KR100801103B1 - Artificial bidirectional promoter for activation of gene expression - Google Patents

Abstract

Bidirectional modules for the activation of gene expression in both directions and for the regulation of transcription are disclosed. The bidirectional module includes a plurality of cis-regulatory DNA sequence elements strategically arranged to provide a Transcription Activating Module that achieves a high level of expression from the Transcription Initiation Module. do. The latter acts like a minimal promotor. The former activates transcription simultaneously from the latter in both directions and also responds to various transcription induction, external stimuli in both directions. Since it is an artificially designed bidirectional transcription module, it does not have a corresponding DNA sequence in the plant genome. This reduces the opportunities for genes to be silencing by homology based mechanisms. Such a bidirectional promoter module can thus be used to develop efficient vectors for genetic engineering in plants.

Bidirectional promoter, plant transformation vector, computational biology, artificially designed promoter, transcription initiation module (TIM), transcription activation module (TAM)

Description

Artificial bidirectional promoter for activation of gene expression

The present invention includes a fully artificial bidirectional expression module, which may also be referred to as a bidirectional promotor module. The bidirectional module comprises a plurality of cis-regulatory DNA sequence elements strategically arranged to provide a Transcription Activating Module that achieves a high level of expression from the Transcription Initiation Module. cis regulatory DNA sequence elements). The latter functions like a minimal promotor. The former activates transcription simultaneously from the latter to both directions and also responds to various transcription inducing external stimuli in both directions. Because the present invention is an artificially designed bidirectional transcription module, it does not have an equivalent DNA sequence in the plant genome. This reduces the likelihood that genes will be silencing by homology-based mechanisms. Such a bidirectional promoter module can thus be used to develop efficient vectors for genetic engineering in plants.

The process of gene expression involves the formation of two construction steps, namely a protein or polypeptide, or in some cases RNA, which has specific functions, including transcription and translation. The process of transcription is the most important regulatory step in gene expression and its regulation. Initiation of transcription and modulation of gene expression in eukaryotic genes is governed by various DNA sequence elements collectively arranged in larger sequences called promoters. The promoter is the portion of the DNA sequence that is present on the 5 'side, i. The promoter contains signals for RNA polymerase machinery that initiate transcription and also regulate the level of transcription. Typical eukaryotic promoters are composed of two parts, one is the minimum, or core promoter (core promoter), the other is in the upstream regulatory sequences (upstream regulatory sequences) or cis-regulatory elements (cis regulatory elements) [Odell, JT , Nagy , F, & Chua N.-H Nature 313, 810-812 (1985) and Benfey , PN & Chua , N.-H. Science 250, 959-966 (1990) ].

The minimum promoter or key promoter is the smallest stretch of contiguous DNA sequence sufficient to direct the exact initiation of transcription by RNA [ Pol II machinery, Smale , ST, genes dev 15, 2503-2508 (2001) . Typical core promoters include initiation sites of transcription with various sequence motifs, and sequence motifs include TATA boxes, Intr sequences, TFIIB recognition elements (BRE), and other key promoter motifs [ Jennifer , EF et. al ., genes & dev 16: 2583-2592 (2002) ]. The core promoter provides basic or common transcription factors such as TFIIA, B, D, E, F and H to the site of action of RNA polymerase II, an enzyme composed of a plurality of subunits. These factors aggregate to form a transcriptional pre-initiation complex (PIC) that catalyzes the synthesis of RNA from a DNA template.

Activation of the core promoter is performed by additional sequences of regulatory DNA sequence elements that various proteins bind to and subsequently interact with the transcription initiation complex to activate gene expression. These regulatory elements individually and / or in combination comprise DNA sequences that determine the pattern of spatio-temporal expression of the promoter [ Benfey , PN, Ren , L. & Chua , N.-H. EMBO 9: 1685-96 (1990) ]. Such short regulatory elements exist at various distances from the point of transcription initiation, so that some regulatory elements (called proximal elements) are adjacent to the core promoter, and others are several kilometers upstream or downstream of the promoter. The bases may be located at distance (enhancers). Both types of promoter elements modulate the level of transcription from the core promoter ( Wasylyk , B. CRC . Critical Rev. Biochem, 23: 77-120 (1988), Johnson & McKinght Ann . Rev. Biochem . 58: 799-839 (1989), Fussler & Gussin , Method in Enzymology 273: 3-29 (1996) ]. Promoters are typically located 5 'upstream of the transcription initiation site of the coding region of the gene of interest.

