WO1991009948A1 - Gene d'actine de riz et promoteur - Google Patents

Gene d'actine de riz et promoteur Download PDF

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WO1991009948A1
WO1991009948A1 PCT/US1991/000073 US9100073W WO9109948A1 WO 1991009948 A1 WO1991009948 A1 WO 1991009948A1 US 9100073 W US9100073 W US 9100073W WO 9109948 A1 WO9109948 A1 WO 9109948A1
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rac1
gene
prac15
rice
sequence
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PCT/US1991/000073
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Ray Wu
David Mcelroy
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Cornell Research Foundation, Inc.
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Publication of WO1991009948A1 publication Critical patent/WO1991009948A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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

Definitions

  • CaMV 35S promoter has been widely used for this purpose in a number of plant transformation systems, however, the CaMV 35S promoter has shown low activity in transforming rice cells, and recent reports suggest that the pattern of CaMV 35S promoter activity in transgenic plants may not be constitutive.
  • the purpose of the present invention to describe an efficient promoter for rice transformation, and in doing so have based such a promoter upon a rice actin gene that displays a constitutive spatial and temporal pattern of transcript abundance.
  • Cytoplasmic actin is a fundamental component of the eukaryotic cell cytoskeleton. fn higher-plant cells a number of cellular process, such as cytoplasmic streaming, extension growth and cell division, are believed to involve cytoskeletal actin protein. Actin has been found to be encoded by gene families in all higher plants studied to date. A unique feature of these plant actin genes, in contrast to the situation for animal actin genes, has been their conservation of gene structure. All of the studied plant actin genes are reported to consist of four coding exons of conserved length, separated by three introns of variable length. In each of these cases the coding regions of the plant actin genes have been deduced following a comparison of their potential translations products with that of previously published animal actin protein sequences.
  • RAc1 actin-like sequences per haploid genome, of which four have been isolated and shown to differ from each other in the tissue and stage specific abundance of their respective transcripts.
  • One rice actin gene, RAc1 was found to encode a transcipt that is relatively abundant in all rice tissues, and at all developmental stages examined.
  • the present invention describes the isolation and partial sequencing of a genomic clone containing the rice RAc1 5' flanking and 5'- transcribed, non-translated, regions.
  • RAc1-GUS GUS stands for ⁇ -glucuromidase gene
  • fusions plasmids were constructed and used in transient expression assays of transformed rice protoplasts.
  • the results from these assays suggest that the regulatory element(s) necessary for maximal RAc1 promoter activity in transformed rice protoplasts are located within a region 1.3 kb upstream of the RAc1 translation initiation codon.
  • the rice RAc1 promoter is 5 times more active than the maize Adh1 promoter in transformed rice cells, suggesting that the RAc1 5' region contains an efficient promoter for rice transformation.
  • RAc1 cDNA clone EBL data bank accession number X16280
  • 5' end mapping the RAc1 transcript EBL data bank accession number X16280
  • Figure 1a is a restriction map of the pcRAc1.3 cDNA insert, according to the present invention
  • Figure 1 b is a restriction map and proposed structure of the RAc1 genomic clone according the present invention
  • Figure 2a is a restriction map of the 15.1 Kb lambda- RAc1 insert, according to the present invention
  • Figure 2b is a restriction map of the pRAc 15'.H3 insert according to the present invention
  • Figure 3 depicts maps of constructs according to the present invention.
  • RAc1-like cDNA clones were identified using a 900-bp BamH1-Hindlll restriction fragment probe from the 3' end of the rice actin genomic clone pRAc1.
  • Actin positive clones from tertiary screening of the Iambda-gt11 cDNA library were further characterized by restriction mapping. Restriction fragments from the cDNA insert of the RAc1 positive clone pcRAc1.3 were subcloned into pBS-KS vectors for nucleotide sequencing. Double-stranded DNA sequencing using 35 S-dATP was carried out using a T7 polymerase sequencing kit following the manufacturer's (Pharmacia) modification of the Sanger et a/ dideoxynucleotide sequencing procedure.
