JP2000504577A - Regulation of gene expression in eukaryotes - Google Patents

Abstract

(57) Abstract: Cells are transformed with a transformation cassette that expresses a eukaryotic active gene or a modulator gene product that controls the product and a modulator gene or a further gene product that controls the product. Eukaryotic active cell genes, characterized in that the two genes, the modulator gene and the promoter of the further gene, are selected from inducible and developmental promoters for the same or complementary tissues. Provide a way to control The lethal gene expressing barnase, a ribonuclease of B. amyloliquefaciens, is placed under the control of a tissue-specific promoter derived from PrMADS1, 2 or 3 of Pinus radiata or EGM1, 2 or 3 of Eucalyptus grandis. The same tissue-specific promoter is used to express the Laclq gene, but the repressor of barnase (barstar) is promoted by a modified 35sRNACaMV promoter containing the lac operon. The cassette is used to transform regenerative plant cells into plants expressing barnase in a target tissue with improved specificity and reduced promoter leakage.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Regulation of gene expression in eukaryotes   The present invention relates to a method for controlling gene expression in eukaryotes.   The present invention relates to the regulation of highly specific expression in target organs or functions of plants. Has a unique but not exclusive application to Will be mentioned. However, the invention does not address the organs and functions of yeast and animal targets. Other regulation, such as regulation of expression of other eukaryotic genes, such as regulation of specific expression in It could also be used in applications.   Eukaryotic organisms of commercial interest including lower animals such as plants, animals and yeast Often convert habitats to non-commercial structures, i.e., Express different genes. Another commercial species introduces features that will increase commercial value. Benefit from your entry.   For example, the development of reproductive structures in forest trees implies a significant burden on tree resources. And reproductive efforts come at the expense of tree growth. Mature tree seeds To prevent competition for soil resources and also minimize the risk of catastrophic wildfires To cause poor growth of young trees that must be removed on a regular basis.   Australian Patent Specification No. 86224/91 increases plant growth in certain plants A method for increasing the size is disclosed. This specification describes the induction by the flowering regulator gibberellin A4 / 7. Induction, early appearance of gene products, and expression characteristics in both male and female reproductive buds. Essentially tissue-specific processes for reproductive structure-specific genes on a heterosexual basis Figure 4 illustrates the method by which the motor (growth promoting substance) was selected. Genes and promoters Is a gene that includes an isolated and characterized tissue-specific promoter for the gene Some of the offspring were fused to a ribonuclease structural gene and the gene was modified .   Transformants show a lack of reproductive structure, but promoter leaks into non-target tissues Are likely to accumulate low levels of lethal ribonuclease in And thus reduced its commercial potential.   Gene expression promoters are generally considered to correspond to one of three broad classes be able to. Constitutive promoters express genes continuously in all tissues. Developmental promoters are expressed in specific tissues or at specific stages . Inducible promoters include the presence of metabolites, exogenous compounds or light, heat or pressure. Respond by switching expression on or off in response to other stimuli.   Barnase barnase under the control of a promoter that is essentially specific to flower tissue Expression of a lethal gene product is dependent on the inhibitor (B 'or "Bars Become more tissue-specific by constitutive coexpression of tar balstar “)” It has been known. Inhibition of gene products expressed in target cells Will occur where it is insufficient to control the action of Non-target cell Barnase activity resulting from some degree of non-specificity or promoter leakage Sex is essentially suppressed.   However, inserted promoters are often leaky and better Stronger promoters have other inhibitors with insufficient inhibition to prevent non-target cell death. Try to be harmful in tissues, which adversely affects commercially important non-target tissues Let's sound. Promoter leaks are less predictable and transformation of, for example, trees Gene production in non-target cells if the body has long growth by the first flowering The resulting activity of the product will eliminate or reduce the benefits of flowering control. Generally more favorable Suitable promoters limit the choice of promoter.   The present invention substantially alleviates at least one of the above disadvantages and provides eukaryotic gene expression. Controlling expression and eukaryotic gene expression that will be reliable and efficient in use Aims to provide a method of control. Other objects and advantages of the invention will be apparent below. Would.   With the foregoing and other objectives in mind, the present invention relates to eukaryotic genes or their production. (Modulator) gene product that regulates a product The gene or its product, the two promoters, the modulator gene and And inducible promoters and developmental promoters for the same or complementary tissues A structure that expresses the product of a further gene that controls a further gene selected from the Active eukaryotic inheritance consisting of transforming cells with structures There are certain aspects of how to control children.   Genes that are active in eukaryotes constitute genes that are innate in the organism of interest, or Or make up the derived gene. Genes are driven by native promoters Or construct a structure that is non-homologous to the gene. Active eukaryotic remains The gene will code for an expression product of any chosen cellular function. example Gene has a lethal effect on ribonucleases in target tissues and nematode resistance in root tissues For active polypeptides including enzymes such as peroxidase that imparts Let's code. Genes include pigments, dyes such as anthocyanins, and Bti toxins. For insecticide compounds or products that allow a route to the production of fragrances Try.   Expression of the reporter gene under the control of an inducible promoter is transmitted to surrounding cells. Show the exact location of target cells in the target tissue without leakage. This is the pathogen Very useful for studying the early stages of interactions between plant and insect and plant cells. Would. GUS, Luc, anthocyanin gene and GFP are candidates for reporters Let's configure. Colorimetry or fluorescence analysis is a small-scale promotion Made from quality.   As used herein, the expression “lethal gene” refers to a peptide or an antigen. Kills target cells by essentially disrupting antisense or ribozymes or target cells Means the gene (or genes) encoding other non-peptides that lead to It is said. The invention provides a reference to an embodiment that constitutes an effective lethal gene in a eukaryote. Along with it will be described below.   A lethal gene can be any gene, or peptide or antisense or A ribozyme, which essentially disrupts the target cells and kills them there. It may be a combination of genes to be encoded. For example, a lethal geneB.amyloliquefaciens Barnase, fromA.aryzaeRN AIDS T1, Bovine RN AIDS  A,E . coli RNases I and RNases H and a set of plants RNases from (A population of S-proteins). . Instead, the lethal gene is a population of restriction endonucleases, 1-4-β-gluca. Enzymes containing glucanase, such as enzyme and 1-3-β-glucanase, ubiquitin, Acidic It may be selected from pyrophosphatase and plant cell wall synthesis inhibitors.   Preferably, the lethal gene is RNase. The chimeric lethal gene is Upstream (promoter) expressed early in development in the male and female parts of the plant reproductive organs -) Under the control of the array,B.amyloliquefaciens Coding region of Barnase from Will be built including   During the first event of a pathogen-host plant interaction on the target cell, Thus, restriction of the expression of the lethal gene may be achieved. For example, in an attacked cell Expression of Barnase or other toxins such as β-glucanase and chitinase It will lead to necrosis of these cells which will prevent the spread of infection to the cells. Artificial The critical step in a hypersensitivity project is the possible promoter in peripheral cells -To shut off the leak.   Expression of the bacterial salicylate-hydroxylate gene is found in all plants Monkeys responsible for systemic expression of the cassette under the control of a pathogen-inducible promoter Will reduce the level of citrate. For example, one pathogen-inducible promoter is Powdery mildewEresfy graminis (Mouradov et al. 1993) Will be separated from strongly expressed barley after being processed. Clones of this gene's genome Were isolated and characterized (Mouradov et al. 1994). This promoter territory The region will be fused to a cassette containing genes that are lethal or of different functionality.   