JP2001501805A - Improvements in or related to gene expression - Google Patents

Abstract

(57) Abstract: A nucleic acid that can be expressed in a plant cell, encodes at least an effective part of a GAL4 DNA binding domain, and has a practically reduced A / T base content relative to a wild-type yeast sequence. An array is disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION Improvement in or Related to Gene Expression Field of the invention The present invention relates to nucleic acid sequences optimized for expression in plant cells, as well as vectors and plant cells containing the nucleic acid sequences. Background of the Invention There is a gene called GAL4 in yeast, and its product acts as a transcriptional activator (Johnson 1987 Microbiol. Rev. 51 , 458-476). This GAL4 gene is very large (3 Kb) and encodes an N-terminal DNA-binding domain, a C-terminal domain having the function of a transcription activator, and an intervening glucose responsive element "GRE" (GAL4 is expressed in the presence of glucose). Is suppressed in). The GAL4 protein has been very well characterized. Amino acid residues 1-147 of this protein bind to DNA in a sequence-specific manner (Keegan et al. 1986 Science). 231 , 6 99-704). The transcriptional activation function is linked to two short parts of the C-terminal domain (Ma & Ptashne 1987 Cell 48, 847-853). The DNA sequence to which GAL4 binds has been identified as a 17-mer, which must exist as a repeat for optimal GAL4 binding (Giniger et al., 1985 Cell 40, 767-774), and respond to GAL4 responses. Sometimes referred to as the sex "upstream activating sequence" (UAS): GAL4 binds to the USA5 'of a gene via a DNA binding domain, and the C-terminal domain results in the up-regulation of transcription of that gene. In recent years, two-element systems have been developed for directing gene expression in Drosophila melanogaster. Brand and Perrimon (1993 Development 118 : 401-415) used a P-element based vector to randomly insert a gene encoding GAL4 into the Drosophila melanogaster genome. A number of stable Drosophila lines have emerged, each expressing GAL4 in a unique pattern in response to adjacent genomic DNA sequences. It was then possible to clone the selected target genes under the control of the GAL4 upstream activating sequence (UAS), transform them separately, and keep them dormant in the absence of GAL4. Was. Genetic crosses between this single system and any library of systems containing GAL4 allow activation of the target gene in many different tissues and cell types, including errors that are fatal to the organism. The phenotypic consequences of the expression could be conveniently studied. In addition, fly libraries, including GAL4, are becoming increasingly characterized and shared resources, providing a common entry point for various "reverse" genetic technologies. Similar systems operable in plants are highly desirable. However, the expression of heterologous eukaryotic genes in plants has been proven to be very problematic in the past (see, eg, green fluorescent protein), and efficient expression of GAL4 appears to be equally difficult, This has been suggested to be due to inefficient translation of GAL4 mRNA in plants (Reichel et al., 1995 Plant Cell Reports). 14 , 773-776). Ma et al. (1988 Nature 334 , 631-633) were able to obtain transient expression of a modified functional GAL4 derivative in tobacco leaf protoplasts, but failed to indicate the presence of functional full-length wild-type GAL4. Similarly, transient expression of GAL4 derivatives has been demonstrated in corn protoplasts (McCarty et al., 1991 Cell. 66 895-905), and in maize aleurone tissue or embryogenic calli when introduced by microprojectile bombardment (Goff et al., 1991 Genes & Dev. 5, 298-309; Goff et al., 1992 Genes & Dev. 6). , 864-875). However, there has been no report on stable and efficient expression of functional GAL4 or a derivative thereof in plant cells. It is an object of the present invention to provide a novel expression system operable in plants using a modified part of the GAL4 gene. Summary of the Invention In a first aspect, the invention relates to a nucleic acid sequence that can be expressed in a plant cell and encodes at least an effective part of the GAL4 DNA binding domain, wherein the A / T base content is relative to the sequence of a wild-type yeast To provide a nucleic acid sequence that is substantially reduced. The A / T content of the sequence of the wild-type yeast encoding the DNA binding domain of GAL4 is about 59%. It is understood that the A / T base content of a sequence of the invention encoding an effective portion of the GAL4 DNA binding domain is substantially reduced when it is less than 50%. Preferably the A / T content is less than 45%, more preferably less than 40%. The sequences of the invention can be generated, for example, by site-directed mutagenesis, or newly generated by chemical synthesis. An “effective portion” of a DNA binding domain is a portion that is sufficient to retain most (ie, greater than 50%) of the DNA binding activity of a full-length DNA binding domain. Typically, the "effective portion" comprises at least 2/3 of the full length sequence of the DNA binding domain. This nucleic acid sequence is very suitable for a substantially smaller portion (about 75 amino acid residues) to retain most of its DNA binding activity (Kraulis et al., 1992 Nature 356 448-450; and Marmorstein et al., 1992 Nature. 