CN101886074B - GhPsbP promoter high-effectively expressed by cotton chlorenchyma - Google Patents

Abstract

The invention discloses a GhPsbP promoter high-effectively expressed by cotton chlorenchyma. A promoter sequence of the GhPsbP gene is obtained by extracting from the cotton. The promoter can drive the gene to be high-effectively specifically expressed in the chlorenchymas including laminas and stems of a plant. The promoter can be used as a regulatory sequence of a functional gene to be applied in transgenic plants.

Description

The GhPsbP promotor that cotton chlorenchyma efficiently expresses

Technical field

The present invention relates to the cotton promotor that a kind of chlorenchyma efficiently expresses, this promoters driven gene comprises in blade and the stem in chlorenchyma and efficiently expressing.This promotor can be applicable to the external source goal gene and in chlorenchyma, efficiently expresses, and does not express or express on a small quantity at positions such as root, germinal tissues, thereby improves the specific aim of exogenous gene expression, saves the energy and the material of plant materials.

Background technology

Genetic expression is important contents of plant genetic engineering research with regulation and control, and promotor is playing a part the transcriptional control center aspect the decision genetic expression, and it is determining time, space and the intensity of destination gene expression to a great extent.The promotor of in the commercialization genetically modified crops, using at present often all is a constitutive promoter, as: cauliflower mosaic virus CaMV35S promotor, rice actin Actl promotor, corn ubiquitin Ubi promotor etc.Yet constitutive promoter drives foreign gene continuous expression in plant; Express the inessential waste that often causes energy and material; Increase the metabolism burden of plant, can cause that sometimes even also the form of plant changes, influence growing of plant; But the deficiency at the position that needs the foreign gene high expression level because of expression amount lacks specific aim, possibly fall flat in some applications.The application organizes specificity promoter is as the relevant expression regulation element of foreign gene in genetically modified crops; Not only can give full play to the correlation function of foreign gene; And make expression of exogenous gene have more specific aim; Avoid other tissue because of energy and material wastage that unnecessary expression causes, alleviated growth and the metabolism burden of transgenic plant.Therefore, replace constitutive promoter with specificity promoter, make foreign gene can be regularly, fixed point, in plant, to express quantitatively be the important content of plant genetically engineered research.

For foreign gene is efficiently expressed in transgenic plant body chlorenchyma; The function that performance is relevant; Reduce simultaneously disadvantageous effect as far as possible to recipient plant; Main purpose of the present invention is to be cloned in the promotor that efficiently expresses gene in the chlorenchyma; Make external source functional gene (for example: anti-herbicide gene, disease-resistant gene etc.), only in the chlorenchyma of its expression of needs, efficiently express, in other non-chlorenchyma, do not express or the only extremely low-level local expression of maintenance by this promoters driven.Make it can be applied to cultivate (for example: transgenic herbicide resistant plants, disease resistance transgenic plant etc.) in the different transgenic plant that need foreign gene specifically expressing in chlorenchyma.Can avoid foreign gene not have like this and efficiently express caused negative effect targetedly for a long time; Can make plant have the characteristic of antiweed and disease etc. again; Thereby solving present foreign gene effectively driven by strong composition type expression promoter; Foreign gene efficiently expresses in plant materials for a long time, influences the problem of plant-growth and output.

Clone's chlorenchyma efficiently expresses the dna sequence dna that promotor, enhanser and the various decision chlorenchyma of gene efficiently express, and can controlling element be provided for foreign gene timing, fixed point, quantitative expression in plant materials.

The GhPsbP promotor is the promotor that in chlorenchyma, efficiently expresses, and in NCBI/nr/BLAST, does not see the log-on message of this promotor as yet at present, and the report of the relevant Glyphosate 62 IPA Salt abduction delivering of Shang Weijian promotor.

Summary of the invention

The purpose of this invention is to provide a kind of GhPsbP gene transcription control region nucleotide sequence or have the analogue development body nucleotide sequence of identical function with it; This promotor is isolating from cotton, is a kind ofly can drive the tissue-specific promoter that the DNA be associated with it efficiently expresses in chlorenchyma.

