CN1131311C - Angrpl gene promotor expressed specifically by plant vascular bundle and its application - Google Patents

Abstract

The present invention relates to a newly discovered DNA sequence which can be used as a promoter adjusting and control purpose gene to be specifically expressed on vascular bundle organizations of plants. In the present invention, an Angrp 1 gene and an upstream sequence thereof from a short foot Nantes rice genome are cloned, the activity of a promoter expressed by a GUS gene driven by a 5'series deletion segment of the upstream sequence of the Angrp 1 gene is analyzed by a tobacco transient expression system on the basis of transcriptional start site measurement, and a segment of 1021 bp is determined as a basic promoter region of the newly discovered DNA sequence. Then, the basic promoter can guide a reporter gene to be specifically expressed on the vascular bundle organizations by the validation of GUS histochemical stain in transgenic tobacco and transgenic rice.

Description

A kind of Angrp1 gene promoter and application thereof of plant vasular bundle specifically expressing

The invention belongs to plant genetic engineering field, specifically, relate to a kind of newfound dna sequence dna, it can be used as the vascular bundle tissue of promoter regulation goal gene specifically expressing in plant, the fusion gene that comprises this dna sequence dna and gus reporter gene, carry the plant expression vector pG6-121 and the pG6-Hpt of this fusion gene, and the transgenic plant of the tissue specific expression gus gene that obtains thus.

The vegetable cell wall-held protein is made up of multiple enzyme and structural protein.The plant cell wall structural protein in the skeleton structure of decision cell walls, the cell walls retractility is provided, satisfies and significance arranged aspect the defense reaction of the cell growth of different developmental phases and anti-infective and damage, mainly can be divided into Extensin, PRP, AGP, GRP four classes ^[1]

Extensins claims that again HPRPs (Hydroxyproline-Rich Proteins) is the alkaline glycoprotein that a class is rich in oxyproline, has Ser-(Hyp) 4 pentapeptide tumor-necrosis factor glycoproteinss, and oxyproline wherein and Serine are usually by glycosylation.PRPs (Proline-Rich Proteins) proline rich has the tumor-necrosis factor glycoproteins of Pro-Pro-Val-X-Y, and its proline residue nearly 50% is by glycosylation.AGP (Arabinogalactan Proteins) is the protein of the high glycosylation of a class solubility, and its sugared content is than Extensins higher (up to 90%), and molecular weight changes greatly, and its glycosyl is D-semi-lactosi and L-arabinose.

GRPs (Glycine-Rich Proteins) is the not sacchariferous cell wall structure albumen of a class, its glycine content can be up to 70%, precursor protein N-end has signal peptide sequence that it is positioned on the cell walls, maturation protein has the repeating unit of (Gly-X) n, wherein the X majority still is Gly, and minority is Ala or Ser.This tumor-necrosis factor glycoproteins has constituted the antiparallel β-lamella secondary structure of rule, needed tension force and elastic force when providing the dimension pipe to grow.GRPs also is a kind of plant defense albumen, but the polyreaction of its tyrosine residues trigger molecule and glycan molecule is crosslinked, synthetic xylogen; (Extensins PRPs) is positioned the different tissues zone of plant organ respectively, forms the spatial defense system for it and other two kinds of cell wall proteins.When plant tissue sustained damage, GRP also may participate in repair process.

Plant cell wall GRP gene has been represented the little multigene family of a class, and its expression is subjected to inducing of different developmental phases and multiple ambient pressure.Physical abuse, fungi infestation, virus infection, ethene, dormin, growth hormone, Whitfield's ointment, mercuric chloride, water logging, mummification, heat, environmental factors such as cold all can influence the GRP expression of gene to some extent.In recent years, show further that about Kidney bean grp1.8 gene, petunia ptGRP1 gene, Arabidopis thaliana GRPs gene and rice Os grp1 expression of gene location and expression regulation research the GRP expression of gene has tangible organ, tissue and cell-specific.In Kidney bean and the soybean, GRP mainly expresses in being about to lignifying and lignified cell and tissue ^[2]The genetic expression of rice Os grp1 then concentrates on young stem, microtubule fasolculus of particularly growing and epidermis position ^[3]To studies have shown that of GRP gene expression regulation element, the cis-regulating element of decision microtubule fasolculus specifically expressing is positioned at the promoter region of this upstream region of gene.For example, Keller etc. are by to 5 of Kidney bean grp1.8 promotor ' serial deletion analysis, found two to the stem vascular tissue special and one to the root vascular tissue special just regulate and control the cis factor and a negative regulation cis factor of controlling the vascular tissue expression activity ^{[4] [5]}