Has homology to other plant MADS-box genes and is named EGM1, 3 and 2. Three Eucalyptus tree genes have been cloned and sequenced. Phylogenetic analysis Performed to test the association of eucalyptus tree genes with other MADS-box genes Was. From now on, EGM2 has been linked to the development of petals and stamens in GLOBOSA MADS-bodies. EGM3 and EGM1 are both MADS-box It was shown to be consistent with the offspring AGL2 group. These genes are most commonly found inside flowers. It is expressed in three ring organisms (petals, stamens and pistils).   The expression pattern of all three EMG genes was characterized. What are the expression of the three genes It could not be found in plant tissues. EGM1 gene is eucalyptus flower petal, stamen And expressed in pistils. EGM3 is expressed in flower growth points and sepals. EGM2 Is expressed in petals and stamens. EGM3 and EGM2 genes were isolated by Southern blotting. one Gene. EGM1 also seems to be a single gene.   Promoters of all three genes to be used in the sterility gene construct The region was isolated from a eucalyptus tree gene library and manipulated into a lethal gene construct. Let's make it.   This eukaryotic active gene, modulator gene and additional genes and Each of these promoters can be used in any manner by which the target organism is known. Let's construct a transformation cassette that can be transformed. Active in eukaryotic cells Promoters for different genes, modulator genes and additional genes (or multiple genes) Are combinations that enhance the specificity of expression of active genes in eukaryotic cells. Is selected. For example, the gene active in eukaryotic cells is a modulator gene. The offspring may be constitutive but driven by an inducible or developmental promoter. Let's get it. The specificity and usefulness of even weak promoters is With a promoter with the same specificity used to drive the gene. It may be enhanced by promoting the gene (or genes) that comprise it.   Instead, tissue-specific promoters for eukaryotic active genes are constructed Is likely to be inhibited by the stimulatory inhibitor gene product, which is then inducible Is controlled by additional gene products that are promoted by   Still further, gene products that are active in eukaryotes may be based on certain tissue-specific processes. For motor, modulator genes or different or complementary tissues Controlled by the expression of additional genes under the control of a specific promoter Modulator of a modulator gene expressed under the control of a controlled product -Be constitutively expressed in response to the effect. In this way, for example, confer insect resistance Constitutively promoted genes are inherited under the control of a seed-specific promoter. Focus on the effect of expressing seed inhibitors and eliminating seeds, and the gene inhibitors themselves Is an additional gene expressed under the control of a promoter specific to non-seed tissues Will be controlled by product.   In a further aspect, the present invention provides a method for producing a eukaryotic gene, comprising the steps of: A modulator gene that expresses the gene or a product that controls the product, and And the modulator genes and their products, the promoters of the two genes, Gene and an inducible promoter and developmental promoter for the same or complementary tissue. Further express certain products that control additional genes selected from To transform a certain cell with a transformation cassette constituting a gene. Thus, there is a wide range of methods for controlling the expression of eukaryotic active genes.   In a further aspect, the invention relates to a gene active in a eukaryote, the eukaryote A modulator gene expressing a gene or a product controlling the product thereof, and The modulator gene and its products, the promoters of the two genes, the modulator Inducible promoter for the promoter gene and developmental promoters for the same or complementary tissues. Further express certain products that control additional genes selected from Are widely present in transformation cassettes that constitute genes.   In a still further aspect, the present invention provides a eukaryotic cell capable of growing Widely present in one method of transformation with an expression cassette containing the following genes:   Cytotoxicity in target cells under the control of a promoter essentially specific for the target tissue A lethal gene expressing a sexual peptide;   A model constitutively expressing an inhibitor of the production and action of the cytotoxic peptide. The durator gene, and   The inhibitor or modulator under the control of the essentially specific promoter of interest. Additional genes that function to block the translator gene.   In a further aspect, the present invention provides an expression cassette comprising the following genes: Exists widely:   Under the control of an inducible promoter essentially specific for the target tissue, the target tissue A gene that expresses a cytotoxic peptide;   Inhibitor gene expressing an inhibitor of the production and action of the cytotoxic peptide And under the control of the inducible promoter, the inhibitor or the inhibitor gene A repressor gene that functions to block offspring.   In a further aspect, the present invention providesB.amyloliquefaciensAnd P from the extract of 2 (for Barstar) and P3 and P4 (for Barnase), and Associated with primers for PCR separation of the resulting Barnase and Barstar genes:   The cytotoxic effect of the lethal gene expression product is determined by the localization signal sequence Would be increased by conjugating For example, more preferred RNase cells Toxic effects target the nucleus where most precursor and mature RNAs exist in unprotected form. Will be increased. For this purpose, for example, the Barnase gene The coding region fuses the nuclear localization signal (NLS) sequence to the RNase gene. Will be qualified by   Therefore, in the following aspects, the present inventionA.tumefaciensNL from VirD2 gene A set of PCR primers for fusing the S sequence to the Barstar gene, comprising: Provide:  Tissue-specific or developmental promoters are used early in flower development in plant reproductive organs. Selected from these tissue-specific promoters that control the gene during the Let's go. These genes are suppressed in all plant organs during the non-flowering period. And is well induced in the reproductive tract during the flowering period.   The developmental promoter could be isolated in any known manner. For example, MRNA that is only present during the development of the target tissue is identified and isolated and these specific cDNA from mRNA is generated and used to search genomic libraries. Cattle then identify parts of the plant genome that contain the promoter region of these genes Let's do it. Tissue-specific promoters may be homologous (unchanged) Same (exotic). For plant reproductive organ tissues, the promoters are male and female In the cells of various flower tissues in both organs: sepals, petals, ovary, style Promoters that are expressed only in specific tissues such as, stigma, ovule, corolla, etc. Let's choose from The production of illegitimate plants occurs early in the development of both male and female organs. Promoters expressed in tissues are more preferred.   For example, homeotics play an important role in flower development Genes encoding the MADS box gene are expressed early in flower primordium development I do. We are known as AGAMOUS AGL-2 and AGL-4 genesArabidopsisE MADS gene showing strong homology to meotic MADS box geneP.radiataFrom separated. These genes are expressed in young flower primordia and are expressed at the inflorescence growth point. Absent.   A gene encoding an inhibitor of a lethal gene product can increase the expression of a lethal gene. It may constitute a repressing gene and a gene that transcribes antisense RNA. Or a gene expressing a protein that can inactivate a lethal protein Will be composed. An integrated operator-repressor system is used. As you might imagine, this inhibitor gene usually produces the inhibitor in non-target tissues. It will be under the control of the expression of the constitutive promoter that appears.   For example, the Barnase gene / reproductive organ tissue-specific promoter system is an anti-Barnase ( B * or Barstar) gene / promoter system.   Instead, the bacteriophage lambda N gene andE.coliExpression of SOS system Could be used with the expression cassette. Bacteriophage lambda The expression of the N gene is^LIt is tightly controlled by the promoter and Controls the expression level from the promoter. This system inhibits target tissue Will be used to provide the necessary negative control for expression of the factor gene.   The SOS system is controlled by the interaction of two proteins. Transcription repressor (LexA) inhibits transcription initiation through specific binding to its operator target . The RecA protein blocks the LexA protein, thus inhibiting its specific site prevent. This would be a one-part lethal gene system and would require a toxin-blocking gene. Will not.   