GhPsbP promoter sequence provided by the invention has the 1st 's to the 2363rd shown in the SEQ ID NO:1 promoter region nucleotide sequence; Because the nucleotide sequence shown in the SEQ ID NO:1 of the present invention is carried out insertion, the replacement of one or more nucleotide sequences and/or lacks resulting functional analogue also reaching the object of the invention; Thereby the present invention also comprises and 50% the homology of having shown in the SEQ ID NO:1 at least; Preferred at least 90% homology, and have the analogue of identical function simultaneously.

" variant of deriving " of the GhPsbP gene 5 ' ending regulating sequence among the present invention is meant in SEQ ID NO:1 sequence on the basis of sequence between 1 to 2363 Nucleotide, adds or an isolating once more part, a few part or its combination through fragment deletion, sequence change, base.The invention provides the roughly section at promotor cis element place, and made up the variant of deriving that lacks step by step from its 5 ' end.The promoter activity of variant of deriving among the present invention can such as beta-Glucuronidase gene (GUS), be able to confirm with the expression of GUS through the chimeric reporter gene in the promotor downstream.

A further object of the present invention provides the analogue that includes above-mentioned chlorenchyma specificity promoter sequence and have identical function derive expression vector and its recombinant host cell of two mutants.Comprise recombinant bacteria, yeast, Agrobacterium and cotton recombinant plant cell.According to chlorenchyma specificity promoter provided by the invention; Can make up the binary expression vector that comprises this promotor; Its promotor downstream can the chimeric interested gene that will express, through transforming plant, realizes the efficient specifically expressing of foreign gene in transgenic plant chlorenchyma.

The explanation of accompanying drawing table

Fig. 1 is a 5 ' RACE electrophorogram of cotton GhPsbP gene.Wherein swimming lane M is a molecular weight marker, and swimming lane 2 is a 5 ' RACE product of GhPsbP gene;

Fig. 2 is the pcr amplification result of cotton GhPsbP total length promotor.Wherein swimming lane M is a molecular weight marker, and swimming lane 1 is the pcr amplification result of GhPsbP total length promotor

Fig. 3 is the restriction enzyme mapping of cotton GhPsbP total length promotor P1, disappearance promotor P2, P3, P4 and P5.Wherein swimming lane M is a molecular weight marker, and the enzyme that swimming lane 7,6,5,3 and 1 is respectively P1, P2, P3, P4 and P5 is cut the result.

Fig. 4 is for changeing the PCR qualification result of P1, P2, P3, P4 and P5 tobacco.Wherein swimming lane M is a molecular weight marker, swimming lane 1 positive contrast, and swimming lane 2 negative contrasts, swimming lane 3-7 is respectively the PCR qualification result that changes P1, P2, P3, P4 and P5 tobacco.

Fig. 5 is for changeing the real-time PCR result of GUS in GhPsbP total length promotor P1 tobacco root, stem and the leaf.

Fig. 6 is for changeing P1, P2, P3, P4 and P5 tobacco GUS histochemical stain result.

Fig. 7 is for changeing the real-time PCR result of GUS in P1, P2, P3, P4 and the P5 tobacco leaf.

Fig. 8 is for changeing gus protein activity in P1, P2, P3, P4 and the P5 tobacco leaf.

Embodiment

Hereinafter further specifies the present invention through embodiment

Embodiment 1 chlorenchyma efficiently expresses GhPsbP promotor clone

1. 5 ' RACE of chlorenchyma specifically expressing GhPsbP gene

It is total RNA that hot borate method extracts cotton Y18

1) the refrigerated blade is put into the mortar of precooling, ground to form powdery;

2) get 200mg in the 1.5ml Eppendorf tube, add the basic extracting solution that 1ml is preheated to 80 ℃, 10 μ l DTT stock solutions and 25 μ l Proteinase K stock solution mixings;

3) 42 ℃ of 100r/min gentlenesses are shaken 1.5h;

4) every pipe adds the Kcl solution of 80 μ l 2mol/L, to Kcl final concentration 160mmol/L, and mixing, ice bath 1h;

5) the centrifugal 20min of 12000r/min gets 900 μ l supernatants, adds 300 μ l 8mol/L Licl, mixing, the ice bath deposition of spending the night;

