WO2008069497A1

WO2008069497A1 - Stress specific promoters

Info

Publication number: WO2008069497A1
Application number: PCT/KR2007/006122
Authority: WO
Inventors: Choon-Hwan Lee; Yong-Hwan Moon; Ji Young Kim; Ji-Sung Han; Hee-Yeon Park; Minkyun Kim; Beak Hie Nahm; Yeon Ki Kim; Min Jeong Kim; Dong-Hun Lee
Original assignee: Pusan National University Industry-University Cooperation Foundation
Priority date: 2006-12-05
Filing date: 2007-11-30
Publication date: 2008-06-12
Also published as: KR20080051437A

Abstract

The present invention relates to promoters, which are linked to target genes in stress environments such as anoxia so as to normally promote the expression of the target genes. According to the disclosed invention, it is possible to obtain transgenic plants having tolerance to stresses such as anoxia.

Description

Stress Specific Promoters

TECHNICAL FIELD

The present invention relates to stress-specific promoters, and more particularly to promoters, which are linked to target genes in stress environments such as anoxia so as to normally promote the expression of the target genes.

BACKGROUND ART

Because plants cannot get away from stress environments, they have developed to have defense mechanisms against the stress environments (Knight, H. et al, Trends in Plant Science, 6:261, 2001; Lexer, C. et al, J. Evol Biol, 18:893-900, 2005). Particularly, rice plants are food resources, which are important worldwide, and the productivity thereof is closely connected with mechanisms responding to abiotic stress. Thus, many researchers have conducted the related studies through molecular biological methods (Kush, G. S., Plant MoI. Biol, 35:25, 1997).

In the productivity of rice, flooding is one of typical stresses, and when the plant root or the whole plant is submerged under water, flooding stress is initiated, and this condition causes hypoxia or anoxia in plants (Subbaiah et ah, Ann. Bot. (Lond), 91 : 119, 2003). Under stresses of hypoxia and anoxia, the expression of various proteins and genes is rapidly regulated. In the case of maize roots, 20 ANPs (anaerobic proteins) are mostly involved in glycolysis, metabolism and fermentation (Sachs et al., Cell, 20:761, 1980). In addition, the transcription of transcription factors (de Vetten et al., Plant J., 7:589, 1995; Hoeren et al., Genetics, 149:479, 1998), signaling factors (Dordas et al., Plant J., 35:763, 2003), nonsymbiotic hemoglobins (Dordas et al, Planta, 219:66, 2004), ethylene biosynthesis (Vriezen et ah, Plant Physiol., 121 : 189, 1999) and nitrogen metabolism (Mattana et ah, Plant Physiol., 106: 1605, 1994) are induced. In the above studies, it was demonstrated that, as elements (ARE) responding to anoxia, the alcohol dehydrogenase gene (ADHl) of maize and Arabidopsis thaliana and the promoter of other genes induced by anoxia are required. Also, it was found that GC and GT motifs binding to AtMYB transcription factors play an important role (Olive et ah, Nucleic Acids Res., 19:7053, 1991; Dolferus et ah, Plant Physiol., 105: 1075, 1994; Hoeren et α/., Genetics, 149:479, 1998).

However, despite such active studies, the molecular background of regulatory mechanisms of genes whose expression is induced by stress such as flooding, has not yet been completely identified.

Accordingly, the present inventors have made many efforts to understand the molecular background of regulatory mechanisms of genes whose expression in rice is induced by an environmental stress. As a result, the present inventors have isolated promoters up-regulating the expression of genes under stress conditions and have found that the isolated promoters are useful to produce transformants having tolerance to stress, thereby completing the present invention.

SUMMARY OF THE INVENTION

It is a main object of the present invention to provide a promoter which increases the expression of a rice-derived target gene in an anoxic environment.

Another object of the present invention is to provide an expression vector, containing mentioned promoter and a target gene, and a transformant transformed with mentioned expression vector. To achieve the above objects, the present invention provides a stress-specific promoter represented by any one base sequence selected from the group consisting of SEQ ID NOs: 1-22.

The present invention also provides an expression vector containing the stress- specific promoter and a target gene, and a recombinant microorganism transformed by the expression vector.