The repressor gene under the control of an inducible promoter is To suppress the action of transcription, translation of RNA transcript, or the peptide product itself Will be selected. More preferably, the repressor gene is the expression of an inhibitor gene. Encodes a peptide product that directly suppresses expression. Therefore, repressor genes and The agent gene is suitable for forming a specific repressor / operator conjugate pair. It is more preferable that they are combined. For example, the same tissue specificity Under motor controlE.coli The laclq gene is inserted by inserting the lac operator sequence. Used to suppress the action of the modified repressor gene promoter. Like.   The repressor gene promoter is the promoter and repressor code 35 having at least one lac operator between the marking region Will be the S-RNA-CaMV promoter. The modified 35S-RNA-CaMV template exemplified below In the selection of the promoters, we used amplification of the modified 35S-RNA-CaMV sequence. Primers have been developed.   Accordingly, in a further aspect, the present invention is referred to as 35S1 and 35S2 and comprises the following: Provides primers for PCR amplification of the modified 35S-RNA-CaMV sequence :   Lac ぺ into both the 3 'and 5' regions of the modified 35S-RNA-CaMV promoter For transfection of the sequencer, the sequence will be amplified by the following PCR primers:   Interaction between lactose operon and DNA binding protein (repressor, laclq) The molecular mechanisms of the promoter and target promoter sequences (operators) are very well understood Have been. Lac operon expression is strong and strictly negative from the laclq repressor Under control. The purified repressor is a tetramer, each containing 360 amino acids (MW 38,350 kDa) formed from four identical subunits. operator The sequence comprises 35 base pairs, including a 28 base pair symmetric sequence.   For maximal suppression of inhibitor gene expression, the repressor protein is Is more preferably targeted in the nucleus. This is a nuclear localization signal to the C-terminus of the repressor. This may be achieved by fusion of the null sequence.   In a further aspect, the invention is adapted for isolating the laclq gene and comprises A set of primers with appropriate restriction sites for cloning into The following primers corresponding to the 5 'and 3' ends of the gene and designated as AM1 and AM2 -2 types, related to:   In yet a further aspect of the invention, an NLS sequence is fused at the 3 'end of the laclq gene. A set of PCR primers is provided for use when combining and consists of: Is:   If desired, the method of the present invention could be the reverse. For example, gene The scade would allow fertility-engineered barren inversions, and thus Use molecular elements that allow the production of offspring. The reverse mechanism is anti-repressor genetics By placing the offspring under the control of a chemically switchable promoter . For example, when fertility needs to be restored, plants can Chemicals that induce the expression of anti-repressor RNA that will block inhibition of offspring expression The quality may be sprayed or administered separately. The accumulation of inhibitory products in flower organs It will inhibit lethal gene function and thus allow normal flower formation.   For example, inhibition of the expression of the modified Barstar gene against the described lac / laclq system Chemicals that induce the expression of anti-laclq RNA that will block U. Accumulation of Barstar products in floral organs may inhibit lethal gene function And thus allow normal flower formation. Corn glutathione-s-to The promoter region of the transferase (GTS II) gene is chemically induced Could actually be used. The expression of the GST II promoter is dichloramine (N, N-diallyl) -2,2-dichloroacetamide) and fluorazole (benzyl-2-chloro-4-trifur) Switch on with a chemical substance such as olomethyl-5-thiazole-carboxylate). Let's get started.   In the case of eucalyptus trees, the EGM3 promoter constituting the upstream sequence of the EGM3 gene is It may be used to express the mortality gene specifically in the eucalyptus flower tissue. This The cytotoxic Barnase gene is used in the early stages of development It will be specifically expressed at the flower growth point. Inhibitor Barstar uses the lac operator sequence It will be expressed under the control of the 35S-RNA-CaMV promoter.   Under the control of the EGM3 promoterE.coli The laclq gene controls the flower during the onset of flowering. As a repressor gene to prevent Barstar expression in meristem tissues Will be used. During flowering, the EGM3 promoter regulates Barnase at the flower growth point. And the modified 35S-RNA-CaMV promoter will Expresses Barstar in all tissues except the growth point of E. coli and protects against promoter leakage Will be. During the non-flowering period, the 35S-RNA-CaMV promoter is evenly distributed on Barstar And will protect against leakage.   Nematodes cause global agriculture to lose more than US $ 100 billion annually. Obviously the most important Pathogens are nematodes that attack a wide range of plantsMeloidogvne incognitaandMeloidogv ne javanica It is. Control methods include the use of hazardous chemicals and cultivation such as rotation. Includes habits and the use of resistant varieties in a limited range of crops. Nematode resistance The advantage of operating is that no special cultivation habits are required and the dangers of the environment It is to reduce.   Lethal gene / inhibitor / repressor approaches manipulate resistance to nematodes Can be used for The ideal site for activating plant defense against nematodes is the plant root In feed cells in the vascular area. In some plant-nematode interactions , A gene promoter specifically expresses the Barnase gene at the site of feeding of nematodes To be identified by differential screening that can be used to Recently ,Meloidogvne incognitaAt a giant cell feeding site in tomato root infected with And two genes (Lemmi9 and Lemmi10) expressed at high levels were identified (Van der Eycken et al., (1996) Plant Jounal 9, 45-54).   Expression of a lethal gene in feed cells kills cells rapidly and, in particular, in females. Will disrupt the feeding process, which is very critical for the occurrence. For non-target tissues Inhibitor gene expression in the root causes damage to other parts of the root due to promoter leakage Will be needed to prevent Pro shows high levels of expression in giant feed cells This is particularly important since motors are expressed at low levels even in non-target cells. It is important. Expression of the repressor gene under the feeding site promoter is Expression of the inhibitor gene in the target tissue to allow the offspring to destroy cells The reality must be cut off enough.   To make the invention easier to understand and have practical effects, Reference is made to the accompanying drawings, which illustrate examples and more preferred embodiments of the invention, in which:   Figure 1 depicts the construction of a lethal gene system localized at the Barnase / NLS gene;   Figure 2 shows the lac operon-modified 35S-RNA-CaMV operator / Barstar inhibitor Represents the gene system;   FIG. 3 depicts the construction of a repressor gene system localized in laclq / NLS;   FIG. 4 shows the mechanism of action of the Barnase / Barstar: lac / laclq cascade in accordance with the present invention. Represent;   FIG. 5 represents a different cascade Barnase: op / LexA / RecA according to the invention;   FIG. 6 shows the reverse Barnase / Barstar: lac / laclq / antisense laclq in accordance with the present invention. Represents the mechanism of action of the cascade;   FIG. 7 illustrates the mode of action of the control cassette and the lethal gene / NLS system of FIG. Express a description;   FIG. 8 shows PrMADS1, PrMADS2, and PrMADS3 in reproductive and plant tissues of P. radiata. Represents the expression of the PrFL1 and PrC0N1 genes;   FIG. 9 is expression of PrMADS3 in male and female cones;   FIG. 10 is a gel mobility shift test of PrMADS1, PrMADS2 and PrMADS3;   FIG. 11 is the nucleotide sequence of the EGM1 promoter;   FIG. 12 is the nucleotide sequence of the EGM2 promoter;   FIG. 13 is the deduced sequence of the EGM3 promoter of the Eucalyptus flower-specific MADS gene;   FIG. 14 shows total RNs extracted from eucalyptus tree tissues probed with EGM1 cDNA (exposed for 6 hours). Schematic representation of Northern blot of A;   FIG. 15 shows the total RNA extracted from eucalyptus tree tissues probed with EGM3 cDNA. Schematic representation of the blot;   FIG. 16 shows the total extracted (from eucalyptus tree tissue) probed with EGM2 cDNA (exposed for 6 hours). Schematic representation of a Northern blot of RNA;   Figure 17 shows the predicted tags of the MADS box region of some plant MADS box genes. Alignment of protein sequences;   FIG. 18 is a phylogenetic tree showing the association of some plant MADS box genes ;   FIG. 19 was digested with EcoR1 and HinDIII and searched for EGM3 and EGM2 genes.Eucalvotus grandis Southern blot of DNA;   Figure 20: Eucalyptus tree extracted from eucalyptus tree and probed with EGM3 cDNA (exposed for 6 hours) Northern blot of total RNA of tissue;   FIG. 21 is a schematic illustration of the plasmid 7prom Barstar;   Figure 22 is a diagrammatic representation of the plasmid pBR Barnase prom Barstar containing the V1 promoter Is an explanation;   Figure 23 is a schematic illustration of a plasmid containing the V2 promoter;   FIG. 24 is a diagrammatic representation of the agarose separation of the Pst1 digest of the EGM3 double bin. E Given that the coR1 cloning site is at base 6772 in the EGM3 bin, The fragment sizes predicted from the map for 4.9, 4.4, 3.3, 1.9, 1.1, and 0 .6 kb.   