6) after centrifugal as stated, deposition is washed 2～3 times with 2mol/LLicl (ice precooling), and is intimate colourless up to supernatant;

7) suspension Licl-RNA adds the 2mol/LKCA (pH 5.5) of 1/10 volume in 400 μ l10mmol/LTris-Hcl (pH7.5), mixing, and behind the ice bath 15min, deposition;

8) the centrifugal insoluble substance (containing RNA in the supernatant) that desalts that removes;

9) spend the night with the absolute ethyl alcohol-20 ℃ deposition of 2.5 times of volumes or-70 ℃ the deposition 2～3h;

10) with 70% cold washing with alcohol RNA deposition, vacuum rapid drying, be dissolved in the DEPC water or the TE damping fluid (10mmol/L Tris-Hcl, 1mmol/L EDTA, pH8);

RACE method amplification GhPsbP 5 ' end

1) use Alkaline Phosphatase (CIAP) that 5 ' exposed among Total RNA phosphate group is carried out the dephosphorization acid-respons.By following component preparation dephosphorization acid-respons liquid, 50 ℃ of reaction 1h obtain CIAP-treated RNA.

Total?RNA(1μg/μl) 1μl

RNase?Inhibitor(40U/μl) 1μl

10×Alkaline?Phosphatase?Buffer(MgCl ₂?Free) 5μl

Alkaline?Phosphatase(Calf?intestine)(16U/μl) 0.6μl

RNase?Free?ddH ₂O?up?to 50μl

2) use Tobacco Acid Pyrophosphatase (TAP) to remove the 5 ' cap sequence of mRNA, keep a phosphate group.By following component preparation " removing cap " reaction solution, 37 ℃ of reaction 1h obtain CIAP/TAP-treated RNA.

CIAP-treated?RNA 7μl

RNase?Inhibitor(40U/μl) 1μl

10×TAP?Reaction?Buffer 1μl

Tobacco?Acid?Pyrophosphatase(0.5U/μl) 1μl

Total?Volume 10μl

3) connection of 5 ' RACE Adaptor.Prepare following solution.

CIAP/TAP-treated?RNA 5μl

5’RACE?Adaptor(15μM) 1μl

RNase?Free?ddH ₂O 4μl

65 ℃ of insulations were placed 2 minutes after 5 minutes on ice, added following reagent then.

RNase?Inhibitor(40U/μl) 1μl

5×RNA?Ligation?Buffer 8μl

40％PEG#6000 20μl

T4?RNA?Ligase(40U/μl) 1μl

16 ℃ were reacted 1 hour, obtained Ligated RNA.

4) reverse transcription reaction.By following component preparation inverse transcription reaction liquid.

Ligated?RNA 6μl

Random?9mers(50μM) 0.5μl

5×M-MLV?Buffer 2μl

dNTP(10mM?each) 1μl

RNase?Inhibitor(40U/μl) 0.25μl

Reverse?Transcriptase?M-MLV(RNase?H-)(200U/μl) 0.25μl

Total?Volume 10μl

The reverse transcription reaction condition is following: 30 ℃ of 10min; 42 ℃ of 1hr; 70 ℃ of 15min.

5) Outer PCR reaction.By following component preparation Outer PCR reaction solution.

Above-mentioned 4 inverse transcription reaction liquid 2 μ l

1×cDNA?Dilution?Buffer?II 8μl

10×LA?PCR?Buffer?II(Mg ²⁺Free) 4μl

MgCl(25mM) 3μl

TaKaRa?LA?Taq(5U/μl) 0.25μl

5’RACE?Control?Outer?Primer(10μM) 2μl

5’RACE?Outer?Primer(10μM) 2μl

ddH ₂O 28.75μl

Total 50μl

The PCR reaction conditions is following:

6) Inner PCR reaction.By following component preparation Inner PCR reaction solution.