  • the 5' and 3' ends of the pcRAc1.3 cDNA insert were further sequenced using two synthetic oligonucleotides; 5' -AAGCGGCCTCTACTACGC-3' and 5'- GAAGCATTTCCTGRGCACAAT-3' respectively and subsequently the sequence data were analyzed.
  • RNA isolation from seven-day-old rice shoots and Northern blotting were performed. 10 ⁇ g of total RNA samples were used in formaldehyde agarose gel electrophoresis and northern analysis was carried out under stringent hybridization conditioned (50% formamide, 50°C). Restriction fragment probes for northern hybridization were isolated from the 5'-untranslated end of the rice actin genomic clone pRAc1 and subcloned into pBS-KS vectors.
  • the primer extension analysis was performed using 1 ⁇ g of a synthetic oligonucleotide primer 5'-CTTCTACCGGCGGCGGC-3' which was annealed to 25 ⁇ g of total RNA from seven-day-old rice shoots.
  • the cDNA library made with mRNA from six-day-old etiolated rice shoots according to Example 1 provided the cDNA clones corresponding to the multiple members of the rice actin gene family.
  • the insert from pcRA1.3 was further subcloned into pBS-KS plasmids. Double stranded DNA was prepared for sequencing.
  • the restriction enzyme map of the pcRAC1.3 insert and the sequencing strategy for determining its base are summarized in Fig. 1a.
  • Fig. 1 describes the determination of the RAc1 gene structure by alignment of the pcRAc1.3 cDNA insert with the pRAc1 genomic clone
  • a restriction map of the pcRAc1 cDNA insert with the strategy used to determine its base sequence.
  • the horizontal arrows indicate the direction of sequencing and their length indicate the actual size of the sequence obtained.
  • Horizontal arrows preceded with a sinusoidal wave indicate the use of synthetic oligonucleotide primers
  • b restriction map and proposed structure of the RAc1 genomic clone.
  • Horizontal lines represent introns and boxes represent exons. Open boxes represent the transcribed but untranslated regions of exons, closed boxes represent the translated regions of exons.
  • the tabulated sequence which depicts the nucleotide sequence of the rice actin gene RAc1 is depicted in the following sequence of 5510 nucleotides 1 GATATCCCTC AGCCGCCTTT CACTATCTTT TTTGCCCGAG
  • AAATAGTCTC CACACCC CGG CACTATCACC GTGAGTTGTC
  • CGTGAGTCG G CCCG GATCCT CGCGGGGAAT GGGGCTCTCG 1 921 GATGTAGATC TTCTTTCTTT CTTCTTTTTG TGGGTAGAAT
  • CTTAGTATAT GCCGGCACAA GCTACCACAA CTCTCAAACT 5361 TGCAGCAGCT GCACTTAGCT ATATTGCCAG AAGTATCATA
  • the analysis also identified an additional intron within the transcribed sequence of RAc1 which is 5' of the region containing the translation initiation codon. This 5'-intron separated a 79 bp GC-rich 5'-noncoding exon from an exon coding the translation initiation codon.
  • the promoter according to the present invention lies within 0 to 2071 nucleotides, more particularly, the efficiency of the promoter appears to lie within about 800 to 2071 nucleotides; nucleotides 811 to 816 and 2066 to 2070 are the Xhol and EcoRV enzyme sites, respectively; 2051 to 3600 nucleotides encompass the coding region DNA sequence for the rice actin gene RAc1; and 1650 to 3841 nucleotides is the RAc1 genomic clone for pRAc1.
  • soybean ⁇ Glycine max actin genes SAc3 and SAc1 the Arabidopsis thaliana actin gene AAc1, the potato ⁇ Soianum tuberosum) actin processed pseudogene PAc-psi, and the maize ⁇ Zea mays) actin gene MAd have regions upstream of their translation initiation codons which bear sequence similarity to the 3'-splice site junction regions of the 5'-introns of the rice actin gene RAc1, a Xenopus borealis cytoplasmic actin gene and the Drosophila melanogaster cytoplasmic actin gene DmA2.
  • a genomic restriction map of the 15.1 kb insert from lambda-RAc1 was prepared by analyzing all possible single and double digests with the enzymes BamHI, EcoRI, Hindlll and Sail.
