgtgctc ccatgatatc cg 1962

<210> 2

<211> 1922

<212> DNA

<213>TPS2 promotor

<400> 2

tactgataag ataagttgcg ctttccctgc atatgaaata gtggccttcg gccacgccgt 60

gaccctctgg gtaagcgagt ctagaagtgt gctgtagtta gagattttga gtttctggga 120

ggcaatggga atgcccaggt atctgaaagg catgtcccct atctggaatc cagtgatggt 180

caaaagctgt tcccgtgtct gagtatctat ccctgtcatg tagatgttgg acttagctat 240

attggccatg aggcttgcta tatgagtaaa ctgtgataag cagtcaacta gctgagtcac 300

cgaagatatg tcctctctgg atagcaagag caggtcatct gcatagatca agtacactat 360

cctaagatgc caaaagctac tgtcgaacag atgaaaggag gaggaattgc gaatagtctt 420

cttgttctag ttgtaagatg tctgaactct catttacttg taattttttc tttcaaattt 480

tttcttgtag cataactcat gcacgacttg ctccaaatgc tggcgttatc ttgaactgga 540

ttgctattga tgttttttcc tgttcttttt ttcaggagct tgtgagttca gtcaaaaatg 600

tgcttccttg gttacaagcg gttctgtcgt tggaaagacc gacggtcaca ctattcatcc 660

gcgctgtgat tttgattgtc aaatacaagt aagttgtacc ctgaaatgat acggttgtta 720

cctttggtta tataaccttt tatgctcaag ctatttagta ctcggttcta aataaaaaaa 780

aatcacacta ttttatttag gatcagctac attgaactta tttcaaatta caatcaaagt 840

gtattcgtcc attgctatca tgctagggca ataatcacct tgtcattcaa cagggaaatg 900

gttggatttg caattgcaat gattgcacta ttactagttg cagcgatact ttgggccaga 960

gtgaatggaa ttggagaaag atgccgagag attgttgtgg acaccacctt tgatcaaaca 1020

atgatggaaa gcatcgttgc agcccagcac agtctgaata acttgcatga gctcgttaag 1080

gaaactaata tcacgatttt gagaatattc tcgataataa tcggaaaggc tccaaaggta 1140

ctgtttcaaa acatccaatt cctccacgac tctctcccaa catttaacag agctattgtc 1200

atatctacag caagctaatg atgtgatgtg ggcaatgatt gcagtagcag tcctgttggt 1260

ggtaatcccg ttcaagtata ttgttatggg gctaactttg tacttgttca tagctaattc 1320

aaggattgcg aagcgcatgt tgaaccaaca gggaaatcga cggctgaaag agtggtggga 1380

gtcgatccct gtcattccag ttcgaacagt gagcagtagt ccttgataga aatggtaaga 1440

ctgatttggg aaaaggtgaa acgatagtct gtttatgaaa cttactgcct catcagcttt 1520

tcttttcaca attgaatttt gcttcatcat ggttttctgt tccttcaatt ctaagttctc 1580

tgtagttttt ttttctttgt gtgcgctcat gtaatcgtct catacgtgca tgttacaagt 1640

atccttgcaa ttttggtcac ccctcgagta aaacttacat aaatcaatat gtagcttgtt 1700

gtaccttgta gagtggcctt tttttttttt cataatttaa aagaagacgg tgttcaattt 1760

gaatcgaatg gtgtactgta atcatcctta taatgacatc tacgcccaaa tccacacgac 1820

aactctgacc tcatcgggta cgattacgaa ctatgaagga caaggctcga aagtagtgat 1880

cgtgtaggtt tattgcaaaa tgaaagaaga agattcaagg tcaggacttg aagttgaaat 1920

tg 1922

<210> 3

<211> 320

<212> DNA

<213> p-320

<400> 3

atacgtgcat gttacaagta tccttgcaat tttggtcacc cctcgagtaa aacttacata 60

aatcaatatg tagcttgttg taccttgtag agtggccttt tttttttttc ataatttaaa 120

agaagacggt gttcaatttg aatcgaatgg tgtactgtaa tcatccttat aatgacatct 180

acgcccaaat ccacacgaca actctgacct catcgggtac gattacgaac tatgaaggac 240

aaggctcgaa agtagtgatc gtgtaggttt attgcaaaat gaaagaagaa gattcaaggt 300

caggacttga agttgaaatt 320

<210> 4

<211> 781

<212> DNA

<213> p-780

<400> 4

ctgtttcaaa acatccaatt cctccacgac tctctcccaa catttaacag agctattgtc 60

atatctacag caagctaatg atgtgatgtg ggcaatgatt gcagtagcag tcctgttggt 120

ggtaatcccg ttcaagtata ttgttatggg gctaactttg tacttgttca tagctaattc 180

aaggattgcg aagcgcatgt tgaaccaaca gggaaatcga cggctgaaag agtggtggga 240

gtcgatccct gtcattccag ttcgaacagt gagcagtagt ccttgataga aatggtaaga 300

ctgatttggg aaaaggtgaa acgatagtct gtttatgaaa cttactgcct catcagcttt 360

tcttttcaca attgaatttt gcttcatcat ggttttctgt tccttcaatt ctaagttctc 420

tgtagttttt ttttctttgt gtgcgctcat gtaatcgtct catacgtgca tgttacaagt 480

