CA2317723A1 - Root-specific promoter - Google Patents
Root-specific promoter Download PDFInfo
- Publication number
- CA2317723A1 CA2317723A1 CA002317723A CA2317723A CA2317723A1 CA 2317723 A1 CA2317723 A1 CA 2317723A1 CA 002317723 A CA002317723 A CA 002317723A CA 2317723 A CA2317723 A CA 2317723A CA 2317723 A1 CA2317723 A1 CA 2317723A1
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- Prior art keywords
- promoter
- construct
- sequence
- root
- gene
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
- C12N15/8227—Root-specific
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Insects & Arthropods (AREA)
- Pest Control & Pesticides (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A DNA which has the sequence shown in Figure 5 and which defines a gene promoter region has been isolated from maize roots. The promoter may be used for driving expression of foreign genes in the roots of plants. This is particularly useful for expressing an insecticidal toxin, such as a deltaendotoxin of Bacillus thuringiensis, to impart resistance to insect attack on the roots of plants by Coleopteran insects.
Description
wo aon~4 Pcrns9siozooo This invention relates to a gene promoter sequence which directs expression of a gene to the root tissue of plants.
In the genetic improvement of plants by molecular techniques,, it is desirable that expression of inserted foreign genes be restricted to tissue where that expression will have significant effect. There are two principal reasons for this. First, restricted expression, rather than total (constitutive) is likely to be less demanding on the metabolism of the plant.
Secondly, it would be good practice to direct expression of the foreign gene to those parts of the plant which are not used for human or animal food when the expressed protein has no effect on such food parts. This second reason may be important when the effect which ingestion of the expressed protein may have is not be fully known.
One widespread target for genetic improvement of crop plants is the introduction of resistance to insect attack. Certain insect species attack green leaf tissue, whereas other, for example Coleoptera, attack the roots. Similarly there are certain disease-inducing microorganisms which attack the below-ground plant tissue and any genetic modification to impart resistance to such organisms will require expression of the resistance-imparting gene in the roots.
_ WO OOI29594 PCT/IB98l02000 An object of the present invention is to provide a root-specific gene promoter sequence and means for isolating same of root DNA.
According to the present invention there is provided a DNA sequence, defining a promoter of a root-expressed plant gene, having the sequence set forth in Figure 5 herewith.
The said DNA may be isolated from the root tissue of a particular target plant species of interest. The preferred species is Zea mays.
The invention a1'~o provides a gene construct comprising, in sequence, the aforesaid gene promoter of the invention, a coding region located downstream and controlled by the said promoter and a 3'-untranslated region including a polyadenylation signal.
Preferably the coding region encodes a protein which is toxic to root-attacking organisms and more preferably the protein is an insecticidal endotoxin of Bacillus thurinaiensis.
Further according to the invention there is provided a plant genome into which the gene construct of the invention has been inserted by transformation.
The promoter sequence of the invention may be isolated from the genomic sequence to which a cDNA
derived from a root-expressed gene hybridises. A
genomic library is screened using the said cDNA as a probe. Those geraomia fragments which hybridise to the cDNA probe carry not~only the structural gene but the promoter sequence associated therewith. The promoter may then be isolated by cleavage of the sequence around the location of the translation start point of the structural gene _ 2 _ _ WO OOIZ9594 PCTIIB98/02000 sequence. The sequences of suitable such cDNAs are shown in Figures 1 and 2 were isolated from maize.
These cDNAs have been deposited (1) in a plasmid designated pMR7 in an E.coli DHSa host and (2) in a plasmid designated pMR7/10.1 in an oli DHSa host, at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, on 15th March 1990, under the Accession Number 40267.
These deposits were made under the terms of the Budapest Treaty on the deposit of microorganisms for patent purposes.'°' Many genes specifying insecticidal proteins, particularly delta-endotoxin genes of Bacillus ~,hurinaiensis have been reported in the literature.
The invention will now be described, by way of illustration, by the following Examples.
Total RNA was extracted from root tissue of five-day old and fourteen-day old maize plants.
For use in certain comparative tests which will be described later, total RNA was also isolated from maize leaf and immature cob.
The RNA samples were purified using the guanidinium thiocyanate/caesium chloride method and poly(a)+mRNA purified on an oligo(dT) column. The corresponding cDNAs were synthesised using the oligo dT priming method and the cDNA cloned into plasmid pUCl3 afte~e=Tinkering.
The success of each of-these stages was monitored by incorporation of a label. Digests of randomly picked clones from the cDNA library showed a size distribution for inserts of between 300 and 1300 base pairs.
In the genetic improvement of plants by molecular techniques,, it is desirable that expression of inserted foreign genes be restricted to tissue where that expression will have significant effect. There are two principal reasons for this. First, restricted expression, rather than total (constitutive) is likely to be less demanding on the metabolism of the plant.
Secondly, it would be good practice to direct expression of the foreign gene to those parts of the plant which are not used for human or animal food when the expressed protein has no effect on such food parts. This second reason may be important when the effect which ingestion of the expressed protein may have is not be fully known.
One widespread target for genetic improvement of crop plants is the introduction of resistance to insect attack. Certain insect species attack green leaf tissue, whereas other, for example Coleoptera, attack the roots. Similarly there are certain disease-inducing microorganisms which attack the below-ground plant tissue and any genetic modification to impart resistance to such organisms will require expression of the resistance-imparting gene in the roots.
_ WO OOI29594 PCT/IB98l02000 An object of the present invention is to provide a root-specific gene promoter sequence and means for isolating same of root DNA.
According to the present invention there is provided a DNA sequence, defining a promoter of a root-expressed plant gene, having the sequence set forth in Figure 5 herewith.
The said DNA may be isolated from the root tissue of a particular target plant species of interest. The preferred species is Zea mays.
The invention a1'~o provides a gene construct comprising, in sequence, the aforesaid gene promoter of the invention, a coding region located downstream and controlled by the said promoter and a 3'-untranslated region including a polyadenylation signal.