  • a 5.3 kb Hindlll-Hindlll restriction fragment from the lambda-RAc1 clone was subcloned into pBluescript-KS to generate the plasmid pRAc15'.H3.
  • a restriction map of pRAc15'.H3 was prepared by analyzing all possible single and double digests with the enzymes BamHI, Bglll, EcoRI, EcoRV, Hincll, Hindlll, Kpnl, Pstl, Smal, Sphl, Xbal and Xhol.
  • the 2.1 kb EcoRV-EcoRV fragment from pRAc15'.H3 was subcloned into the Smal site of pBluescript-KS, in both orientations, to produce the plasmids pRAc15'.21V and pRAc15'.21V r .
  • GUS promoterless ⁇ -glucuronidase
  • NOS nopaline synthase
  • the plasmids pRAc15'.09RGI ⁇ 8, pRAc15'.09RGI ⁇ 1 and pRAc15'.09RGl ⁇ 12 were generated by cleavage of pRAc15'.09RG at its Bglll site followed by exonuclease III deletion and S1 nuclease treatment to remove different amounts of the RAc1 first intron.
  • the intronless 0.6 kb EcoRV fragment from pBRAc15'.09RI- was excised and cloned between the EcoRI and Smal sites of pBS-KS to yield the plasmid pRAc15'.09RI-.
  • Protoplasts were isolated from rice ⁇ Oryza sativa L. v Nipponbare) cell suspension cultures and resuspended in MaMg medium to a final density of 10 6 protoplasts/ml.
  • 1 ml of protoplast suspension was incubated with 10 ⁇ g of circular plasmid DNA, 50 ⁇ g/ml of calf thymus carrier DNA and an equal volume of 30% polyethylene glycol 4000. The mixture was incubated for 30 minutes, diluted with CPW13 medium, washed a further 3 times in CPW13 media, with centrifugation between each wash, before being resuspended in simplified KPR liquid media to a final density of 10 6 protoplasts/ml. 0.1 ml aliquots of this final suspension were incubated by thin layer culture in 96-well plates prior to subsequent assays for GUS activity.
  • This figure also indicates the position of the RAc1 coding and noncoding exons, as previously determined.
  • a 5.3 kb Hindlll fragment from the lambda-RAc1 insert, spanning a region from 3.9 kb upstream of the RAc1 coding sequence to a point within its third coding exon was isolated and cloned into the Hindlll site of the plasmid pBluescript-KS to produce the plasmid pRAc15'.H3.
  • a restriction map of the pRAc15',H3 insert is shown in Fig. 2B.
  • Restriction maps of the lambda-RAc1 (A) and pRAc15'.H3 (b) inserts were determined by single and double restriction enzyme digestion, as detailed in the methods.
  • the enzyme sites are abbreviated as follows: BamHI, B; Bglll, G; EcoRI, E; EcoRV, R; Hindi, H2; Hindlll, H3; Sphl, Sp; Sstl S; Xhol, Xh.
  • the position of the 5.3 kb Hind III fragment within the lambda-RAc1 insert is indicated by dashed lines between (A) and (B).
  • Noncoding and coding portions of RAc1 exons are depicted by open and filled boxes respectively.
  • the strategy used to sequence the 2.1 kb EcoRV fragment within the pRAc15'.H3 insert is indicated by horizontal arrows. The length of the horizontal arrows are indicative of the size of the sequence obtained.
  • the restriction map of the pRAc15'.H3 insert shown in Fig. 2B also indicates the strategy used to sequence an EcoRV fragment which covers a region 2.1 kb upstream of the translation initiation codon of the RAc1 gene. This sequence is as follows: T
  • AAATAGTCTC CACACCCCGG CACTATCACC GTGAGTTGTC
  • CGCCGCCGGT AACCACCCCG CCCCTCTCCT CTTTCTTTCT 1 761 CCGT T T T T T T T TTTOGTCTCG GTCTCGATCT TTGGCCTTGG
  • nucleotides are numbered with the A of the RAc1 transcription initiation site designated in bold. Restriction sites used in the subsequent construction of the various RAc1-
  • GUS fusion plasmids and structural regions described in detail are underlined and named or designated by lower case Roman numerals and noted at the right margin of the sequence table.