Outer PCR reaction solution 1 μ l

10×LA?PCR?Buffer?II(Mg ²⁺Free) 5μl

MgCl(25mM) 5μl

dNTP?Mixture(2.5mM?each) 8μl

TaKaRa?LA?Taq(5U/μl) 0.5μl

5’RACE?Control?Inner?Primer(10μM) 2μl

5’RACE?Inner?Primer(10μM) 2μl

dH ₂O 26.5μl

Total 50μl

2) the PCR reaction conditions is following:

7) after reaction finishes; Get 5 μ l PCR reaction solutions and carry out 1% agarose gel electrophoresis; Reclaim the purpose band of No. 2 swimming lane 0.3kb among Fig. 1, be connected to transformed into escherichia coli behind the PGEM-T, extract plasmid after enzyme is cut evaluation; With the plasmid sequencing analysis of positive colony, obtain the sequence information of GhPsbP5 ' end.

2. the clone of chlorenchyma specifically expressing GhPsbP gene promoter

After accomplishing 5 ' RACE of GhPsbP gene, confirmed this gene transcription initiation site.The G2-J-15BAC that comprises GhPsbP gene and promotor thereof according to the cotton Y18 that has checked order clones, and 5 ' end upper reaches 2363bp-1bp at GhPsbP genetic transcription initiation site designs primer respectively:

P1F：5’-CCCAAGCTTTGGATGACTATAGTTGCTCCGACC-3’；

PR：5’-CGGATCCCGCGCATTAGTGCCAGTGGCAG-3’。

With cotton Y18 genome is template, and amplification obtains GhPsbP gene promoter total length.

The PCR reaction system is following:

Y18 genomic templates DNA 1 μ l

P1F(10μmol) 1μl

PR(10μmol) 1μl

dNTP?Mixture(2.5mmol) 4μl

buffer 5μl

Taq enzyme 1 μ l

ddH ₂O 37μl

Total 50μl

The PCR reaction conditions is following:

After reaction finishes, the PCR reaction solution is carried out 1% agarose gel electrophoresis, reclaim the purpose band of 2.3kb among Fig. 2; Be connected to transformed into escherichia coli behind the Pmd-18T carrier, extract plasmid, enzyme is cut evaluation; See Fig. 3 the 7th swimming lane, cut out the band of 2.3kb, with the plasmid sequencing analysis of positive colony; Obtain the sequence information of GhPsbP promotor,, will clone called after pMDGhPsbPP this section sequence called after P1.Its sequence is shown in SEQ ID NO:1.NCBI/nr/BLAST result shows: no any and GhPsbP promotor homologous sequence in nucleic acid (nr) storehouse.

Embodiment 2 cotton GhPsbP promoter expression specificity analysises

1. make up the plant expression vector pBIGhPsbPP of GhPsbP promoters driven GUS

1) plasmid of extraction pMDGhPsbPP and pBI121, with HindIII and BamHI double digestion plasmid, 37 ℃ of enzymes are cut 5h.It is following that enzyme is cut system:

PMDGhPsbPP plasmid 5 μ l pBI121 carrier 30 as one kind μ l

HindIII 3μl HindIII 3μl

BamHI 3μl BamHI 3μl

BufferR 5μl BufferR 5μl

ddH ₂O 34μl ddH ₂O 6μl

Total 50μl Total 50μl

2) reclaim GhPsbPP and pBI121 carrier sequence respectively, carry out ligation, 16 ℃ connect 12h, and linked system is following:

PBI121 carrier 1 μ l

Two mutants fragment 3 μ l

T4-ligase 1μl

Buffer 1μl

Deionized water 4 μ l

Total system 10 μ l

3) transform the connection product, the picking clone, enzyme is cut evaluation.Enzyme is cut all correct expression vector called after pBIGhPsbPP that identifies and check order.

2. plant expression vector pBIGhPsbPP transforms agrobacterium tumefaciens LB4404

In 200 μ l LBA4404 competent cells, add 2 μ g recombinant plasmid dnas, ice bath 5min goes to freezing 8min in the liquid nitrogen then; In 37 ℃ of water-baths, behind the warm 5min, add 800 μ l YEB liquid nutrient mediums rapidly; 28 ℃, 250rpm prefiguration reach to be cultivated 4～5h hour, and being coated with the shop then, to contain the YEB of kantlex, Streptomycin sulphate dull and stereotyped, and 28 ℃ bacterium colony can occur after cultivating 24～48h; Picking colony is bacterium colony PCR and is identified, confirm to be stored in after correct-70 ℃ subsequent use.