Preferably the coding region encodes a protein which is toxic to root-attacking organisms and more preferably the protein is an insecticidal endotoxin of Bacillus thurinaiensis.
Further according to the invention there is provided a plant genome into which the gene construct of the invention has been inserted by transformation.
The promoter sequence of the invention may be isolated from the genomic sequence to which a cDNA
derived from a root-expressed gene hybridises. A
genomic library is screened using the said cDNA as a probe. Those geraomia fragments which hybridise to the cDNA probe carry not~only the structural gene but the promoter sequence associated therewith. The promoter may then be isolated by cleavage of the sequence around the location of the translation start point of the structural gene _ 2 _ _ WO OOIZ9594 PCTIIB98/02000 sequence. The sequences of suitable such cDNAs are shown in Figures 1 and 2 were isolated from maize.
These cDNAs have been deposited (1) in a plasmid designated pMR7 in an E.coli DHSa host and (2) in a plasmid designated pMR7/10.1 in an oli DHSa host, at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, on 15th March 1990, under the Accession Number 40267.
These deposits were made under the terms of the Budapest Treaty on the deposit of microorganisms for patent purposes.'°' Many genes specifying insecticidal proteins, particularly delta-endotoxin genes of Bacillus ~,hurinaiensis have been reported in the literature.
The invention will now be described, by way of illustration, by the following Examples.
Total RNA was extracted from root tissue of five-day old and fourteen-day old maize plants.
For use in certain comparative tests which will be described later, total RNA was also isolated from maize leaf and immature cob.
The RNA samples were purified using the guanidinium thiocyanate/caesium chloride method and poly(a)+mRNA purified on an oligo(dT) column. The corresponding cDNAs were synthesised using the oligo dT priming method and the cDNA cloned into plasmid pUCl3 afte~e=Tinkering.
The success of each of-these stages was monitored by incorporation of a label. Digests of randomly picked clones from the cDNA library showed a size distribution for inserts of between 300 and 1300 base pairs.
_ WO 00/29594 PCT/IB98/02000 Recombinants were individually transferred to microtitre wells, in total the library consisted of about 7,000 clones.
Clones representing genes with root enhanced expression were identified by differential screening. Identical filters were prepared from the microtitre plates and hybridised separately with probes prepared by first strand synthesis of root mRNA and four week old leaf mRNA. The autoradiographs were superimposed and recombinants showing root enhanced expression were selected as showing a more intense signal with the root probe than with the leaf probe. Interestingly, none of the selected clones showing differential hybridisation fell into the highly expressed category; all examples of this type showed equally intense signals to both probes.
By this procedure, 235 clones were selected as potentially showing a degree of differential hybridisation after the first screen. This number was reduced to thirteen after further screens.
The cDNA inserts of these thirteen clones ranged from 300 to 1100 base pairs as judged by restriction digestion or PCR. The inserts of each of the thirteen candidate inserts were then used in Northern hybridisations to confirm their tissue specificity.
RNAs from the five-day and fourteen day old root tissue and, fer comparison, from leaf and cob tissue were probed to identify any which were expressed in root tissue but not in leaf or cob.
By these procedures, the clone designated pMR7 showed enhanced expression in both the five and the fourteen day old root and only insignificant WO.00/29594 PCT/IB98I02000 expression in leaf and cob.
Figure 3 herewith shows the autoradiograph of a Northern blot probed with pMR7. Fox comparison purposes, Figure 4 shows the auto- radiograph of a Northern blot probed with pMRl2 which was typical of those clones which do not show root enhanced expression. Comparison of Figures 3 and 4 shows that whereas pMR7 hybridised to both five- and fourteen-day old root RNA with little hybridisation to either leaf-or cob RNA,~pMR2 gave strong signals on five-day old root~RRA, much reduced signal on fourteen day old root but strong signals to both leaf and cob RNA.
Thus pMR7 has been selected for further analysis. The insert of pMR7 is 700 base pairs in length and has been fully sequenced by walking through its length by synthesising oligonucleotides at approximately 200 base pair intervals and performing direct plasmid sequencing. There is a poly(A)+ tail. The sequence of the pMR7 insert is given as Figure 1 herewith.
Maize genomic DNA digests have been probed using pMR7 as a probe. Southern blots have indicated that the corresponding gene is of low copy number, that is, only a small number of hybridising bands are detectable at the level of stringency used.
From the screen of a partial M~gl genomic library a number of-gutative positives have been identified and from these the-upstream promoter sequence which directs expression to root tissue can be isolated and sequenced.
The pMR7 insert was used to screen a second maize seedling root cDNA library constructed in the cloning vector 1ZAP II. From a number of positively hybridising clones, one, pMR7/10.1, was selected for further analysis. DNA sequencing indicated that pMR7/10.1 was completely homologous with pMR7 but was of longer length, perhaps representing the full length cDNA clone. The sequence of pMR7/10.1 is given as Figure 2 herewith.
A 'genespecific' probe, representing the entire 3' untranslated region of the MR7 gene, was radioactively labelled and used to screen a commercial corn genomic library obtained from Clontech, USA (line W22). The probe, obtained by PCR using the cDNA as a template, was 350bp in length and of lower G+C content than the. entire cDNA, thereby reducing the chances of non-specific hybridisation.
Five clones were selected for further analysis after three rounds of plaque purification. Each.
hybridised strongly to oligonucleotide probes designed throughout the length of the pMR7 cDNA, confirming that they were closely related to the original cDNA. Restriction analysis of purified DNA obtained from these lambda clones indicated that 4 of them (numbers 7, 11, 14 and 15) were clearly related on the basis of similarity of restriction profiles. The other clone, number 10 had a different pr'bfile. Hybridisation of the MR7 gene-specific probe confirmed this relationship.
Single or few hybridisation bands resulted from probing digests of each of the 5 lambda isolates, number 10 having a different profile than the other f our .
- fi -_ WO OOIZ9594 PCT/IB98/02000 Of the four more. closely related lambda clones, number 7 was chosen for further analysis on the basis of ita larger insert size of approximately l6kb,~estimated from restriction analysis (the other inserts ranging in size from 9.0 to 13.5kb).