356 408-414), but advantageously encodes substantially all of the GAL4 DNA binding domain (ie, amino acid residues 1-147 of the yeast polypeptide). In one particular embodiment, the sequence comprises the nucleotide sequence shown in the 5 'portion of Figure 1 (nucleotides 1 to about 460), which encodes the same amino acid sequence, but which is identical to the sequence of a wild-type yeast. The A / T content is substantially reduced (38%). The GAL4 DNA binding domain has no transcriptional activation function by itself. Thus, in a preferred embodiment, the sequence encoding the active portion of the GAL4 DNA binding domain is replaced with another one or more nucleic acid sequences which may be structural (ie, encode a functional polypeptide) and / or regulatory. Operatively connected to Typically, the sequence encoding the DNA binding domain is operably linked (eg, fused in frame) to a sequence encoding a peptide or polypeptide having a regulatory function, preferably a transcriptional activator. The transcriptional activator may be the activation domain of the GAL4 protein, and the sequence encoding it is preferably optimized for expression in plants (eg, by reducing their A / T content). Should be Alternatively, the transcriptional activator is known to those skilled in the art to be active in plants, such that the sequence of the invention encodes a chimeric polypeptide comprising at least an effective portion of the GAL4 DNA binding domain and a transcriptional activation domain. May be any one of a number of known proteins. Particularly suitable transcriptional activator domains are those obtainable from herpes simplex virus (HSV) VP-16 (Greaves and 0'Hare 1989 J. Virol 63, 1641-1650; see also VP also known as VMW65). ). Other transcriptional activation domains include specific peptides encoded by E. coli genomic DNA fragments (Ma & Ptashne 1987 Cell 51 , 113-119) or a synthetic peptide designed to form an amphipathic α-helix (Giniger & Ptashne 1987 Nature). 330 , 670-672). A common feature is either positive (Gill & Ptashne 1987 Cell 51, 121-126) or, particularly for the plant activation domain, an excess negative charge (Estruch et al., 1994 Nucl. Acids Res. 22, 3983-3989). Requires an excess charge of In view of this, one skilled in the art can readily synthesize or encode a naturally occurring sequence that encodes a peptide or polypeptide having transcriptional activation activity in plants. In any case, such activator sequences can be modified for optimal activity in plant cells. In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence as defined above. In one specific embodiment, the nucleic acid construct can be used to "catch" a plant enhancer sequence as an "enhancer-trap" (Sundaresan et al., 1995 Genes & Dev. 9 1797-1810). In such an embodiment, the construct preferably includes right and left Ti-DNA to allow random and stable insertion into the genome of the plant cell host. This construct preferably contains the native plant promoter sequence. The term refers to a promoter (operable in plant cells) that requires the presence of appropriate enhancer sequences to produce substantial levels of transcription. Such "native" promoters can be considered "enhancer-dependent" promoters, which essentially correspond to the TATA box region of known plant promoters. "Plant" promoters include references to viral and bacterial promoter sequences that are active in plant cells (eg, nucleotides -48 to +1 of the CaMV 35S promoter). Desirably, this native promoter is substantially adjacent to one of the Ti boundaries. In so doing, the promoter is directed in plants to direct the expression of the GAL4 DNA binding domain and the transcriptional activation domain when the promoter becomes inserted into the genome of the plant host cell in a functional relationship with the enhancer sequence. It has a competent relationship with the sequence encoding the GAL4 DNA binding domain fused to the operable transcription activation domain. The construct may further include a plant selectable marker (eg, kanamycin resistance) for convenience. In addition, the constructs include a reporter gene (eg, green fluorescent protein, or β-glucuronidase “GUS”) that is active in a plant, operably linked to a GAL4 responsive upstream activating sequence (UAS). A gene may be included that is expressed in response to synthesis of a GAL4 DNA binding domain / transcriptional activator fusion protein. Preferably, the reporter gene is a modified GFP as disclosed in WO 96/27675, or GUS (conveniently also having a nuclear localization signal). Preferably, the UAS comprises one or more repeats of the 17-mer to which GAL4 binds (Giniger et al., 1985 Cell. 40 , 767-774). As an alternative to introducing the GAL4 binding domain / transcriptional activator sequence into the genome of a plant host cell, the construct may include a Ds ("dissociation") element, which is then transiently expressed. Can be transferred to the periphery of the genome by the action of an Ac (activator) enzyme (see, eg, Cocherel et al., 1996 Plant Mol. Biol. 30 , 539-55 1). In a further aspect, the invention provides plants or parts thereof (eg, plant host cells or cell lines) comprising a construct as defined above. Typically, this construct will be integrated into the genome of the plant cell, where it will be stably maintained. In particular, the present invention provides a plurality of plants or parts thereof (e.g., isolated plant cells, or cell lines, or tissue cultures) comprising a library, each plant or part thereof. Has a nucleic acid sequence that is stably maintained, encoding an effective portion of the GAL4 DNA binding domain as defined above. Conveniently, the nucleic acid sequence will be integrated into the genome of the plant cell present in the library. Thus, libraries of plants or parts thereof provide a very useful resource. The library may be, for example, from Arabidopsis thaliana, or any other plant routinely used in research. Each plant or part thereof in this library may have a unique pattern of expression of the integrated reporter gene (eg, Sundaresan et al., 1995 Genes & Dev. 9, 1797-1810; Klimyuk et al., 1995 Molec. Gen. Genet. 