The present invention also provides plants or plant cells transformed by the expression vector.

Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a systematic diagram showing a gene group associated with oxidative stress (HL: high light stress; HO: high oxygen stress; and LO: low oxygen stress).

FIG. 2 shows the results of RT-PCR conducted to examine the reproducibility of genes whose expression is induced or increased in an anoxia environment, in which the genes were selected by a microarray method (CON: control, HL: high light stress, HO: high oxygen stress; and LO: low oxygen stress).

FIG. 3 shows differentially expressed genes (DEG) responding to high light stress, high oxygen stress and low oxygen stress using annealing control primers (ACPs) in rice.

FIG. 4 shows the results of RT-PCR conducted to examine the reproducibility of genes whose expression is induced or increased in an anoxia environment, in which the genes were screened by a differentially expressed gene (DEG) analysis method.

FIG. 5 shows the results of RT-PCR conducted to examine the expression of seven genes screened by DEG, after treating the genes with anoxia stress at varying times.

FIG. 6 shows the effects of treatment time and concentration of stress-inducing factors on the expression of genes, screened by DEG, under various stress conditions (A: drought stress; B: cold stress; C: salt stress; D: ROS stress; and E: ABA).

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In the present invention, in order to examine how plants respond to environmental stress, changes in gene expression, which occur during periods of environmentally induced stress, was examined in a large amount using the rice 6OK microarray. In a process of analyzing this information, genes whose expression has been changed by anoxia could be selected.

In the present invention, rice-derived promoters inducing an increase in gene expression in an anoxia environment were found in a procedure of selecting genes whose expression has been increased in an anoxia environment, using not only a microarray method, but also a differentially expressed gene analysis method. The selected genes were analyzed by RT-PCR and, as a result, it was identified that the expression of 16 genes selected in the microarray experiment and 7 genes selected in the DEG experiment was induced or increased due to the influence of environmental stress such as anoxia, and among these genes, one gene overlapped. Accordingly, a total of 22 genes were finally selected as genes associated with stress tolerance. The 23 selected genes include the following loci:

LOC_Osl lgl0510, LOC_Os03gl3150, LOC_Osl lgl0480, LOC_Os08g25720,

LOC_Os04gl7660, LOC_Os01g60850, LOC_Os01g08370, LOC_Os01g68680, LOC_Os04g57600, LOC_Os08g06280, LOC_Os07g47790, LOC_Os03g08460,

LOC_Os06g07000, LOC_Os01g52980, LOC_Os04g44820, LOC_Os02gl0380,

LOC_Osl lgl0480(AK069330(AF172282)), LOC_Os08g04440(AK070704(AP003876)),

LOC_Os02g55420(XM_468277(AK105059)), LOC_Os06g04990(AK105832(AP000399)),

LOC_Os02g07260(AK100371(XM_464267)), LOC_Os02g39930(AK071108(XM_466697)), and LOC_Os05g39610(AK101630(AC135924)).

The selected genes are characterized in that the expression thereof is increased in an environment of anoxia stress, and factors imparting this characteristic to the genes are promoters. Thus, when the promoter is inserted into a vector and linked to a target gene, the target gene can be expressed in a stress environment.

The promoters according to the present invention determine the initiation of transcription or the intensity of expression, because they include domains for the expression and regulation of genes.

As used herein, the term "stress-specific promoters" refers to promoters which induce or increase the expression of genes in stress environments.

In the present invention, the promoters that can increase the expression of target genes in an anoxia environment are the promoters of the 22 genes and have base sequences represented by SEQ ID NOs: 1-22.

Vectors for plant transformation, which contain the inventive promoters in order to facilitate the expression of genes in a stress environment, may be plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or other cloning systems. Such vectors commonly contain an expression cassette, which may contain the inventive promoter for expression, a cDNA or DNA fragment capable of expressing a target gene, a polylinker, a termination codon, a marker allowing the selection of a regulatory gene and a transformant, and the like. The insertion of the promoter, the target gene and the like into the vector can be easily performed by any technique known in the art.