  • a number of potential regulatory sequences were identified in the 5'-flanking region of RAc1 gene.
  • a 12 bp direct repeat, GGTTTTAAGTT (region i) is located between bases 1027 to 1038 and 1078 to 1088.
  • a tandem (imperfect) direct repeat of 16 bp, AA(G/C)CCC(T)AAAGT(G/C)CTA (region ii), is located between bases 1301 and 1333.
  • 20 bp downstream of this tandem direct repeat are eight tandem copies of an imperfectly repeating pentamer with the consensus sequence CCCAA (region iii).
  • the 5'-intron of the rice RAc1 gene is 313 bp long. Its 5'- acceptor splice site [(G/G)TA], and 3'-donor splice site [TTTTTTGTA(G/G)], follow the consensus sequence previously determined for a number of plant genes.
  • a putative branch point site for mRNA splicing between bases 2008 and 2012 (region vii) was identified whose sequence, GTGAC, and distance from the 3'- donor splice site bears similarity to the location and consensus sequence for animal mRNA branch point splice sites, YTRAC.
  • RAc1-GUS fusion plasmids were constructed to determine the minimum amount of RAc1 5'-flanking sequence required for maximal ⁇ -glucuronidase (GUS) gene expression; these are shown in Fig. 3.
  • the plasmid pRAc15'.21VG has the 2.1 kb EcoRV restriction fragment from pRAc15'.H3 fused to the GUS coding region and 3'-noncoding transcript terminator region of the nopaline synthase (NOS) gene.
  • NOS nopaline synthase
  • the plasmids pRAc15'.13XG and pRAc15'.09RG were created by the deletion of 0.8 and 1.2 kb of sequence, respectively, from the 5' end of pRAc15'.21VG.
  • the plasmid pRAc15'.21 VR r G has the 5' 1.2 kb EcoRI fragment cloned in the opposite orientation to that in pRAc15'.21 VG.
  • the plasmid pRAc15'.09RGI ⁇ 8 contains a deletion of 9 bp around the Bglll site of the RAc1 5'-intron.
  • the plasmid pRAc15',09RGI ⁇ 1 contains a deletion of 133 bp between bases 1868 and 2003 of the sequence depicted above, reducing the distance between the 5' acceptor and mRNA branch point from its 5'-intron.
  • the plasmid pRAc15'.09RGI ⁇ 12 has a deletion of 157 bp between bases 1868 and 2027, removing the putative mRNA branch point from its 5'-intron.
  • the plasmid pRAc15'.O ⁇ RGIDS- was created by excision of a 170 bp BamHI fragment from pRAc15'.09RG, removing the putative mRNA branch point and 3'- donor splicing sites from the RAc1 5'-intron.
  • the plasmid pRAc1 5' .09RGI- is identical to pRAc15'.09RG except that it lacks the entire RAc1 5'-intron.
  • the plasmid pRAc15'.21V r G contains the 2.1 kb EcoRV fragment from pRAc15'.H3 cloned in the opposite orientation to that in pRAc15'.21VG. It was postulated that the RAc1 5'-region would not show bidirectional promoter activity because the 5'- most 1.2 kb EcoRV-EcoRI restriction fragment in pRAc15.21VG did not show any binding when used as a probe in Northern hybridization against total RNA from 7 day old rice shoots. As a positive control, the plasmid pAI 1 GusN was used. This plasmid contains the promoter, first exon and first intron of the maize Adh1 gene fused to a GUS coding sequence with the 3' noncoding region of the NOS gene.
  • restriction enzyme sites are abbreviated as follows: BamHI, B1 ; Bglll, B2; EcoRI, R1 ; EcoRV, RV; Hindi, H2; Hindlll, H3; Sstl, S1 ; Xhol, Xh1 ; Xbal, Xb1.
  • Table I also shows the results of a determination of GUS expression efficiencies, defined as the percent of intact cells displaying visible GUS activity 10 days after PEG-mediated transformation of rice protoplasts with the RAc1 -GUS fusion constructs.
  • the plasmids pRAc15'.21VG and pRAc15'.13XG displayed the highest GUS expression efficiencies at 8.4 and 7.9% respectively.