249, 357-365). Thus, by introducing an additional gene with a GAL4-responsive UAS into these cell lines, the introduced gene will be expressed in the same temporal / spatial pattern as the reporter gene, and researchers will be of particular interest. Genes can be expressed in a predictable manner in selected tissues and / or at selected times. Accordingly, the present invention is a method of expressing a gene of interest in a known (predictable) pattern in a plant or a part thereof, wherein the gene of interest is introduced into the plant or a part thereof. Wherein the gene of interest has a GAL4 responsive upstream activating sequence (UAS) and the plant or a part thereof is GAL4 DNA encoded by a sequence according to the invention as defined above. A reporter gene expressed in a known pattern under the influence of a transcriptional activator comprising an effective portion of a binding domain, such that binding of the transcriptional activator to UAS results in transcriptional activation of the gene of interest. A method characterized in that it occurs. Thereby, the temporal / spatial pattern of expression of the introduced gene or genes of interest reflects what is known for the reporter gene. Typically, the plants or parts thereof are Arabidopsis, but can be any other plants routinely studied (eg, corn, rice, potatoes, etc.). Typically, the gene or genes of interest are introduced by crossing a GAL4-expressing plant cell line with a plant cell line that contains the gene (s) of interest linking a GAL4-responsive UAS. I do. The nucleic acid sequences of the present invention have other useful uses in addition to being used as "enhancer traps" or to direct expression of a gene or genes of interest in a predictable manner. This modified GAL4 DNA binding domain / transcriptional activator can also be used to regulate the expression of multiple genes of interest. In many situations, for research purposes or for agricultural / industrial motivation (eg, to study metabolic pathways or to manipulate the synthesis of desired products, such as plant dyes or lipids), plants , It is desirable to express a plurality of genes simultaneously. Accordingly, the present invention is a method for regulating the expression of a plurality of genes of interest in a plant or part thereof, wherein each of the genes of interest is functionally associated with a GAL4-responsive UAS. Wherein said plant or part thereof comprises a sequence according to the first aspect of the invention as defined above, and is capable of expressing a transcriptional activator comprising an effective part of a GAL4 DNA binding domain, Thus, there is provided a method characterized in that binding of the transcriptional activator to UAS results in simultaneous transcriptional activation of all the genes of interest. Conveniently, these multiple genes of interest may all be associated with a single UAS in a polycistronic configuration, which facilitates their introduction into plants or parts thereof (The gene of interest is typically a gene not naturally found in plants or parts thereof, such as a mammalian or bacterial gene, or a gene from a different plant or various modifications. Is probably a natural plant gene). Alternatively, one or more genes can be operably linked to each UAS. The transcriptional activator may be encoded by a sequence already present in the plant or a part thereof, or may be introduced simultaneously with (or subsequent to) the introduction of a plurality of genes of interest. The present invention is further described by way of illustrative examples and with reference to the accompanying drawings. Among these accompanying drawings: FIG. 1 shows the nucleotide sequence according to the invention encoding a modified GAL4 DNA binding domain fused in frame to a sequence encoding a modified transcriptional activation domain derived from HSVVP16, vertical The line represents the end of the GAL4 DNA binding domain; FIG. 2 is a schematic diagram of a nucleic acid construct comprising a sequence according to the present invention; FIG. 3 is a fragment of the nucleic acid construct schematically represented in FIG. 4 shows the nucleotite sequence; and FIGS. 4a-4d are photomicrographs of the roots of plants containing the sequence according to the invention. Example We have used an approach similar to that described by Brand & Perrimon (1993, cited above), but using Agrobacterium tumefasciens-mediated transformation for use in Arabidopsis. It was adapted to produce an "enhancer trap" plant cell line with a transcriptional activator comprising a GAL4 DNA binding domain (mGAL4-VP16) fused to the activation domain of HSV VP16. Example 1-Construction of mGAL4-VP16 gene Eight oligonucleotides corresponding to the modified sequence of the GAL4 DNA binding region were synthesized. GAL-5 '(28 mer) GGC AAC AAT GAA GCT ACT GTC TTC TAT C (SEQ ID NO: 3) VP16-3' (31 mer) GGC AGA TCT ACC CAC CGT ACT CGT CAA TTC C (SEQ ID NO: 4) MGAL1 (109 GGC AAG CTT GGA TCC AAC AAT GAA GCT CCT GTC CTC CAT CGA GCA GGC CTG CGA CAT CTG CCG CCT CAA GAA GCT CAA GTG CTC CAA GGA GAA GCC GAA GTG CGC CAA G (SEQ ID NO: 5) MGAL2 (108 mer) TCC TCT CGA GGG AAG ATC AGG AGG AAG AGC TGC TCC AGG CGC TCC AGG CGG GAC TCC ACT TCG GTG AGG TGG GCG CGG GTC AGC GGG GAG CGC TTG GTT TTG GGA GAG (SEQ ID NO: 6) MGAL3 (81 mer) TTC CCT CGA GAG GAC CTC GAC ATG ATC CTG AAA ATG GAC TCC CTC CAG GAC ATC AAA GCC CTG CTC ACC GGC CTC TTC GTC (SEQ ID NO: 7) MGAL4 (89-mer) GGG TGA GGG GCA TGT CGG TCT CCA CGG AGG CCA GGC GGT CGG TGA CGG CGT CTT TGT TCA CGT TGT CCT GGA CGA AGA GGC CGG TGA GC (SEQ ID NO: 8) MGAL5 (80 mer) GGA GAC CGA CAT GCC CCT CAC CCT GCG CCA GCA CCG CAT CAG CGC GAC CTC CTC CTC GGA GGA GAG CAG CAA CAA GGG CC SEQ ID NO: 9) MGAL6 (83 AGT GGA GCT CGT CCC CCA GGC TGA CGT CGG TCG GGG GGG CCG TCG AGA CGG TCA ACT GGC GCT GGC CCT TGT TGC TGC TCT CC (SEQ ID NO: 10) Full-length oligonucleotide was prepared using 7M urea and 90M tris-borate 1 mM EDTA pH8. 