In plant transformation, as a transformant selection marker, hygromycin phosphotransferase (hpt), phosphinothricin acetyl transferase gene (bar), 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS), neomycin 3'-O- phosphotransferase (NPT) or acetolactate synthase (ALS) may be used, but the scope of the present invention is not limited thereto.

Target genes that can be inserted into the vector may include a functional structural gene or a regulatory gene, and preferably a functional structural gene or a regulatory gene, which enhances tolerance to stress.

The structural gene that enhances stress tolerance is a gene expressing a protein, which is expressed in plants and directly imparts environmental stress tolerance to the plants, and examples thereof may include: LEA proteins; water channel proteins; synthases for compatible solutes; detoxification enzymes of tobacco, synthases for osmoregulatory substances (e.g., sugar, proline or glycinebetaine); genes encoding w-3 fatty acid desaturase of Arabidopsis thaliana and D9- desaturase, which are modification enzymes of the cell membrane lipid; P5CS for proline synthesis and AtGolS3 for galactinol synthesis.

Also, the regulatory gene that enhances tolerance to stress is a gene expressing a protein which regulates the activity of a stress-induced promoter or a tolerance- inducing gene, and examples thereof include: Arabidopsis thaliana-derived transcription factors such as DREBlA, DREB2A, DREBIB, and DREBIC genes; rice-derived transcription factors such as OsDREBlA, OsDREBlB, OsDREBlC, OsDREBlD, and OsDREB2A genes; and NCED (9-cis epoxicarotenoid; 9-cis epoxicarotenoid dioxygenase) genes which are key enzymes for the biosynthesis of the plant hormone, ABA (absicisic acid). In addition to the target gene for enhancing tolerance to environmental stress, various genes for imparting genetic characteristics advantageous for plants and seeds can be inserted, and the scope of the present invention is not limited thereto.

A method for plant transformation or gene regeneration with the vector is well known in the art. For example, the vector, containing the target gene and the inventive promoter, can be transformed into plants either using a method of introducing DNA directly into cells, such as a method of introducing DNA by thermochemical treatment, a liposome method, an electroporation method, a microinjection method or a particle gun method, or using an indirect vector transfer method which utilizes bacteria such as Agrobacterium.

Plant transformation can be performed by a direct gene transfer technique, such as a method of transferring the DNA vector into protoplasts using polyethylene glycol (PEG), an electroporation method, or a particle gun method. Protoplast- mediated transformation has been described for Japonica-type and Indica-types (Zhang et al, Plant Cell Rep., 7:379, 1988; Shimamoto et al, Nature, 338:274, 1989; Datta et al, Biotechnology, 8:726, 1990). In addition to the two methods, the particle gun method can be conventionally used for transformation (Christou et al, Biotechnology, 9:957, 1991).

The indirect DNA vector transfer method for plant transformation can be performed using Agrobacterium sp.. Mentioned bacteria include Agrobacterium tumefaciens and Agrobacterium rhizogenes, and Agrobacterium tumefaciens (for example, LBA4404 or EHA 105) is particularly useful, because it is most widely used for plant transformation. As the method for introducing the vector into Agrobacterium sp., a method of directly introducing DNA by chemical or thermal treatment (Holsters et ah, MoI. Gen. Genet., 163: 181, 1978), a method of introducing the vector by electroporation (Wen-jun and Forde, Nucleic Acids Res., 17:8385, 1989), a triparental conjugational transfer method of transferring the vector from E. coli to Agrobacterium mediated by Tra+ (Ditta et ah, Proc. Natl. Acad. Sci. USA, 11:12>A1, 1980) or a direct conjugational transfer method can be generally used.

The Agrobacterium sp. transformed by the above method can be used to infect the tissue of plants to be transformed, thus obtaining transgenic plants. More particularly, mentioned transgenic plants can be prepared by following steps: (a) pre-culturing explants of target plant, and then co-culturing the pre-cultured explants with Agrobacterium sp. into which a target gene have been introduced; (b) culturing the co-cultured explants in a selection medium so as to form callus and selecting the formed callus; and (c) cutting the callus and culturing the cut callus in a shoot induction medium so as to form shoots.