As mentioned above, the GRP gene provides a good model for the defensive raction of tissue specificity, etap specificity and the response environment pressure of research gene expression in plants.The research that plant tissue is specific expressed, become an important research field of molecular biology of plants in recent years, particularly in the practicability of plant genetic engineering in the stage, preferentially express at specific tissue site and specific etap for making goal gene, more make it to become the focus that various countries are competitively studied, but the research to the promotor of controlling plant vascular bundle organizing specific expression is few in number, and the research of the trans transcription factor that acts on mutually with cis-regulating element in the promotor still is in the starting stage.Do not see report so far as yet to paddy rice GRP gene promoter analyzing in detail.Paddy rice GRP gene promoter has comprised the cis element of controlling gene expression specificity, it is furtherd investigate the molecular mechanism that will help to illustrate genetic expression, and can be the controlling element that plant genetic engineering provides usefulness, have important theoretical significance and application value.

The object of the present invention is to provide a kind of promoter sequence of new paddy rice GRP gene (Angrp1), comprise negative regulation zone and one in this sequence and just regulating and control the zone, the basic promoter region that comprises a 1021bp in this sequence, can be in transgenic plant, concrete is to instruct gus reporter gene at the vascular tissue specifically expressing in transgene tobacco and transgenic paddy rice.Agricultural pests (as aphid, leaf buddhist, planthopper etc.) with piercing-sucking mouthparts all are the juice of sucking plant from vascular tissue, and the growth of serious harm crop, growth and maturation bring about great losses to agriculture production.Make anti insect gene can prevent and treat this class pest targetedly, efficiently by the Angrp1 gene promoter, also can reduce the selection of insect is pressed, alleviate the metabolism burden of expression of plants anti insect gene at the vascular tissue specifically expressing of crop.When plant virus infects in the formation system, must make antiviral gene can control the generation and the harm of virus disease effectively at the vascular tissue specifically expressing of crop with the Angrp1 gene promoter by the long-range propagation of vascular tissue.

The present invention is based on the cDNA clone who from short pin Nan Te (An) rice cDNA library that rice yellow dwarf virus (RYSV) infects, has obtained the Angrp1 gene, and prove that this gene is a newfound rice cell wall GRP gene with the differential sieve method ^[6]Infer that from nucleotide sequence gene product N end is the signal peptide sequence that 23 amino-acid residues are formed, maturation protein contains 66% glycine, has the tumor-necrosis factor glycoproteins of (Gly-X) n, belongs to typical plant cell wall GRP.By the cDNA sequence is that the Northern hybridization analysis of probe shows that the expression of Angrp1 gene in the An paddy rice has tangible organ specificity and etap specificity, and physical abuse and RYSV infect also can obviously strengthen this expression of gene.This provides the foundation for further promoter function fragment analysis.

The present invention has cloned Angrp1 gene and 5 ' upstream sequence thereof from the An rice genome, and is located on No. 10 karyomit(e) of paddy rice.On the basis of having measured transcription initiation site, the serial deletion fragment that the present invention has analyzed Angrp1 gene 5 ' upstream sequence by tobacco moment expression system drives gus gene expression promoter activity, obtained one and just regulated and control zone and a negative regulation zone, and determined that the fragment of 1021bp is its basic promoter region.The present invention subsequently confirms that by the GUS histochemical stain this basic promotor can instruct reporter gene at the vascular tissue specifically expressing in transgene tobacco and transgenic paddy rice.It is as follows to implement concrete steps of the present invention:

CDNA with An paddy rice GRP is a probe, and the λ genomic library of screening An paddy rice obtains a phage clone that contains GRP gene (called after Angrp1).Relevant range (the SalI endonuclease bamhi of a 3kb) is cloned in (called after pBS5111) on pBlueScriptII SK (-) plasmid vector, and it is carried out subclone and sequencing (Fig. 1), Fig. 2 is seen in interpretation of result.

-568～+ 1 promoter fragment is made probe, is that 17 hybridization DH colony carries out the RFLP mapping with narrow leaf blue or green 8 and capital, with the Angrp1 assignment of genes gene mapping on No. 10 karyomit(e) of paddy rice (Fig. 3).

Total RNA makes primer extension assay to the young An paddy rice in seedling stage of process damage inductive, and son leaves and stems RNA in seedling stage is done 5 ' RACE experiment.Two result of experiment show that this gene has only a transcription product, and transcripting start point is A (Fig. 4), apart from the TATA box 25bp (Fig. 2) that infers.