3 Purified by electrophoresis on a 5% polyacrylamide gel containing (TBE) buffer. The fractionated oligonucleotide was detected and excised by simple staining with filtered 0.05% toluidine blue dye and extracted overnight at 37 ° C. in 0.5 M ammonium acetate, 0.1% SDS and 0.1 mM EDTA. The extracted DNA was precipitated, washed with ethanol, and phosphorylated using T4 polynucleotide kinase. Separate DNA (0.5 μg each) from 50 mM Tris-HCl pH 9.0, 10 mM MgCl _Two , 10 mM DTT containing 1 mM ATP, and 5 units of T4 polynucleotide kinase and incubated at 37 ° C. for 30 minutes. Plasmid pCMVGal65 (Cousens et al., 1989 EMBO J. 8 , 2337-2342) was used as the source of the GAL4-VP16 sequence of the unmodified codon use. Two restriction endonuclease fragments were isolated from this plasmid. The SacI-SacI fragment containing the sequence encoding the DNA binding element of Saccharomyces cerevisiae GAL4 protein and the SacI-KpnI fragment encoding the activation domain of the herpes simplex virus VP16 protein were reduced to a low gelling temperature (LGT) 1. Purified by fractionation and extraction on a% agarose gel. The 5 'portion of the mGAL4-VP16 sequence was amplified by polymerase chain reaction (PCR) using GAI-5' and MGAL2 oligos as primers and a SacI-SacI GAL4 DNA fragment as a template. The primers and template were incubated with VENT DNA polymerase (New England Biolabs) under standard conditions, 30 minutes at 94 ° C, 1 minute at 50 ° C, 1 minute at 72 ° C. The cycle was performed 30 times. The product was cut with XhoI restriction endonuclease and purified by electrophoresis on a 1.5% LGT agarose gel. The MGAL4 and MGAL5 oligonucleotides were annealed by heating to 65 ° C. and gradually cooled to room temperature. Next, MGAL3 and MGAL6 oligonucleotides were added and also annealed to the mixture. These oligonucleotides were then combined with the Klenow fragment of DNA polymerase 1 and T4 DNA ligase in 50 mM Tris-HCl pH 7.5, 10 mM MgCl2. _Two , 10 mM DTT, 1 mM dNTP and 1 mM ATP at 37 ° C. for 60 minutes. Excess MGAL3 and MGAL6 oligonucleotides were added and the mixture was subjected to PCR amplification as described above. The product was cut with the restriction endonucleases XhoI and SacI and purified by electrophoresis on a 1.5% LGT agarose gel. These three gel-purified restriction fragments: (1) the GAL-5 '/ MGAL2 PCR amplification product corresponding to the 5' portion of the GAL4 DNA binding domain, and (2) the MGAL3-- equivalent to the 3 'portion of the GAL4 DNA binding domain. The 6 annealed amplification products and (3) the SacI-KpnI fragment from pCMVGal65 corresponding to the VP16 activation domain are mixed and, using T4 DNA ligase, 50 mM Tris-HCl pH 7.5, 10 mM MgCl _Two Ligated in 10 mM DTT and 1 mM ATP at 20 ° C. overnight. The ligated product was PCR amplified (as described above) using MGAL1 and VP16-3 ′ as primers. The product containing the entire modified GAL4-VP16 sequence is cut with BamHI, cut with SacI, blunt-ended by treatment with T4 DNA polymerase, and recut with BamHI (Jefferson et al.). EMBO J. 6: 3907, 1987). This recombinant plasmid (pBIN 35S-mGAL4-VP16) contained the mGAL4-VP16 gene downstream of the structural 35S plant promoter. In FIG. 1, the lower DNA sequence is that of the fusion gene (SEQ ID NO: 1) and encodes the 5 'portion of the GAL4 DNA binding domain and encodes the 3' portion of the transcription activation domain derived from HSV VP16. I do. The wild-type sequence is shown above for comparison. The wild-type GAL4 DNA binding domain coding sequence was found to be A / T-rich (% A / T content is shown to the right of each column in the sequence), causing inefficient expression in plants Believe it. In particular, A / T-rich DNA is believed to contain one or more regions that when transcribed are recognized as mRNA splice sites in plant cells. The modified nucleotides are outlined in FIG. The encoded polypeptide sequence is shown below (SEQ ID NO: 2). Modifications to the nucleic acid sequence were carefully chosen to ensure that no changes in the sequence of the encoded amino acids occurred. The numbers above the sequence represent the number of the nucleotide or amino acid at that position. Specific restriction endonuclease sites are also indicated. The 35S promoter and mGAL4-VP16 gene were excised by digestion with the restriction endonucleases HinDIII and EcoRI, purified by 1% LGT agarose gel electrophoresis, and subcloned into a vector containing a GAL4-responsive mgfp5-ER marker gene ( See Example 2 below). The resulting construct is shown in FIG. 2, where R 1 and L represent right and left Ti plasmid border sequences. The GAL4-VP16 fusion gene is located downstream of the TATA box region (nucleotides -48 to +1) of the CaMV 35S gene inserted adjacent to the right Ti border. The plasmid also contains a kanamycin resistance (Kan R) selectable marker, and a reporter gene (GUS or GFP) operably linked to the synthetic GAL4-UAS promoter sequence. Specific restriction endonuclease sites are also indicated. FIG. 3 shows the nucleic acid sequence of this plasmid (SEQ ID NO: 15), which crosses the junction of the Ti right border, the CaMV35S TATA box, and the start of the GAL4 DNA binding domain coding region. The mGAL4-VP16 gene was directly assayed for activity in Arabidopsis plants transformed by Agrobacterium-mediated transformation (Valvekens et al., Proc. Natl. Acad. Sci. USA 85: 5536-5540, 1988). Example 2 Construction of GAL-GFP enhancer trap vector Modification of right boundary of T-DNA Plasmid pBIN19 (Bevan, M. Nuc. Acids Res. 12: 8711-8721, 1984) was digested with restriction endonuclease SacII, and Agrobacterium T -The 2.6 Kb fragment containing the DNA right