3. leaf dish method transformation of tobacco

1) agrobacterium mediation converted tobacco

Get the leaflet tablet of tobacco aseptic seedling, be cut into the dice of about 0.5cm by knife, in the Agrobacterium suspension, soak 5～10min, blot with aseptic filter paper then; Cultivate altogether: the leaf piece is placed in the common culture medium (top shop 2 metafiltration paper) cultivates 3d altogether in 25 ℃ of lucifuges; Select to cultivate: blade is transferred to selected in 25 ℃ of illumination boxs (illumination 12h, dark 12h), to be cultured to the appearance of resistant buds in the substratum; The acquisition of resistance budlet: the resistant buds that 2～3cm is long moves on in the root media, sends out roots gradually about 7d.Treat seedling length to 5～when 6cm is high, be transplanted in the little polypots that contains nutritious soil after about one month;

2) the PCR Molecular Identification of transgene tobacco

Design GUS primer:

GUSF：5’-GTGGGCATTCAGTCTGGATCG-3’；

GUSR：5’-TTGCCGTTTTCGTCGGTAATC-3’；

With the tobacco gene group is template, carries out pcr amplification with GUS primer, promoter primer and GUS and promotor pairing primer respectively, adopts 50 μ l systems; Contain template DNA 1 μ l; Each 1 μ l of 10 μ mol primers, 2.5mmoldNTP 4 μ l, buffer 5 μ l; Taq enzyme 1 μ l adds deionized water and supplies 50 μ l; The reaction condition of carrying out is 94 ℃, 3min; 94 ℃, 30s, 60 ℃, 30s, 72 ℃, 1min, 35 circulations; 72 ℃, 10min.Qualification result is seen Fig. 4 the 3rd swimming lane, shows the tobacco plant that has obtained to change pBIGhPsbPP.

3. the real-time PCR of transgene tobacco GUS

1) design tobacco actin primer respectively and the gus gene primer is used for real-time PCR:

actinF：5’-TTCCTGGCATTGCAGATCGTAT-3’；

actinR：5’-ATCCTCCAATCCAGACACTGTACTTG-3’；

GUSF：5’-GTTTCGATGCGGTCACTCATTA-3’；

GUSR：5’-GTCTGCCAGTTCAGTTCGTTGTT-3’。

Standard is with reference to fluorescent quantitation design of primers principle, and designed primer amplified production length range is from 150-250bp;

2) reaction system 20 μ l:10 μ l 2 * Realtime PCR Master Mix (TOYOBO of employing) of quantitative fluorescent PCR, 10uM forward and reverse primer, and the cDNA of 2 μ l, each reacts three repetitions.Pcr amplification should carry out under MJR DNA Engine Opticon 2 ContinuousFluorescence Detector; Response procedures is following: 94 ℃ of sex change 15s; 57 ℃ of annealing 15s; 72 ℃ are extended 15s, read plate 2s respectively for 82 ℃ and 85 ℃, and melting curve analysis subsequently proves the specificity of the product of amplification;

3) analyze the quantitative PCR product with software Opticon Monitor Version 2.02 (MJ Research);

4) data analysis, that the relative quantification expression of gene adopts is Δ C _TMethod, β-actin is as the internal reference gene, Δ C _TBe the average C of sample _TValue deducts the average C of internal reference gene _TThe numerical value of value gained, it is used for the different influences that brought of sum and RT reactions step reaction efficiency of different cDNA templates in each reaction of homogenization.Relatively two samples are just used the Δ C of two samples respectively _TValue is subtracted each other, and the numerical value that obtains is Δ Δ C _T, the relative value of GUSmRNA copy number can use 2 ^{-Δ Δ CT}Calculate.The relative expression quantity of GUS is seen Fig. 5 in transgene tobacco root, stem, the leaf, and as can be seen from the figure the transcriptional level of GUS is respectively 15 times and 11 times in the root in transgene tobacco leaf, the stem.

