JP2001524315A - Methods and compositions useful for silent transgene activation - Google Patents

Abstract

  (57) [Summary] A composition and method for inducing the expression of a desired DNA sequence in a stably transformed plant that expresses a hybrid transcription factor, comprising an effector of expression of a desired DNA sequence controlled by a synthetic promoter. Comprising a fusion of a DNA binding domain and a transcriptional activation domain, wherein the synthetic promoter preferably comprises a concatemer copy of a cis-acting site recognized by the DNA binding domain of the hybrid transcription factor fused to the promoter. A composition and method comprising:

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition for controlling the expression of a gene sequence, and a method for using the same. More particularly, the invention relates to the induction of the expression of an antisense nucleic acid sequence that specifically inactivates the expression of a target gene.

A fundamental problem with current techniques for inactivating gene expression is relying on the use of a constitutive promoter to constantly drive expression of the incoming transgene (or gene fragment). . That is, transformed cells that have received the incoming transgene are immediately committed to their expression. If the incoming transgene is capable of inhibiting the expression of an intrinsic endogenous gene, all transformed cells will die after transformation. Therefore, it is desirable to control the expression of the introduced DNA sequence in an inducible manner.

However, strict inducible control of transgene expression has not yet been achieved in whole plants. Such controls may have several uses, such as being practical, such as regulatory genes for controlling fertilization, and more basic, such as probing functions by knocking out novel genes.

[0004] Many aggressive transcription factors are modules, consisting of a DNA-binding domain and an activation domain that interacts with components of the transcription machinery assembled at the promoter (Ptashne, 1988; Swaffield et al., 1995). . Fusion combinations of such elements from different kingdoms have led to the production of a variety of hybrid factors with specific DNA binding specificities and transcriptional activation functions for the target organism of interest. For example, in a transient expression experiment in tobacco protoplasts, a transcription factor derived from the yeast GAL4 transcriptional activator is controlled by a synthetic promoter consisting of multiple GAL4 DNA binding sites and a TATA element derived from a promoter recognized by plant cells. It has been shown to activate transcription from (Ma et al., 1988). The function of hybrid transcription activators and activator mutants has also been investigated through rapid microprojectile transfer of genes into the aleurone layer of corn seeds. GA fused to the acidic activation domain of the herpes simplex virus VP16 protein or a functionally related maize regulatory protein C1
The L4 DNA binding domain has been shown to stimulate GAL4-dependent reporter gene expression when both transactivator and reporter genes are introduced with microprojectiles (Goff et al., 1991). A chimeric transcriptional activator consisting of the DNA binding domain of bacteriophage 434 fused to the VP16 activation domain, when transiently introduced into tobacco protoplasts, has a minimum of 35
It has been shown to activate gene expression of a reporter gene driven by a synthetic promoter consisting of 434 operators fused to the S promoter (Wilde
Et al., 1994). Taken together, these studies indicate that a DNA binding domain from a heterologous factor can bind to a synthetic promoter containing the appropriate binding site on a naked DNA template introduced into plant cells, and that the non-plant activation domain is DNA It is established that if covalently bound to the binding domain, it can interact with plant transcription machinery in many ways.

[0005] While analysis of transgenes transiently introduced into host cells may be useful in conducting preliminary studies of genetic function, stable transformants are more widely used to study plant gene expression. It would be an applicable system. However, it has been reported that the GAL4 DNA binding domain is not efficiently expressed in plants (Reichel, C
Et al. (1995) "Inefficient Expression of the DNA-Binding Domain of GAL4 i
n Transgenic Plants "Plant Cell Reports 14 (12): 773-776). An ideal regulatory system for controlling transgene expression in plants is one that has little or no background expression in the absence of a functional transcription factor and is highly expressed in the presence of a functional transcription factor. is there.

[0006] Therefore, what is needed is to introduce the introduced DN in stably transformed plants.
Compositions and methods useful for providing inducible expression of the A sequence.

The present invention provides a heterologous DNA for stably introducing a plant hybrid transcription factor and a synthetic promoter to induce the expression of an activating DNA sequence such as a transgene.
Providing methods, compositions and transgenic plants containing sequences addresses a long-felt but unmet need in the art. Importantly, the invention described herein makes the insertion of the transgene efficiently independent of its activity, thus allowing recovery of the naturally lethal transformants by subsequent activation. The utility of the present invention lies in the investigation of gene function, for example, by identifying genes that are important for plant growth, development and survival. A crucial feature of the present invention is that the expression of an antisense DNA sequence or a dominant inhibitor, such as a translatable or non-translatable sense sequence capable of interrupting gene function, in a stably transformed plant, Ability to positively identify one or more genes that are essential for successful growth and development.

The present invention includes hybrid transcription factor genes, synthetic promoters, activating DNA sequences, and cells, tissues and plants containing such genes, promoters and DNA sequences, and methods of using the same to determine plant gene function. I do. The hybrid transcription factor gene and the synthetic promoter are selected such that the DNA binding domain of the hybrid transcription factor can specifically bind to the synthetic promoter so as to activate the expression of an activating DNA sequence driven by the synthetic promoter. Or be designed.

More particularly, the present invention relates to a first cell or tissue, preferably a plant cell or plant tissue or plant, comprising a sequence encoding a hybrid transcription factor necessary for activating a synthetic promoter, and said synthetic promoter And preferably a plant cell or plant tissue, or a plant, wherein the first and second cells, tissue or plant are both sequences encoding the hybrid transcription factor and the synthetic promoter. May be manipulated so as to construct a cell, tissue or plant containing If desired, the second cell, tissue or plant comprises a synthetic promoter operably linked to the activating DNA sequence, and expression of the DNA sequence is activated when the promoter is activated by a hybrid transcription factor. It is driven by this synthetic promoter. In another embodiment, the hybrid transcription factor is designed or selected to be controllable by an independent factor, such as estrogen.