To amplify Angrp1 gene promoter fragment from pBS5111 and carry out pcr amplification with different restriction enzyme digestion or with different primers respectively, obtain the promoter fragment of a series of 5 ' disappearances.They are cloned into respectively among the pBI221, to replace original 35S promoter, be built into 9 Angrp1 genes 5 ' series disappearance promotor and gus gene transcribe fusion cloning (pG1-GUS～pG9-GUS, Fig. 2).

Set up protoplastis moment expression system with tobacco BY-2 suspension cell, with contain 35S promoter-luciferase (LUC) gene with reference to plasmid pFFO respectively with each the test plasmid (pGn-GUS or positive control pBI221) carry out cotransfection, measure and proofread and correct the GUS activity (Fig. 5) of lysate, the result shows: there is positive regulating and controlling sequence in G2-G5 district (2283～-1404), have negative regulatory sequence in G5～G6 district (1404～-1020).

To plant expression vector pBI121, make the basic promotor G6 of Angrp1 replace the 35S promoter among the pBI121 G6-GUS fusion gene cloning, obtain tobacco conversion carrier pG6-121 (Fig. 6).With agriculture bacillus mediated leaf dish method transformation of tobacco K326, vein and the stem of getting the positive transfer-gen plant of PCR carry out the GUS staining analysis.The vein transverse section show dyeing part mainly phloem (interior tough and outside tough, Fig. 7 A), the longitudinal section demonstration dyeing part of stem mainly is at endophloem (Fig. 7 B).

The G6-GUS fusion gene cloning to improved paddy rice stable conversion carrier p35S-Hpt-Nos, is built into the rice conversion carrier pG6-Hpt (Fig. 8) of zonal tide mycin resistant gene, spends paddy rice No. one in transforming with particle bombardment.Blade to the transgenic rice plant of PCR test positive carries out GUS tissue staining (Fig. 9 A), and does the square section and observe (Fig. 9 B).The result shows that dyeing part is mainly in the vascular bundle of rice leaf.

Above result shows that the basic promotor G6 of paddy rice Angrp1 gene (1021bp) has similar vascular bundle tissue expression specificity in dicotyledon tobacco He in the monocotyledon rice.

The present patent application people is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center May calendar year 2001 with the intestinal bacteria XL1Blue transformant that the basic promotor G6 of paddy rice Angrp1 gene merges the plasmid pG6-Gus of reporter gene GUS.Preservation registration number is: CGMCCNo.0579.

Below in conjunction with accompanying drawing the present invention is described in further detail:

Clone, subclone and the sequencing of Fig. 1 .Angrp1 gene

Angrp1 gene coding region upstream sequence and 3 ' non-coding region

The Angrp1 gene coding region

The used probe sequence in screening library

With universal primer subclone is checked order

With special primer subclone is checked order

B:Bcl I, C:Cla I, H:Hind III, K:Kpn I, S:Sal I, Sp:Spe I, the complete sequence of X:Xba I Fig. 2 .Angrp1 gene

Be numbered for+1 pair of Nucleotide with genetic transcription starting point (see figure 4).At 5 ' upstream,

Heat shock protein factor binding sequence (HS) marks with double underline; Being used to prepare 5 ' series disappearance opens

The restriction endonuclease of mover fragment (G1-G9) (Sal I, Hind III, Spe I, Kpn I, Xba I) identification

(F2, F3 F4) mark with underscore or arrow line respectively, open substantially for sequence or PCR primer

The sequence of mover G6 marks with shading, and the TATA box and the CAAT box of supposition show with frame table.

At gene coding region, the aminoacid sequence of supposition is listed below codon, is used for transcription initiation

Primers F 5 and P2 that the site is measured represent with arrow line.The location of Fig. 3 .Angrp1 gene on rice chromosome

Be recombination value (%) in the bracket of the left side, left-hand digit is genetic distance (cM), and the right is RFLP

Mark, arrow are depicted as the position of Angrp1 gene.Determining of Fig. 4 .Angrp1 genetic transcription initiation site

(A): to coagulating of primers F 5 extension products (W) that paddy rice RNA is done in young seedling stage of damage

Gel electrophoresis figure.The left side (CTAG) is anti-for the order-checking of the Angrp1 gene being done with same primer

Should, in order to determine the terminating point of primer extension product.(B): primers F 5 and transcription initiation site

(+1) position in the Angrp1 gene.Fig. 5 .Angrp1 gene 5 ' relative reactivity of series disappearance promotor in tobacco moment expression system

(A): 5 ' series disappearance promotor and GUS transcribe the structure of fusion cloning

(B): the GUS relative reactivity (%) in the tobacco protoplast moment expression system

F2, F3, F4, GUS2:PCR primer.