FIG. 25 shows Pst1 deletion of EGM3 bin showing possible different orientations of the cloned insert. 2 is a schematic illustration of two gel exposures of a compound. The expected fragment size from the digest 7.4, 4.9, 4.4, 1.1, 0.7, and 0.6 kb.   FIG. 26 is a plasmid diagram of the EGM3 double bin.   FIG. 27 is a plasmid diagram of EGM3 V1 sense.   FIG. 28 is a plasmid diagram of plasmid V1 (Barnase-Barstar).   FIG. 29 is a plasmid diagram of plasmid V2 (LaclqNSL-35s promoter Op-Barstar). It is.   Figure 30 is a representation of the promoter seeker strategy;   FIG. 31 is the nucleotide sequence of the PrMADS2 promoter;   FIG. 32 is the nucleotide sequence of the PrMADS3 cDNA promoter;   FIG. 33 is the amino acid sequence of the PrMADS3 protein;   FIG. 34 is the nucleotide sequence of the PrMADS promoter;   FIG. 35 is the nucleotide sequence of the PrFL1 cDNA clone;   FIG. 36 is the amino acid sequence of the PrFL1 protein;   FIG. 37 is the nucleotide sequence of the PrFL1 promoter;   FIG. 38 is the nucleotide sequence of the PrCon1 gene (part);   FIG. 39 shows the total RNA extracted from eucalyptus tree tissues probed with EGM2 cDNA (exposed for 3 days). It is a Northern blot.   Example 1   A chimeric reproductive organ-specific expression cassette was constructed. This is a reproductive organ specific pro Under the positive control of the genes encoding the motor and barnase inhibitor Barnase gene from T. amyloliquefaciens, transfected lac operation Bacillus under a modified constitutive promoter (35SRNA CaNV) having a ・ Balster from Amilorique Efficience (B^*), And lethal as shown in FIG. Control of the same reproductive organ-specific promoter used to express the gene Including the negative control of the E. coli laclq gene under the roll.   Reproductive organ-specific promoters are produced during the early stages of flower development, as described below. Includes upstream sequences of genes expressed in reproductive organs. The cytotoxic effect of barnase is Barnase nuclei where most of the reRNA and mature RNA are in unprotected form Increased by targeting. For this purpose, as shown in FIG. The nuclear localization signal (NLS) sequence from the VirD2 gene of A. tumefacieus strain C58 was The coding region of the B gene was modified by fusing to the 3'-region of the B gene. .   5'-, 3'- and 5'-, 3'- of the 35S-RNA-CaMV promoter region Inhibitor Balster (B^*) Gene part The reactor was constructed as shown in FIG.   The same reproductive organ-specific promoter used to express the lethal gene The E. coli laclq gene under the troll is the C-terminal of the laclq protein as shown in FIG. The nucleus was labeled by fusing NLS from the VirD2 gene of A. tumefacieus It has its targeted laclq protein.   Example 2   Another chimeric reproductive organ-specific expression cassette was constructed as in FIG. Where, The reproductive organ-specific promoter and barnase gene are 353-RNA-CaM Under the control of the V promoter, under the positive transcriptional control of the LexA product, and Of the same reproductive organ-specific promoter used to express the lethal gene Under negative control of RecA gene product under troll.   Example 3   A reversal system was constructed substantially according to Example 1. Furthermore, antisense L Maize glutathione S-transferr regulates aclq RNA gene A promoter that can be chemically switched from the zeta (GSTII) gene was included. this thing Results in seed production as shown in FIG. Fertility is restored by dichloramine (N , N-diaryl-2,2-dichloracetamide) or fluorazole (benzyl) -2-chloro-4-trifluoromethyl-5-thiazole-carboxylate) It is achieved by spraying with an inducer such as. This chemistry of anti-laclqRNA Induced expression was determined using the modified 35S-RNA-CaMV-B^*Prevents inhibition of gene expression . B in flower organs^*Accumulation inhibits lethal gene function and normal flower formation Will bring.   Example 4   Modification and insertion of laclq repressor gene into plant expression vector   The pRT99GuS vector was digested with XbaI and KpnI to encode the GUS gene. Release of the region results in a vector suitable for expression in plants.   The laclq repressor can be used in almost all commercial E. coli strains. How many Such commercial plasmids also contain a repressor gene. This remains in plants The prokaryotic translation initiation codon (GTG) was changed into ATG to express the gene. To isolate the laclq gene, two plasmids corresponding to the 5 'and 3' ends of the gene were used. Immers were used (AM1 and AM2). These two primers are contained in a plant vector It has a restriction enzyme cleavage site suitable for cloning into E. coli.   After amplification, the PCR fragment was digested with Xba1 and Kpn1 and the GUS gene (pB R-35Slaclq) (pBR-35SGUS digested with the same enzymes to replace) Connected inside.   Several PCRs were performed to fuse the NLS sequence to the 3 'end of the laclq gene. Was.   Primer   From the VirD2 gene from A. tumefacieus fused to the coding region of the laclq gene The final PCR fragment containing NLS was digested with XbaI and KpnI and Plant expression vector pBR32 digested with the same enzyme (pBR-35Slaclq-NLS) Linked into 2-355 GUS. This vector contains potential homologous recombination XL1Blue was transformed into a JC8697 (recA) E. coli strain to eliminate .   Example 5   Barnase and barstar genes   P1 and B2 (for Barstar), and P3 and P4 (for Barnase) About the Bacillus amyloliquefaciens crude extract using primers These genes were isolated by PCR.   The P1-P2 PCR fragment (barstar) was digested with EcoRI and HindIII. (BS-B)^*) And ligated into Bluescript SK vector digested with Was. BS-B^*Protein expression of BamHI-HindIII fragment of vector Vector pQE32 (pQE3-B^*).   The P3-P4 PCR fragment (Barnase) is a PCR cloning vector Ligation was performed in P / GEM-T (pGEM-TB). NLS sequence at 3 'end of B gene Several PCRs were performed to fuse.   Primer   VirD2 gene from A. tumefacieus fused to the coding region of barnase gene The final PCR fragment containing NLS from the offspring is cloned into a PCR cloning vector. -Ligation was performed in pGEM-T (pGEM-TB-NLS).   Example 6   Introduction of lac operator sequence into 35S-RNA-CaMV promoter   The commercially available plasmid pQE32 contains two lac operators (operators I and II). Have the ideal combination. In the first step, HindII from pRT99GUS vector The I fragment was introduced into the Bluescript SK vector (A1). 35S-RN GUS gene fused into A-CaMV promoter and terminator region The EcoRI-SalI fragment of the retained A1 vector was digested with the same enzymes. It was introduced into the pQE32 vector.   The resulting vector has two lac operons in the upstream region (Dp + 35S-GUS). 35-RNA-CaMV promoter.   lac operator with 35S RNA-CaMV promoter and GUS gene coding region First, an A1 vector carrying the GUS gene without a promoter was introduced. BamHI-SalI fragment of pUC19 vector digested with the same enzymes. Connected in the heater. In the next step, the pUC19-promoter GUS vector Is digested with EcoRI and SalI enzymes and digested with the same enzyme (pQE-promoter GUS). And ligated into pQE32 digested with. 5 'of 35S-RNA-CaMV promoter PCR using the 35S1 and 35S2 primers corresponding to the Thus, the 35S-RNA-CaMV promoter region was amplified.   The PCR fragment was digested with XhoI and SalI, and pQE digested with Xho1. Introduced into the promoter GUS. Clones containing the promoter in the correct orientation The selected plasmid (35S-GUS) was converted from XL1Blue to VC8692 (recA). E. coli strains were transformed to eliminate possible homologous recombination.   A lac operator was used in both the 5 'and 3' regions of the 35S-RNA-CaMV promoter. 35S-GUS vector operator 35S promoter Regions OP1 and 35S2 corresponding to the 5 'and 3'-regions of the 35S promoter. Amplified by PCR using primers.   The PCR fragment was digested with XhoI and SalI and the pQE plasmid digested with XhoI. Introduced into the Motora GUS. Select clones containing the promoter in the correct direction And the resulting plasmid (35S-promoter GUS) was transferred from XL1Blue to JC869. 7 (recA) E. coli strain to eliminate possible homologous recombination. Was.   The bulster gene was ligated into the pQE promoter GUS vector by EcoRI and Kpnl. After digestion of the individual, it was cloned.   One difficulty with tissue resection is leakage and expression of the promoter in other tissues. The effect is. To overcome this, a control system (gene cascade) Developed. This allows the expression of lethal genes to be counted in non-target tissues. (FIG. 7). Barnase (V1) and Barstar plus these cascades Two vectors carrying the receptor (laclq) (V2) portion were designed.   To clone the promoter region into vectors V1 and V2, And EcoRI control for the SalI restriction site at the end (SalI primer) and V2. Amplification was performed using a primer set having a restriction site (EcoRI primer). V1 EcoRI restriction site to look for promoter orientation in V2 and V2 vectors. Position is designed downstream from the SalI site to the SalI promoter, and the SalI site is An EcoRI primer downstream from I was designed.   