In order to identify a genomic fragment containing the MR7 promoter, the insert from lambda clone 7 was subcloned into pUClB vectors. pMRPl represents a--lbkb EcoRI fragment subcloned from lambda clone number 7:r Partial sequencing with an internal primer confirmed that this fragment contained DNA related to that of pMR7/10.1 cDNA, as opposed to any related but distinguishably different classes of the MR7 gene.
Utilising restriction sites identified at the 5' end of the pMR7 cDNA, the upstream region of the MR7 gene contained within lambda clone 7 was identified and subsequently isolated on the basis of hybridisation to specific oligonucleotide probes designed against sequence in the cDNA upstream of the aforementioned sites. A 4.2kb NcoI fragment was subcloned into pUClB (pMRP2) which represents the region of the gene immediatly upstream of the ATG translation startpoint (the ATG being a part of the 3' Ncol restriction site).
From within the insert of pMRP2, a l.9kb XbaI
fragment Was also identified which represented a region expected to-contain an active gene promoter.
The entire 4.2kb region of pMRP2 was sequenced. The sequence is given in Figure 5 herewith. Short sequences sharing homology with a number of promoter 'sequence motifs' described in the literature ca be recognised.
_7_ _ WO 00/29594 PCT1IB98/02000.
The technique of primer extension was utilised to identify the transcription start point within the promoter region. A possible transcription start point was identified 25 nucleotides downstream, of the A+T region thought to represent the 'TATA' box of the MR7 promoter.
To confirm the activity of the putative promoter regions, both the 4.2kb NcoI fragment and the l.9kb XbaI fragment Were cloned into a 'promoter assay construct', in which they were fused to a the easily'assayable B-glucuronidase (GUS) gene. In the former case (pMRP3), there was precise fusion through the ATG of the NcoI site.
In the latter case (pMRP4), the fusion was a transcriptional one, the resulting expression construct also containing the 'enhancing' maize AdhI Intron I sequence within the transcribed region.
Plasmid DNA of both pMRP3 and pMR.P4 were used in transient expression experiments in maize protoplasts derived from several sources, including root, leaf and endosperm tissue. In each case, expression of GUS from the constructs was classifed as 'high', being greater than control plasmids in which GUS expression was driven by 'standard' promoters such as 35S and maize Adh. High level GUS expression from these two constructs was also demonstrated by bombardment of root, leaf and coleoptile tissues of. maize seedlings.
SE(~UENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ADVANTA B.V.
(ii) TITLE OF INVENTION: ROOT-SPECIFIC PROMOTER
(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSe.,E: PILLSBURY MADISON & SUTRO, L.L.P.
(B) STREET: 1100 New York Avenue, N.W.
(C) CITY: Washington (D) STATE: D.C.
(E) COUNTRY: U.S.A.
(F} ZIP: 20005-3918 (v) COMPGTER REi-.DABLE FORM:
(A) MEDICii~' TYPE: Diskette (B) COMPUTER: IBM PC compatible (C) OPERATI\G SYSTEM: PC-DOS/MS-DOS
(D) SOFTh~R: PatentIn Release #1.0, Version #1.25 (vi) CURRE\T AP?T_.T_CATION DATA:
(A) ~PPL~:,~iION NUMBER: PCT/IB98/02000 (B) =ILING SATE: November 16, 1998 (C) ;.LASSI:ICATION:
(viii} ATTORNEY/~~ENT INFORMATION:
(A) \AME: AUL N. KOKULIS
(3) REGISTRt:T ION NUMBER: 16, 773 (C) REFEn=.\:.~/DOCKET NUMBER: SEE35669 PCT
(ix) TELECOMMUN=C=.TION INFORMATION:
(A) TELEF_=0\E: 1 (202) 861-3000 (B) TELEF=.1: 1 (202) 822-0944 (C) TELEX: c714627 CUSH
(2) INFORMATIC_~: FOR ~=Q ID NO:1:
(i) SEQU=::CE
CH_--_~~CTERISTICS:
(A) TENGTH: 1333 Lase pairs (B) TYPE: -:u~leic acid (C) STRA\l:=CHESS:
single i~) TOPOT_~~': linear ( ii ) MOLEC::~LE
T'_'FC
: cDNE
(xi) SEQUEtdCE
i.E~CRIPTION:
SEQ
ID N0:1:
ACTGAAGCCAGTC~ATAGC~ :GTTCTAGAACTAGTAGATAGCCTGCTGAT CTGT=CTGTT60 GTTiAGTTCGCAzaGCC'=T~. 'GTTTCGGCGACCATGGAGGATGAGAGGAA CACCCAGCAG120 CACCAGGGCGGTVAGGCCvR uCAGGACGCTGCCGGTCAGGTGGAGGTGAA GGATiGGGGG180 CTCCTGGACAGCCTTCTC:::: CAGGAAGAAGCACGACGACGACCAGGAGAA GAAG.3AGCAG240 ACGGAGGAGCTGGCGACCG:: CATGG~:GAAGGTCACGGTGTCCGAGCCCGA GAAGCACGGG300 CACP.AGGAGGAGG-GCACA GGTCGTCGGCGAGAAGAAGGAGGGCCTTTT CGCCAzIGCTG360 CACCGCACCAGTTCCAGCTC CAGCT~GTCGAGCGACGAGGAAGAGGAGGC GATCGATGAG420 AACGGCGAGATTATCAAG=G GAAGAAGAAGAAGGTGGGCCTCAAGGAGAA GATC:jAGGAG480 AAGCTGCCGGGCGCACGP.AG GACGGCCACCACACGGCCGCACCGTCCCCG GCGCCCGCGC540 SUBSTtTttrE SHEET (RULE 26) CCGCGCCCGTGGAGACGCATGCCCACCACCAGGAGGAAGCGNATCACNGG~CCGCACGTCG 600 TCGTCGTCCAGr..AGGTCGAC:GACGACGTGAAGACCGAGACCCCGCCGCATGCACCGGGGG 720 AGGAGAAGAAAG~CCTGCT~GACAAGATCAAGGAGAAGCTCCCC~GTGGCCACAACAAGA 780 AGCCTGAAGCC~CTGCCGC=CCGGCTCCGCCCGTCCACGCGCCGGCGCCAGCGCCGCACG 840 CCGAGt~ACGTG=::CAGCCCGGATGGCAAGGAGAAGAAGGGTTTGCTGGGCAAGATCATGG 900 ACAAGATAGCCGGCTACCA:.AAGAGCTCAGGTGAGGAAGCAGACCACAAGGCGGACGCCT 960 GCCGGCGAGCAC=:GGACCA~CTCCTCTAATTAAGGTCGCAGTCC~AGCGTGTCCTGGCCG 1020 TGGGCGGCGGAT~'AGAAGCTAGCTAGCGTTGGCATGTGTGTTGGuTTCTGGTTTGCTTTT 1080 ACCAA_~AGTTTGTTTCAAGGTGGATCGCCTGGTCAAGGTCCGTGTGCTCTATTAAGGTGG 1140 ATCGCGTGAC TCTGGCAGzC-. AGTGT ,GCTG CTTGTGTAGG ACGT~GTACG TACGGGCTTT 1200 ATTTTGGTCC C=~.AGTCAA~.~=i GTCACGGTCG GTCTGGATGT TGTGTACTTG GGTTTGTTGA 1260 ATTATGAGCA GCTGCGTGrT GTAAT_CGGC TGGGCTACCT GGA~'GCGGTT AATAATTGCT 1320 (2) INFORMAT=,"'.N FOR SEQ ID _~0:2:
Vii) SEQWNCE
CHARACTERISTICS:
(A; LENGTH: 758 base pairs (B! TYPE: nucleic acid (C; STRANCEDNESS:
double (D? TOPOLOGY: llncar (ii) MOLECiILE
T'_'~E:
cDN
(xi) SEQ:IE~~CE
DE~CRIPTi~;t:
SEQ ID
N0:2:
CGCCP~ GCGCTGCACC TCGAC'_'CTAGAGGATCCCCGGGCG!GCTCG AATTCCTTTT60 GCTT
TTTTT=TTTTTIT?P.TGATA ATTGC:.ATATATATATACACGCTAACACGC TCGCGCGCTG120 GGCAG F_L~:GCAATTH TTAACGCATCCAGGTAGCCCAGCCGAATTA CAACACGCAG180 ~~ACC
CTGCTCATAATTC?ACAAAC CCAAG=ACACAACATCCAGACCGACCGTGA CTTTTGACTT240 GGGACCAAAATAAHGCCCC:I ACGTr~CACGTCCTACACAAGCrCCAACAC TCACTGCCAG300 AGTCACGCGATCCACCTTLA TAGA6;.ACACGGACCTTGACCAGCCGATCC ACCTTGAAAC360 AAACT~TTGGTAP_~AGCAAH CAGAtCC:CAACACACATGCC=.AC6CTAGCT AGCTTCTAAT420 CCGCCGCCNACCCCCAGG .C ACGCT~-CGACGCGACCTTAA~T~~AGGAGC TGGTCCTGTG980 CTCGCa?7NGNC:.GCCTTGTC GTCTGCTTCCTCACCTGAGT~TTGTGGTAG CCGGCTATCT540 TGTCC~'TGATCTTGCCCAGC AAACC~'TTCTTCTCCTTGCCATCCGGGCGC TCACGTTCTC600 CACTGGCCGT CGTTTTCAAC GTCGTGACTG GGAAAACC 75g (2) I14FORMAT=0~9 FOR SJQ ID Iv0:3:
SUB~nTUTE SHEET (RULE 26) (i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:9203 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY:
linear (ii) OLECULE
M TYPE:
cDNA
(xi) EQUENCE
S DESCRIPTION:
SEQ ID
N0:3:
CTGA~ACCATTAGTTCCTTTCTAACATAGCATGTTCCTAGGTTGCTTTCTTATTTGTCTG180 CTCCATTCCACTCCTATGAaATTACTATGACTTAATTTCAATCGAGGTCATCTTCTTGCT540 CCTTCGCTTGCTTAGCAGTAAGACATAACTTCCTTTACCTTGCTCAATAGTTTGCCTiTT600 AATTTGAACAAAAATCTAATCACCTGACATTGCATGGGAGGTAAGCTCCTGTTTTTCF.CA660 TCATATGTTTCCATCTATAi-~TGGTTGTCTTGGTATTCTCAATCAGTGGACTTGTGCP.FCT,960 ATGTAATTTGCAGTCTCCArAAGGATGCTAATGATAGGTCCTCAACACAAGCCTTATTGG1020 TAAGCTGAAA:~P.CAACTTChCACCTTCATTTCATTTCAATAATCGTCTACAAGACTP.PAC1080 CACTTATCTTaTCCTTCCCTTCCTGTTGTCTTTGATGCAGGACCATCCATTCTTGAGCGT1140 ACTTGTTCTT~TAATCTTT~CTTTTTATGTATCCATCGTTGTGCACATAAGTG=TAC~.TT1260 TTATTTTACGTTTCAGGCA%:CTCTAATATTTATCCTCCTTATTAAGCAAAGAGTGTGGTG1320 ACACATTTCCCTTTTGGGC~:AGGGTTGGGTTGTGTACTGAGCTGTAATGATTCrCAAiTC1380 ACCTGATATC:~TGATTTi~GATGGTTTTCTGAAAGTGCATTGAG.~.CATTAGGAAt~CACP.AG1440 TGGGF.iGTAGT.GATAACAP=~TCTT'fT'!'AGTCACAAAGATTTTTTTTCTTGGAACCATiAA1500 TAGTTGGCTAACAGCTACAuTGATACAAGCGTTTGTTTTAATATGTTGTGAATTGCAATG1560 GTTTCCACCCCTATCTTTTCTCAACCTACCCATTTTCTCTAAAATACAATAAAP.AGCTTT1790 SUBSTITUTE SHEET (RULE 26) GAAATGCAGCAGAAACTTTCAGAGCCAATGGAGATATAA~.TAGACTTATATCACACTGTA 2580 ACTGGTTCTAAAATACA~TGCTCTCTTGTCTCATCATTTTGTGACGTGCCAGCCAATATT 2990 TTTTTCCTCTTTTAGATTGAGAGAGTTATGGAAATATGGAACP.AAAACGAGGACTTCCGC 3000 CACATTGTTTCGATTCTCTGTGGTCTGTACTTAATAAGT.3GTCTATTTATTTCGTGTGAT 3300 TGATCAGACACCGTTCTCTGCATGCCAACATC~AGCTGA!GP.~:GCACCCTCCTGAAGTTA 3360 TTTGATATTGTATACTGATAAGTAATAAACTAGATTATGTAGiTCCTATAATTTTTATCA 3420 ATATAACACA=GTGCATATATAAGTTATCGAGATATTATCGTCTCTCGTTGCAACGCACG 3540 TGCACTGACCTATAAAAGTATAACACJiCATTTGTACATAGTT'_'ATCGTGGTTTTATACGT 3600 SUBSTITUTE SHEET (RULE 26) - wo oon9s9a PcTnB9aroZOOo TGG
SUBSTITUTE SHEET (RULE 26~
ATAGTCAGGTAAATC
Clones representing genes with root enhanced expression were identified by differential screening. Identical filters were prepared from the microtitre plates and hybridised separately with probes prepared by first strand synthesis of root mRNA and four week old leaf mRNA. The autoradiographs were superimposed and recombinants showing root enhanced expression were selected as showing a more intense signal with the root probe than with the leaf probe. Interestingly, none of the selected clones showing differential hybridisation fell into the highly expressed category; all examples of this type showed equally intense signals to both probes.