A：Asp?718，B：BamH?I，H：Hind?III，P：Pst?I，S：Sal?I，Sp：Spe?I，

X:Xba I Fig. 6. the vascular-specific expression of tobacco stably express carrier pG6-121 Fig. 7 .Angrp1 gene promoter in tobacco

(A): the square section of transgene tobacco petiole, (B): the profile of transgene tobacco stem

Ip: endophloem, op: outer phloem, p: marrow, x: xylem Fig. 8. the vascular-specific expression of paddy rice stably express carrier pG6-Hpt Fig. 9 .Angrp1 gene promoter in paddy rice

(A): the blade of transgenic paddy rice, (B): the square section of transgenic paddy rice blade, lv: vein

Clone, location and the transcripting start point of embodiment 1:Angrp1 gene determined the clone of 1.Angrp1 gene

At first extract the genomic dna of An paddy rice: change the 50ml centrifuge tube over to after an amount of paddy rice vanes is ground (in liquid nitrogen), add extracting solution (500mM NaCl, the 1.25%SDS of 15ml 65 ℃ of preheatings, 100mM Tris, pH8.0,5mM EDTA), 65 ℃ are incubated 30 minutes behind the mixing; Add 5ml 5M KAc, placed 20 minutes on ice; Add isopyknic chloroform and put upside down mixing several times, 4 ℃ 12, centrifugal 15 minutes of 000rpm; Supernatant liquor is transferred to new pipe, adds the Virahol of 0.6 times of volume, mixing; Cotton-shaped DNA is moved on in the new pipe, dry after washing once with 70% ethanol, use an amount of ddH ₂The O dissolving.

With the genomic dna of An paddy rice with Sau3A part enzymolysis, with test kit (Stratagene company, Cat#400766) fragment of separation 9-23kb from agarose electrophoresis glue, after substrate only adds in the reaction solution of G and A with Klenow part polishing with λ FixII/Xho I part polishing carrier (Stratagene company test kit, Cat#248211) connect, according to test kit specification sheets operation, to be packaged to be a titre be 3.5 * 10 through external ⁵Genomic library.The cDNA fragment (Fig. 1) of the short pin special paddy rice GRP in south is made probe with the PCR method of mixing, screen this genomic library, obtain a phage clone λ 5-1 who contains GRP gene (called after Angrp1) by in situ hybridization.

The SalI endonuclease bamhi of the 3kb of λ 5-1 is cloned on pBlueScriptII SK (-) plasmid vector (called after pBS5111) and according to shown in Figure 1 it is carried out subclone and sequencing.The zone of sequencing is 3138bp altogether, the Angrp1 gene upstream sequence of 2401bp, 5 ' non-coding region of 41bp, the coding region of 552bp and 3 ' non-coding region of 138bp have been comprised, together with the 3 ' non-coding region end sequence 111bp that comprises among the Angrp1 cDNA, known Angrp1 gene order is 3249bp (Fig. 2) altogether.2.Angrp1 the location of gene in rice chromosome

With Angrp1 cDNA fragment (Fig. 2) is that probe is made the Southern hybridization analysis to An paddy DNA Hind III enzymolysis sample, proves that this gene is single the copy in genome.Further-568～+ 1 promoter fragments are made probe, be that the different enzymes of 17 genomic dna are cut sample (BamH I with narrow leaf blue or green 8 and capital respectively, Bgl I, Dra I, EcoR I, Hind III, Sca I and Xba I) hybridize, the result shows when cutting with Hind III enzyme to have polymorphism between them.Therefore be that the genomic dna of 17 DH colony (127) is cut the back with Hind III enzyme and made Southern with above-mentioned probe and hybridize with narrow leaf blue or green 8 and capital, carry out the assignment of genes gene mapping.Data processing is to utilize Mapmaker software to carry out on the MacintoshII computer, carry out the linkage group that two point analysiss obtain Angrp1 gene place with Two point and Group order earlier, do the multiple spot analysis with the Compare order then, convert recombination value to map unit (cM) with the Kosambi function at last.The result with the Angrp1 assignment of genes gene mapping on No. 10 karyomit(e) of paddy rice (Fig. 3).3.Angrp1 determining of transcription initiation site