Sa1I primer       PrMADS2 promoter: PrMADS3 promoter: PrFL1 promoter:       EcoRI primer:       PrMADS2 promoter: PrMADS3 promoter: PrFL1 promoter:   The PCR fragment of the SalI primer is digested with SalI restriction enzyme and digested with SalI. It was introduced into the digested V1 vector. Direction of the promoter to digestion with EcoRI enzyme It examined using.   The PCR fragment of the EcoRI primer was digested with EcoRI restriction enzyme, It was introduced into the V1 vector digested with RI. Turn off promoter direction with SalI enzyme It was examined using the chemical formula. A map of the resulting plasmid is shown in FIGS.   These vectors were linearized with the HindIII enzyme and the Hin19-digested Bin19 plasmid. Introduced into the Inari vector. Colonies carrying the modified Biu19 vector Selected on plates with namycin and ampicillin antibiotics. The next step To transfer the Bin19-V1-promoter and B19V2-promoter vectors. Several Agrobacterium strains were transformed. Arabidopsis Sariana, You Agrot embryos and explants from Calyptus grandis and Pitus radiata Co-transformed with the bacterial strain.   Plasmid pRT99qus (Topfer et al. NuclEic Acids Research (198 8) The HindIII fragment from 16 (17): 8725) was converted to the Hind of pBR322. It was cloned into the III site. This insert corresponds to an insert that is cloned in both directions. Two types of plasmids were generated. The insertion area is cauliflower mosaic Rus (CaMV) 35S RNA promoter, β-glucoronidase (GUS) gene And a CaMV35S terminator region. These plasmids are called pBrGus 1 and pBrGus2 were used as the basis for the lethal gene construct. In the insertion The orientation was determined by BamHI-digestion. pBrGus2 is a 2.5 kb and 4.4 kb BamHI plasmid. Fragment, pBrGus1 produces fragments of 6.1 kb and 0.8 kb.   The DNA encoding the laclq nuclear localization signal protein comprises the 5 'primer Lac I (with EcoRI site) and 3 'primer D23 with KpnI site It is amplified from the plasmid pGEMlaclqNLS by PCR. Amplified DNA flag Were restricted with EcoRI and KpnI and pBRGus cut with the same enzymes. 2 cloned. The resulting plasmid was named pBRLac. pBRLac Rasmids are cut with SphI and then run on agarose gel with SphI, Apart from the small SphI fragment containing the restriction site to be removed, the Laclq gene Enables purification of plasmids containing ampicillin resistance gene and replication origin I do. The resulting plasmid was named pBRAcBH-.   The second step in plasmid construction involves the modification of the modified CaMV plus lac operator promoter. Preparation for the cloning of the Balstar gene under regulation. This is a professional Promoter modified to remove EcoRI site between motor and coding sequence From the plasmid p35S-op-barstar EcoRI containing the promoter / gene sequence Amplified. This amplification was performed by digestion of the 35S-op balster plasmid with EcoRI. Of blunting reaction with T4 DNA polymerase and reconnection of blunt plasmid It was made by the conclusion. PCR was performed using 5'-primer-pQE-F and 3'-primer Performed using Bar-3 '. Cut PCR fragment with Xhol and Kpn1 did. This was cleaved with Xhol and Kpnl to give 35S-op balster-EcoRI plus. Cloned into mid. From there, purify unwanted genes by gel electrophoresis Removed. This allows for the removal of restriction sites at the 3 prime end of the balster coding region. Functioned. This plasmid was named p35S-op-barstar EcoRI-2. Was.   The p35S-op-barstar EcoRI-2 was then replaced with Xhol and SalI. The Balstar gene was excised and cloned into pBrLacBH- with SalI. Barstars can enter the SalI site in both directions, but only in one direction. Insert Direction was confirmed by Kpnl digestion. In the plasmid pBRacOp Ballstar 1 Only the 1 kb band was seen as an indication of the direction of the insertion seen in the E. coli. This gained The rasmid is called V2.   The plasmid is, in this case, 5 prime of the laclq gene of the flower-specific promoter. You are now ready to clone into the unique EcoRI for site cloning. The EGM3 promoter from the Eucalyptus MADS gene EGM3 for this purpose Using. This promoter was cloned in both directions and the resulting plasmid was Named M3 sense and V2EGM3 antisense. V2EGM3 sense Regulates laclq and CaMVop balstar genes for plant transformation vectors Was used as the source of the purified EGM3. XbalPst1 digestion of EGM3 promoter direction Determined by The presence of the 2.3 and 0.9 kb bands is an indicator of the correct direction. Was.   R2 construct V1 contains the barnase gene without promoter, and as a starting point Constructed using pBrGus2. The barnase gene is a primer barnase 5 Gene Bacillus amylo using Prime Sal and Barnase 3 Prime Kpn Amplified from the request faculty. The resulting PCR products were KpnI and Sa1I. Restriction was performed for pBrGus1 and ligation. Rolls obtained using amplification primers Plasmid from knee was used as a template for sequencing. Plasmid SB4 Is a barna with the same sequence as the Bacillus amyloliquefaciens barnase gene. It was found to contain a DNA fragment, which was named pBR barnase and Used for construction.   Previous studies using barnase as a lethal gene in plants show that The presence of the antitoxin gene for the star requires the presence of the promoter region. Same as barnase before can be 5 prime of cloned barnase gene Expressed from a plasmid (Paul et al. 1992 Plant Mo1ecu1ar Biolo gy 19: 611-622).   This is from Bacillus amyloliquefaciens with a barnase sequence. This was accomplished using a plasmid containing the bulster gene and promoter. By this end, the promoter and bulster DNA region must be Bacillus amylolic using the Mbalstar promoter and Bar3 prime Amplified from Efacience gene DNA. The PCR fragment is converted to Kpnl and p Ligation was performed with restriction with GEM3f. The resulting white colony is screened for insertion. I knew it. DNA from colony 7 was used for sequencing and the gene Bacillus As in Amyloliquefaciens DNA, the bulster promoter and It was found to contain a Luster sequence. As shown in FIG. Prom named Barstar.   The 7prom barstar plasmid was restricted with Kpnl and pBR barnase, Cloning of the bartarstar fragment into the Kpnl site of pBR barnase DNA did. This resulted in a plasmid known as V1. EGM3 promoter The pEGM3 was excised using EcoRI, and DNA polymerase (Klenow fragment) was excised. (Unique) and V1 DNA units already restricted and smoothed. Cloned into the Sac I cloning site. Promo inserted correctly The plasmid with the data was named EGM3VI Sessos (FIG. 27).   EM3V1 sense DNA was linearized with HindIII and also linearized with HindIII. It was cloned into the plant transformation vector Bin19. Two types corresponding to two directions of insertion Plasmids EGM3V1Bin1 and 2 were obtained. In direction 2, EGM3V The 2 EcoRI sites present in 1 Bin were approximately 80 bp apart. EGM3V1Bi n2 is cut with EcoRI and DNA polymerase I (Klenow fragment) is used. And smoothed. EGM3 promoter laclq gene and CaMV35Sop balsta The gene was excised from EGM3V2 sense using AatII and HindIII. . This fragment was blunted, EcoRI cut and smooth EGM3V1Bin2 And the plasmid EGM3Double BinI corresponding to the two possible orientations And II (FIG. 26). These plasmids are in plant form in A. tumefacieus Used for quality conversion.   This procedure was repeated for the Eucalyptus MAD promoter. EGM2 There were long, short and short EGM3.   Primer  Example 7   Isolation of reproductive organ-specific promoters   Arabidopsis thaliana and Anthirrhinum majus flower meristems and organs Five corn-specific genes showing strong homology to the identified genes were identified in immature female and male Isolated from a Pita radiata cDNA library made from sex corn . Three of these, PrMADs 1, 2 and 3, are of the MADS-box gene. Arabidopsis AGL-2, AGL-4 and AGL6 genes belonging to the family Offspring, and other non-angiospellus, d from Picece abies (Noway spruce) Each shows homology to the alI gene. The PrF1 gene is derived from Arabidopsis Leafy (Lfy) and Pine and antithirrhinum genes from Floricaula (Flo) It is lithology. PrCon1 is strong against Arabidopsis CONSTANS (co) gene High homology. To elucidate the function of these genes, Characterization of the expression patterns of male and female corn at different stages was performed.   Significantly lower levels of expression were found in the growing tissues: buds, needles, stems and roots. According to in situ hybridization, the expression of these genes was Only detectable in cells.   Expression analysis showed that all five genes differed in male and female corn development. Different patterns of expression were shown at different stages (FIGS. 8 and 9). PrMADS The 1, 2 and 3 genes are corn specific. Expression of the two genes is a reproductive organ Limited to the underlying tissue. Growth tissue: buds, needles, stems, roots, these are detected No possible expression was found. For PrFL1 and PrCon1, grow pots Only low detectable expression was seen.   Low levels of expression of both genes in the reproductive tract indicate that corn development (50 mg corn) Detected in the early stages of corn development (5 mg corn) increased during the period. Male Expression of both genes in corn is restricted to micropropagated reproduction, including the first transmitted germ cells Was decided. In female corn, expression of both genes was restricted to immature eggs.   