atccttgcaa ttttggtcac ccctcgagta aaacttacat aaatcaatat gtagcttgtt 540

gtaccttgta gagtggcctt tttttttttt cataatttaa aagaagacgg tgttcaattt 600

gaatcgaatg gtgtactgta atcatcctta taatgacatc tacgcccaaa tccacacgac 660

aactctgacc tcatcgggta cgattacgaa ctatgaagga caaggctcga aagtagtgat 720

cgtgtaggtt tattgcaaaa tgaaagaaga agattcaagg tcaggacttg aagttgaaat 780

t 781

<210> 5

<211> 1921

<212> DNA

<213> p-1928

<400> 5

tactgataag ataagttgcg ctttccctgc atatgaaata gtggccttcg gccacgccgt 60

gaccctctgg gtaagcgagt ctagaagtgt gctgtagtta gagattttga gtttctggga 120

ggcaatggga atgcccaggt atctgaaagg catgtcccct atctggaatc cagtgatggt 180

caaaagctgt tcccgtgtct gagtatctat ccctgtcatg tagatgttgg acttagctat 240

attggccatg aggcttgcta tatgagtaaa ctgtgataag cagtcaacta gctgagtcac 300

cgaagatatg tcctctctgg atagcaagag caggtcatct gcatagatca agtacactat 360

cctaagatgc caaaagctac tgtcgaacag atgaaaggag gaggaattgc gaatagtctt 420

cttgttctag ttgtaagatg tctgaactct catttacttg taattttttc tttcaaattt 480

tttcttgtag cataactcat gcacgacttg ctccaaatgc tggcgttatc ttgaactgga 540

ttgctattga tgttttttcc tgttcttttt ttcaggagct tgtgagttca gtcaaaaatg 600

tgcttccttg gttacaagcg gttctgtcgt tggaaagacc gacggtcaca ctattcatcc 660

gcgctgtgat tttgattgtc aaatacaagt aagttgtacc ctgaaatgat acggttgtta 720

cctttggtta tataaccttt tatgctcaag ctatttagta ctcggttcta aataaaaaaa 780

aatcacacta ttttatttag gatcagctac attgaactta tttcaaatta caatcaaagt 840

gtattcgtcc attgctatca tgctagggca ataatcacct tgtcattcaa cagggaaatg 900

gttggatttg caattgcaat gattgcacta ttactagttg cagcgatact ttgggccaga 960

gtgaatggaa ttggagaaag atgccgagag attgttgtgg acaccacctt tgatcaaaca 1020

atgatggaaa gcatcgttgc agcccagcac agtctgaata acttgcatga gctcgttaag 1080

gaaactaata tcacgatttt gagaatattc tcgataataa tcggaaaggc tccaaaggta 1140

ctgtttcaaa acatccaatt cctccacgac tctctcccaa catttaacag agctattgtc 1200

atatctacag caagctaatg atgtgatgtg ggcaatgatt gcagtagcag tcctgttggt 1260

ggtaatcccg ttcaagtata ttgttatggg gctaactttg tacttgttca tagctaattc 1320

aaggattgcg aagcgcatgt tgaaccaaca gggaaatcga cggctgaaag agtggtggga 1380

gtcgatccct gtcattccag ttcgaacagt gagcagtagt ccttgataga aatggtaaga 1440

ctgatttggg aaaaggtgaa acgatagtct gtttatgaaa cttactgcct catcagcttt 1520

tcttttcaca attgaatttt gcttcatcat ggttttctgt tccttcaatt ctaagttctc 1580

tgtagttttt ttttctttgt gtgcgctcat gtaatcgtct catacgtgca tgttacaagt 1640

atccttgcaa ttttggtcac ccctcgagta aaacttacat aaatcaatat gtagcttgtt 1700

gtaccttgta gagtggcctt tttttttttt cataatttaa aagaagacgg tgttcaattt 1760

gaatcgaatg gtgtactgta atcatcctta taatgacatc tacgcccaaa tccacacgac 1820

aactctgacc tcatcgggta cgattacgaa ctatgaagga caaggctcga aagtagtgat 1880

cgtgtaggtt tattgcaaaa tgaaagaaga agattcaagg tcaggacttg aagttgaaat 1920

t 1921

<210> 6

<211> 30

<212> DNA

<213>primer sp1

<400> 6

cggatatcat gggagcacag atagtagtgc 30

<210> 7

<211> 34

<212> DNA

<213>primer sp2

<400> 7

ctacacgat cactactttg gagccttgtc cttc 34

<210> 8

<211> 39

<212> DNA

<213>primer sp3

<400> 8

atgagg tcagagttgt cgtgtggatt tgggcgtag 39

<210> 9

<211> 18

<212> DNA

<213>primer LAD-1

<400> 9

acgatggact ccagagcggc cgcvnvnnng gaa 18

<210> 10

<211> 18

<212> DNA

<213>primer LAD-2

<400> 10

acgatggact ccagagcggc cgcbnbnnng gtt 18

<210> 11

<211> 19

<212> DNA

<213>primer LAD-3

<400> 11

acgatggact ccagagcggc cgcvvnvnnn ccaa 19

<210> 12

<211> 19

<212> DNA

<213>primer LAD-4

<400> 12

acgatggact ccagagcggc cgcbdnbnnn cggt 19

<210> 13

<211> 16

<212> DNA

<213>primer AC1

<400> 13

acgatggact ccagag 16

<210> 14