With primers F 5 (Fig. 2,5 '-CTAAGGAGGACAAGGATGGC-3 ') the 5 ' terminal T4-Kinase mark of using ³²Behind the P to making primer extension assay through the total RNA of the damage inductive short pin special paddy rice in south in young seedling stage: total RNA is got 8.5 μ l (about 40 μ g) through 85 ℃ of sex change join and contain after 5 minute ³²In the extension mixture of P-F5, annealed 30 minutes for 42 ℃; Add dNTP mixture (every kind of 10mM) 1 μ l and AMV ThermoScript II 1 μ l then, 42 ℃ were extended 60 minutes; Add 3MNaOH 2 μ l, 42 ℃ of insulations were also used phenol and chloroform extracting with degradation of rna with 0.18M HCl 34 μ l neutralization in 20 minutes successively, and supernatant liquor is resuspended in the 8 μ l sequencing reaction stop buffers after with ethanol sedimentation; Get the sequencing reaction electrophoresis of 2.5 μ l with 5 couples of pBS5111 of same primers F.The result shows that this gene has only a transcription product, and transcripting start point is A, and both sides are C (Fig. 4), apart from the TATA box 25bp (Fig. 2) that infers, meets the rule of eukaryotic gene transcription initiation region.

Press Gibico-BRL company 5 '-method of RACE test kit (Cat#18382-010) specification sheets, with primer P2 (Fig. 2,5 '-ACGGATCCATAACCTAGGAGGGT-3 ') in full accord to 5 ' RACE experimental result (unlisted) that young seedling stage, leaves and stems RNA was done with The above results, further verified Angrp1 gene transcription starting point.Activity 1.5 ' series disappearance the promotor of embodiment 2:Angrp1 gene 5 ' series disappearance promotor in tobacco protoplast merges the structure of GUS plasmid

At first use primer T3 (Fig. 1,5 '-AATTAACCCTCACTAAAGGG-3 ') and F2 (Fig. 2,5 '-CTGGGATCCGTGGTTGGAGTGAAGGGGAG-3 ') amplify the promoter fragment of Angrp1 gene 2.4kb from pBS5111, carry out pcr amplification (Fig. 2) with different restriction enzyme digestion or with different primers respectively then, obtain the promoter fragment of a series of 5 ' disappearances.They are cloned into respectively among the pBI221, and to replace original 35S promoter, what be built into 9 Angrp1 genes 5 ' series disappearance promotor and gus gene transcribes fusion cloning (pG1-GUS～pG9-GUS), 5 ' starting point of its promoter fragment is respectively G1:-2401, G2:-2283, G3:-2035, G4:-1793, G5:-1404, G6:-1021, G7:-941, G8:-568, G9:-182 (Fig. 2, Fig. 5 A).2. the preparation of protoplastis

With tobacco BY-2 suspension cell subculture after 3-5 days, centrifugal collecting cell (4 ℃, centrifugal 3 minutes of 100 * g, as follows), wash one time with the N.F,USP MANNITOL of 0.4M; Be resuspended in the 0.4M N.F,USP MANNITOL that contains 1% cellulase and 0.1% polygalacturonase, 30 ℃ of water-baths 1 hour, therebetween once and observation of cell enzymolysis situation every 15 minutes jogs; Enzymolysis solution is used the screen filtration of 100 μ m and 60 μ m, centrifugal collecting cell successively; With W5 solution (154mM NaCl, 125mM CaCl ₂, 5mM KCl, 5mM sucrose pH5.9) is given a baby a bath on the third day after its birth time, is resuspended among the 2-5ml W5, and 4 ℃ left standstill 2-6 hour.3.PEG the transfection of the protoplastis of mediation

Centrifugal collecting cell is resuspended in MaMg solution (0.4M N.F,USP MANNITOL, 15mM MgCl ₂, pH5.6), adjust protoplastis concentration to 2 * 10 ⁶Individual/ml; Add 0.3ml in dividing the centrifuge tube that DNA (what contain 35S promoter-LUC gene is pGn-GUS or positive control pBI221 with reference to plasmid pFFO and each test plasmid) is housed, rotate centrifuge tube gently with mixing; Slowly add 0.3ml PEG solution (40%PEG4000,0.4mM N.F,USP MANNITOL, 0.1mM Ca (NO after 5 minutes ₃) ₂, pH7.9), rotate centrifuge tube gently with mixing, left standstill 15 minutes; Dropwise add W5 solution, centrifugal collecting cell after 5 minutes is resuspended in the 5ml liquid nutrient medium, cultivates 24 hours for 28 ℃; With PBS damping fluid (0.8%NaCl, 0.02%KCl, 0.144%Na ₂HPO ₄, 0.024%KH ₂PO ₄, pH7.4) to wash once, centrifugal collecting cell is resuspended in 0.3ml Promega cell pyrolysis liquid (Cat.#E1531); In 4 ℃, 12, centrifugal 15 minutes of 000rpm, supernatant liquor is used for the determination of activity of LUC and GUS.4.GUS active mensuration and correction