Transgenic plants are developed to improve the desired properties (eg yield, disease resistance, phytoremediation, etc.) and use them as protein plants. Often, it is necessary to introduce a plurality of genes to achieve the goals of genetic engineering. For example, almost always one gene (eg, resistance to antibiotics such as kanamycin) is used to enable the selection of transformed cells and tissues and improve the properties of the transgenic plant. A second gene is used as a target gene to be used (e.g., a gene that enhances yield or confers resistance to a disease). Each such gene must be normally expressed from its own promoter sequence. The constitutive promoters most widely used in the development of transgenic plants are the cauliflower mosaic virus 35S transcript promoter and promoters of nopaline and octapine synthase, ubiquitin promoters, etc. to be. However, the introduction of a plurality of useful genes into plants using the same promoter repeatedly results in gene silencing. Several strategies have been developed to introduce multiple genes through genetic engineering, sequential transformation using multiple gene constructs with different selection markers, and co-transformation by multiple constructs. ), Genetic crosses between plants transformed with different constructs, and the like. One way to achieve the expression of a plurality of genes while at the same time reducing the size of the plant transformation vector and the number of promoters required would be to develop transcriptional regulatory signals that can initiate and regulate transcription in both directions.

The use of bidirectional promoters can clearly overcome the limitations of sequential transformation and co-transformation by two genes. [ Xie et al ., Nature Biotechnology , 19: 677-679 (2001) , invented a method of converting a natural unilateral or polar promoter into a bidirectional promoter. Such promoters may govern the expression of two genes or gene fusions at a time. Only some types of powerful promoters, such as the CaMV 35S promoter, have been reported and can be bidirectional.

There is no conventional report on building a fully artificially designed promoter that will not have a long range of homology with native genomic DNA. Developing the ability to design an artificial promoter does not have any sequence homology with the genome is very important, because such are the promoter will not be because the Cylons Singh homology. [Davies et al ., The Plant Journal 12, 791-804 (1997) ].

We have previously established a technique for designing artificial synthetic promoters [ ( US Patent application 09/263692; EP 99301419.0-2106, Sawant et al ., 2001, Theor. Appl. Genet . 102, 635-644) . Such conventional synthetic promoters are described in detail in the above references and are considered to be incorporated herein by reference:

The present invention is partially based on SEQ ID NO: 1.

It is based on the discovery that a portion of the artificial promoter indicated in can be used as an enhancer sequence or 'transcriptional activation module' (TAM).

 Another portion of SEQ ID NO: 1 may be used as a transcription initiator or transcription initiation module (TIM). The present invention teaches that TAM activates and regulates gene expression in both directions from the TIM.

Artificial synthesis of a powerful gene expression module provides a tool for avoiding the repetitive use of so-called powerful promoters because various artificial promoters can be designed. In addition, developing a powerful bidirectional promoter with a plurality of tissue-specific or inducible features will have major applications in the improvement of desired crop properties in plants. Designing expression vectors for gene conversion using so-called bidirectional expression modules would be of great use for the transformant development and biotechnology industries. As demonstrated herein, the technique of designing bidirectional promoters in entirely computational methods provides considerable flexibility in genetic engineering.

Construction of synthetic gene expression modules is an important alternative to the dependence on natural promoters. This allows you to bypass the gene silencing and also gives the ability to regulate the expression levels of the proteins useful in the plant, or a compound of a special economic value and improvements. [(Rance, I. et al ., Plant Science 162: 833-842 (2002), combined three viral promoter sequences to generate highly active promoters that allowed strong transgene expression in plants ) ].

The inventors of the present invention (Sawant S. et al., Theor. Appl. Genet. 102: 635-644) provided the only example to develop a fully artificial synthetic promoter designed for high levels of expression in plants. The promoter (US Patent Application No. 09/263692, EP Application No. 99301419.0-2106) was developed by computational methods and demonstrated to be expressed at high levels in various plants, thus achieving design goals.