Characterize MAD protein as a DNA binding protein in vitro To this end, both proteins were expressed in E. coli and characterized for their DNA binding properties. These DNA binding consensus sequences are similar to those of the AGAMOUS protein are doing. These three proteins are PrMADS1 and PrMADS2. And the consensus sequence of the CArG box TT (A / T) CC (A / T) (A / t)_Two( T / A) NNGG (-G) (A / T)_Twoligates DNA sequences that match (oligo A) (FIG. 32). Mutations in these consensus sequences (oligo B) were due to their PrMAD1, Reduces binding of 2 and 3 proteins. Competition with non-radioactive oligos was determined by CAr It did not reduce the binding of any protein to the G consensus. This means This indicates that they are dealing with specific DNA-protein interactions.   The upstream sequence was isolated using a promoter finder strategy (FIG. 30). EcoRV From Pita radiata by ScaI, DraI, PuuII and SspI Special adapter at the end of each DNA fragment created by DNA digestion To Connected. Since the enzyme used has a 6 base recognition site and generates blunt ends, Selected. After the adapter ligation reaction, the first adapter, primers AP1, AP2 And these DNA flags using the gene-specific primers GSP-1 and GSP-2. Was used as a template for PCR.   Adapter (first sequence), adapter primer and polymerase inhibitor The sequence of the immer is shown below. Adapter primer AP1 is 22 salts of adapter Corresponds to bases 1-22, and adapter primer AP2 corresponds to bases 13-31. It should be noted that polymerase inhibition corresponds to bases 41-48. adapter:   The presence of an amino group on the 3 end of the low strand indicates that the -Blocking polymerase catalyzed elongation from the molecule, and Must extend the DNA strand to the upper strand of the adapter Prevents the occurrence of primer binding sites.   The primary PCR was performed using Avantage Tth Polymerase Mix (Clontech) and PrM Adapter primers AP1 and A1 for ADS2, 3 and PrFL1 genes And GSP-1. GSP-1 sequence:   PCR two-step cycle parameters:   7 cycles: 94 ° C for 25 seconds                      72 ° C for 4 minutes   32 cycles: 94 ° C for 25 seconds                      67 ° C for 4 minutes   Add an additional 4 minutes at 67 ° C after the last cycle.   GSP with same cycle parameters and AP2 adapter primer 1 ml of the primary PCR was used in the secondary PCR using a second set of GSP-2 primer sequence:   Cloning the 1-2 kb PCR fragment into a TA cloning vector Was sequenced. CD for PrMADS2, PrMADS3 and PrF1 The sequences of NA, protein and promoter are shown in FIGS. 31, 34 and 37.   Example 8   Introduction of chimeric DNA sequence into plant   The chimeric DNA sequence was co-transferred to plant tissue using A. tumefacieus. Selective culture The transformant was maintained by growing on the ground. Standard molecular biology techniques (Southern, Presence of two plasmids using Northern hybridization (PCR) Proved. Under the control of the plant promoter, plasmid A The plasmid B has the bulster gene. This is a co-transformation Create additional choices for   The specified vector system, under the positive control of a reproductive organ-specific promoter, And E. coli lactase (lac) repressor operator system negative control Includes a cascade of sequence genes under the roll. During the non-flowering period, In the absence of the tumor protein, the operator must Alternatively, it allows for a constitutive sequence of the antisense RNA. Reproductive organ-specific promotion Low levels of lethal gene products may be present in various plant organs due to potential leakage of And present in cells.   Barstar protein in the cytoplasm or antisense R in the nucleus of these cells Accumulation of NA blocks the cytotoxic effects of lethal gene products. Early stage of flowering B (or other RNase) and laclq genes in appropriate cells of the reproductive organs Offspring expression increases dramatically. Laclq proteins in the nucleus of these reproductive organ cells Accumulation of protein can alter expression of the bulster gene (or antisense RNA) to target Lac repressor increases the level of lethal gene products in reproductive organ cells Will block through operator interaction.   Example 9   Three Eucalyptus inheritances with homology to other plant MADS-box genes Offspring (EGM1,2,3) were cloned and sequenced (FIGS. 10-13 and 17). To investigate the relationship between the eucalypt gene and other MADS box genes, Embryological analysis was performed (FIG. 18). According to this, EGM2 is found in flower pieces and stamens. Appears to be homologous to the GLOBOSAMADS-box gene involved in development You can see that. EGM3 and EGM1 are both MADS box gene AGLs. Join two groups. These genes are responsible for the internal three-wheeled body of the flower (flower pieces, male core and heart). Most commonly expressed in bark, but its function in flower development is still unknown Not.   The expression patterns of all 3EGM genes were characterized (FIGS. 14, 15 and 1). 6). Expression of these genes was not detected in any of the growing tissues. The EGM1 gene is expressed in flower pieces, male core and carpel of Eucalyptus flowers. EGM3 Is expressed in flower meristems and cups. EGM2 is expressed in flower pieces and male wicks. E Southern analysis revealed that GM3 and EGM2 were single genes. (FIG. 19). EGM1 also appears to be a single gene.   The promoter regions of all three genes are used in barren gene constructs and -Isolated from the Calypto Genome Library. EGM3 promoter is deadly Used for the gene construct.   To examine the tissue specificity of the expression of the three EGMMADS box genes, A Northern blot was performed on the box. R from roots, shoots, shoot leaves and mature flowers The tissues used for NA isolation were obtained from Eucalyptus granges plants. Flower bed, flower pieces, Tissue for the isolation of RNA from flower tissue, including male wick, carpel and style, was obtained from Euca Collected from Lips globulus flowers. This species produces very large flowers and RN A was used because a sufficient amount of A could be easily collected. Blocks containing different flower tissues Eucalyptus grandi containing 10 mcg of total RNA 20 mcg was used in the bacterial blot. Suitable for removing the MADS-box The blot was probed with three EGM genes digested with different restriction enzymes .   By Northern blot, all three EGM genes were found in Eucalyptus flowers. And was found to be expressed at a high level. Northern is exposed to film for a long time Then, weak expression of EGM2 and EG1 was observed in the growing tissue. EGM The three genes were specifically expressed in growing tissues. In flowers, the EGM2 gene is male-core And expression in flower pieces. EGM3 gene is flower bed, flower piece, male core, heart It is expressed in skins and styles.   Example 10   The first part of the cascade (factor 1) has a lac operator (LEAFY-op) Flower meristem identification gene promoter (ie, LEA from Arabidopsis thaliana) The reporter GUS gene is included under the control of FY). The second construct (factor 2) And the receptor la under control of the corn GST-27 gene promoter. Including clq. This promoter uses the safener dichlormid and R-29. Upregulated by treating the plant at 148. Arabidopsis plants have two factors Was co-transformed.   Another strategy involves the Arabidopsis line, which separately holds factors 1 and 2 to be crossed About Expression only from Factor 1 produces blue flowers after coloring with X-gal. Chemical transfer of a factor 2 expression into a plant that expresses factor 1 results in the blue color seen in the flower. Disappear.   Example 11   The first factor 1 of the cascade is a flower meristem identification gene vector with a lac operator. Control of the lomoter (ie LEAFY from Arabidopsis thaliana) Under the barnase gene. The second factor is the maize GST-27 gene Includes the receptor laclq gene under the control of the motor. Arabidopsis plant Was co-transformed with the two factors. Only one factor 1 is expressed in uninduced plants Produces barren Arabidopsis plants. Factor 2 in plants expressing factor 1 Introduction of expression from E. coli stops expression from the LEAFY-op promoter and is initially Fertility was restored in haired Arabidopsis plants.   Example 12   In the following, some abbreviations are used. These are commonly used in plant tissue culture Is what is being done. BA: benzyladenine, also known as 6-benzylaminopurine IBA: indole-3-butyric acid NAA: 1-naphthaleneacetic acid TDZ: 1-phenyl-3- [1,2,3-thiadiazol-5-yl] urea Thidiazuron, also known as   The following shows the production of transformed Eucalyptus from shoots and seedling explants. This is a description of a preferred process for the above.Shoot explants Shoots are subcultured monthly in a solid KG medium containing 1.0.2 mM BA. weak Light (irradiated for 16 hours, 100-350 Lux or 1-8 mmolm^-2s^-1PAR) and 225 ° C. 2. All shoots after 3-4 weeks of subculture are used. 3. Leave the upper 4-6 leaves and remove the lower leaves. 4. Injure leaves 5-6 times with a needle (for example, 25G), and use 10-100 mM acetosyline. Gon-containing Agrobacterium suspension (about 1 × 10⁸cfu-ml) for 10 minutes-2 hours Put all shots. Agrobacterium with optical density of 1.0 at 1/200 dilution at 600nm About 1 × 10⁸cfu ML^-1It is. Best when you hurt the base of the leaf The result is obtained. Alternatively, damaged shoots in Agrobacterium suspension Agrobacterium at 40-100 Kpa for 10-30 minutes instead of leaving for 1 hour The vacuum can infiltrate the chute with the system. Chute hurts due to vacuum infiltration You don't need to. However, when wounded with a needle, it is more calculable than with vacuum infiltration. The size of the formation increases. 