  • the plasmids pRa c15'.09RG and pRAc15'.21VR r G showed GUS expression efficiencies of 5.7 and 6.1%, respectively. All four of the aforementioned plasmids displayed significantly higher GUS expression efficiencies than was found for pAI 1 GusN.
  • the effects of the various RAc1 intron deletions could be seen when their GUS specific activities were compared to that of their progenitor plasmid, pRAc15'.09RG.
  • the small 9 bp deletion in the RAc1 5'-intron of plasmid pRAc15'.09RGI ⁇ 8 had no effect on the quantitative level of GUS activity.
  • the plasmid pRAc15'.09RGI ⁇ 1 containing a deletion of 133 bp between the 5'- acceptor splice site and the putative mRNA branch point splice site, displayed a GUS specific activity that was less than 57% of that observed for pRAc15'.09RG.
  • the plasmid pRAc15'.09RGI ⁇ 12 which had the putative mRNA branch point site removed, showed a further decrease in GUS specific activity of 17% of that observed for pRAc15'.09RG.
  • the plasmid pRAc15'.09RGIDS- which lacks the putative mRNA splicing branch site and 3'-splicing donor site of the RAc1 5'-intron, and pRAc15'.09RGI _ , which lacks the entire RAc1 5'-intron, displayed no significant GUS specific activities over that observed for untransformed samples.
  • the present invention has described the isolation and characterization of the 5'-regions of the rice RAc1 gene. Within the 5'-flanking sequence, the occurrence of a long poly(dA) stretch located between bases 1465 and 1505 which appears to play a role in the constitutive activation of RAc1 gene expression was found. Based upon chemical analysis, it has been suggested that a minimal affinity for histone cores and nucleosome formation is provided by homogeneous tracts of purines located on one strand of the double helix.
  • Poly(dA) regions within recombinant DNA molecules can prevent nucleosome formation in vitro, and it has also been found that naturally occurring poly(dA) tracts act as 5'-promoter elements for the constitutive expression of different yeast genes. It is believed that poly(dA) stretches may change the chromatin structure, enabling general transcription factors to access the DNA template and activate constitutive transcription in the absence of more specific transcription factors.
  • the sequence of RAc1 untranslated mRNA 5'-region is unusually GC rich, with an AT content of only 26.5%.
  • RAc1-GUS fusion constructs The construction of a number of RAc1-GUS fusion constructs has provided the determination that the plasmid pRAc15'.13XG, containing 0.83 kb of 5'-flanking sequence, the noncoding exon and 5'-intron of the RAc1 gene, has the minimal amount of RAc1 sequence necessary for maximal GUS expression in transient assays of transformed rice protoplasts; the additional 0.8 kb of RAc1 5'-sequence presents in the pRAc15'.13VG did not significantly increase GUS expression above that of the plasmid pRAc15'.13XG.
  • RAc1 5'-flanking sequence was more active than the maize Adh1 5'-flanking region in stimulating GUS expression in transformed rice protoplasts. Deletion of a 0.4 kb region from the 5'-end of the pRAc15'.13XG insert resulted in a 52% reduction in GUS activity.
  • RAc1-intron-deletion-GUS plasmids By constructing RAc1-intron-deletion-GUS plasmids, it was able to be shown that GUS expression in transformed rice protoplasts was dependent on the presence of an intact RAc1 5'- intron. Deletion of the RAc1 intron reduced GUS specific activity to levels that were not significantly greater than that of untransformed protoplasts. In transient assays of transformed maize protoplasts a maize Adh1 -S gene lacking the nine Adh1 -S introns was expressed at levels 50- to 100-times lower than that of the intact gene.
  • the small deletion in the RAc1 intron in the plasmid pRAc15'.09RGI ⁇ 8 caused no significant reduction in GUS specific activity relative to that of pRAc15'.09RG. However, a significant difference was observed between the GUS specific activities of the plasmids pRAc15'.09RG and pRAc15'.09RGI ⁇ 1.
  • the 133 bp deletion in the plasmid pRAc15'.09RGl ⁇ 1 while not removing any of the sequences previously implicated in intron splicing, did cause a reduction in the distance between the 5'-acceptor and putative mRNA branch point splice sites with an associated 44% reduction in GUS specific activity.