border sequence was subcloned into SacII cut pGEM5Zf (Genebank Accession No. X65308: purchased from Promega). The bacteria containing this recombinant phagemid were superinfected with helper phage to generate single-stranded phagemid DNA. A synthetic mutagenic oligonucleotide ("TR +": ATA TCC TGT CAA ACA CTG GAT C CG AGC TCC AAT TCA TAG TTT AAA CTG AAG GCG GG; SEQ ID NO: 11) was subjected to kinase treatment, annealed to purified phagemid DNA, Extended with T7 DNA polymerase above and ligated with T4 DNA ligase. Bacteria were transformed with this DNA and the recombinant plasmid (pTr +) was screened for insertion of new sites for the restriction endonucleases BamHI, SmaI and EcoRI immediately adjacent to the right border of T-DNA. Insertion of TATA box region The TATA region of the 35S promoter was ligated with an oligonucleotide (oligonucleotide Δ35SBglII; GGC AGA TCT TCG CAA GAC CC T TCC TCT ATA TAA GG; SEQ ID NO: 12) complementary to the −48 region of this promoter, PCR amplification from pBI121 using the glucuronidase gene (oligonucleotide GUSSnaBI; CAC ACA AAC GGT GAT ACG TA; SEQ ID NO: 13). The PCR product was cut with the restriction endonucleases BglII and BamHI and ligated into BamHI cut pTr +. The resulting recombinant plasmid (pTr + Δ35S) contained a minimal (native) promoter located adjacent to the right border of T-DNA. Insertion of the 35S promoter-driven mGAL4-VP16 gene An unmodified codon U.S.A.GAL4 derivative was inserted into the pTr + Δ35S plasmid in a unsuccessful trial, and one of these constructs (insertion of the shortened GAL4 gene from pMA236). Ma et al., 1988 Nature 334, 631-633) resulted in the insertion of a unique HindIII site immediately adjacent to the BamHI site in pTR + Δ35S. This plasmid, pTr + ΔGAL4, was digested with restriction endonucleases HindIII and EcoRI, ligated with a HindIII-EcoRI fragment derived from pBIN 35S-mGAL4-VP16, and the 35S-driven mGAL4-VP16 gene was introduced. Next, to test the Agrobacterium sequence derived from this plasmid (pTr + 35S-mGAL4-VP16) in in planta, a binary plant transformation vector containing a GAL4 responsive marker gene (see below) was used. Recloned. Construction of Synthetic GAL4 Responsive Promoter A HindIII-XbaI restriction endonuclease fragment containing the five optimized GAL4 binding sites was excised from plasmid pUAST (Bran & Perrimon, 1993 Development, 118, 401-415) and the GUS gene upstream. Was ligated to HindIII-XbaI-cut pBI101 (Jefeerson et al., 1987 EMBO J. 6, 3901-3907) (to produce pBINΔUAS-GUS). The sequence corresponding to the −90 region of the 35S promoter was ligated using oligonucleotide DELAS1 (GAA CTC TAG AAG CTA CTC CAC GTC CAT AAG GGA CAC ATC ACA ATC CCA CTA TCC TTC GC; SEQ ID NO: 14) and GUSSnaBI (see above) PCR amplified. This product was cut with XbaI-BamHI and cloned into similarly cut pBINΔUAS-GUS. The resulting plasmid (pBIN UAS (-90-AS1) GUS) contains a synthetic GAL4 promoter upstream of the β-glucuronidase (GUS) coding sequence. Next, the GUS gene was replaced with that of green fluorescent protein (GFP). GAL4-responsive mgfp5-ER gene To optimize expression in plants, we have extensively mutagenized green fluorescent protein. The optimized gene is called mgfp5-ER (Siemering et al., Current Biology 6: 1653-1663, 1996; WO96 / 27675). The mgfp5-ER gene was cloned into a plant transformation vector pBI121, a BamHI-SacI fragment containing the coding sequence was excised, and inserted into a BamHI-SacI cut pBIN UAS (-90-AS1) GUS to replace the GUS gene. did. This plasmid (pBIN UAS (-90-AS1) mgfp5-ER) was cut with SacII and ligated with a SacII fragment containing the modified T-DNA right border and the mGAL4-VP16 gene derived from pTr + 35S-mGAL4-VP16. Thus, pBIN35S-GAL4-VP16 + UAS-mgfp5-ER (abbreviated as pBIN35S-GAL-GFP) was obtained. Testing of the pBIN35S-GAL-GFP vector The pBIN35S-GAL-GFP plasmid was introduced into Agrobacterium tumefaciens strain LBA4044 by electroporation followed by selection on plates containing kanamycin. This Agrobacterium strain was used for the transformation of Arabidopsis thaliana using the technique described below. After transformation, plants were scored for GAL4-dependent induction of GFP fluorescence. Construction of GAL-GFP enhancer trap vector The pTR + 35S-mGAL4-VP16 plasmid was treated with BamHI (to excise the 35S promoter sequence) and ligated again using T4 DNA ligase. This plasmid (pTR-mGAL4-VP16) contains a modified GAL4-VP16 located within the minimal promoter adjacent to the right border of T-DNA. Next, the T-DNA sequence was cut out by digestion with SacII, and ligated to pBIN UAS (-90-AS1) mgfp5-ER that was similarly cut. This produced pBIN GAL-GFP, which contains a highly active GAL4-VP16 transcriptional activator and a GAL4-dependent GFP gene, which responds to adjacent enhancer elements. GFP fluorescence occurs in response to GAL4-VP16 activation. Enhancer trap screening This enhancer trap vector pBIN GAL-GFP was introduced into Agrobacterium tumefaciens (see details below) LBA4044 and used to generate over 7,500 transformed Arabidopsis seedlings. Transgenic seedlings were screened directly for GAL4-mediated GFP expression and seeds were collected from the expressing plants. We have a library of over 250 Arabidopsis thaliana lines that show a stable genetic pattern of GAL4-mediated GFP expression in roots. Transactivation To demonstrate GAL4-mediated transactivation of exogenous genes, transgenic Arabidopsis thaliana containing a T-DNA sequence derived from pBIN UAS (-90-AS1) GUS was generated. In the absence of GAL4-VP16, these plants do not express the GUS gene. However, when genetically crossed with a system that expresses the mGAL'4-VP16 gene, the GUS gene is activated with an expression pattern that accurately reflects that of the mGAL4-VP16 gene from the donor plant. (See Example 3 