Confirming of embodiment 3 cotton GhPsbP promoter functions zone

According to the controlling element distribution situation of prediction, it is following to design the deletion mutantion promoter primer respectively:

P2F：5’-CCCAAGCTTTTCAAAGGCTATGAGTTTTG-3’

P3F：5’-CCCAAGCTTCTTGCTACCATTTCTCTGCG-3’

P4F：5’-CCCAAGCTTTGGTACTTGAAGGTAACAATG-3’

P5F：5’-CCCAAGCTTGGCAAATCAACAATATGTTAAC-3’

P2F, P3F, P4F and P5F are increased with PR respectively; Adopt the PCR method of amplification P1 to obtain 4 GhPsbP promotors 5 ' end change deletion fragment, called after P2 (corresponding to the 286th to 2363 Nucleotide of SEQ ID NO:1), P3 (corresponding to the 740th to 2363 Nucleotide of SEQ ID NO:1), P4 (corresponding to the 1693rd to 2363 Nucleotide of SEQ ID NO:1) and P5 (corresponding to the 2154th to 2363 Nucleotide of SEQID NO:1) respectively.After adopting above-mentioned method to be cloned into P2, P3, P4 and P5 on the pMD18-T carrier respectively; Enzyme is cut the back sequence verification; Enzyme is cut the result and is seen Fig. 3 the 6th, 5,3,1 swimming lane, and enzyme is cut and is connected on the pBI121 carrier again, passes through leaf dish method transformation of tobacco behind the conversion Agrobacterium; Identify the tobacco that obtains to change P2, P3, P4 and P5 respectively through PCR, the PCR qualification result that changes P2, P3, P4 and P5 tobacco is seen 4-the 7th swimming lane of Fig. 4.

1. transgene tobacco GUS histochemical stain.

1) staining fluid consists of: 0.1M sodium phosphate buffer (pH7.0) 20ml, 0.5M EDTA solution 1ml, Triton X-100 0.5ml; Yellow prussiate of potash (Pataxium ferrocyamide) 42.5mg; The Tripotassium iron hexacyanide (Pataxium ferricyamide) 33mg, paraxin (Chloramphenicol5mg, X-Gluc50mg; Methyl alcohol (Methanol) solution 10ml adds water to 50ml.Destainer consists of: 50% ethanol, acetic acid 5%, 3.7% formaldehyde solution. prepare 4 ℃ of preservations of GUS dye liquor lucifuge;

2) handle tobacco seedling 2-24h with staining fluid at 37 ℃;

3) dyeing back sample is fixed in 70% the ethanol and decolours; Coloration result is seen Fig. 6, explain that P1, P2, P3, P4 and P5 all can drive gene efficient specifically expressing in the chlorenchyma of plant, but expression level is P1＞P2＞P3＞P4＞P5.

2. the real-time PCR of transgene tobacco GUS

Take the method for above-mentioned real-time PCR; Respectively the blade that changes P1, P2, P3, P4 and P5 tobacco is carried out real-time PCR; The result sees Fig. 7; Can see that promotor total length and deletion mutant all can drive GUS and transcribe, but transcriptional level is that P1＞P2＞P3＞P4＞P5 and transgene tobacco GUS histochemical stain result are consistent.