Prior to the present invention, there are some representative patents summarized below, which describe bidirectional promoters. US Pat. No. 58,146,18 discloses a bidirectional promoter having a plurality of tet operator sequences. This patent claims that the seven repeats of prokaryotic tet repressor / operator / inducer sequences found naturally when flanked by two minimal promoters in the presence of a tetracycline inducer are eukaryotic. It can direct the expression of two genes in cells. U.S. Pat.No. 595564 discloses a bidirectional heterogeneous but naturally occurring construct for the expression of transgenes in plants. U. S. Patent No. 5,359, 142 discloses natural promoter sequences, which can be engineered to allow for variations in enhancement of gene expression. US Pat. No. 5837849 discloses another natural plant enhancer element that enhances the level of transcription of a plant expressible gene. U. S. Patent No. 5627046 discloses a natural bidirectional promoter. Techniques for making bipolar promoters of eukaryotic cells bidirectional are disclosed in US Pat. No. 6,388,170. The inventors of the patent used a technique to bi-directionally specific groups of natural plant promoters such as CaMV 35S, PCISV, OPR, and SAG12.

It is an important object of the present invention to provide a method for computationally designing and artificially synthesizing a bidirectional transcriptional activation DNA module that modulates the expression level of a plurality of transgenes in plants simultaneously.

Another object of the present invention is to artificially design and chemically synthesize a 'transcription initiation module' that activates or initiates transcription and use it for expression from any 'transcriptional activation module'.

It is another object of the present invention to provide a 'transcription initiation module' which will determine the direction and initiation point of transcription in a bidirectional promoter.

Another object of the present invention is to provide a completely artificially designed 'bidirectional' design by placing a so-called transcription initiation module on one or both sides of a so-called 'transcription activation module' to modulate the expression of one or both genes in one or both directions. Expression module '.

It is still another object of the present invention to provide computationally designed promoters that do not have a stretch that is homologous to genomic DNA and thus stably express the transgene in the transgenic population and do not exhibit silencing.

Another object of the present invention is to view by artificially designing and developing a novel 'bidirectional expression module' based solely on nucleic acid sequence analysis of a database of genes selected based on the potential to be expressed at high levels in plants. To verify the functional potency of sequence features identified by the inventors.

Another object of the present invention is to select markers such as nptII (kanamycin resistance) or hptII (hygromycin resistance) or one useful gene in one direction, and GUS A, GFP, or any other useful protein in the other direction. It is to provide a plant transformation vector using a so-called 'bidirectional expression module' that expresses a reporter gene such as a coding gene.

Another object of the present invention is to demonstrate the usefulness in developing transformed plants using the designed plant transformation vector and improving the desired agronomically important features in the plants.

Summary of the Invention

The present invention is directed to an improvement on the applicant's prior invention cited above. More specifically, the present invention relates to novel artificially synthesized and strategically designed bidirectional gene expression modules capable of modulating the expression of one or two genes, respectively, together in one or two directions. This novel bidirectional expression module was chosen as part of the entire DNA sequence that has been reported in the prior invention, including chemically synthesized and theoretically designed DNA sequences. The present invention provides a portion of a DNA sequence, called 'transcriptional activation module' (TAM), represented by SEQ ID NO: 1 below:

Is a second component DNA sequence called 'transcription initiation module' (TIM) represented by SEQ ID NO: 2 below:

It is based on the discovery that if placed in one or both directions it can activate transcription in one or both directions. Thus, if the 'transcription initiation module' (TIM) is strategically placed, the first portion, i.e., the 'transcription activation module' (TAM), can modulate the expression of genes simultaneously in both sense and antisense directions. Thus, TIM is essential for using TAM. The former functions as a minimal or key promoter and is referred to as P _tim in the embodiments given herein.

The 'transcriptional activation module' identified in the present invention is designed entirely based on nucleotide sequences in a database of genes selected based on their potential to be expressed at high levels in plants. This activation module contains a plurality of cis-regulatory factors, which have been identified in the region 100 to 500 bp upstream to the left or in the 5 'direction of the transcription initiation site of highly expressible plant genes.

These cis-elements have been computationally arranged to design an artificial 'transcriptional activation module' that can enhance the expression level of genes in an orientation-independent manner in transformed plants. Thus, the 'transcriptional activation module' functions like a bidirectional transcription enhancing sequence.