In the method of the present invention, a stable transgenic plant containing both a hybrid transcription factor gene and a synthetic promoter is formed by combining the first and second cells, and the activating DNA sequence is contained in the plant. Is expressed. In one embodiment, the skilled artisan encodes a transcription factor that specifically activates the expression of an otherwise silent gene (or gene fragment) of interest, such as an antisense, from a synthetic promoter that controls the gene of interest. Through the introduction of the gene to be crossed into the mating system. If the gene of interest is capable of inactivating the expression of an endogenous gene, progeny of a cross between a plant containing the gene of interest and an effector cell will not be able to express the endogenous gene normally. Plant genes essential for normal growth or development can thus be identified. Identification of such genes is a useful target for screening compound libraries for effective herbicides.

The invention described herein demonstrates that hybrid transcription factors can function to efficiently regulate gene expression in stably transformed plants. The following detailed description shows that hybrid transcription factor can efficiently activate gene function in whole plant,
It is therefore primary evidence that a useful system is provided for actively identifying genes essential for plant growth.

The present invention provides an important new method for examining the function of a gene in a plant. The present invention relates to endogenous plant DNA that is necessary or important for growth, development or survival.
Very useful for sequence identification. Identification of such DNA sequences provides essential information for the rational and efficient development of safe, effective, economically expressible, and environmentally sound products to solve important agricultural problems I do. In one embodiment, the invention can be used to test the function of an endogenous gene by knocking out its expression. In particular, it can be used to identify potential herbicidal target genes by identifying whether the gene of interest is essential for normal plant growth and development. This serves as an early and essential bridge in the screening and development chain, and motivates them to rank resources to identify and test the most competent compounds, leading them to invest in their research and therefore to invest in their research. Immediately in the public interest.

[0013] In order to provide a thorough, clear, concise and accurate description of the present invention, the following terminology definitions are used herein.

Activating DNA construct—refers to a DNA sequence, or a recombinant construct and a synthetic promoter containing the DNA sequence, which, when introduced into a cell, and, if desired, a plant cell, become the synthetic promoter. It is not expressed, ie, silent, unless there is a complete hybrid transcription factor capable of binding and activating. This activating DNA construct is then introduced into a cell, tissue or plant, as described in more detail below.
A stable gene transfer system capable of expressing the activating DNA sequence is formed.

Activating (activatable) DNA sequence-refers to a DNA sequence that regulates the expression of a gene in the genome, if desired the genome of the plant. The activating DNA sequence is complementary to a target DNA sequence in the genome. When the activating DNA sequence is introduced into a cell and expressed, it inhibits expression of the target DNA. Activating DNA sequences useful in the present invention positively identify those that encode or act as dominant inhibitors, for example, one or more genes essential for normal growth and development of plants. And a translatable or non-translatable sense sequence capable of interrupting gene function in a stably transformed plant. A preferred activating DNA sequence is an antisense DNA sequence. The mechanism by which the antisense sequence works is not well understood, but probably the antisense RNA
It will bind to NA and inhibit the expression of the target DNA gene product. Such R
NA: RNA complexes can inhibit the binding or function of the translation machinery, while such RNA: RNA complexes can be rapidly degraded. Other mechanisms are possible. If desired, the target gene may be a protein, such as a biosynthetic enzyme, receptor, signaling protein, essential for plant growth or survival.
Encodes a structural gene product or transport protein. Interaction of the antisense RNA sequence with the target gene RNA results in a substantial inhibition of the expression of the target DNA sequence, thus killing the plant or inhibiting normal plant growth or development.

“Expression” refers to the transcription and / or translation of an endogenous gene or transgene in a plant. For example, in the case of an antisense construct, expression may mean only transcription of the antisense DNA.

“Gene” means a coding sequence and associated regulatory sequences, wherein the coding sequence is an RNA, such as an mRNA, rRNA, tRNA, snRNA, sense RN.
Transcribed to A or antisense RNA. Examples of regulatory sequences are promoter sequences,
5 'and 3' untranslated sequences, and termination sequences. Further elements that may be present are, for example, introns.

A “gene of interest” refers to a desired characteristic of a plant when introduced into a plant, such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, Any gene that confers enhanced nutritional value, enhanced performance in an industrial process, or altered regenerative capacity is meant. A “gene of interest” may be one that is introduced into a plant for the production of a commercially valuable enzyme or metabolite in the plant.

Heterologous DNA sequence-means a DNA sequence that is not naturally associated with the host cell to be introduced, including non-naturally occurring multiple copies of the native DNA sequence.

“Marker gene” refers to a gene that encodes a selectable or screenable feature. "... operably linked / coupled" means that the regulatory DNA sequence is located if the two sequences are arranged such that the regulatory DNA sequence governs the expression of the DNA sequence encoding the RNA or protein. "Linked to operable expression /
"Joined".

By “minimal promoter” is meant a promoter element, particularly a TATA element, which is inactive without upstream activation or has significantly reduced promoter activity. In the presence of appropriate transcription factors, this minimal promoter functions to allow transcription.

“Plant” means the structural and physiological units of a plant, including protoplasts and cell walls. The plant cells may be in the form of isolated single cells or cultured cells, or may be part of a higher unit, such as a plant tissue or plant organ. "Plant cell" means any plant, especially a seed plant.

“Plant material” refers to a leaf, stem, root, flower or organ of flower, fruit, pollen, pollen tube, ovule, embryo sac, egg cell, zygote, embryo, seed, cutting, cell or tissue culture Or any other plant organ or product.

“Promoter” means a DNA sequence that initiates transcription of a bound DNA sequence. The promoter region also contains elements that act as regulators of gene expression, such as activators, enhancers and / or repressors.