3. transgene tobacco gus protein determination of activity

Get the tobacco appropriate amount of material of changeing P1, P2, P3, P4 and P5 respectively and use the liquid nitrogen grinding powdered, get about 0.6 and restrain powder and pack in the 1.5mL centrifuge tube; Add the 1mL protein extract, shake up, put on ice to deposition; Centrifugal 15 minutes of 4 ℃ of 13000rpm; Draw supernatant, preserve in the refrigerator; Adopt the Bradford method to measure protein content, 100 μ g/mL BSA standardized solution accordings to the form below are processed BSA gradient liquid, see table 1; The BSA solution 1mL that gets above-mentioned concentration respectively shakes up to the 10mL centrifuge tube that 3mL Xylene Brilliant Cyanine G G-250 solution is housed, and room temperature was placed 5 minutes; Measure the absorbancy of each sample under 595nm with spectrophotometer Du640; With BSA concentration is X-coordinate, and absorbancy is an ordinate zou, the drawing standard curve; Get gus protein 20 μ L and be diluted to 1mL, be added in the 3ml Xylene Brilliant Cyanine G G-250 solution, shake up, room temperature was placed 5 minutes, measured the absorbancy under the 595nm, calculated the concentration of protein example according to typical curve; In the 10mL centrifuge tube, add 400 μ L gus proteins and extract damping fluid, 100 μ gGUS albumen, 10 μ L40mM 4-MUG, 37 ℃ were reacted 1 hour; Add the 1.6mL reaction terminating liquid; The preheating spectrophotofluorometer is measured its reading under exciting light 365nm, emission light 455nm condition; Pressing table 2 concentration gradient preparation 4-MU solution, is blank solution with the reaction terminating liquid, the drawing standard curve; Enzyme activity unit is defined as: the enzyme amount of PM hydrolysis 4-MUG generation 1nmol or 1mg, 1 μ g, 1ng 4-MU is a unit of activity, obtains the enzyme activity of each sample according to definition.Gus protein content is seen Fig. 8 in the tobacco leaf of commentaries on classics P1, P2, P3, P4 and P5; Can see that promotor total length and deletion mutant all can drive GUS and transcribe; But it is the highest to change P1 and P2 tobacco plant gus protein activity, and the chances are changes 4 times of the P5 tobacco plant, secondly for changeing the tobacco plant of P3 and P4; Its gus protein activity is that to change the real-time PCR result of 2 times of the P5 tobacco plant and transgene tobacco GUS histochemical stain result and transgene tobacco GUS consistent.

The preparation of table 1BSA standardized solution

Table1?Standard?curve?for?the?measurement?of?BSA

The preparation of table 24-MU standardized solution

Table2?Standard?curve?for?the?measurement?of4-MU

The preservation information of related cotton GhPsbP promotor is following in this specification sheets:

1. depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center

2. depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica

3. preservation date: on June 1st, 2010

4. deposit number: 3880

5. classification name: ETEC (Escherichia coli).