Another feature of the invention is the identification of a 'transcription initiation module' which activates the transcription of genes located downstream of the transcription initiation site from the prior invention. The so-called 'transcription initiation module' was designed based on the unique characteristics of the nucleotide sequence in the TATA box, transcription initiation site, untranslated leader and translation initiation regions of plant genes that are highly expressible in the database.

The present invention describes a fully artificially designed 'bidirectional expression module' consisting of a 'transcription activation module' and a 'transcription initiation module' based on the computational analysis of a data set of plant genes that are highly expressed from a nucleic acid sequence database. Such artificially designed sequences do not have sequences that are substantially equivalent or homologous to the plant. Thus, artificially designed sequences are more stable in expression because they are not silencing by silencing of genes by homology ( G. J Davies, MA Sheikh, OJ Ratcliffe , G. Coupland). and IJ Furner , The Plant Journal 12, 791-804 (1997). The results presented by this bidirectional expression module demonstrate the additional and very useful function of the features previously identified by the inventors. The present invention is directed to the design and development of highly expressive bidirectional gene expression cassettes for tightly regulated, tissue specific, constitutive and inducible expression of a plurality of transgenes in transgenic plants. Demonstrate the potential of computational biology.

Another important aspect of the present invention is the simultaneous enhancement of expression of genes placed in both directions of artificially designed synthetic 'transcriptional activation modules' by various cellular and environmental agents. For example, regulation of gene expression corresponding to salicylic acid, indole acetic acid, sodium chloride, and the like in transformed tobacco is presented as part of the present invention. This property of the bidirectional expression module shows that the transcriptional activation module of the present invention also functions as a chemically inducible bidirectional enhancer.

Thus, the present invention provides a bidirectional promoter as:

a) a transcriptional activation module comprising a chemically synthesized and strategically engineered artificial nucleotide sequence having a sequence as shown in SEQ ID NO: 1 or a sequence showing up to 70% homology therewith, designed to enhance the expression level of genes in plants;

b) a minimal sequence comprising a chemically synthesized and strategically engineered artificial nucleotide sequence having a sequence as shown in SEQ ID NO: 2 or a sequence showing up to 70% homology therewith that initiates transcription of the gene placed downstream A transcription initiation module designed to function.

Preferably, the transcription initiation module is located on one or both sides of the 'transcriptional activation module' to simultaneously express one or two genes, one at a time, to develop genetically engineered plants.

In another preferred embodiment, the transcription initiation module is disposed in the 5 'to 3' direction on one or both sides of the transcriptional activation module.

Preferably, one or more important genes are disposed downstream of the transcriptional activation module for expression purposes from one or both of the transcriptional activation module.

In another preferred feature of the invention, the transcriptional activation module is a statistically identified fingerprint sequence that is commonly found in plant genes that are highly expressed within 100 to 500 nucleotide positions upstream of the transcription initiation site in plants. DNA sequence of SEQ ID NO: 1).

In another aspect of the invention, the transcription initiation module comprises the DNA sequence of SEQ ID NO: 2 of the fingerprint sequences statistically identified in plants as being within 100 nucleotides upstream of the transcription initiation site of natural promoters.

The invention also relates to transgenic plants developed after being stably transformed by the bidirectional promoter described above for the purpose of improving agricultural or industrially important plant characteristics.

The present invention also expresses a selection marker, such as nptII , bar, hpt, etc., or any other such gene from one direction, and gusA , gfp , luc , or the like , from another direction , which can be conveniently monitored. Plant transformation vectors comprising a bidirectional promoter as described above expressing a reporter gene such as any gene and the use of such vectors for the development of transgenic plants.

In a preferred embodiment, the invention is:

a) a transcriptional activation module comprising a chemically synthesized and strategically engineered artificial nucleotide sequence having a sequence as shown in SEQ ID NO: 1 or a sequence showing up to 70% homology therewith, designed to enhance the expression level of genes in plants ;

b) a minimal sequence comprising a chemically synthesized and strategically engineered artificial nucleotide sequence having a sequence as shown in SEQ ID NO: 2 or a sequence showing up to 70% homology therewith and initiating transcription of the gene placed downstream A transcription initiation module designed to function as;

c) The transcription initiation module is directed to a bidirectional promoter, wherein the transcription initiation module is located on one or both sides of the 'transcriptional activation module' to develop genetically engineered plants one or two genes at a time, or simultaneously.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

1 shows a number of gene constructs made in accordance with the present invention.