  • results of the present invention clearly demonstrate that the RAc1 5'-flanking sequence between 0 and 2070 nucleotides and more particularly between 800 and 2070 nucleotides in the genomic sequence contains an efficient promoter for rice transformation. Furthermore, the expression of a foreign gene in transformed plant cells can be dependent upon, but not necessarily an absolute function of, the presence of an intact 5'-intron sequence. It appears that a functional requirement for the presence of the 5'-intron may be correlated with the conservation of the 5' noncoding exon during this rice actin gene's structural evolution.
  • results of the present invention indicate that the maize 5'-intron is located between the first and second coding exons of the Adh1 gene, while the rice 5'-intron is located between a 5' noncoding exon and the first coding exon of the RAc1 gene, suggesting that there may be a common positional component to the intron mediated stimulation of gene expression observed for the maize and rice 5'-intron.
  • the preceding description of the present invention clearly demonstrates that a 2.1 kbp 5' of the Act1 gene's translation initiation codon, containing 1.3 kb of 5' untranscribed sequence, the 5' transcribed but untranslated exon, 5'-intron and part of the first coding exon of the rice Act1 gene, is capable of conferring high level expression of foreign gene in transformed rice material.
  • this region can be used to activate the constitutive expression of foreign genes in transgenic plants of rice and other agronomically important plants; the 5'-intron of the rice Act1 gene can stimulate the expression of a foreign gene in transformed rice material [thus this (and the other introns of the rice Act1 gene) will be able to increase the expression of foreign genes in transformed plants of rice (and other agronomically important plants) when ligated in front of such a gene in between a promoter and the gene]; and although no specific function for the 3'-end of the rice Act1 gene in the regulation of foreign gene expression in rice has been demonstrated. It appears from the present invention that the 3'- end of the rice Act1 gene should also stimulate the expression of such foreign genes in transformed plants of rice and other agronomically important crops.

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Abstract

L'invention concerne la séquence génomique de nucléotides, ainsi que l'isolation d'un promoteur fort indépendant des introns, à partir de gènes d'actine de riz.
PCT/US1991/000073 1990-01-05 1991-01-04 Gene d'actine de riz et promoteur WO1991009948A1 (fr)

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Cited By (22)

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EP0666921A1 (fr) * 1991-10-02 1995-08-16 Cornell Research Foundation, Inc. Monocot presentant un promoteur dicot pouvant etre induit par une blessure
US5684239A (en) * 1990-01-05 1997-11-04 Cornell Research Foundation, Inc. Monocot having dicot wound-inducible promoter
US5866793A (en) * 1996-06-03 1999-02-02 National Research Council Of Canada Promoter for expressing foreign genes in monocotyledonous plants
FR2772787A1 (fr) * 1997-12-24 1999-06-25 Rhone Poulenc Agrochimie Promoteur h3c4 de mais associe au premier intron de l'actine de riz, gene chimere le comprenant et plante transformee
US6040497A (en) * 1997-04-03 2000-03-21 Dekalb Genetics Corporation Glyphosate resistant maize lines
WO2000020571A2 (fr) * 1998-10-06 2000-04-13 Pioneer Hi-Bred International, Inc. Promoteurs du maïs
WO2000070068A1 (fr) * 1999-05-14 2000-11-23 Dekalb Genetics Corporation Promoteur rs81 et procedes d'utilisation de ce promoteur
WO2000070066A1 (fr) * 1999-05-14 2000-11-23 Dekalb Genetics Corporation Promoteur rs324 du mais et procedes d'utilisation de ce promoteur
WO2000070067A1 (fr) * 1999-05-14 2000-11-23 Dekalb Genetics Corporation Promoteur actine 2 de riz et intron, procedes d'utilisation associes