Recombinant DNA—refers to a molecule that is a combination of DNA sequences linked together using recombinant DNA technology.

[0026] Recombinant DNA engineering is described, for example, in Sambrook et al., 1989, Cold Spring Harbor, NY: C
It refers to the procedure used to link DNA sequences together as described in the Old Spring Harbor Laboratory Press.

“Selectable marker gene” means a gene whose expression in a plant cell provides a selective advantage. The selectivity advantage retained by cells transformed with this selectable marker gene may be based on the ability to grow in the presence of a negative selection agent, such as an antibiotic or herbicide, as compared to the growth of non-transformed cells. . This selective advantage retained by the transformed cells over untransformed cells may be due to their enhanced or new ability to utilize added compounds as nutrients, growth factors or energy sources. A selectable marker gene also refers to a gene or combination of genes whose expression in a plant cell provides both negative and positive selective advantages.

By “stably transformed” is meant a cell containing at least one heterologous DNA sequence, preferably a plant cell, wherein the heterologous DNA sequence is maintained in the cell, and It is functional under conditions appropriate for its intended use, and is inheritable to subsequent generations. The term specifically includes, by whatever means, the cell into which the heterologous DNA sequence has been first introduced, and the subsequently derived cells, tissues, plants and seeds containing the heterologous DNA sequence. The latter is sometimes called a stable transgenic line.

“Synthetic” refers to a nucleotide sequence that comprises structural features not found in the native sequence. For example, an artificial sequence approximated by the G + C content and normal codon distribution of dicotyledonous and / or monocotyledonous genes is referred to as "synthetic."

“Transformation” (r) refers to the introduction of a nucleic acid into a cell. Specifically, it refers to the stable integration of DNA molecules into the genome of the organism of interest.

As described in more detail below, the present invention encompasses hybrid transcription factor genes, synthetic promoters, activating DNA sequences, and cells, tissues and plants containing such genes, promoters and DNA sequences, and methods of using the same. . The hybrid transcription factor gene and the synthetic promoter are the DNA of the hybrid transcription factor.
The binding domain is selected or designed so that it can specifically bind to the synthetic promoter and turn on the expression of the activating DNA sequence driven by the synthetic promoter.

In one embodiment, the invention is based on a cross that achieves inducible gene activation that relies on a transcription effector system to activate expression of a silent transgene under the control of a synthetic activatable promoter. I do. Traits whose expression can be controlled by this system include knocking out the expression of a non-vegetable gene for a transcription control element such as an endogenous promoter, a translated or untranslated sense gene, or a post-transcription control element, and an endogenous gene. An antisense gene (for example, AdSS described below) is included. In one case, the transcription effector system comprises a DNA sequence encoding a hybrid transcription factor, described more fully below,
It is crossed with a cell, tissue or plant system containing a synthetic promoter operably linked to the activating DNA sequence. In another case, the transcription effector system comprises a DNA sequence that encodes a portion of a hybrid transcription factor, wherein the protein encoded thereby binds via a peptide-peptide interaction with a junction of the hybrid transcription factor. A hybrid transcription factor can be formed. This synthetic promoter,
Activating DNA sequence and D encoding the corresponding junction of this hybrid transcription factor
The NA sequence is housed in an independent system. In yet another case, the hybrid transcription factor is enabled by an inhibitor, thus preventing activation of the synthetic promoter by, for example, functional binding of the hybrid transcription factor to the synthetic promoter. As a result, the activating DNA sequence is not expressed unless and until the hybrid transcription factor inhibitor is removed.

Based on this disclosure, those skilled in the art will expect that any DNA sequence or gene of interest can be controlled in this way.

This system is extremely useful for allowing the expression of traits that cannot be recovered as constitutively driven transgenes. For example, foreign genes with potentially lethal effects, or antisense genes or dominant negative mutants designed to abolish the function of essential genes, have received much attention in basic research in plant biology. Have inherent experimental problems. Although reduced transformation rates are often cited as lethal evidence for specific constitutively driven transgenes,
This type of negative result is accompanied by another boring interpretation. Systems of the type described herein allow for the maintenance and reproduction of stability of the test transgene independent of its expression. The ability to make the transgene insertion independent of expression is important for sound conclusions about evolving the nature of gene function. Thus, the present invention has a significant contribution to the art.

[0035] Variations in the degree of phenotype can be achieved by examining the phenotype of multiple independently activating systems crossed to a single activator. By relying on position effects governing different levels of expression ability from different transloci, it is possible to obtain phenotypes of allelic systems for specific traits. This provides plant lines with varying degrees of phenotype with varying levels of expression from antisense genes designed to knock out essential metabolic functions, as described in Example 2 below. Indicates that Similarly, other traits may not be activatable to alter the phenotype in independent systems.

Optionally, the expression of a particular phenotype is regulated at a suitable promoter of the hybrid activator gene, eg, during development or in space. Depending on the stringency of control of the promoter of interest, it is possible to evaluate the function of a particular cell type, tissue or organ, or at a particular time. For example, the requirement for gene function in embryonic development can be tested by activating an antisense transgene having a factor driven by a promoter known to be strongly expressed in developing seeds. Such an approach is known as Drosophila (Droso).
(Phila) has been widely used by randomly inserting GAL4 effector constructs to obtain fusions to various genomic enhancers that direct expression in various cell and tissue types (Brand and Perrimon,
1993). Similarly, a DNA construct of the present invention can be provided via a chemically-inducible promoter.
For example, Schena et al. (Proc. Natl. Acad. Sci., USA, Vol. 88, pp 10421-10425, 19
Dec. 1991) provides additional control of gene expression by utilizing steroid-induced gene expression as exemplified.