<211> 32

<212> DNA

<213>primer p-320-F

<400> 14

ggccaagctt atacgtgcat gttacaagta tc 32

<210> 15

<211> 34

<212> DNA

<213>primer p-780-F

<400> 15

ggccaagctt ctgtttcaaa acatccaatt cctc 34

<210> 16

<211> 28

<212> DNA

<213>primer p-1928-F

<400> 16

ggccaagctt actgataaga taagttgc 28

<210> 17

<211> 32

<212> DNA

<213>downstream primer R

<400> 17

ggcggatccc aatttcaact tcaagtcctg ac 32

<210> 18

<211> 33

<212> DNA

<213>TPS2 promotor special primer P1

<400> 18

ggcgagctcc tgtttcaaaa catccaattc ctc 33

<210> 19

<211> 32

<212> DNA

<213>TPS2 promotor special primer P2

<400> 19

ggccaagctt aatttcaact tcaagtcctg ac 32

<210> 20

<211> 24

<212> DNA

<213>Totomycin primer p1

<400> 20

cttctacaca gccatcggtc caga 24

<210> 21

<211> 24

<212> DNA

<213>Totomycin primer p2

<400> 21

gatgtaggag ggcgtggata tgtc 24

<210> 22

<211> 29

<212> DNA

<213>HcTPS1 special primer p1

<400> 22

gcaactagta tgtctctctt ccaacctgc 29

<210> 23

<211> 32

<212> DNA

<213>HcTPS1 special primer p2

<400> 23

gggcggatcc ttacaatagg atagggttga tc 32

<210> 24

<211> 540

<212> DNA

<213>hygromycin phosphotransferase gene

<400> 24

tggtgtactg taatcatcct tataattaca tctacgccca aatccacacg acaactcgga 60

cctcatcggg tacgattacg aactatgaag gacaaggctc caaagtagtg atcgtgtaga 120

tttattgcaa aatgaaggaa gaagattcaa ggtcaggact tgaagttgaa attgatggca 180

aaatgcacta ctgtctgtgc tcccatgata tccgccctcc ctcgtcggcc gatgatcgtt 240

gctgctgtcg agcatcgagg cctgcagatg ttccggcgaa ctctgcaggt acgatcatgc 300

agtggcacta gtcatgtagc tcccttgcgg cggtcggcga attatcaacc aagcatatgg 360

acggacgagc gtgtgcaatc cctcacgagc gcctccacgg tgcaacaaga agagaatcgt 420

gagagaataa acgtactcaa ggagcatacc aggaatctga tacgcgagaa gcaacaagtt 480

gcagagcagc ttcaactaat cgaccacctg caacagctcg gcgtggcata ccacttcaaa 540

Claims (9)

1. the TPS2 promotor that ginger portion is special and injury, insect pest are induced, is characterized in that, nucleotide sequence is as shown in SEQ ID NO:2.

2. TPS2 promotor according to claim 1, is characterized in that, object fragment p-320, p-780 and p-1928 that described TPS2 promotor contains 35 ' end series disappearance, and the sequence of p-320, p-780 and p-1928 is as shown in SEQ ID NO:3～5.

3. a recombinant vectors, is characterized in that, described recombinant vectors is TPS2 promoter sequence described in the multiple clone site insertion claim 1 at the carrier that sets out.

4. a recombinant vectors, is characterized in that, described recombinant vectors is object fragment p-320, p-780 or the p-1928 sequence of 5 ' end series disappearance of TPS2 promotor described in the multiple clone site insertion claim 2 at the carrier that sets out.

5. a recombinant bacterium that comprises recombinant vectors described in claim 3 or 4.

6. a clone that comprises recombinant vectors described in claim 3 or 4.

7. the application of TPS2 promotor in cultivating the transgenosis fragrance of a flower or resistance plant kind described in claim 1.

8. a method of cultivating the transgenosis fragrance of a flower or resistance plant, is characterized in that, the recombinant vectors containing described in claim 3 or 4 is imported to the vegetable cell that sets out, thereby obtains the transgenosis fragrance of a flower or resistance plant that TPS2 promotor starts destination gene expression.

9. method according to claim 8, is characterized in that: described goal gene is gus gene.

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