By this procedure, 235 clones were selected as potentially showing a degree of differential hybridisation after the first screen. This number was reduced to thirteen after further screens.
The cDNA inserts of these thirteen clones ranged from 300 to 1100 base pairs as judged by restriction digestion or PCR. The inserts of each of the thirteen candidate inserts were then used in Northern hybridisations to confirm their tissue specificity.
RNAs from the five-day and fourteen day old root tissue and, fer comparison, from leaf and cob tissue were probed to identify any which were expressed in root tissue but not in leaf or cob.
By these procedures, the clone designated pMR7 showed enhanced expression in both the five and the fourteen day old root and only insignificant WO.00/29594 PCT/IB98I02000 expression in leaf and cob.
Figure 3 herewith shows the autoradiograph of a Northern blot probed with pMR7. Fox comparison purposes, Figure 4 shows the auto- radiograph of a Northern blot probed with pMRl2 which was typical of those clones which do not show root enhanced expression. Comparison of Figures 3 and 4 shows that whereas pMR7 hybridised to both five- and fourteen-day old root RNA with little hybridisation to either leaf-or cob RNA,~pMR2 gave strong signals on five-day old root~RRA, much reduced signal on fourteen day old root but strong signals to both leaf and cob RNA.
Thus pMR7 has been selected for further analysis. The insert of pMR7 is 700 base pairs in length and has been fully sequenced by walking through its length by synthesising oligonucleotides at approximately 200 base pair intervals and performing direct plasmid sequencing. There is a poly(A)+ tail. The sequence of the pMR7 insert is given as Figure 1 herewith.
Maize genomic DNA digests have been probed using pMR7 as a probe. Southern blots have indicated that the corresponding gene is of low copy number, that is, only a small number of hybridising bands are detectable at the level of stringency used.
From the screen of a partial M~gl genomic library a number of-gutative positives have been identified and from these the-upstream promoter sequence which directs expression to root tissue can be isolated and sequenced.
The pMR7 insert was used to screen a second maize seedling root cDNA library constructed in the cloning vector 1ZAP II. From a number of positively hybridising clones, one, pMR7/10.1, was selected for further analysis. DNA sequencing indicated that pMR7/10.1 was completely homologous with pMR7 but was of longer length, perhaps representing the full length cDNA clone. The sequence of pMR7/10.1 is given as Figure 2 herewith.
A 'genespecific' probe, representing the entire 3' untranslated region of the MR7 gene, was radioactively labelled and used to screen a commercial corn genomic library obtained from Clontech, USA (line W22). The probe, obtained by PCR using the cDNA as a template, was 350bp in length and of lower G+C content than the. entire cDNA, thereby reducing the chances of non-specific hybridisation.
Five clones were selected for further analysis after three rounds of plaque purification. Each.
hybridised strongly to oligonucleotide probes designed throughout the length of the pMR7 cDNA, confirming that they were closely related to the original cDNA. Restriction analysis of purified DNA obtained from these lambda clones indicated that 4 of them (numbers 7, 11, 14 and 15) were clearly related on the basis of similarity of restriction profiles. The other clone, number 10 had a different pr'bfile. Hybridisation of the MR7 gene-specific probe confirmed this relationship.
Single or few hybridisation bands resulted from probing digests of each of the 5 lambda isolates, number 10 having a different profile than the other f our .
- fi -_ WO OOIZ9594 PCT/IB98/02000 Of the four more. closely related lambda clones, number 7 was chosen for further analysis on the basis of ita larger insert size of approximately l6kb,~estimated from restriction analysis (the other inserts ranging in size from 9.0 to 13.5kb).
In order to identify a genomic fragment containing the MR7 promoter, the insert from lambda clone 7 was subcloned into pUClB vectors. pMRPl represents a--lbkb EcoRI fragment subcloned from lambda clone number 7:r Partial sequencing with an internal primer confirmed that this fragment contained DNA related to that of pMR7/10.1 cDNA, as opposed to any related but distinguishably different classes of the MR7 gene.