[0037] A further example of regulation of expression is achieved by selecting an activation domain of appropriate strength for a particular application. Recently, a net positive or negatively charged plant transcriptional activation domain was identified in a yeast functional screen (Estruch et al., 1994). GA
Fusion of these domains to the DNA binding domain of L4 yields a protein that can activate a GAL4-dependent promoter gene when both are introduced into corn or tobacco cells with microprojectiles. Thus, a wide variety of activation domains can be identified by direct function or structural screening.

Although the work described herein is exemplified in Arabidopsis, the essential transactivation is not limited to this one species. Utilizing a suitable promoter, the system described herein can be used for any species, including, for example, without limitation, commercially important plants, such as corn, rice, wheat, sugar beet, barley, rye, cotton, rapeseed, Oats, sorghum, millet, grass, and ornamental plants.

Hybrid Transcription Factor Gene The hybrid transcription factor gene comprises a DNA sequence encoding 1) a DNA binding domain; and 2) an activation domain that interacts with components of the transcription machinery assembled at the promoter. Gene fragments typically have a DNA binding domain facing the 5 'end and an activator domain facing the 3' end,
The expression is ligated to form a hybrid gene that produces a hybrid transcription factor. One of skill in the art can routinely combine various DNA sequences encoding a DNA binding domain with various DNA sequences encoding an activation domain to provide a wide array of hybrid transcription factor genes.

Examples of DNA sequences useful for making hybrid transcription factors useful in the present invention include, but are not limited to, GAL4, bacteriophage 434, lexA,
lacI and those encoding the DNA binding domain of the phage lambda repressor.

Examples of DNA sequences useful for providing a hybrid transcription factor useful in the present invention include, but are not limited to, herpes simplex VP16, corn C1 and P1.
Encoding the acidic activation domain of Further, a suitable activation domain fuses a DNA fragment from the selected organism to a suitable DNA binding domain,
It can then be isolated by direct screening for function (Estruch et al., 1994, incorporated herein by reference). Transcriptional activator protein domains may be interchangeable between proteins of various origins (Brent and Ptas
hne (1985) Cell 43: 729-736).

The desired hybrid transcribed gene is a G gene fused to the corn C1 activation domain.
Comprising a DNA sequence encoding an AL DNA binding domain. One skilled in the art can utilize conventional molecular biology and recombinant DNA engineering to create the desired hybrid transcription factor gene.

Synthetic Promoters A synthetic promoter comprises at least one DNA binding site recognized by a DNA binding domain of a hybrid transcription factor, and a minimal promoter, preferably a TATA element derived from a promoter recognized by plant cells. More specifically, the TATA element is derived from a promoter that is recognized by the plant cell type into which the synthetic promoter has been incorporated. If desired, the DNA binding site is repeated many times in the synthetic promoter, so that the minimal promoter is more efficiently activated, and thus the activating DNA sequence attached to the synthetic promoter is less. Expressed efficiently.

Examples of DNA binding sites that can be used to create synthetic promoters useful in the present invention include, without limitation, upstream acting sequences (UAS _G ) recognized by GAL4 DNA binding proteins, lac operators and lexA binding site. Examples of promoter TATA elements recognized by plant cells include:
Without limitation, CAMV35S, corn Bz1 promoter, UBQ
3 promoters, Agrobacterium nopaline synthase or mannopine synthase promoters, such as Super MAS
, The maize Bz1 promoter, the UBQ3 promoter, the hsp80 from Brassica oleracea, and the Arabidopsis actin-2 promoter.

The desired synthetic promoter was truncated about 10 concatemers TATA element fused at its 5 'end for direct repeat of the upstream-action sequence (UAS _G) that is recognized by the GAL4 DNA binding site CaMV35
The S sequence (nucleotides -59 to +48 around the start of transcription). One skilled in the art will appreciate that conventional molecular biology and recombinant DNA techniques are used to create the desired synthetic promoter.
Engineering available.

Activating DNA Sequences Activating DNA sequences include any desired DNA sequence for stable introduction and expression in plant cells. Particularly desirable activating DNA sequences are sense or antisense sequences, the expression of which reduces the expression of its endogenous counterpart gene and thus inhibits normal plant growth or development.

The activating DNA sequence can be operably linked to a synthetic promoter to form an activating DNA construct. The activating DNA sequence in the activating DNA construct is not expressed, ie, silent, in the transgenic system unless there is also a hybrid transcription factor capable of binding and activating the synthetic promoter. The activating DNA construct is then introduced into a cell, tissue or plant to form a stable transgenic system that expresses the activating DNA sequence, as described in more detail below.

Cells, Tissues or Plants Comprising a Hybrid Transcription Factor or a Synthetic Promoter and an Activating DNA Sequence The present invention further comprises a first and a second cell comprising a hybrid transcription factor gene and an activating DNA construct, respectively. Or a tissue, preferably a plant cell or plant tissue, or a plant. The first cell, tissue or plant, and the second cell, tissue or plant comprise the hybrid transcription factor gene and express the hybrid transcription factor, and further comprise a synthetic promoter of the activating DNA construct; The plant is selected so that it can be manipulated to construct a plant cell, plant tissue or plant that is activated to express the activating DNA sequence driven by this promoter.

The above-described hybrid transcription factor genes and activating DNA constructs are well known in the art and are commonly used in the art, including, but not limited to, mating, Agrobacterium-mediated transformation, Ti plasmid vectors, direct DNA uptake, For example, they are introduced into cells, tissues or plants by microprojectile bombardment, liposome-mediated uptake, microinjection, and the like.