The present invention is directed to a 'bidirectional transcription activator DNA module' comprising a plurality of transcriptional cis-regulatory elements identified upstream (100-500) from the transcription initiation site of highly expressible plant genes. Artificial design and chemical synthesis. Detailed claims regarding designs based on computational analysis of highly expressing plant promoters have been previously filed in pending US patent application Ser. No. 09/263692 or European patent application 99301419.0-2106.

These so-called cis-regulated conserved sequence elements have their percentage occurrence, copy number and most commonly occur in a data set of highly expressed plant genes to form a completely novel transcriptional activation module (SEQ ID NO: 1). It was strategically arranged on the basis of the location. TAM is a 312 bp long sequence whose core function is activation or augmentation of transcription.

This so-called 'transcription activation module' of the present invention is a completely new artificially synthesized 'composition that can enhance transcription in sense and antisense directions when the' transcription initiation module 'is placed in both directions of the' transcription activation module '. Bidirectional expression modules'.

The so-called 'transcription initiation module' used in the present invention is also theoretically designed and chemically synthesized, and is a unique feature of the nucleotide sequences of the TATA-box proximal region of a series of highly expressed genes in plants. Redesigned based on (SEQ ID NO: 2). TIM is 130 bp in length and its key function is to initiate transcription of the gene when placed downstream of the gene.

We used so-called 'transcription activation modules' and 'transcription initiation modules' to produce fully artificial 'bidirectional expression modules' that modulate expression of genes from sense and antisense directions.

SEQ ID NO: 1 is 312 bp in length and SEQ ID NO: 2 is 130 bp in length. A number of genetic constructs used in the examples are shown in FIG. 1. Embodiments illustrate the bidirectional functionality of the present invention. The present invention describes a fully artificially designed 'bidirectional expression module' comprising a 'transcription activation module' and a 'transcription initiation module' based on the computational analysis of a dataset of highly expressed plant genes from a nucleic acid sequence database. Such artificially designed sequences do not have substantially corresponding or homologous sequences in the plant. Thus, artificially designed sequences are more stable because they are not silenced due to the silencing of homologous genes (GJ Davies , MA Sheikh , OJ Ratcliffe , G. Coupland). and IJ Furner , The Plant Journal 12, 791-804 (1997) ]. The results presented by this bidirectional expression module demonstrate the additional and very useful function of the features previously identified by the inventors. The present invention provides a highly expressible bidirectional gene expression cassette for at a time expression of a plurality of closely controlled, tissue specific, constitutive and inducible transgenes in transformed plants of the two sequences. Demonstrate the potential of computational biology in their design and development. However, as is known to those skilled in the art, up to 30% of sequence variations may not affect the function of TAM and TIM. In addition, as illustrated in the preceding patent applications (US Patent Application Serial No. 09/263692 and European Patent Application No. 99301419.0-2106), there are several variations of naturally designed sequences in nature.

The invention is described below with reference to non-limiting embodiments, the purpose of which is to merely illustrate the invention.

Example  One

Expression of Reporter Gene ( gusA ) Placed in the Sense Direction of the Transcription Activation Module (TAM) in Transgenic Tobacco Plants

Table 1

The gene for glucuronidase ( gusA ) is placed downstream of an artificially designed 'transcription initiation module' ( P _tim ) and then such cassettes are artificially designed 'transcription activation module' in the 5 'to 3' direction. It was arrange | positioned to the right side (sense direction) of ( TAM ). In this case, the gene cassette was not arranged on the left side (antisense direction). Gene constructs are shown in FIG. 1. TAM is P _tim on the left Or to demonstrate that activating transcription to the right even in the absence of the gene. Transformed tobacco plants were kanamycin resistance genes cloned into the known and commercially available (Clontech, USA) binary vector pBI 101 as the selection marker ( nptII ) and the construct TAM / P _mec cloned. Agrobacterium tumeo Pacifico Enschede (Agrobacterium containing the gusA tumefaciens ) was developed using LBA 4404. The results of gusA expression in five transformed plants are given in Table 1. As can be seen, TAMs express the reporter gene gusA very efficiently in the leaves of transformed tobacco plants. As expected, the activity varies from 10.8 for plant # 527-2 to 324.2 for plant # 527.1. Such changes in expression levels are expected and known because of the integration of the transgene ( gusA ) at different positions on tobacco chromosomes.