Utilising restriction sites identified at the 5' end of the pMR7 cDNA, the upstream region of the MR7 gene contained within lambda clone 7 was identified and subsequently isolated on the basis of hybridisation to specific oligonucleotide probes designed against sequence in the cDNA upstream of the aforementioned sites. A 4.2kb NcoI fragment was subcloned into pUClB (pMRP2) which represents the region of the gene immediatly upstream of the ATG translation startpoint (the ATG being a part of the 3' Ncol restriction site).
From within the insert of pMRP2, a l.9kb XbaI
fragment Was also identified which represented a region expected to-contain an active gene promoter.
The entire 4.2kb region of pMRP2 was sequenced. The sequence is given in Figure 5 herewith. Short sequences sharing homology with a number of promoter 'sequence motifs' described in the literature ca be recognised.
_7_ _ WO 00/29594 PCT1IB98/02000.
The technique of primer extension was utilised to identify the transcription start point within the promoter region. A possible transcription start point was identified 25 nucleotides downstream, of the A+T region thought to represent the 'TATA' box of the MR7 promoter.
To confirm the activity of the putative promoter regions, both the 4.2kb NcoI fragment and the l.9kb XbaI fragment Were cloned into a 'promoter assay construct', in which they were fused to a the easily'assayable B-glucuronidase (GUS) gene. In the former case (pMRP3), there was precise fusion through the ATG of the NcoI site.
In the latter case (pMRP4), the fusion was a transcriptional one, the resulting expression construct also containing the 'enhancing' maize AdhI Intron I sequence within the transcribed region.
Plasmid DNA of both pMRP3 and pMR.P4 were used in transient expression experiments in maize protoplasts derived from several sources, including root, leaf and endosperm tissue. In each case, expression of GUS from the constructs was classifed as 'high', being greater than control plasmids in which GUS expression was driven by 'standard' promoters such as 35S and maize Adh. High level GUS expression from these two constructs was also demonstrated by bombardment of root, leaf and coleoptile tissues of. maize seedlings.
SE(~UENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ADVANTA B.V.
(ii) TITLE OF INVENTION: ROOT-SPECIFIC PROMOTER
(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSe.,E: PILLSBURY MADISON & SUTRO, L.L.P.
(B) STREET: 1100 New York Avenue, N.W.
(C) CITY: Washington (D) STATE: D.C.
(E) COUNTRY: U.S.A.
(F} ZIP: 20005-3918 (v) COMPGTER REi-.DABLE FORM:
(A) MEDICii~' TYPE: Diskette (B) COMPUTER: IBM PC compatible (C) OPERATI\G SYSTEM: PC-DOS/MS-DOS
(D) SOFTh~R: PatentIn Release #1.0, Version #1.25 (vi) CURRE\T AP?T_.T_CATION DATA:
(A) ~PPL~:,~iION NUMBER: PCT/IB98/02000 (B) =ILING SATE: November 16, 1998 (C) ;.LASSI:ICATION:
(viii} ATTORNEY/~~ENT INFORMATION:
(A) \AME: AUL N. KOKULIS
(3) REGISTRt:T ION NUMBER: 16, 773 (C) REFEn=.\:.~/DOCKET NUMBER: SEE35669 PCT
(ix) TELECOMMUN=C=.TION INFORMATION:
(A) TELEF_=0\E: 1 (202) 861-3000 (B) TELEF=.1: 1 (202) 822-0944 (C) TELEX: c714627 CUSH
(2) INFORMATIC_~: FOR ~=Q ID NO:1:
(i) SEQU=::CE
CH_--_~~CTERISTICS:
(A) TENGTH: 1333 Lase pairs (B) TYPE: -:u~leic acid (C) STRA\l:=CHESS:
single i~) TOPOT_~~': linear ( ii ) MOLEC::~LE
T'_'FC
: cDNE
(xi) SEQUEtdCE
i.E~CRIPTION:
SEQ
ID N0:1:
ACTGAAGCCAGTC~ATAGC~ :GTTCTAGAACTAGTAGATAGCCTGCTGAT CTGT=CTGTT60 GTTiAGTTCGCAzaGCC'=T~. 'GTTTCGGCGACCATGGAGGATGAGAGGAA CACCCAGCAG120 CACCAGGGCGGTVAGGCCvR uCAGGACGCTGCCGGTCAGGTGGAGGTGAA GGATiGGGGG180 CTCCTGGACAGCCTTCTC:::: CAGGAAGAAGCACGACGACGACCAGGAGAA GAAG.3AGCAG240 ACGGAGGAGCTGGCGACCG:: CATGG~:GAAGGTCACGGTGTCCGAGCCCGA GAAGCACGGG300 CACP.AGGAGGAGG-GCACA GGTCGTCGGCGAGAAGAAGGAGGGCCTTTT CGCCAzIGCTG360 CACCGCACCAGTTCCAGCTC CAGCT~GTCGAGCGACGAGGAAGAGGAGGC GATCGATGAG420 AACGGCGAGATTATCAAG=G GAAGAAGAAGAAGGTGGGCCTCAAGGAGAA GATC:jAGGAG480 AAGCTGCCGGGCGCACGP.AG GACGGCCACCACACGGCCGCACCGTCCCCG GCGCCCGCGC540 SUBSTtTttrE SHEET (RULE 26) CCGCGCCCGTGGAGACGCATGCCCACCACCAGGAGGAAGCGNATCACNGG~CCGCACGTCG 600 TCGTCGTCCAGr..AGGTCGAC:GACGACGTGAAGACCGAGACCCCGCCGCATGCACCGGGGG 720 AGGAGAAGAAAG~CCTGCT~GACAAGATCAAGGAGAAGCTCCCC~GTGGCCACAACAAGA 780 AGCCTGAAGCC~CTGCCGC=CCGGCTCCGCCCGTCCACGCGCCGGCGCCAGCGCCGCACG 840 CCGAGt~ACGTG=::CAGCCCGGATGGCAAGGAGAAGAAGGGTTTGCTGGGCAAGATCATGG 900 ACAAGATAGCCGGCTACCA:.AAGAGCTCAGGTGAGGAAGCAGACCACAAGGCGGACGCCT 960 GCCGGCGAGCAC=:GGACCA~CTCCTCTAATTAAGGTCGCAGTCC~AGCGTGTCCTGGCCG 1020 TGGGCGGCGGAT~'AGAAGCTAGCTAGCGTTGGCATGTGTGTTGGuTTCTGGTTTGCTTTT 1080 ACCAA_~AGTTTGTTTCAAGGTGGATCGCCTGGTCAAGGTCCGTGTGCTCTATTAAGGTGG 1140 ATCGCGTGAC TCTGGCAGzC-. AGTGT ,GCTG CTTGTGTAGG ACGT~GTACG TACGGGCTTT 1200 ATTTTGGTCC C=~.AGTCAA~.~=i GTCACGGTCG GTCTGGATGT TGTGTACTTG GGTTTGTTGA 1260 ATTATGAGCA GCTGCGTGrT GTAAT_CGGC TGGGCTACCT GGA~'GCGGTT AATAATTGCT 1320 (2) INFORMAT=,"'.N FOR SEQ ID _~0:2:
Vii) SEQWNCE
CHARACTERISTICS:
(A; LENGTH: 758 base pairs (B! TYPE: nucleic acid (C; STRANCEDNESS:
double (D? TOPOLOGY: llncar (ii) MOLECiILE
T'_'~E:
cDN
(xi) SEQ:IE~~CE
DE~CRIPTi~;t:
SEQ ID
N0:2:
CGCCP~ GCGCTGCACC TCGAC'_'CTAGAGGATCCCCGGGCG!GCTCG AATTCCTTTT60 GCTT
TTTTT=TTTTTIT?P.TGATA ATTGC:.ATATATATATACACGCTAACACGC TCGCGCGCTG120 GGCAG F_L~:GCAATTH TTAACGCATCCAGGTAGCCCAGCCGAATTA CAACACGCAG180 ~~ACC
CTGCTCATAATTC?ACAAAC CCAAG=ACACAACATCCAGACCGACCGTGA CTTTTGACTT240 GGGACCAAAATAAHGCCCC:I ACGTr~CACGTCCTACACAAGCrCCAACAC TCACTGCCAG300 AGTCACGCGATCCACCTTLA TAGA6;.ACACGGACCTTGACCAGCCGATCC ACCTTGAAAC360 AAACT~TTGGTAP_~AGCAAH CAGAtCC:CAACACACATGCC=.AC6CTAGCT AGCTTCTAAT420 CCGCCGCCNACCCCCAGG .C ACGCT~-CGACGCGACCTTAA~T~~AGGAGC TGGTCCTGTG980 CTCGCa?7NGNC:.GCCTTGTC GTCTGCTTCCTCACCTGAGT~TTGTGGTAG CCGGCTATCT540 TGTCC~'TGATCTTGCCCAGC AAACC~'TTCTTCTCCTTGCCATCCGGGCGC TCACGTTCTC600 CACTGGCCGT CGTTTTCAAC GTCGTGACTG GGAAAACC 75g (2) I14FORMAT=0~9 FOR SJQ ID Iv0:3:
SUB~nTUTE SHEET (RULE 26) (i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:9203 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY:
linear (ii) OLECULE
M TYPE:
cDNA
(xi) EQUENCE
S DESCRIPTION:
SEQ ID
N0:3:
CTGA~ACCATTAGTTCCTTTCTAACATAGCATGTTCCTAGGTTGCTTTCTTATTTGTCTG180 CTCCATTCCACTCCTATGAaATTACTATGACTTAATTTCAATCGAGGTCATCTTCTTGCT540 CCTTCGCTTGCTTAGCAGTAAGACATAACTTCCTTTACCTTGCTCAATAGTTTGCCTiTT600 AATTTGAACAAAAATCTAATCACCTGACATTGCATGGGAGGTAAGCTCCTGTTTTTCF.CA660 TCATATGTTTCCATCTATAi-~TGGTTGTCTTGGTATTCTCAATCAGTGGACTTGTGCP.FCT,960 ATGTAATTTGCAGTCTCCArAAGGATGCTAATGATAGGTCCTCAACACAAGCCTTATTGG1020 TAAGCTGAAA:~P.CAACTTChCACCTTCATTTCATTTCAATAATCGTCTACAAGACTP.PAC1080 CACTTATCTTaTCCTTCCCTTCCTGTTGTCTTTGATGCAGGACCATCCATTCTTGAGCGT1140 ACTTGTTCTT~TAATCTTT~CTTTTTATGTATCCATCGTTGTGCACATAAGTG=TAC~.TT1260 TTATTTTACGTTTCAGGCA%:CTCTAATATTTATCCTCCTTATTAAGCAAAGAGTGTGGTG1320 ACACATTTCCCTTTTGGGC~:AGGGTTGGGTTGTGTACTGAGCTGTAATGATTCrCAAiTC1380 ACCTGATATC:~TGATTTi~GATGGTTTTCTGAAAGTGCATTGAG.~.CATTAGGAAt~CACP.AG1440 TGGGF.iGTAGT.GATAACAP=~TCTT'fT'!'AGTCACAAAGATTTTTTTTCTTGGAACCATiAA1500 TAGTTGGCTAACAGCTACAuTGATACAAGCGTTTGTTTTAATATGTTGTGAATTGCAATG1560 GTTTCCACCCCTATCTTTTCTCAACCTACCCATTTTCTCTAAAATACAATAAAP.AGCTTT1790 SUBSTITUTE SHEET (RULE 26) GAAATGCAGCAGAAACTTTCAGAGCCAATGGAGATATAA~.TAGACTTATATCACACTGTA 2580 ACTGGTTCTAAAATACA~TGCTCTCTTGTCTCATCATTTTGTGACGTGCCAGCCAATATT 2990 TTTTTCCTCTTTTAGATTGAGAGAGTTATGGAAATATGGAACP.AAAACGAGGACTTCCGC 3000 CACATTGTTTCGATTCTCTGTGGTCTGTACTTAATAAGT.3GTCTATTTATTTCGTGTGAT 3300 TGATCAGACACCGTTCTCTGCATGCCAACATC~AGCTGA!GP.~:GCACCCTCCTGAAGTTA 3360 TTTGATATTGTATACTGATAAGTAATAAACTAGATTATGTAGiTCCTATAATTTTTATCA 3420 ATATAACACA=GTGCATATATAAGTTATCGAGATATTATCGTCTCTCGTTGCAACGCACG 3540 TGCACTGACCTATAAAAGTATAACACJiCATTTGTACATAGTT'_'ATCGTGGTTTTATACGT 3600 SUBSTITUTE SHEET (RULE 26) - wo oon9s9a PcTnB9aroZOOo TGG
SUBSTITUTE SHEET (RULE 26~
ATAGTCAGGTAAATC
Claims (16)
1. An isolated DNA sequence which codes for a promoter of a root-expressed plant gene, said gene having the sequence of FIGS. 5A-F (SEQ ID NO:3).