5. Dry the shoots between sterile filter papers and place vertically in KG medium containing 0.2 mM BA. Insert a shoot. Co-culture for 2 days in the dark. 6.0.2 mM BA and 200 mgL^-1Shoe on KG medium containing cefotaxime Transfer (still vertical). Keep in low light for 5 days (irradiation 16 hours, 100-350 Lux or 1-8 mmolm^-2s^-1PAR). The upper leaves of 7.4-6 were collected and 2 mM BA, 2.5 mM NAA, 200 mgL^-1 Cefotaxime and 5-10 mgL^-1Solid callus induction medium containing geneticin The leaves are planted on top, with the axial side up. 8.2 mM BA, 2.5 mM NAA, 1.0 mM TDZ, 200 mgL^-1Sefo Taxim and 15mgL^-1Transfer explants to G22 medium containing geneticin. darkness And subculture every 2 weeks. Incubation in light turns brown phenolic Produce a compound. After 9.6 weeks, 200 mg L^-1Cefotaxime and 15mgL^-1Geneticin included With a shoot-inducing medium GBA (ie containing 5 mM BA and 0.5 mM NAA) Transfer the explants to G22). Place in dark for 5-6 days, then in light (irradiate for 16 hours , 100-350 Lux or 1-8 mmolm^-2s^-1PAR). Every 2 weeks Perform subculture. 10.200mgL^-1Cefotaxime and 15mgL^-1On Geneticin's GBA 200mgL after 8-10 weeks^-1Cefotaxime and 30mgL^-1Geneti Callus pieces with buds and callus formed on original explants in Shin GBA Transfer. 11. After 4-6 weeks on this medium, 0.01 mM BA, 50 mgL^-1Sef Otaxime and 5mgL^-1Shoot for 2 weeks in liquid KG medium containing geneticin Regenerate, then high geneticin (10 mg L^-12) Transfer to medium for 2-4 weeks. Liquid culture The substrate is shaken at 100-120 rpm with 8 ml of liquid in a 70 ml container. 12. Solid medium (200mgL^-1Cefotaxime and 10mgL^-1Geneticin included Yes, hormone-free KG) and strong light environment (16 hours irradiation, 450 Lux or Is 10 mmolm^-2s^-1Transfer surviving shoots and calli to PAR). Every two weeks Subculture. 13. The putative transformed shoots are assayed for marker gene activity. 14． Regenerate the plant from the confirmed positive shoot material. 10mgL^-1Geneticin included Rooting is induced by transferring shoots on KGs with and without hormones. I However, if there is no root after 3 weeks, 0.2 mg IBA^-1(9.8 mM) Transfer to the same medium.   The method described in detail above is performed for 1-8 months from transformation to regenerating shoot.Seedling explants 1. Seeds are disinfected and germinated on KG containing 0.2 mM BA. 2. Roots were collected using seedlings aged 10-12 days and contained 50 mM acetosyringone. Overnight grown Agrobacterium suspension (about 1 × 10⁸cfu ml). 600n Agrobacterium suspension having an optical density of 1.0 at 1/20 m dilution is about 1 × 10⁸ cfu ML^-1It is. Gently pierce the 30 gauge syringe needle under the microscope, Injure cotyledons and hypocotyls. Incubate for 1 hour, remove seedlings from suspension, The seedlings are dried between sterile filter papers to remove excess liquid. 1 hour Agrobacterium Instead of leaving the damaged cotyledons in the suspension, 95 KPa (28 mgHg) for 20 minutes ) Allows the cotyledons to be vacuum infiltrated with Agrobacterium. Vacuum infiltration method Then it is not necessary to hurt the cotyledons. However, the hypocotyl is hurt even when vacuum infiltrated Need to be removed. 3. Co-culture on KG medium (containing 0.2 mM BA) for 2 days in the dark. Seedlings in medium Be careful to be upright. 4.200mgL^-1Seedling on cefotaxime-containing KG medium (containing 0.2 mM BA) Transfer. 5 days weak light (16 hours irradiation, 100-350 Lux or 1-8 mmolm^{- Two} s^-1PAR). 5. Cotyledons and hypocotyls are excised. 0.5 mM BA, 1.0 mM NAA, 1.0 mMTDZ, 200 mgL^-1Cefotaxime and 10mgL^-1Geneticin-containing Transfer hypocotyls to callus induction medium. 1.0 mM BA, 1.0 mM NAA, 0.3 mM   TDZ, 200mgL^-1Cefotaxime and 10mgL^-1G with geneticin Transfer cotyledons to medium 22. Continue incubation in the dark for 2 weeks. 6.15mgL^-1Geneticin and 200mgL^-1Same medium with cefotaxime Transfer explants to Continue incubation in the dark. Every 2 weeks until approximately 6 weeks Subculture for a week. 7.6 weeks later, 15mgL^-1Shoot-inducing medium GBA containing geneticin To G22) containing 5 mgM BA and 0.5 mM NAA. 5-7 days dark The culture is left in the dark and then in a light environment (irradiated for 16 hours, 100-350 Lux or 1-8 mmolm^-2s^-1PAR). 8.15mgL^-1Geneticin and 200mgL^-1About on Cefotaxime's GBA After 10 weeks, many explants produce shoots. Collect these shoots And 30mgL^-1Geneticin and 200mgL^-1Cefotaxime KG medium (0 .2 mM BA). 15 mgL of callus formed on the original explant^-1Neti Syn and 200mgL^-1Return to cefotaxime GBA and subculture and place for 1 month And more shoots, 30mgL^-1Geneticin and 200mgL^-1C Transfer to fotaxime KG medium (containing 0.2 mM BA). 9.30mgL^-1Geneticin and 200mgL^-1New KG culture of Cefotaxime All shoots and calli were transferred to the ground (containing 0.2 mM BA) for an additional month. You. 10. After 4-6 weeks in this medium, 0.01 M BA, 50 mg L^-1Cefotaxime And 5mgL^-1Put the regenerated shoots for 2 weeks in liquid KG medium containing geneticin, Advanced Geneticin (10mgL^-12) Transfer to medium for 2-4 weeks. 11. Solid medium (200mgL^-1Cefotaxime and 10mgL^-1Geneticin included Yes, but hormone-free KG), high optical environment (16 hours irradiation, 450 Lux Or 10 mmolm^-2s^-1Transfer surviving shoots and calli to PAR). Every 2 weeks Subculture. 12. The putative transformed shoots are assayed for marker gene activity. 13. Regenerate the plant from the confirmed positive shoot material. 10mgL^-1Geneticin included Rooting is induced by transferring shoots on KGs with and without hormones. I However, if there is no root after 3 weeks, 0.2 mg IBA^-1(9.8 mM) Transfer to the same medium.   Seedlings 12 days or less are optimal for transformation, but seedlings up to 20 days Can be used for reproduction.   The shoot regeneration rate of untransformed cotyledons and hypocotyls without selection is about 80%. is there.   Removal of cefotaxime from the culture medium, even after 3 months, Experience teaches that it will result in rapid overgrowth of um. Of cefotaxime The concentration varies between liquid and solid media.   Shoot material grows rapidly in liquid medium, and in comparison to solid medium, A shoot was formed.   This liquid selection process reduces false positives by increasing the selection pressure and reduces residue Agrobacterium and reduce the amount of shoot tissue compared to solid growth media Has the advantage of increasing   Regenerated rooted transgenic plants are transferred to soil-based media and transformed into trees of a certain size. It grows and becomes suitable for tree planting.   The media used in this study were G22, G described in Laine and David (1994). BA and KG. However, various basic cultures, including MS, B5 and P24, The ground was tested and found to support shoot regeneration. Plant growth regulation is La In general, it differs from that of ine and David (1994), in terms of callus induction from leaves. Are 1-3 mM BA, 0.05-2 mM TDZ and 0.5-2.5 mM NAA For cotyledons, 1-3 mM BA, 0.05-1 mM TDZ and 0.5-2. 5 mM NAA, 1-3 mM BA, 0.05-2 mM TDZ for hypocotyl and And 0.5-2.5 mM NAA. Differentiation medium is generally GBA medium And this is G22, but with 2.5-5 mM BA and 0.5 mM NAA (L aine and David, 1994). KG medium containing 0.2 mM BA Generally used as a subculture medium for cloned material. 0.25% G for all media Solidified with e1rite or Phytage1. Autoclave at 121C for 15 minutes Prior to application, the pH was adjusted to 5.7-5.8 with potassium hydroxide. Aggro The preferred method of making the bacterial inoculum is described below. 1. Selectively place a construct-containing Agrobacterium strain on a plant, for example, LBA4404, EHA105 and AGL containing pBin GUSINT structure YEP + rifampicin (50 mg / L) + kanamycin (100 mg / L). 2. Incubate the plate at 280C for 2 days. 3. Select and pick a single colony into YEP broth, shaker overnight at 28OC Grow on top. 4. Using overnight culture, select and inoculate fresh medium (1% inoculum) at 280C Grow overnight on a shaker. This additional step allows for continuous inoculation by plant transformation. Produce. 5. Agrobacterium is harvested, washed with water and resuspended in tissue culture medium. Transformation Dilute to an appropriate concentration. 6. Resuspended Agrobacterium is linearly inoculated on a lactose yeast medium plate And incubate the plate at 280C for 2 days. Bened About Colony Perform an ict test (Bernaerts and Deley, 1963) and be Agrobacterium Check.   This method yields an excellent, dense culture of Agrobacterium on the day of transformation. Will.   Overnight growth culture incubated from plate or glycerol stock Things will have a variety of consequences.   AG grown as above and diluted to an optical density (600 nm) of 0.98 The L1 culture had a change of 2.37 × 10⁹Has cfu / ml.   The foregoing has been made for the purpose of describing the present invention and all Other modifications and changes will occur to those skilled in the art in the scope and scope of the invention as claimed. What should be inside will be understood, of course.