As used herein, the term "explants" refers to tissue segments excised from plants, which includes cotyledons or hypocotyls. In the present invention, as mentioned explants of plants, cotyledons or hypocotyls can be used, more preferably, cotyledons obtained by the following steps can be used: sterilizing seeds, washing mentioned seeds, and germinating in MS medium

Plants to be transformed, which can be used in the present invention, may include tobacco, tomato, red pepper, bean, rice and maize plants, but the scope of the present invention is not limited thereto.

Examples Hereinafter, the present invention will be described in further detail with reference to examples. It will be apparent to one skilled in the art that these examples are for illustrative purpose only and are not construed to limit the scope of the present invention.

Example 1 : Induction of stress in rice

Rice seeds (O. sativa var. Japonica Dong/in byeό) were first sterilized with 3% sodium hypochloride, and then washed several times with sterilized distilled water to disinfect the surface thereof. Then, the seeds were germinated in water for 7 days and transferred and cultivated in soil. The plant cultured in a greenhouse for 1 month was used in the experiment, and the plant was placed in a dark place for 3 hours before treatment with stress.

As stress conditions, high light, high oxygen and anoxia conditions were applied. In a condition of high light stress, the plant was treated with a light intensity of 2000 μmol'm ^s ¹ at 28 "C for 4 hours. In a condition of high oxygen stress, the plant was placed in a chamber containing nitrogen and oxygen at a ratio of 50:50 and was maintained in the same light and temperature conditions as those of the high light stress. In a condition of anoxia stress, the plant was placed in a chamber containing 100% nitrogen and was treated with a light intensity of 200 μmol'm^'^s^"1 at 28 ^°C for 4 hours.

The plants and genes whose expression has been increased in the stress induction conditions were analyzed by a microarray and RT-PCR for verifying the microarray results, and were subjected to differentially expressed gene (DEG) analysis and RT-PCR for verifying the analysis results.

Example 2: Selection of stress-induced expressed genes by microarray From the plants treated with stress, RNA was extracted using Trizol (Invitrogen, Carlsbad, CA). The extracted RNA was subjected to a reverse transcription using Oligo(dT), dNTP, MMLV, RNasin (Promega, Madison, WI, USA), thus constructing cDNA.

The rice 6OK microarray (GreenGene Biotech Inc, Korea), which is a cDNA chip, is a product constructed for the recurrence of all genes in rice. The rice 6OK microarray is composed of two slides, and a total of 64896 spot addresses include blank spots. Fluorescent labeling, hybridization, image analysis, data normalization and hierarchical clustering were performed in GreenGene Biotech Inc (Oh, SJ. et al., Plant physiology, 138:341, 2005).

The 6OK microarray was used to analyze the expressed genes and the promoters. Because about 45000 of the spots on the gene chip are associated with locus identifiers of TIGR Pseudomolecules V4 rice annotation database (http://www.tigr.org), important genes in a gene group was classified based on this tentative biological function using gene ontology (GO) annotation. The recurrence of classes having a difference in GO between the differentially expressed genes was performed through calculation using Microsoft excel (Microsoft, Co.) and access (Microsoft, Co.) programs. Also, for the analysis of the promoters, PLACE (http://www.dna.affrc.go.jp/htdocs/ PLACE/) was used.

As a result, it was shown that a number of genes were expressed by oxidative stress, and these genes had similar regulatory patterns. For example, when one gene was associated with two or more oxidative stresses, and the expression thereof was up-regulated in one stress, the expression was also up-regulated in the other stresses (FIG. 1). The anoxia stress column showed a great difference from other oxidative stresses. Specifically, a total of 1775 genes showed specific expression patterns in the anoxia stress. Among them, 1314 genes were induced, and 461 genes were inhibited. Among the genes whose expression has been specifically up-regulated, only 280 genes were annotated, and the functional classification of each gene was performed using gene ontology (GO) capable of describing the corresponding biological mechanism, molecular function and cellular location of each gene.

Example 3: Verification of expression of genes selected by microarrav

In order to verify the results obtained in the microarray, mRNA was extracted from the rice plants treated with stresses in the same conditions as in Example 1 and was used to construct cDNA in the same manner as in Example 2. In the analysis of microarray information, the genetic sequences of genes of interest were obtained in the rice database consortium

(http://www.tigr.org/tdb/e2kl/osal/), primers for RT-PCR were constructed using the Primer 3 program, and an actin gene was used as a control group (Table 1).