2. The isolated DNA sequence of claim 1 wherein said promoter has the sequence of a 1.9 Kb XbaI
fragment of the sequence of FIGS. 5A-F (SEQ ID NO:3).
fragment of the sequence of FIGS. 5A-F (SEQ ID NO:3).
3. The isolated DNA sequence of claim 1 wherein said sequence is isolated from the root tissue of a target plant species.
4. The isolated DNA sequence of claim 3, wherein the target plant species is Zea mays.
5. An isolated DNA construct comprising, in sequence, the DNA sequence of claim 1 which comprises a promoter, a coding region located downstream and controlled by said promoter and a 3'-untranslated region including a polyadenylation signal.
6. An isolated DNA construct comprising, in sequence, the DNA sequence of claim 2 which comprises a promoter, a coding region located downstream and controlled by said promoter and a 3'-untranslated region including a polyadenylation signal.
7. The construct of claim 5, wherein the coding region encodes a protein which is toxic to root-attacking organisms.
8. The construct of claim 6, wherein the coding region encodes a protein which is toxic to root-attacking organisms.
9. The construct of claim 7, wherein the protein is an insecticidal endotoxin of Bacillus thuringiensis.
10. The construct of claim 8, wherein the protein is an insecticidal endotoxin of Bacillus thuringiensis.
11. A plant genome into which the construct of claim 5, has been inserted such that said promoter is transcriptionally active.
12. A plant genome into which the construct of claim 6 has been inserted such that said promoter is transcriptionally active.
13. A plant genome into which the construct of claim 7 has been inserted such that said promoter is transcriptionally active.
14. A plant genome into which the construct of claim 8 has been inserted such that said promoter is transcriptionally active.
15. A plant genome into which the construct of claim 9 has been inserted such that said promoter is transcriptionally active.
16. A plant genome into which the construct of claim 10 has been inserted such that said promoter is transcriptionally active.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IB1998/002000 WO2000029594A1 (en) | 1998-11-16 | 1998-11-16 | Root-specific promoter |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2317723A1 true CA2317723A1 (en) | 2000-05-25 |
Family
ID=11004788
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002317723A Abandoned CA2317723A1 (en) | 1998-11-16 | 1998-11-16 | Root-specific promoter |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1047790A1 (en) |
| JP (1) | JP2002537760A (en) |
| AU (1) | AU1347999A (en) |
| CA (1) | CA2317723A1 (en) |
| WO (1) | WO2000029594A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1409669B1 (en) | 2001-03-23 | 2009-10-21 | E.I. Du Pont De Nemours And Company | Root specific, stimulant inducible promoter and its use |
| CA2442024A1 (en) * | 2001-03-29 | 2002-10-10 | Evogene Ltd. | Methods, platforms and kits useful for identifying, isolating and utilizing nucleotide sequences which regulate gene expression in an organism |
| CA2462615C (en) * | 2001-11-07 | 2012-06-26 | Syngenta Participations Ag | Promoters for regulation of gene expression in plant roots |
| FR2848570B1 (en) | 2002-12-12 | 2005-04-01 | Bayer Cropscience Sa | EXPRESSION CASSETTE ENCODING A 5-ENOL PYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) AND HERBICIDE TOLERANT PLANTS CONTAINING THE SAME |
| BRPI0408735A (en) | 2003-03-12 | 2006-03-07 | Evogene Ltd | isolated polynucleotide, nucleic acid construction, transgenic cell, transgenic organism, transgenic plant, method for producing a transgenic plant, method for expressing a polynucleotide of interest in a cell, and method for co-expressing two polynucleotides of interest in a cell |
| KR101450398B1 (en) | 2012-10-12 | 2014-10-15 | 경희대학교 산학협력단 | Root-specific promoter, expression vector comprising the same, transformed plants thereby and method for preparation thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837848A (en) * | 1990-03-16 | 1998-11-17 | Zeneca Limited | Root-specific promoter |
| US5633363A (en) * | 1994-06-03 | 1997-05-27 | Iowa State University, Research Foundation In | Root preferential promoter |
| ATE280224T1 (en) * | 1996-05-17 | 2004-11-15 | Pioneer Hi Bred Int | PROMOTER ELEMENTS WHICH PROVIDE ROOT PREFERRED GENE EXPRESSION |
-
1998
- 1998-11-16 AU AU13479/99A patent/AU1347999A/en not_active Abandoned
- 1998-11-16 EP EP98957061A patent/EP1047790A1/en not_active Withdrawn
- 1998-11-16 WO PCT/IB1998/002000 patent/WO2000029594A1/en not_active Application Discontinuation
- 1998-11-16 JP JP2000582576A patent/JP2002537760A/en active Pending
- 1998-11-16 CA CA002317723A patent/CA2317723A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP1047790A1 (en) | 2000-11-02 |
| JP2002537760A (en) | 2002-11-12 |
| AU1347999A (en) | 2000-06-05 |
| WO2000029594A1 (en) | 2000-05-25 |
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