Example  2

Expression of a Second Reporter Gene ( gfp ) in Transgenic Tobacco Plants Positioned in the Sense Direction of the Transcription Activation Module (TAM)

TABLE 2

In this case, a reporter gene different from that taken in Example 1 was chosen to demonstrate that high levels of expression from the right side of TAM are a common phenomenon and not limited to gusA . In this case, the gene for the green fluorescent protein ( gfp ) is placed downstream of the artificially designed transcription initiation module ( P _tim ) and such cassettes are artificially designed 'transcriptional activation module' ( TAM) in the 5 'to 3' direction. ) To the right side (sense direction). In this case, not place the gene cassette is left (antisense orientation) of the TAM TAM sikindaneun to demonstrate that even in this case there is no gene transcription activation in the right to left. Selection marker ( nptII ) suitable for binary vector pBI 101 and the structure TAM- P _tim described above It was developed using Agrobacterium tumeo Pacifico Enschede with gfp. Gene constructs are shown in FIG. 1. As can be seen, TAMs express the reporter gene gfp very efficiently in the leaves of transformed tobacco plants. As expected, the activity varied from 165.5 for plant # 528.32 to 881.0 for plant # 528.12. Such changes in expression levels are expected and known because of the integration of the transgene ( gfp ) at different positions on the tobacco chromosomes.

Example  3

Expression of gusA and gfp disposed in opposite directions of the transcriptional activation module to demonstrate the bidirectional function of the 'transcriptional activation module' in transformed tobacco plants

TABLE 3

In this example, the expression of the two reporter genes described in Examples 1 and 2 showed that they are activated when placed on opposite sides of the artificially designed bidirectional module TAM . Both genes were expressed simultaneously in the leaves of the transformed tobacco. The reporter gene gfp was placed in the sense direction (right side of the TAM ) with artificially designed P _tim and gusA was placed in the antisense direction (left side of the TAM ) with Ptim . Both were in the 5 'to 3' direction along the TAM. As in the two examples described above, transformed tobacco plants were developed using Agrobacterium tumerfaciens and selected based on kanamycin resistance. The results show that two reporter genes were simultaneously expressed in both directions in all plants. The expression levels of the two genes showed variations, as expected due to the different positions of integration of the transgene in the genome. This example establishes the bidirectional nature of the module TAM viz., SEQ ID NO: 1 when used in the P _tim (antisense) -TAM - P _tim (sense) format as shown in FIG. 1.

Example  4

Simultaneous enhancement of expression of genes arranged in two directions in response to induction of transcription by salicylic acid in transformed tobacco plants.

Table 4

                        1301-5 78850 3110

This example demonstrates that an artificially designed transcriptional activation module ( TAM ) not only activates gene expression in both directions, as shown in Example 3, but also provides additional enhancement of expression in response to external stimuli as shown in Example 4. see. In this example, salicylic acid was used as an external stimulus. The results in Table 4 establish that TAM can be multiplied by salicylic acid simultaneously in both directions. For example, glucuronidase activity ( gusA ) was enhanced about 57-fold in transformed plant # 1301-3. At the same time, in the same plants, green fluorescent protein ( gfp ) was also enhanced 8-fold. As shown in the gene construct in FIG. 1, the former was in the antisense direction and the latter was in the sense direction. Similarly, in plant # 1301-5 an increase of about 788 fold was observed for gusA (antisense direction) and a 31 fold increase for gfp (sense direction). The results establish that TAM responded to salicylic acid in both directions. However, the degree of enhancement could be different in both directions.

Example  5

Simultaneous enhancement of expression of genes arranged in two directions corresponding to induction of transcription initiation by NaCl and IAA.