A transgenic plant system containing the hybrid transcription factor gene is constructed using, for example, Agrobacterium-mediated transformation. Primary transformant (T1 generation)
Screen for their ability to activate expression from the corresponding synthetic promoter (ie, a synthetic promoter activated by a hybrid transcription factor) by transiently transforming them with a reporter construct. The transgenic nature of this system is due to kanamycin resistance (or inserted DNA) as a single locus after selfing.
The T2 generation is further tested for isolation of the other suitable selectable marker genes carried above). The presence of a single T-DNA insert is confirmed by genomic DNA gel blot analysis in a system showing a 3: 1 separation. Such a system could be further analyzed for expression of the hybrid transcription factor gene by RNA gel blot analysis.
Some T2 plants are selfed to obtain T3 progeny, which are screened for homozygosity of the inserted hybrid transcription factor gene.

A transgenic system containing a synthetic promoter that is activatable by the desired hybrid transcription factor is prepared in a manner similar to Agrobacterium-mediated transformation. Synthetic promoter and activating DNA sequence (activating DNA construct)
A transgenic system, optionally containing a selectable marker, is prepared by standard methods. Optionally, the transgenic system is selected for a marker to confirm the presence of the activating DNA construct.

F1 plant containing hybrid transcription factor and activating DNA construct F containing plant hybrid activator gene and activating DNA construct
One plant is made by cross-pollination and selected for the presence of a suitable marker such as kanamycin. In contrast to plants containing only the activating DNA construct, F1
Plants produce high levels of activating DNA sequence expression products comparable to those obtained with strong constitutive promoters such as CaMV35S.

Useful assay methods of the invention include: a) priming a first stably transformed plant comprising a hybrid transcription factor gene encoding a hybrid transcription factor capable of activating a synthetic promoter; A) crossing a second stably transformed plant comprising an activating DNA sequence and a synthetic promoter activatable by said hybrid transcription factor, wherein in a) the synthetic promoter is Present in a plant, and the plant of a) is homozygous for said hybrid transcription factor, and b)
Wherein the DNA sequence is expressed in the presence of the hybrid transcription factor, thereby providing an F1 plant that expresses the DNA sequence; and c) determining the effect of expression of the DNA sequence on the F1 plant. Comprising.

Here, the above-mentioned hybrid transcription factor gene is derived from yeast GAL4 gene.
It preferably encodes an NA binding domain and a transcriptional activation domain derived from the maize C1 gene.

Here, the minimum promoter is preferably selected from the group consisting of a CAMV35S minimum promoter, a corn Bz1 promoter and a UBQ3 promoter.

Here, the synthetic promoter sequence may comprise a TATA element-containing CaMV 35S minimal promoter fused at its 5 ′ end with 10 concatemer copies of the upstream acting sequence recognized by the GAL4 DNA binding domain. preferable.

Here, the above-mentioned hybrid transcription factor gene is derived from yeast GAL4 gene.
The NA-binding domain and a transcriptional activation domain derived from the maize C1 gene, and wherein said activating DNA construct has 10 concatemer copies of the upstream acting sequence recognized by the GAL4 DNA-binding domain and its 5 'end It preferably comprises a synthetic promoter sequence comprising the CaMV 35S minimal promoter containing the TATA element fused in.

Another useful method of the present invention is to use essential plant gene inhibitors, such as plant AdS
A method for identifying an S gene, comprising: a) reacting a plant AdSS enzyme with an AdSS substrate in the presence of a putative inhibitor of plant AdSS enzyme function; and b) reacting the plant with an AdSS enzyme in the presence of the putative inhibitor. Is compared to the rate of a plant AdSS enzymatic reaction under the same conditions in the absence of the putative inhibitor to determine whether the putative inhibitor inhibits AdSS.

A preferred embodiment of the present invention is a plant comprising a hybrid transcription factor and an activating DNA construct, wherein the hybrid transcription factor encoded by the hybrid transcription factor gene comprises A synthetic promoter can be activated to induce expression of an operably linked antisense DNA sequence, wherein the plant is stably transformed with the hybrid transcription factor and the activating DNA construct. And plants.

Here, the hybrid transcription factor gene is a DNA derived from a gene selected from the group consisting of yeast GAL4 gene, bacteriophage 434, lexA, lacI and lambda phage repressor.
A binding domain; a transcription activation domain derived from a gene selected from the group consisting of herpes simplex VP16, corn C1 and P1; a CaMV35S minimal promoter, a corn Bz1 promoter and UB.
An activatable DNA construct comprising a minimal promoter selected from the group consisting of the Q3 promoter.

Here, the synthetic promoter sequence may comprise a TATA element-containing CaMV 35S minimal promoter fused at its 5 ′ end with 10 concatemer copies of the upstream acting sequence recognized by the GAL4 DNA binding domain. More preferred.

Here, the above-mentioned hybrid transcription factor gene is derived from yeast GAL4 gene.
The NA-binding domain and a transcriptional activation domain derived from the maize C1 gene, and wherein said activating DNA construct has 10 concatemer copies of the upstream acting sequence recognized by the GAL4 DNA-binding domain and its 5 'end It is further preferred to comprise a synthetic promoter sequence comprising the CaMV 35S minimal promoter containing the TATA element fused in.

Here, it is further preferred that the activating DNA sequence is an AdSS antisense sequence.

EXAMPLES The examples disclosed below demonstrate that hybrid transcription factors can function to efficiently regulate gene expression in stably transformed plants. The present invention may be better understood with reference to the following non-limiting examples.

Example 1 Expression of Silent Reporter Gene in Stable Transgenic Plants This example demonstrates transgenic expression of a GAL4 / C1 hybrid factor against a system containing a reporter transgene controllable by a suitable synthetic promoter. Shows that mating plants results in strong induction of reporter gene expression. Suitable genes for testing this system in Arabidopsis were constructed as described in more detail below.