WO2000073474A1 (fr) * 1999-05-14 2000-12-07 Dekalb Genetics Corporation Promoteur rs81 du mais et procedes d'utilisation correspondants
EP1217073A2 (fr) * 1995-07-19 2002-06-26 Aventis Cropscience S.A. Plantes transformées à tolérance accrue aux herbicides de la famille des phosphométhylglycines contenant un gène codant pour une 5-énol pyruvylshikimate-3- phosphate synthase mutée
US6762344B1 (en) 1997-04-03 2004-07-13 Dekalb Genetics Corporation Method of plant breeding
WO2004058979A1 (fr) * 2002-12-27 2004-07-15 Centro De Ingenieria Genetica Y Biotecnologia Promoteur artificiel pour l'expression de sequences d'adn dans des cellules vegetales
WO2005030968A2 (fr) * 2003-09-25 2005-04-07 Monsanto Technology Llc Elements regulateurs de l'actine, destines a des plantes
EP1614754A1 (fr) * 2004-07-06 2006-01-11 Biogemma Procédé pour améliorer l'expression de gènes dans des plantes
WO2007069894A3 (fr) * 2005-12-16 2007-08-16 Keygene Nv Promoteurs de plante constitutifs
EP2107118A1 (fr) * 1993-08-25 2009-10-07 DeKalb Genetics Corporation Plantes de maïs transgénique fertiles et leurs procédé de production
WO2009149304A2 (fr) * 2008-06-04 2009-12-10 Edenspace Systems Corporation Éléments régulateurs de gène de plante
US9139838B2 (en) 2011-07-01 2015-09-22 Monsanto Technology Llc Methods and compositions for selective regulation of protein expression
US9192112B2 (en) 2005-10-13 2015-11-24 Monsanto Technology Llc Methods for producing hybrid seed
WO2016146833A1 (fr) 2015-03-19 2016-09-22 F. Hoffmann-La Roche Ag Biomarqueurs de résistance à la nad(+)-diphtamide adp-ribosyltransférase
EP3184547A1 (fr) 2015-10-29 2017-06-28 F. Hoffmann-La Roche AG Anticorps anti-tpbg et procédés d'utilisation

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JOURNAL OF MOLECUALR AND APPLIED GENETICS, Volume 2, No. 1, issued 1983, SHAH et al, "Genes Encoding Actin a Higher Plants: Intron Positions are Highly Conserved but the Coding Sequences are Not", pages 111-126. *
NUCLEAR ACIDS RESEARCH, Volume 17, Number 2, issued 1989, NG et al, "Regulation of the Human Beta-Actin Promoter by Upstream and Intron Domains", pages 601-615. *
NUCLEIC ACIDS RESEARCH, Volume 17, Number 1, issued 1989, FREDERICKSON et al, "5' Flanking and First Intron Sequences of the Human Beta-Actin Gene Required for Efficient Promoter Activity", pages 253-270. *
PLANT CELL REPORTS, Volume 8, issued 1989, OARD et al, "Chimeric Gene Expression Using Maize Intron in Cultured Cells of Breadwheat", pages 156-160. *
PLANT MOLECULAR BIOLOGY, Volume 14, issued 1990, MCELROY et al., "Structural Characterization of a Rice Actin Gene", pages 163-171. *
PROTOPLASMA, Volume 141, issued 1987, STAIGER et al; "Actin Localization and Function in Higher Plants", pages 1-12. *
THE PLANT CELL. Volume 2, issued February 1990, MCELROY et al, "Isolation of an Efficient Actin Promoter for Use in Rice Transformation", pages 163-171. *

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EP1217073A2 (fr) * 1995-07-19 2002-06-26 Aventis Cropscience S.A. Plantes transformées à tolérance accrue aux herbicides de la famille des phosphométhylglycines contenant un gène codant pour une 5-énol pyruvylshikimate-3- phosphate synthase mutée
US5866793A (en) * 1996-06-03 1999-02-02 National Research Council Of Canada Promoter for expressing foreign genes in monocotyledonous plants
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WO1999034005A1 (fr) * 1997-12-24 1999-07-08 Aventis Cropscience S.A. Promoteur h3c4 de mais associe au premier intron de l'actine de riz, gene chimere le comprenant et plante transformee
WO2000020571A3 (fr) * 1998-10-06 2000-09-21 Pioneer Hi Bred Int Promoteurs du maïs
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WO2004058979A1 (fr) * 2002-12-27 2004-07-15 Centro De Ingenieria Genetica Y Biotecnologia Promoteur artificiel pour l'expression de sequences d'adn dans des cellules vegetales
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