Table 5

This example shows that the 'bidirectional expression module' designed as in Example 5a increased expression in response to NaCl (sodium chloride) treatment. The results in Table 5 show that TAM in transformed line 1301-3 showed about a 10-fold increase in antisense direction (GUS activity) and a 12-fold increase in GFP in the sense direction. Different expression levels of gusA and GFP in different transformed lines change due to the integration position of the transgene in the genome. Gene constructs are shown in FIG. 1. Transgenic plants are as described in Table 4.

Example  6

Enhancement of expression of genes arranged in two directions corresponding to induction of transcription by indole acetic acid (IAA) in transformed tobacco plants.

Table 6

This example shows an increase in gusA and gfp activity upon IAA (indole acetic acid) treatment on transformed tobacco leaves. Glucuronidase activity ( gusA ) was enhanced about 7-fold in transgenic plant # 1301-4. In the same plant, gfp was enhanced 22-fold. In plant # 1301-5 (antisense direction), there was a 4-fold increase in gus activity and an 8-fold increase in gfp activity (sense direction). The results shown in Examples 4, 5 and 6 show the chemically inducible behavior of the synthetic 'transcriptional activation module' which modulates expression in both directions.

Example  7

Development of crop-enhanced tobacco plants by expressing insecticidal protein from a bidirectional enhancer.

TABLE 7

This example establishes that an artificially designed transcriptional activation module can be used to develop vectors for the transformation of plants and for the development of improved transgenic plants for better crop performance. In this example, a known selection marker gene ( npt1 ) for kanamycin resistance was placed to the left of the TAM (antisense strand) along with P _tim to enable selection of transformed plants based on resistance to kanamycin. . Crystal protein gene was placed with a very useful genes to crop cryIAc ever known (from Schiller's Bar turing group N-Sys (Bacillus thuringiensis) bacteria in the soil in the first ningdoem Croix) to the right of the TAM (sense orientation) P _tim. cryIAc encodes a protein highly toxic to larvae of insects such as man-hour neck helicase Kobe fail-fast (Helicoverpa) species. Transformed plants were selected based on their resistance to kanamycin. All five selected transgenic plants showed expression of the insecticidal protein CryIAc in the range of 0.07 to 0.16% of total soluble leaf protein, as estimated by ELISA. All five plants were toxic to larvae and resulted in a killing rate of 89 to 100% of the 1 ^st instar larvae that ate leaves of transformed tobacco plants. The results show that the fully artificially designed sequence TAM in combination with P _tim can be used to develop bidirectional promoter-based vectors that can be used for the development of industrially valuable transformed plants. Similar results were obtained when transformed cotton lines were developed. Insect toxicity Coker cotton lines expressing δ-endotoxins were obtained from similar bidirectional modules. They are very resistant to patronize Kobe fail-fast kind of sports and helping Terra species (Spodoptera s p.) As two major boll worm (bollworm), ingestion damage (feeding damage) caused by pests, including the Insignia mokryu called.

conclusion

To demonstrate the usefulness and features of the designed 'bidirectional expression module', the inventors have reported reporter genes glucuronidase A ( gusA or GUS), Green, placed in the sense and antisense directions of the expression module (FIG. 1). Various gene constructs were prepared using fluorescent protein ( gfp ), δ-endotoxin ( cry ), and used in the examples. Expression patterns of reporter genes can be one at a time (Examples 1 and 2) or two at a time (Examples 3, 4, 5, 6, 7) to the extent of expected variation in expression levels within different lines maintaining the same construct. ) Is taken, is similar and comparable in both sense and antisense directions. Variations in such expression levels are expected and are known because of the integration of transgenes at different locations on the chromosomes of the target plant.

Another important feature shown by the inventors is that in addition to enhancing gene expression in both directions as seen in Examples 4, 5, and 6, salicylic acid treatment (Example 4) or other environmental factors (Example 5) is a chemically inducible behavior of the designed 'transcriptional activation module', showing further enhancement of expression. The results show an increase in fold by salicylic acid in both directions simultaneously. Similar results were observed for NaCl (Example 5) and IAA (Example 6) showing the chemically inducible behavior of the transcriptional activation module. We further utilized bidirectional expression to develop a plant gene expression vector module. Such vectors include bidirectional promoters expressing a selection marker such as kanamycin resistance ( nptII ) in one direction and crylAc genes encoding insecticidal δ-endotoxin in one direction (Example 7). The examples clearly demonstrate the experimental usefulness of such developed vectors in developing crop-improved transformed plants.