The hybrid transcribed gene is constructed from components of the GAL4 and C1 genes previously shown to contain DNA binding and transcriptional activation functions, respectively. (The construct pAT53 used for plant transformation was left coupled to a 35S promoter operably linked to a hybrid transcription factor comprising a GAL4 DNA binding domain coupled to a C1 activation domain. Including the border sequence, with the 35S 3 'terminator sequence and the pNOS / NPT / nos 3' selectable marker cassette flanking the right border sequence). The 147 amino acids N-terminal to the encoded protein are from GAL4, and the 101 amino acids C-terminal are from the carboxy-terminal amino acids 173-273 of C1. Similar combinations have previously been shown to work in transient assays (Goff et al.,
1991).

The synthetic promoter designed to be activatable by this factor is G
Utilizing a Bz1 TATA element fused at its 5 'end to 10 concatemer copies of the upstream acting sequence (UAS _G ) recognized by the AL4 protein (optionally, a TATA element Nucleotides -59 to +
48) using a truncated CaMV 35S promoter comprising (construct pAT73 comprises a left border sequence coupled to a 35S promoter operable linked dihydrofolate reductase coding sequence linked to a 35S 3 'terminator; It is a GU with 35S3 'terminator
Ligation to a 10-fold concatameric GAL4 binding site construct containing a TATA element operably linked to the S reporter element, all of which are flanked by right border sequences). To evaluate the efficiency of this system in stable transformants, modified E. coli driven by a reporter gene, such as a synthetic UAS _G / TATA promoter, as the activating DNA sequence. Coli uidA (β-glucuronidase; GUS
) Select the code sequence. The GUS gene is considered a model reporter gene for expression in that its products can be easily detected and quantified.

A transgenic Arabidopsis plant line containing this hybrid transcription factor gene was constructed utilizing Agrobacterium-mediated transformation. Primary transformant (T1
Generations) were screened for their ability to activate expression from a synthetic UAS _G / TATA promoter by transiently transforming them with a luciferase reporter construct. About half of the T1 transformants showed luciferase activity after microprojectile bombardment. RNA gel blot analysis confirmed that these transformants expressed the GAL4 / C1 gene.
These lines were used in the T2 generation to isolate kanamycin resistance as a single locus after selfing (T
-Selectable marker gene carried on DNA). Single T-
The presence of the DNA insert is confirmed by genomic DNA gel blot analysis in a system showing a 3: 1 separation (data not shown). These systems are further analyzed for GAL4 / C1 gene expression by RNA gel blot analysis. RNA blot analysis showed that both systems expressed detectable levels of stable RNA from this transgene. A single effector system, designated pAT53-103, was selected for further experiments and some T2 plants were selfed to obtain T3 transgenic progeny, which were screened for T-DNA insert homozygosity. .

[0069] Transgenic Arabidopsis plant lines containing the UAS _G / TATA / GUS genes were constructed utilizing Agrobacterium-mediated transformation and selected with methotrexate and screened for homozygosity. Two lines, designated pAT73-309 and -346, were analyzed for GUS activity and showed only trace amounts of activity that were not significantly different from the assay background (Table 1). F1 plants containing both the hybrid transactivator gene and the activating reporter gene were made by cross-pollination and selected with kanamycin. In contrast to plants containing only the reporter gene, F1 plants provided very high levels of GUS activity comparable to those obtained with strong promoters such as CaMV35S (Table 1). Table 1. Β-glucuronidase activity of F1 plant Transformant GUS activity ^* (nmol MU / min / mg protein) 35S / GUS line 7 17.6 ± 4.3 line 105 19.1 ± 7.2 line 115 12.1 ± 3 0.7 Untransformed Nossen 0.03 ± 0.01 pAT53-103 0.01 ± 0.0 pAT73-309 0.01 ± 0.01 pAT73-309 × pAT53-103F1 4.97 ± 3.41 pAT73-346 0.01 ± 0.0 pAT73-346 × pAT53-103F1 6.02 ± 2.3 ^* GUS activity was measured on 20 plants per line or F1 cross line (mean ± standard deviation).

Example 2-Silent antisense DN in stable transgenic plants
Expression of A Sequence In addition, the present invention can be used to examine gene function by inducing the expression of an antisense gene that specifically inactivates the expression of a test gene. The invention is used to drive the expression of antisense genes that eliminate gene function. de nov
o A gene encoding adenylosuccinate synthetase (AdSS) that converts IMP to AMP during one of the two stages of purine synthesis is used as the activating DNA sequence. AdSS has recently been considered a potential target of the natural product hydantosidine (
Cseke et al., 1996; Fonne'-Pfister et al., 1996; Siehl et al., 1996). AdSS activity is determined by standard enzyme assays well known in the art, such as reacting an AdSS enzyme with an AdSS substrate, allowing the AdSS enzyme to catalyze a product that is significantly different from the substrate, and catalyzing the substrate to this product. It is determined by measuring the conversion rate. This conversion can be performed directly by measuring the amount of substrate or product or both present in the reaction at various times, or a label, such as a radioactivity or color indicator, attached to one of the substrates or products. Can be measured indirectly by measuring. Southern blot analysis indicated that the full-length cDNA used for antisense construction here was the sole gene in the Arabidopsis genome.

Based on the expression of the effective antisense DNA sequence achieved in the stable transgenic plant of Example 1, a stable transgene containing both the hybrid transcription factor gene and a synthetic promoter driving the expression of the AdSS antisense sequence It was estimated that effective expression in transgenic plants would result in inactivation of endogenous AdSS gene expression. If the AdSS antisense sequence is not effectively expressed, the plant will die based on effects similar to the herbicidal effect of Hindatocidin. However, before conducting such experiments, it is not clear to those skilled in the art whether the expression of antisense in stably transformed plants will occur and will result in inactivation of the AdSS gene. As described in more detail below, AdS
The S antisense sequence is effectively expressed in stable transgenic plants,
And the expression of the endogenous AdSS enzyme was inhibited.