below). Example 3 The Arabidopsis stable transformation system (J2302), which forms part of the library described above, expressed the modified GFP (under the influence of the mGAL4-VP16 activator) in the most advanced cells of the root. FIG. 4a is a low magnification micrograph showing GFP-mediated epifluorescence (400 nm excitation) at the root tip. FIG. 4b is a higher magnification confocal micrograph (488 nm excitation) showing the area of GFP-mediated fluorescence in more detail. This J2302 line was crossed (using standard techniques) with another Arabidopsis line containing a stable dormant GUS reporter gene operably linked to a GAL4-responsive UAS. The products of this cross continued to express GUS, as expected, under the influence of the GAL4-VP16 transcriptional activator, in the same pattern as the GFP reporter gene in J2302 (ie, at the very tip of the root). Appropriately stained samples imaged by brightfield microscopy are shown in FIGS. 4c and 4d, which show the activity of the GUS reporter gene at the root tip (highest staining). This means that the modified GAL4 DNA binding domain sequence can be used successfully in (i) constructing enhancer trap vectors; (ii) predict the gene of interest (as exemplified by GUS) And (iii) can regulate the co-expression of multiple genes of interest (as exemplified by GFP and GUS). Example 4 Transformation of Arabidopsis thaliana The method used was Valvekens et al. 1988, Proc. Natl. Acad Sci. 85, 5536-5540. Media Unless otherwise stated, all procedures were performed aseptically using sterile solutions and equipment. All media is used in standard plastic disposable Petri dishes or, at a later stage, in a Magenta GA7 pot (Magenta Corp., USA). Hormones are dimethyl sulfoxide as x1000 stock solution. (DMSO) Hormones and antibiotics are added after autoclaving the medium and cooling to 65 ° C. 1. Germination medium (GM): 1 × Murashige and Skoog salt mixture; 100 mg / l inositol; 1.0 mg / l thiamine; 0.5 mg / l pyridoxine; 0.5 mg / l nicotinic acid; 0.5 g / l 2- (N-morpholino) ethanesulfonic acid (MES); (pH 5.7 with 1 M KOH) And 0.8% Difco Bacto agar (for solid GM media) 1a.GM K50 Same as GM, but supplemented with 50 mg / l kanamycin (Sigma) 2 Callus induction medium (CIM): 1 × Gamborg's B5 medium; 2% glucose; 0.5 g / l MES (pH 5.7, adjusted with 1N KOH); 0.5 mg / l 2,4-D (Sigma); 0.05 mg / l kinetin ( And 0.8% agar (for solid CIM medium) 3. Sprout induction medium containing vancomycin (SIM V750K50): Gamborg's B5 medium; 2% glucose; 0.5 g / l MES (pH 5.7) 0.8% agar; 5 mg / l N6- (2-isopentenyl) adenine (2 ip); 0.15 mg / l indole-3-acetic acid (IAA); 750 mg / l vancomycin; and 50 mg / l kanamycin 3a SIM V500K50 Same as SIM, but supplemented with 500 mg / l Bancoma isin; and 50 mg / l kanamycin 4. Root induction medium (RIM): Gamborg's B5 medium; 2% glucose; 0.5 g / l MES (pH 5.7); 0.8% agar; 12.5 mg / l indolebutyric acid (IBA); and 50 mg / l cefotaxime. (I) Put the seeds in a 15 ml polypropylene centrifuge tube (ii) Add ethanol for 2 minutes, remove the ethanol with a pipette (iii) 5% commercial bleach containing 1 drop of NP40 per 50 ml (~ 0.25% available chlorine). Leave for 15 minutes with regular stirring. (Iv) Wash the seeds with sterile distilled water at least three times. (V) After the last wash, add sterile seeds to 100 ml of GM in a conical flask. Grow in a culture room for 2-4 weeks with shaking at 20-25 ° C. Agrobacterium-mediated transformation Day 1 (i) Using a scalpel, cut out all of the green (upper) portion of the seedling, leaving only the root system. (Ii) lay the roots on the plate containing the solid CIM. Gently press the unity roots on each to ensure that they make contact with the surface of the agar. (Iii) Incubate in growth chamber for 3 days (22 ° C., continuous light). 2． Day 4 (i) After ensuring no visible signs of contamination, the callus-induced roots are collected in an empty Petri dish. (Ii) Cut the roots into 0.5 cm explants. (Iii) Add 3-5 ml of Agrobacterium culture grown overnight at 28 ° C. in Luria broth and wash by centrifugation. Stir with blunt tweezers. Leave for 2 minutes for co-culture. (Iv) Dry the roots with double-thick sterile filter paper (Whatman No. 1) in a Petri dish. (V) Place these explants on solidified CIM medium in a Petri dish and gently press against the surface to ensure contact with the medium. (Vi) Incubate plates in growth chamber for 2 days to allow co-cultivation of Agrobacterium. 3． Day 6 (i) Transfer the explant to a Petri dish in 20-25 ml water. Agrobacterium is washed off by stirring with blunt forceps. The bath medium should be somewhat turbid. Transfer root pieces to sieve and repeat washing. Thereafter, the sieve is withdrawn from the buffer to drain the water. The explant is pressed into a mesh to remove as much buffer and Agrobacterium as possible. The semi-dried implants are pressed together to clump. (Customized, autoclavable and reusable sieves are used for Agrobacterium infection and graft retention during washing of root explants. These sieves are 100 ml plastic triangular beakers and 100 um nylon mesh. Cut the top 3-4 cm of the beaker, hold the mesh in place by passing the mesh to the bottom of the part and pushing a 2 cm high loop cut just above the base of the beaker into the bottom of the top The two parts are sealed to each other by pressing a hot metal rod through them in several places.) (Ii) Moisten a small bundle of root material on a double-thick sterile filter paper in a Petri dish until dry. Remove and transfer to solidified SIM V750K50 medium, taking care that the root explants are in close contact with the medium. (Iii) Incubate in growth chamber. Regeneration (i) In the growth chamber, small green kanamycin resistant calli appear on the yellowish root explants after 3 weeks. (Ii) Every two weeks, transfer