At first measure the GUS activity: contain in the cell pyrolysis liquid (PromegaCat#E1531) of 1mM MUG at 0.5ml, add 20 μ l-50 μ l cell extracts, 37 ℃ after water-bath 30-60 minute, get 100 μ l and be added to 900 μ l 0.2M Na ₂CO ₃In, at Hoefer DyNA Quant ^TMMeasure GUS activity (4-MU with 50nM is a standard) on the instrument.Use the luciferase assay kit (Cat.#1500) of Promega company to measure the LUC activity then: to get 5 μ l cell extracts and the rapid mixing of 50 μ l LUC reaction substrates, (parameter is made as: Tritium to insert the liquid flashing counting device immediately, windows0005-1024,30 seconds) record CPM and CLM% (chemoluminescence percentage ratio).Proofread and correct the GUS activity of each time mensuration at last with the LUC activity of FFO, respectively to be tested the relative comparatively accurately GUS activity of plasmid, formula is: GUSn=GUS1 * LUCn/LUC1.5. interpretation of result

Moment, the result of expression analysis showed (Fig. 5), and 9 the 5 ' series disappearance promotor of Angrp1 all has transcriptional activity in the protoplasm somatocyte of dicotyledon tobacco.Wherein, the activity of promoter fragment G2 is the highest, is about the active twice of 35S promoter.Reduce gradually to the G5 activity from G2, but the activity of G6 is higher than G5, constantly descends again from G6 to the G9 activity.There is positive regulating and controlling sequence in (2283～-1404) in the G2-G5 district in this explanation, and the heat shock protein factor land in-1874～-1796 zones may be relevant in just regulating and control; Have negative regulatory sequence in G5～G6 district (1404～-1020), three inverted repeats in-1321～-1109 zones may be relevant with the negative regulation function in this district.We select G6 (1021bp) promoter fragment is the basic promotor of Angrp1, and does the analysis of transgenic plant as main research object.

The sequence of G6 promoter fragment is as follows :-1021 ACTAGTATAT ATATTATTCC CTCCGTTTCA TAATGTAAGA CTTTCTAGCA TTGCTCATAT

-961?TTATATAGAT?GTTAATGAAT?CTAGATGCAT?ATATATGTCT?AGATTTATTA?ATATCTGTAT

-901?GAATATGGGT?AATATGCTAA?AAAGACTTGC?ATTATGAAAC?GGAGGAAGTA?TGTCGCCAGG

-841?CTCACTTAAC?ATGTTGGTCT?AGATCGGTTT?AAAATGCTGC?ATTTAAGTCG?GAATCAGATC

-781?ATGTATACAT?GTAACACAAT?ACTGGTAAGC?AGTAAGAAAT?GCCCGATCTA?GGGATGAAAA

-721?AGAATAACTG?AGATAATAAC?TTACCATGCA?ATTTTATTAT?TGCACAAATT?TAATTAACTG

-661?TTTAGTTTGA?TACTTGGCCT?CATATATAGT?ACGTAGTAAT?TAATTAAACC?ATCGATCTCA

-601?CTAGGCTCCA?ACTAGAAATC?AGATCCACGT?CTACTAGTAC?AGATATGTGA?TGAGCAGATA

-541?TAAGTGCATC?TGCCACACAT?CACTGAACCA?GCACATGGAT?CACATTAAGC?ACGTCAATGT

-481?ATGCTAATTA?ATTGATTAAC?TATACCATGC?CTAATTGCTT?AATTAGCAGC?CAACGGTATT

-421?CGATTGATCC?CATTCGCCAA?AACCGGCCGG?AGTCGCTCTC?ATGAAAACAG?CCTCAGATGA

-361?CCTTGAAGTA?TGGACAGAAT?ACAATAGCAA?ACCGATCGAA?TAATTCTATC?TCTGCTTACA

-301?TAAAAGCAAA?AGCAAAAGTA?AACACACACA?CACACAAAGC?AAAAACAATA?AATCAGTATT

-241?CTAAAAATAA?ATCAGTAACA?TCACCACACC?GCTCTCCAAG?TGTCAAAAAG?AATTAAAGGA

-181?CAACAGCTAC?TGGCACTCTC?CATGAACTCT?CCTGGTCATC?ATTTCACACC?CATATACTGT

-121?GTCCAAAGTC?CAAAAGCTCT?AACGTCTCTA?GTTTTGGACC?ATGCAGACTG?ATGAACATCT

-61 GATGAGATAG?CCATACACGA?GATGCACCCT?ATAAATACCC?CAGCTCCCCT?TCACTCCAAC

-1 CAC embodiment 3: the vascular-specific expression of Angrp1 promotor in the transgene tobacco

Plant expression vector pBI121 with HindIII and EcoRI double digestion, is reclaimed the big fragment of carrier; PG6-GUS is also used HindIII and EcoRI double digestion, reclaim the G6-GUS fragment, be connected with carrier segments and obtain pG6-121.Wherein, the basic promotor G6 of Angrp1 has replaced the 35S promoter among the pBI121 (Fig. 6).