Fifteen transgenic plants containing the UAS _G / TATA / antisense AdSS construct were generated by Agrobacterium transformation. (Construct pJG2
61 AntiAdSS is an AdSS terminated by 35S3 'and right border sequence
10-fold concatamer G containing a TATA element linked to an antisense coding sequence
(Including the right border sequence coupled to the pNos / BAR / gene 73 'selectable marker cassette linked to the AL4 binding site construct). Flowers that bloomed on the primary transformants were crossed with pollen from the homozygous transactivator line pAT53-103. F1 seeds were plated on kanamycin and outbred offspring were selected. These primary transformants are hemizygous for the introduced T-DNA (including the antisense gene), which is often a single Mendelian trait. Thus, this antisense gene always contains a transactivator in a hemizygous state (except for rare contaminants due to selfing; it is selected by germination with a selectable marker such as kanamycin): Will be separated by one. In the six lines, the growth of about 50% of the seedlings was severely delayed, and some failed to germinate perfectly. The other five lines survived the true leaf breeding season, but showed various growth abnormalities after transfer to soil. The last four lines showed little or no abnormal phenotype.

To determine if severe growth retardation and death was due to the presence of the antisense transgene, a primer designed to amplify the polymerase chain reaction to amplify the region between the 5 'end of the AdSS cDNA and the minimal 35S promoter Was carried out using Gel electrophoresis showed that a one-to-one relationship was observed between the abnormal seedling and the antisense gene. To examine the phenotypic variation between different antisense systems, we performed RNA gel blot hybridization on F1 plants derived from different antisense systems. Untransformed Col-0 plant, p
Gel blots probed with AdSS probes containing RNA from AT53-103 plants and F1 plants from crosses of pAT53-103x antisense AdSS show that AdSS RNA is hardly detectable in systems with a severe phenotype. (The most severe seedling-death line was excluded from analysis because it yielded less tissue than RNA extraction).

This embodiment can be better understood from the following technical description.

The recombinant plasmid pSGZL1 was constructed by ligating the GAL4-C1 EcoRI fragment from pGALC1 (Goff et al., 1991) into the EcoRI site of pIC20H. The GAL4-C1 fragment of pSGZL1 was ligated with BamHI-
Cut with BglII and BamH of pCIB770 (Rothstein et al., 1987).
Insertion into the I site created pAT53.

[0076] Ten UAS _G sites and a minimum 35S promoter (-59 to +1) were added to pGA
LLuc2 (Goff et al., 1991) was excised as an EcoRI-PstI fragment and inserted into the corresponding site of pBluescript to yield pAT52. pAT66 is a HindIII-PstI fragment of pAT52, pCIB1
716 PstI-EcoRI fragment (including 35S untranslated leader, GUS gene, 35S terminator) and HindIII-EcoRI cut pUC
Constructed by 18 three-way ligation. PAT66 35S leader P
Cut with stI-NcoI and PCR construction 35 from +1 to +48 35
Replaced with S-leader to obtain pAT71.

PCIB921 contains the dihydrofolate reductase (dhfr) plant selectable marker gene inserted into the BamHI site of pCIB710 (Rothstein et al., 1987). The 35S promoter / dhfr gene cassette of pCIB921 was excised with XbaI-EcoRI and the corresponding site of pCIB730 (Roth
stein et al., 1987) to create pAT58. pAT73 has 10 U
P, including the AS _G site / minimal 35S promoter / GUS / 35S terminator
It was constructed by inserting the EcoRI fragment from AT71 into the EcoRI site of pAT58.

The plasmid pBS SKt (Stratagene, La Jolla, CA) was linearized with SacI and Treatment with bean nuclease removed the SacI site and religated with T4 ligase to create pJG201. UAS _G / CAMV35
SAT promoter / GUS gene / CaMV terminator cassette
71 was removed with KpnI and cloned into the KpnI site of pJG201 to create pJG304. Restrict pJG304 to endonuclease Asp
The full length linear fragment was isolated by partial digestion at 718. This fragment is combined with a molar excess of the oligonucleotide GTACCTCGAGTCTAGACTCGAG.
Ligation with 3 '. Restriction analysis was used to identify clones with this linker inserted 5 'to this site, and this plasmid was used to construct pJG3
04 / EXhoI.

A fragment of a previously published AdSS synthase cDNA clone (Fonn
e'-Pfister et al., 1996) (GenBank accession number U49389) was PCR amplified with the following oligonucleotide primers: 5'GATTCGAGCTCATGT.
CTCTCTCTTCCCTCC 3 'and 5' GATTCCCATCATGTGGA
CCTGAACCAACTC 3 '. The vector pJG304ΔXhoI was transformed into Sac
Digestion with I and NcoI cut out the GUS genetic coding sequence. AdSSPCR
The fragment was digested with SacI and NcoI and pJG304ΔXhoI
To make pJG304 AntiAdSS.

The vector pGPTV (Becker et al., 1992) was digested with EcoRI and HindIII to remove the nopaline synthase promoter / GUS cassette. At the same time, a superlinker was added from pSE380 (Invitrogen, San Diego, CA) to EcoRI.
And HindIII, and EcoRI / HindIII linearized pG
It was cloned into PTV to create pJG261.

The pJG304 AntiAdSS was cut with XhoI and the cassette containing the UAS _G / 35S minimal promoter / antisense AdSS / CaMV terminator fusion was cut out. This cassette was ligated into XhoI digested pJG261 such that its transcription was independent of that of the bar selectable marker.
1 AntiAdSS was made.