────────────────────────────────────────────────── ─── 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), UA (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 (72) Inventor Southerton, Simon George             Australia 4520 Queensland             Samford, Castlewood Court             No. 1 (72) Inventor Sawbridge, Timothy Iver             Australia 4067 Queensland             St. Lucia, Carmody Road 142             Number, unit 1 [Continuation of summary] Used to let.

Claims (1)

[Claims] 1. Transforming the cell with a construct that expresses a eukaryotic active gene or a modulator gene product that regulates the product thereof and a further gene product that regulates the modulator gene or the product thereof; and A method of regulating a eukaryotic activation gene, wherein the promoters of the modulator gene and the additional gene are selected from an inducible promoter and a developmental promoter for the same or complementary tissue. 2. The method for regulating a eukaryotic active gene according to claim 1, wherein the eukaryotic active gene is selected from a natural gene or a related organism or an inducible gene. 3. The method for regulating a eukaryotic active gene according to claim 1, wherein the eukaryotic active gene encodes an expression product selected from active polypeptides, enzymes or possible gene products for the synthetic pathway. 4. 4. The method of regulating a eukaryotically active gene according to claim 3, wherein the synthetic pathway has been selected for the production of pigments and dyes. 5. 5. The method for regulating a eukaryotic active gene according to claim 4, wherein the dye is such as anthocyanin. 6. 5. The method of regulating a eukaryotic active gene according to claim 4, wherein the synthetic pathway has been selected for the production of pesticidal compounds. 7. 7. The method for regulating a eukaryotically active gene according to claim 6, wherein the insecticidal compound is butitoxin. 8. The method for regulating a eukaryotic active gene according to claim 3, wherein the enzyme is selected from ribonuclease having a lethal effect on a target tissue and peroxidase which imparts nematode resistance to root tissue. 9. 9. The method of regulating a eukaryotic active gene according to claim 8, wherein the ribonuclease gene is that of barnase from B. amyloliquefaciens. 10. The gene is configured to include a coding region for barnase from Bacillus amyloliquefaciens under the control of the promoter sequence of the gene expressed in the male and female parts of the plant reproductive tract at an early stage of development, A method for regulating a eukaryotic active gene according to claim 9. 11. The eukaryotic active gene according to any of the preceding claims, wherein the eukaryotic active gene is constituted with a promoter selected from the promoters for EGML1 and 2 eucalypt genes in Eucalyptus grandis. How to adjust. 12. The eukaryotic activity according to any one of claims 1 to 10, wherein the eukaryotic activity gene is constituted with a promoter selected from promoters for the PrMADSL1, 2 and 3 pine genes in Pinus radiata. How to regulate genes. 13. Modulating a eukaryotic active gene according to any of the preceding claims, wherein the eukaryotic active gene, modulator gene and further genes and their promoters can comprise a transformation cassette for transforming the target organism. how to. 14． 14. The method for regulating a eukaryotic active gene according to claim 13, wherein the eukaryotic active gene is promoted by a developmental promoter, and the promoter of the modulator gene is constitutive. 15. 14. The method according to claim 13, wherein the promoter for the eukaryotic active gene is tissue-specific, inhibited by a constitutively promoted inhibitor gene product, and thus controlled by an inducibly promoted additional gene product. A method of regulating a eukaryotic active gene. 16. A eukaryotic active gene product is constitutively expressed by the modulator effect of a modulator gene expressed under the control of a tissue-specific promoter, wherein the modulator gene or product is itself specific for a different or complementary tissue 14. The method for regulating a eukaryotic active gene according to claim 13, which is controlled by expression of a further gene under the control of a specific promoter. 17． Transformation of a cell comprising a eukaryotic active gene, a modulator gene that expresses a substance that modulates the eukaryotic active gene or a product thereof, and a further gene that expresses the modulator gene or a substance that modulates the product thereof Eukaryotic activity, characterized by being transformed with a cassette, and wherein the promoters of the two genes, the modulator gene and the further gene, are selected from inducible and developmental promoters for the same or complementary tissues. A method of regulating gene expression. 18. Method for transforming a proliferative eukaryotic cell with an expression cassette comprising: a lethal expression of a cytotoxic peptide in the target tissue under the control of a promoter essentially specific for the target tissue A modulator gene that constitutively expresses an inhibitor of the production or activity of the cytotoxic peptide; and a modulator that is under the control of the essentially specific promoter and blocks the inhibitor or modulator gene. Additional genes that work. 19. The eukaryotic active gene is selected from the barnase and barstar genes from Bacillus amyloliquefaciens extract and defined as P1 and P2 (for barstar) and B3 and B4 (for barnase) The method for regulating a eukaryotic active gene according to any one of claims 1 to 16, wherein the eukaryotic gene is isolated by PCR with a primer comprising the following sequence: 20. The eukaryotic active gene is a lethal gene wherein the cytotoxic effect of the lethal gene expression product is enhanced by coupling a localization signal sequence to the construct. A method for regulating the eukaryotic active gene according to the above. 21. The eukaryotic activation gene according to claim 20, wherein the eukaryotic activation gene encodes a nucleus-targeted ribonuclease by fusing a nuclear localization signal (NLS) sequence to a ribonuclease (Rnase) gene. how to. 22. The gene is a barnase gene, the fusion is performed by PCR, a set of PCR primers for fusing the NLS sequence from the A. tumefaciens VirD2 gene to the B gene is used, and comprises the following sequences: Item 21. A method for regulating a eukaryotic active gene according to item 21: 23. 23. The method for regulating a eukaryotic active gene according to any of claims 1 to 22, wherein the repressor gene and the inhibitor gene are suitable for forming a specific repressor / inhibitor couple. 24. The repressor / inhibitor couple comprises the E. coli laclq gene under the control of the same promoter as the eukaryotic active gene construct and is used to suppress the action of the repressor gene promoter modified by insertion of a lac operator sequence. Item 23. A method for regulating a eukaryotic active gene according to any one of Items 1 to 22. 25. 25. The method of regulating a eukaryotic active gene according to claim 24, wherein the repressor gene promoter is a modified 35S-RNA-Ca MV promoter having at least one lac operator between the promoter and the coding region of the repressor. 26. 26. The method of regulating a eukaryotic active gene according to claim 25, wherein the modified 35S-RNA-CaMV sequence is amplified by PCR using primers 35S1 and 35S2 having the following sequences: 27. The modified 35S-RNA-CaMV sequence is amplified by PCR using primers OP1 and 35S2 with the following sequences to introduce a lac operator between the 3 'and 5' regions of the modified 35S promoter. 25. A method of regulating a eukaryotic active gene according to 25: 28. The laclq gene is isolated with an enzyme cleavage site for cloning into a plant vector using two primers AM1 and AM2 having the following sequences corresponding to the 5 'and 3' ends of the gene: Item 24. A method for regulating a eukaryotic active gene according to item 24: 29. 29. The method for regulating a eukaryotic active gene according to claim 28, wherein the 3 'end of the laclq gene is fused to the NLS sequence by the following set of PCR primers: 30. It has a eukaryotic active gene, a modulator gene that expresses a substance that regulates the eukaryotic active gene or a product thereof, and a further gene that expresses a substance that regulates the modulator gene or a product thereof. A transformation cassette, wherein the promoter of one modulator gene and the additional gene is one selected from an inducible promoter and a developmental promoter for the same or complementary tissue. 31. A gene that expresses a peptide that is cytotoxic to the target tissue under the control of an inducible promoter essentially specific to the target tissue, an inhibitor gene that expresses an inhibitor of the production or activity of the cytotoxic peptide And an expression cassette comprising a repressor gene under the control of the inducible promoter and functioning to block the inhibitor or inhibitor gene.