the graft to fresh SIM V750K50. Over the next few weeks, shoots, often initially vitreous, emerge from green calli. (Iii) transfer these shoots to RIM. Here, roots develop over a couple of weeks. (Iv) After roots are formed, transfer the plants directly to GM or soil in a ventilated magenta GA7 pot. Ensure that the humidity is kept low to ensure good seed set. (V) Harvesting the pods when they turn yellow-brown and therefore mature. Germination and Screening of F1 Progeny F1 seedlings are germinated on GM K50 to test for transformation. (I) Place the seeds on GMK50 (if necessary, sterilize the seed surface). (Ii) Put the Petri dish in a dark place at 4 ° C (refrigerator) for 3-5 days to release the dormant state of the seeds. This is unnecessary if the seed has been stored for more than a month. (Iii) Incubate the Petri dish in the growth chamber for 2 weeks. Susceptible seedlings do not form roots or leaves and have white cotyledons. The transformed seedlings are phenotypically normal. (Iv) Introducing the transgenic calli and shoots with an inverted fluorescence equipped with a filter set (Leitz-D excited BP355-425, dichroic 455, emitted LP460) suitable for the main 395 nm excitation and 509 emission peak of GFP. Screened for GFP expression using a microscope (Rights DM-IL). A longer working distance is obtained by using a 7 mm threaded extension tube with a 4 × objective (EF 4 / 0.12), allowing convenient direct observation of the tissue in an inverted sealed petri dish. A 100 watt long wavelength portable UV lamp (UV Products, B100AP) was also used for routine observation of transgenic shoots and plants. Transgenic Arabidopsis F1 seedlings were grown in sterile agar culture for 5 days and mounted in water under a coverslip for microscopic examination. The sample was subjected to a krypton-argon laser and a filter set suitable for the detection of fluorescence and Texas red dyes (BioRad Ki / K2), and a Nikon 60 × PlanApo NA. 1.2 Investigated using a Bio-Rad MRC-600 laser scanning confocal microscope equipped with a water immersion objective.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] April 17, 1998 (April 17, 1998) [Correction contents]                                The scope of the claims 1. Expressable in plant cells and effective at least for GAL4 DNA binding domain And the percentage of A / T bases is substantially reduced relative to the sequence of the wild-type yeast. Wherein the A / T base content% is less than 45%. Nucleic acid sequence. 2. Claimed in claim 1, characterized in that the% A / T base content is less than 40% Nucleic acid sequence. 3. A structure operably linked to a sequence encoding an effective portion of a GAL4 DNA binding domain. 3. The sequence according to claim 1 or 2, further comprising a construction and / or regulatory sequence. 4. The nucleic acid is operable with a sequence encoding an effective portion of a GAL4 DNA binding domain Encoding a peptide or polypeptide having a transcription activating activity, The sequence according to any one of claims 1 to 3. 5. The sequence has transcriptional activation activity in a plant cell, GAL4, VPA16, GCN 4 or a peptide or polypeptide that is a modified part of a designed sequence that does not occur in nature The sequence according to any one of claims 1 to 4, which encodes a tide. 6. A nucleic acid construct comprising the sequence according to any one of claims 1-5. 7. Insert the sequence according to any one of claims 1-5 into the genome of the plant host cell 7. The construct of claim 6, wherein the construct is capable of: 8. A reporter gene operably linked to the GAL4-responsive upstream activation sequence (UAS) 8. The construct according to claim 6, further comprising: 9. Encodes GUS or green fluorescent protein (GFP) suitable for expression in plant cells 9. The construct according to any one of claims 6, 7 or 8, comprising a sequence that: Ten. The structure according to any one of claims 6 to 9, comprising a T-DNA border or a Ds element. Built body. 11． The expression of the sequence according to claim 1 is an enhancer-dependent plant promoter. 11. The construct according to any one of claims 6 to 10, which is under the control of a human. 12． 12. The method according to claim 6, further comprising a selectable marker gene. A construct according to claim 1. 13. It comprises or comprises the nucleic acid sequence according to any one of claims 1 to 5. A plant transformed with the construct according to any one of Items 6 to 12, or a plant transformed therewith. Part of. 14. The method according to claim 13, wherein the sequence is stably maintained in a plant genome. Plants or parts thereof. 15． A library containing a plurality of plants or parts thereof, wherein each plant or Contains the nucleic acid sequence according to claim 4 or claim 5, a part of which is stably incorporated. The sequence of the reporter gene in a known temporal and / or spatial pattern. The library that produces 16． The nucleic acid construct according to any one of claims 7 to 12, wherein a plurality of plants or 16. The library according to claim 15, wherein the library is produced by introducing a part of the library. 17． The library according to claim 15 or 16, comprising a plurality of Arabidopsis plants. . 18． Expresses genes of interest in known patterns in plants or parts of them And introducing the gene of interest into the plant or a part thereof. Wherein the gene of interest is a GAL4-responsive upstream activation sequence (UAS) Wherein the plant or a part thereof is the sequence according to any one of claims 1 to 5. Of a transcriptional activator containing an effective portion of the GAL4 DNA binding domain encoded by A reporter gene expressed in a known pattern under the influence, and Binding of a transcriptional activator to UAS results in transcriptional activation of the gene of interest The method as described above. 