The transgenic plant pJG261 AntiAdSS was transformed into Agrobacterium. Agrobacterium tumefaciens strain GV3101 (pMP90) (Koncz and Sc
hell, 1986) and the resulting strain was used to transform Arabidopsis plants (ecotype Columbia) by invasion (Bechtold et al., 1993). Glufosinate (Basta; Agr Ev
o) at 15 mg / L (Murashige-Skoog salt 4.3 g / L, MES
0.5 g / L, sucrose 1%, thiamine 10 μg / L, pyridoxy 5 μg /
L, nicotinic acid 5 μg / L, myo-inositol 1 mg / L, pH 5.8).

An Arabidopsis root explant (ecotype Nossen) was transformed with pAT53 as described (Valvekens et al., 1988).

[0084] Fifteen transgenic plants containing the UAS _G / minimal CaMV 35S promoter / antisense AdSS construct were planted in soil and grown in a greenhouse to maturity. Flowers that bloomed on the primary transformants were crossed with pollen from the homozygous GAL4 / C1 transactivator line pAT53-103. 5 seeds
Plates were plated on a germination medium containing 0 mg / L kanamycin.

Nucleic Acid Analysis RNA was isolated as described by phenol / chloroform extraction followed by LiCl precipitation (Lagrimini et al., 1987). RNA gel blots were performed as published (Ward et al., 1991). The hybridization probe was prepared with ³² P-dCTP using Prime Time Kit (International Biotechnologie).
s, Inc., New Haven, CT). Hybridization conditions were 7% sodium dodecyl sulfate (SDS), 0%
. 5M NaPO ₄ , pH 7.0, 1 mM EDTA, 1% bovine albumin 65 ° C
And After overnight hybridization, filters were filtered with 1% SDS, 50 mM
NaPO ₄ and 1 mM EDTA at 65 ° C. (Church and Gilbert, 1984)
.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 C12N 15/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), 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 GD, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK , MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Borleis, Sandra Lin United States, North Carolina 27707, Durham, Pin Oak Drive 4225 (72) Inventor Gerlach, Jorn United States of America, North Carolina 27707, Durham, King Charles Road 3907

Claims (11)

[Claims] 1. An assay method comprising: a) a first stably transformed plant comprising a hybrid transcription factor gene encoding a hybrid transcription factor capable of activating a synthetic promoter; b) Crossing a second stably transformed plant comprising an activating DNA sequence and a synthetic promoter activatable by said hybrid transcription factor, wherein in a) said synthetic promoter is a plant of a) And the plant of a) is homozygous for said hybrid transcription factor, and b)
Wherein the activating DNA sequence is expressed in the presence of the hybrid transcription factor, thereby providing an F1 plant that expresses the activating DNA sequence; and c) providing the activating DNA sequence to the F1 plant. Determining the effect of the expression. 2. The assay according to claim 1, wherein the hybrid transcription factor gene encodes a DNA binding domain derived from the yeast GAL4 gene and a transcription activation domain derived from the maize C1 gene. 3. The assay according to claim 1, wherein the minimal promoter is selected from the group consisting of a CAMV35S minimal promoter, a corn Bz1 promoter and a UBQ3 promoter. 4. The synthetic promoter sequence comprises a CaMV 35S minimal promoter containing a TATA element fused at its 5 'end with 10 concatemer copies of the upstream acting sequence recognized by the GAL4 DNA binding domain. The assay of claim 1. 5. The hybrid transcription factor gene encodes a DNA binding domain derived from the yeast GAL4 gene and a transcription activation domain derived from the maize C1 gene, and the activating DNA construct comprises a GAL4 DNA binding domain. Concatemer copies of the upstream acting sequence and its 5
2. The assay of claim 1, comprising a synthetic promoter sequence comprising a CaMV 35S minimal promoter containing a TATA element fused at the 'end. 6. A method for identifying an AdSS herbicide inhibitor, comprising: a) plant AdS herbicide inhibitor in the presence of a putative herbicide inhibitor of plant AdSS enzyme function.
Reacting the S enzyme with the AdSS substrate; and b) determining the percentage of the plant's AdSS enzyme reaction in the presence of the putative herbicide inhibitor under the same conditions in the absence of the putative herbicide inhibitor. Plant Ad
The putative herbicide inhibitor was compared to the rate of the SS enzymatic reaction,
Determining whether or not the inhibitory activity is inhibited. 7. A plant comprising a hybrid transcription factor and an activating DNA construct, wherein the hybrid transcription factor encoded by the hybrid transcription factor gene activates a synthetic promoter of the activating DNA construct. To induce expression of an operably linked antisense DNA sequence, wherein the plant has been stably transformed with the hybrid transcription factor and the activating DNA construct. ,plant. 8. The hybrid transcription factor gene comprises: a) the yeast GAL4 gene, bacteriophage 434, lexA, lacI.
And a lambda phage repressor, derived from a gene selected from the group consisting of
B) a transcription activation domain derived from a gene selected from the group consisting of herpes simplex VP16, corn C1 and P1; c) a minimal promoter selected from the group consisting of a CaMV35S minimal promoter, a corn Bz1 promoter and a UBQ3 promoter. 8. The plant of claim 7, comprising an activating DNA construct. 9. The synthetic promoter sequence comprises a CaMV 35S minimal promoter containing a TATA element fused at its 5 'end with 10 concatemer copies of the upstream acting sequence recognized by the GAL4 DNA binding domain. 7. The plant according to 7. 10. The hybrid transcription factor gene encodes a DNA binding domain derived from the yeast GAL4 gene and a transcription activation domain derived from the maize C1 gene, and the activating DNA construct comprises a GAL4 DNA binding domain. The plant according to claim 7, comprising a synthetic promoter sequence comprising a CaMV 35S minimal promoter containing a TATA element fused at its 5 'end with 10 concatemer copies of the upstream acting sequence recognized by the following. 11. The plant according to claim 7, wherein said activating DNA sequence is an AdSS antisense sequence.