Plasmid pG6-121 electric shock is transformed Agrobacterium LBA4404, use agriculture bacillus mediated leaf dish method transformation of tobacco K326 then: will be cut into small pieces behind aseptic seedling blade margins of excision and the middle arteries and veins, put into MS substratum dilution 20-40 Agrobacterium incubated overnight liquid doubly doubly with , take out after about 5 minutes and remove unnecessary bacterium liquid with filter paper, be tiled on the common culture medium, 28 ℃ of dark cultivations are changeed two days later on division culture medium, illumination cultivation 3 all left and right sides regeneration buds are grown up, in time cut out and change on root media, can transplant after a large amount of roots grow in the greenhouse.

Regrowth is with extracting miniprep dna with the CTAB method: 100 μ l CTAB extracting solutions (100mMTris, pH8.0,1.4M NaCl, 20mM EDTA grinds the plant tissue of about 50mg in 2%CTAB), adds 400 μ l extracting solutions, 65 ℃ of water-baths 30 minutes.Add the cold chloroform extracting of 500 μ l, centrifuged supernatant adds the cold Virahol mixing of equal-volume, and centrifugal sediment is resuspended in 50 μ l TE, gets 1 μ l as the PCR substrate.

With primers F 4 (Fig. 2,5 '-TTAGTCGACAACAGCTACTGGCACTC-3 ') and GUS2 (be positioned at the reverse primer of 450-469 bp behind the gus gene ATG, 5 '-GGGATAGTCTGCCAGTTCA-3 ') carry out PCR and identify to determine transfer-gen plant.

Transfer-gen plant is transplanted in the greenhouse after 25 days, get vein and stem and carry out the GUS staining analysis: get transgenic plant rhizome leaf (stem cuts out cross section or profile with blade) respectively, immerse stationary liquid (0.3% formaldehyde, 10mM MES, fix 1 hour 0.3M N.F,USP MANNITOL), vacuumize after 3-5 minute room temperature; Give a baby a bath on the third day after its birth time with 50mM phosphoric acid buffer (pH7.0), immerse the above-mentioned damping fluid that contains 1mM X-Gluc, vacuumize after 3-5 minute 37 ℃ of reactions and spend the night; Make paraffin section with 65 ℃ of decolourings of 70% ethanol after 1 hour, microscopic examination is taken a picture.

The vein transverse section show dyeing part mainly phloem (interior tough and outside tough, Fig. 7 A), the longitudinal section demonstration dyeing part of stem mainly is at endophloem (Fig. 7 B).This just shows that the basic promotor G6 (1021bp) of paddy rice Angrp1 gene is specific expressed, mainly is expressed in the phloem position of vascular tissue in transgene tobacco.Embodiment 4: the vascular-specific expression of Angrp1 promotor in the transgenic paddy rice

At first the multiple clone site of pcr amplification plasmid pBluescript SK (+) is connected on the paddy rice stable conversion carrier p35S-Hpt-Nos; This recombinant vectors is cut with Sal I enzyme, and polishing is cut with Hind III enzyme again, reclaims carrier segments.PG6-GUS is cut with EcoR I enzyme, and polishing is cut with Hind III enzyme again, reclaims the G6-GUS fragment, is connected with carrier segments, is built into the paddy rice stably express carrier pG6-Hpt (Fig. 8) of zonal tide mycin resistant gene.

Therefrom spend in the seed of a paddy rice and separate rataria, evoked callus on the NB substratum.Plasmid pG6-Hpt and bronze are mixed and made into little bullet suspension,, (comprise pre-differentiation screening through hygromycin selection with particle gun (BioRad company) bombardment callus, the differentiation screening, the screening in strong sprout) obtains transgenic seedling, extract miniprep dna, carry out PCR with primers F 4 and GUS2 and identify with the CTAB method.