Sequence table

< 110>Biological Technology institute, Chinese Academy of Agricultural Sciences

< 120>cotton GhPsbP total length promotor

<160>1

<170>PatentIn?version?3.5

<210>1

<211>2363

<212>DNA

<213>G.hirsutum

<400>1

tggatgacta?tagttgctcc?gaccagtatg?acagcatatc?tttatctcta?ctctaatgga 60

gaagaatcat?tggctgctac?ccaactagtg?taatagatgt?aattttacca?tatattaaat 120

tttaagatca?acaatgtaat?tttatcgtat?attaatttat?aattctatct?tttttaaagg 180

tactaaacat?atttttttaa?tattttgggg?gctaaagtgc?aattttacca?tagatcaatt 240

tataatttta?ttctttctaa?agggactgaa?ttgaaaatat?tttatttcaa?aggctatgag 300

ttttgatttt?ttatttcaca?tatgaaaaga?actaatgtta?ctaaaattta?gtaattttta 360

tgtaaaattt?aaaggttttt?atttcaccaa?aagattgcta?taaattattc?atttattaaa 420

gagtatacct?tataattaat?tgttgttatc?atgttattat?aatggataaa?ataaaatata 480

aaaatatgtt?ttctattaaa?ataaaatgtt?attagaggat?tagtatatcg?tctgttaaac 540

tcattttaat?attggcgatt?atgacctaag?agaacatatg?tgagaggcta?ggcaaatata 600

ttaggtattg?aaatattctt?aataataatt?ttatattaaa?aatgctatta?aattataaat 660

tattttatta?aattgtattt?aataataatt?atgttaaaat?atgattaaat?tattcctcac 720

atttctctaa?catgtttaac?ttgctaccat?ttctctgcgc?caactctgtt?taagaagggt 780

gttcattgac?ttctcttatt?cctcttttct?ccttttatca?gtttgtattt?tcttaaattt 840

gtcttctatc?atttaaatct?ggtcttaaaa?ggctataaaa?gaataaatgt?ttgatgaata 900

gttagattta?tgttcattta?tcaaattaaa?taaataaata?tgaacaagat?ttcaaggctc 960

gtttattaaa?cgaattgata?cgaacaaaac?ttattcaatt?tatttatgct?aatttaaaac 1020

tcatttaata?aattatttaa?agtttattat?tcgattatta?aattattatt?gtttgtgtac 1080

ttattttttt?tataatatac?ttttttaata?cttatatttg?attattttat?atctaaaaat 1140

aatatatatg?tttgtttatt?tatttattgt?taattatcca?tgaacattat?ttgtgaatat 1200

attcaattat?gtgttcatga?atatgttcgt?tcaacttaaa?cgaatgaaca?tgatatatat 1260

atacatataa?ttttaaacaa?acgaacatga?atagaaattt?gaaattctta?acaagcatga 1320

atcaaatata?aatacatttt?aaagaataaa?caaatgacta?taattagaag?ttattcattt 1380

aaactcaatg?agttatatgt?ttttgccaat?taattgcaat?tttcaattaa?aaagttcctt 1440

ggtatgatgt?aaaattattt?tacgaaggct?aaattgattg?ttaatttttg?attaactact 1500

taattaattg?taatttttaa?ttaaaaatta?attattttta?gttattttac?aaaagtttcg 1560

ttttatcatt?taagctttcc?ttttgggttt?aatatataat?ttggtactct?aatttgacat 1620

ttttttctaa?tgcaatactt?gtatttttct?ttgcctaata?tggtacctat?attttacaaa 1680

agttatacat?tttggtactt?gaaggtaaca?atgttaattt?ttttagcgtc?tcattcgtat 1740

tttaaaatta?atttgatgaa?aattattaac?aaataatatt?ataaattaac?ttttatcgaa 1800

tcaatcttaa?aatctaaaat?aaatcatatt?tataagcttt?tgtttgataa?attaaatgca 1860

aaactaaata?aattcacaat?caaaactaaa?tttattttat?taacaaaatc?atgaaaaatt 1920

caaatttaat?agtattaata?attaactttt?atcaaatcaa?tactaaaatc?cgaattaaac 1980

attaaaaatt?taacaccatt?atcattaaat?atcaaaatat?ataattttta?tcaaatgcat 2040

acattaaatt?agataataat?aaaaaaaaaa?gaagaagaag?aagaactcaa?attccccatt 2100

aaaaaaataa?aaaatgatat?gttaagcgtc?attttactac?gcattatttc?tttggcaaat 2160

caacaatatg?ttaacatagc?tgaaaatcaa?gataaattga?gctgttttcg?accgttggat 2220

cagccacaac?acaattaaga?taaccaatca?gaaactgcca?cgcatgcatc?gcatcacaca 2280

atttgatcca?acccaaaact?ctatcctttt?gctgctgccc?tctcttgtcc?tcgcctgctg 2340

ccactgccac?tggcactaat?gcg 2363

Claims (5)

1. a transcription regulatory region dna fragmentation that derives from cotton is characterized in that, said dna fragmentation is P1, the 1st 's to the 2363rd shown in the SEQ ID NO:1 promoter region complete nucleotide sequence, and deletion mutant: P2, P3, P4 and P5; P2 is corresponding to the 286th to 2363 Nucleotide of SEQ IDNO:1; P3 is corresponding to the 740th to 2363 Nucleotide of SEQ ID NO:1; P4 is corresponding to the 1693rd to 2363 Nucleotide of SEQ ID NO:1; P5 is corresponding to the 2154th to 2363 Nucleotide of SEQ ID NO:1.

2. transcription regulatory region dna fragmentation as claimed in claim 1 is characterized in that, said dna fragmentation can drive with its associated base because of in the green plant tissue, transcribing.

3. a recombinant DNA that contains any described transcription regulatory region dna fragmentation of claim 1 is characterized in that: after it is imported plant again, can start the gene transcription that is associated, expression.

4. the expression vector that contains the said transcription regulatory region dna fragmentation of claim 1.

5. the application of the described transcription regulatory region dna fragmentation of claim 1 is characterized in that: the arbitrary dna fragmentation of P1, P2, P3, P4 and P5 separates regulating and controlling sequence again as molecule marker from cotton.

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