19. The library according to any one of claims 15, 16 or 17, wherein the gene of interest is 19. The method of claim 18, wherein the method is introduced into one or more plants or parts thereof from Lee. . 20. Regulate the expression of multiple genes of interest in plants or parts thereof Wherein each of the genes of interest functionally associates with a GAL4-responsive UAS. Wherein the plant or a part thereof is according to any one of claims 1 to 5. Expresses a transcriptional activator that contains the sequence and contains an effective portion of the GAL4 DNA binding domain Do It is therefore possible to bind the transcriptional activator to UAS and Wherein the transcriptional activation of all genes occurs simultaneously. twenty one. If at least some of the genes of interest are arranged bolicitronically, 21. The method of claim 20, wherein said method is associated with one single GAL4-responsive UAS. twenty two. Claim that the gene of interest is functionally associated with each GAL4-responsive UAS Item 20. The method according to Item 20,

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), EA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, G E, HU, IL, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, P L, PT, RO, RU, SD, SE, SG, SI, SK , TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU

Claims (1)

[Claims] 1. Expressable in plant cells and effective at least for GAL4 DNA binding domain And the A / T base content is substantially reduced relative to the sequence of the wild-type yeast. Nucleic acid sequence. 2. A structure operably linked to a sequence encoding an effective portion of a GAL4 DNA binding domain. The sequence according to claim 1, further comprising a construction and / or regulatory sequence. 3. The nucleic acid is operable with a sequence encoding an effective portion of a GAL4 DNA binding domain Encoding a peptide or polypeptide having a transcription activating activity, 3. The sequence according to claim 1 or 2. 4. The sequence has transcriptional activation activity in plant cells, GAL4, VP16, GCN4 Or a peptide or polypeptide that is a modified part of a designed sequence that does not occur in nature 4. The sequence according to any one of claims 1, 2 or 3, which encodes a code. 5. A nucleic acid construct comprising the sequence according to any one of claims 1-4. 6. Insert the sequence according to any one of claims 1-4 into the genome of a plant host cell 6. The construct of claim 5, wherein the construct is capable of: 7. A reporter gene operably linked to a GAL4-responsive upstream activation sequence (UAS) The construct according to claim 5, further comprising: 8. Encodes GUS or green fluorescent protein (GFP) suitable for expression in plant cells 8. The construct according to any one of claims 5, 6 or 7, comprising a sequence that: 9. The construct according to any one of claims 5 to 8, comprising a T-DNA border or a Ds element. Ten. Expression of the sequence according to claim 1-4 is an enhancer-dependent plant promoter. 10. A construct according to any one of claims 5-9, which is under control. 11． The method according to any one of claims 5 to 10, further comprising a selectable marker gene. The above-mentioned construct. 12． It comprises the nucleic acid sequence according to any one of claims 1-4, or alternatively, it comprises 12. A plant transformed with the construct according to any one of 11 or a part thereof. 13. The method according to claim 12, wherein the sequence is stably maintained in a plant genome. Plants or parts thereof. 14. A library containing a plurality of plants or parts thereof, wherein each plant or Contains the nucleic acid sequence according to claim 3 or 4, wherein a part thereof is stably incorporated. The sequence of the reporter gene in a known temporal and / or spatial pattern. The library that produces 15． A nucleic acid construct according to any one of claims 6 to 11, 15. The library according to claim 14, which is produced by partially introducing the library. 16． The library according to claim 14 or 15, comprising a plurality of Arabidopsis plants. . 17． Expresses genes of interest in known patterns in plants or parts of them And introducing the gene of interest into the plant or a part thereof. Wherein the gene of interest is a GAL4-responsive upstream activation sequence (UAS) Wherein the plant or a part thereof is according to the sequence of any one of claims 1-4. Of a transcriptional activator containing an active portion of the GAL4 DNA binding domain encoded by And a reporter gene that is expressed in a known pattern below Binding of an activator to UAS results in transcriptional activation of the gene of interest. The method as described above. 18． The library according to any one of claims 14, 15 or 16, wherein the gene of interest is 18. The method of claim 17, wherein the method is introduced into one or more plants or parts thereof from Lee. . 19. Regulate the expression of multiple genes of interest in plants or parts thereof Wherein each of the genes of interest functionally associates with a GAL4-responsive UAS. Wherein the plant or a part thereof is a sequence according to any one of claims 1-4. And a transcriptional activator that contains an effective portion of the GAL4 DNA binding domain It is therefore possible to bind the transcriptional activator to UAS and Wherein the transcriptional activation of all genes occurs simultaneously. 20. If at least some of the genes of interest are polycistronically arranged, 20. The method of claim 19, wherein said method is associated with one single GAL4-responsive UAS. twenty one. Claim that the gene of interest is functionally associated with each GAL4-responsive UAS Item 19. The method according to Item 19.