PCR is accredited as the male rice plant, gets its blade and carry out GUS tissue staining (Fig. 9 A), and do the square section and observe (Fig. 9 B).The result shows that dyeing part mainly in the vascular bundle of rice leaf, illustrates that the 1021bp promotor of Angrp1 can instruct foreign gene to carry out the special expression of vascular bundle in transgenic paddy rice.

Main reference document 1.Showalter AM. (1993) .Structure and function of plant cell wall proteins. Plant Cell.5:9-23.2.Keller B, Sauer N, Lamb CJ. (1988) .Glycine-rich cell wall proteins in bean:gene structure and association of the protein with the vascular system. EMBOJ.7:3625-3633.3.Xu D, Lei M, Wu R. (1995) .Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation.Plant Mol Biol.28:455-471.4.Keller B, Baumgartner C. (1991) .Vascular-specific expression of the bean GRP 1.8 gene is negatively regulated.Plant Cell.3:1051-1061.5.Keller B, Heierli D. (1994) .Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence.Plant Mol Biol.26:747-756.6.Fang RX, Pang Z, Gao DM, Mang KQ, Chua NH. (1991) .cDNA sequence of a virus-inducible, glycine-rich protein gene from rice.Plant Mol Biol. 17:1255-1257

Claims (9)

1. Angrp1 promotor of expressing at the plant vasular bundle, its nucleotide sequence

1021bp is as follows :-1021 ACTAGTATAT ATATTATTCC CTCCGTTTCA TAATGTAAGA CTTTCTAGCA TTGCTCATAT

-961?TTATATAGAT?GTTAATGAAT?CTAGATGCAT?ATATATGTCT?AGATTTATTA?ATATCTGTAT

-901?GAATATGGGT?AATATGCTAA?AAAGACTTGC?ATTATGAAAC?GGAGGAAGTA?TGTCGCCAGG

-841?CTCACTTAAC?ATGTTGGTCT?AGATCGGTTT?AAAATGCTGC?ATTTAAGTCG?GAATCAGATC

-781?ATGTATACAT?GTAACACAAT?ACTGGTAAGC?AGTAAGAAAT?GCCCGATCTA?GGGATGAAAA

-721?AGAATAACTG?AGATAATAAC?TTACCATGCA?ATTTTATTAT?TGCACAAATT?TAATTAACTG

-661?TTTAGTTTGA?TACTTGGCCT?CATATATAGT?ACGTAGTAAT?TAATTAAACC?ATCGATCTCA

-601?CTAGGCTCCA?ACTAGAAATC?AGATCCACGT?CTACTAGTAC?AGATATGTGA?TGAGCAGATA

-541?TAAGTGCATC?TGCCACACAT?CACTGAACCA?GCACATGGAT?CACATTAAGC?ACGTCAATGT

-481?ATGCTAATTA?ATTGATTAAC?TATACCATGC?CTAATTGCTT?AATTAGCAGC?CAACGGTATT

-421?CGATTGATCC?CATTCGCCAA?AACCGGCCGG?AGTCGCTCTC?ATGAAAACAG?CCTCAGATGA

-361?CCTTGAAGTA?TGGACAGAAT?ACAATAGCAA?ACCGATCGAA?TAATTCTATC?TCTGCTTACA

-301?TAAAAGCAAA?AGCAAAAGTA?AACACACACA?CACACAAAGC?AAAAACAATA?AATCAGTATT

-241?CTAAAAATAA?ATCAGTAACA?TCACCACACC?GCTCTCCAAG?TGTCAAAAAG?AATTAAAGGA

-181?CAACAGCTAC?TGGCACTCTC?CATGAACTCT?CCTGGTCATC?ATTTCACACC?CATATACTGT

-121?GTCCAAAGTC?CAAAAGCTCT?AACGTCTCTA?GTTTTGGACC?ATGCAGACTG?ATGAACATCT

-61?GATGAGATAG?CCATACACGA?GATGCACCCT?ATAAATACCC?CAGCTCCCCT?TCACTCCAAC

-1?C。

2. plant expression vector that contains the described promotor of claim 1.

3. plant expression vector according to claim 2 is pG6-121 shown in Figure 6.

4. plant expression vector according to claim 2 is pG6-Hpt shown in Figure 8.

5. transformant that contains the promotor of the described nucleotide sequence of claim 1.

6. transformant according to claim 5 is the intestinal bacteria XL1Blue transformant of plasmid pG6-GUS, and its preservation registration number is CGMCC No.0579.

7. the application of Angrp1 gene promoter according to claim 1 in transgenic plant.

8. according to the application of described each plant expression vector of claim 2 to 4 in transgenic plant.

9. according to claim 5,6 application of described each transformant in transgenic plant.

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