The results presented by expression modules designed in transformed tobacco establish the functional utility of the features and methods used by the inventors for the development of this novel 'bidirectional gene expression module'. The present invention clearly shows the potential application of computational biology, which can be used to design artificial bidirectional expression cassettes for control, tissue-specific, constitutive and inducible transgene expression in plants. These will have important applications in biotechnology.

Other Cited References

Wasylyk, B, "Transcription elements and factors of RNA pol B promoters in highereukaryotes", CRC, Critical Rev. Biochem. (1988) 23: 77-120

Johnson, P. F. and McKnight, S. L. "Eukaryotic transcription regulatory proteins", Ann. Rev. Biochem. (1989) 58: 799-839.

Fassler, J. S. and Gussin, G. N. "Promoter and basal transcription machinery in bacteria and eucaryotes: Concept, definitions and analogies", Method in Enzymology (1996) 273: 3-29.

Smale, S. T. "Core promoters active contributes of combinatorial gene regulation", Genes & Dev. (2001) 15: 2503-2508.

Sawant, S. Singh, P. K. Madnala R. Tuli, R. "Designing of an artificial expression cassette for high-level of expression of transgenes in plants", Theor. Appl. Genet. (2001) 102: 635-644.

Iann R., Frederic N., Veronique G., Manfred T., "Combination of viral promoter sequences to generate highly active promoter for heterologous therapeutic protein over expression in plants", Plant Science (2002) 162: 833-842.

Benefy, P. N. Ren, L. and Chua, N.-H., "Combinatorial and synergestic properties of CaMV 35S enhances sub doamins", EMBO, (1990) 9: 1685-1696.

Benfey, P. N. and Chua, N. -H., "The cauliflower mosaic virus 35S promoter: Combinatorial regulation of transcription in plants". Science (1990) 250: 959-966.

Odell, J. J., Nagy, F. and Chua, N.-H., "Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter", Nature (1985) 313: 810-812.

Xie, M., He, Y. and Gan, S., "Bidirectionalization of polar promoter in plants." Nature Biotechnology (2001) 19: 677-679.

Attach sequence list electronic file

Claims (12)

As a bidirectional promoter: a) a transcriptional activation module comprising a chemically synthesized and strategically engineered artificial nucleotide sequence having the sequence set forth in SEQ ID NO: 1 and designed to enhance the expression level of genes in plants; b) a transcription initiation module comprising a chemically synthesized and strategically engineered artificial nucleotide sequence having the sequence set forth in SEQ ID NO: 2 and designed to function as a minimal sequence that initiates transcription of a gene disposed downstream; Bidirectional promoter. The bidirectional promoter according to claim 1, wherein the transcription initiation module is located on one or both sides of a 'transcriptional activation module' that expresses one or two genes one at a time, or simultaneously, to develop genetically engineered plants. The bidirectional promoter of claim 1, wherein the transcription initiation module is disposed in the 5 ′ to 3 ′ direction on one or both sides of the transcriptional activation module. The bidirectional promoter of claim 2, wherein the transcription initiation module is disposed in the 5 ′ to 3 ′ direction on one or both sides of the transcriptional activation module. The bidirectional promoter of claim 2, wherein one or more useful genes are disposed downstream of the transcriptional activation module to express them from one or both sides of the transcriptional activation module. delete delete A transformed plant developed after stable transformation by the bidirectional promoter claimed in claim 1 for the purpose of improving plant characteristics useful for agriculture or industry. A plant transformation vector comprising a bidirectional promoter according to claim 1 expressing a reporter gene from which a selection marker and expression product from one direction can be conveniently monitored. delete 10. The method of claim 9, wherein the selection marker nptII (neomycin phosphotransferase II), bar (bialaphos resistance) and hpt (hygromycin phosphotransferase) vector conversion of the plant transformed is selected from the group consisting of a gene. The plant transformation vector of claim 9, wherein the reporter gene is selected from the group consisting of gusA (β-glucuronidase), gfp (green fluorescent protein), and luc (luciferase) genes.

Images