All the PCR reactions were performed using a 25 μi reaction mixture, containing 1 μi of cDNA, 1 μi of forward primer (10 pmol), 1 μi of reverse primer (10 pmol), 12.5 μi of 2x Taq premixture (Cosmo Genetech Co., Korea) and 9.5 μi of distilled water. Also, the PCR reaction consisted of predenaturation at 95 °C for 5 minutes, and then 25-30 cycles of denaturation at 95 "C for 40 sec, annealing at 56-59 °C for 40 sec, and extension at 72 ^°C for 30 sec, followed by final extension at 72 ^°C for 10 minutes, and was finally maintained at 4°C . The PCR products were electrophoresed on 1.5% agarose gel stained with ETBR (ethidium bromide, Sigma, USA), and then visualized.

Table 1

As a result, it was confirmed that the genes induced by anoxia stress in the microarray data had tendencies similar to the results of RT-PCR. On the basis of this fact, 16 genes whose expression has been increased in anoxia stress were confirmed again (Table 2 and FIG. 2).

Table 2

Example 4: Selection of stress-induced expressed genes by PEG analysis

In order to identify and analyze genes whose expression level would be increased or reduced due to environmental conditions (photoinhibitory and photooxidative stresses), the expression patterns of 30-day-old wild-type plants treated with high light, high oxygen and low oxygen stresses were examined using 20 kinds of annealing control primers (ACPs). As a result, 37 DEGs showed reaction (FIG. 3). Among them, 14 DEGs whose expression has been increased due to low oxygen stress were selected, sequenced and analyzed through BLAST (Basic Local Alignment Search Tool). Among them, 13 genes, including early nodulin, temperature stress-induced lipocalin and the like, were selected (Table 3).

Table 3

Example 5: Verification of expression of selected genes by PEG analysis

The 13 genes selected in Example 4 were subjected to semi-quantitative RT-PCR using primers shown in Table 4 below. The results of RT-PCR were compared with the DEG analysis results, and as a result, it was confirmed that the expression patterns of 7 genes coincided with the results of APC experiments (FIG. 4).

Table 4

In the results of RT-PCR, the expression of all the 7 genes showed a tendency to increase in low oxygen stress. Among them, the expression of DEGlO and DEG27 was strongly increased not only in response to low oxygen stress, but also in response to the other stresses, the expression of DEGl 5, DEG25 and DEG29 was increased in response to high light stress in addition to low oxygen stress, and the expression of DEG6 and DEG20 was strongly increased only in response to low oxygen stress (FIG. 4).

The expression levels of the 7 selected genes in response to low oxygen stress at varying times points were analyzed by RT-PCR. As a result, it could be seen that the expression of the genes was increased with the passage of time, and particularly, the expression was the strongest at 3 hours after treatment with stresses (FIG. 5). Also, the expression patterns of the genes in other kinds of stresses, including drought, cold, salt, ROS and ABA stresses were examined. As a result, it could be seen that the expression of DEGlO, DEG27 and DEG29 showed a tendency to increase, suggesting that the expression patterns coincided with the expression patterns shown in FIG. 4 (FIG. 6).

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention provides promoters, which are linked to target genes in stress environments such as anoxia so as to normally promote the expression of the target genes. The promoters of the present invention can be inserted into a vector together with target genes and used as tools for the transformation of plants. Also, these promoters can be used to provide transformants having tolerance to stress such as anoxia, and thus they can maintain the growth of plants in low-oxygen conditions such as flooding and contribute to an increase in the production of plants.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS

What is Claimed is:

L A stress-specific promoter represented by any one base sequence selected from the group consisting of SEQ ID NOs: 1-22.

2. The stress-specific promoter according to claim 1, wherein the stress is anoxia.

3. An expression vector containing the stress-specific promoter of claim 1 and a target gene.

4. A recombinant microorganism transformed by the expression vector of claim 3.

5. Plants or plant cells transformed by the expression vector of claim 3.