WO2006080598A9

WO2006080598A9 - Anther-specific genes derived from malus x domestica, promoters thereof and a method for preparing transformed plant using the same

Info

Publication number: WO2006080598A9
Application number: PCT/KR2005/000284
Authority: WO
Inventors: Soon-Kee Sung; Sung Soo Kim; Gibo Yoon
Original assignee: Dongbu Hannong Chemical Co Ltd; Soon-Kee Sung; Sung Soo Kim; Gibo Yoon
Priority date: 2005-01-31
Filing date: 2005-01-31
Publication date: 2007-01-25
Also published as: WO2006080598A1

Abstract

The present invention relates to anther-specific genes, promoters thereof and transformed plants using the same, more precisely, novel genes MdASGl, MdASG2 and MdASG3 which are represented by SEQ. ID. NO: 1, NO: 2 and NO: 3 and expressed specifically in the anther derived from an apple plant {Malus x domestica cv. Fuji), and their promoters PMdASGl, PMdASG2 and PMdASG3 containing the whole or a part of sequences represented by SEQ. ID NO: 4, NO: 5 and NO: 6 involved in transcriptional activity and a method for preparing transgenic plants using the same. The anther-specific genes of the present invention can be effectively used to examine the pollen development mechanism involved in plant fertilization. The promoters PMdASGl, PMdASG2 and pMDASG3 of the present invention have a high activity to express a target gene specifically in the anther or pollen of a transgenic plant, so that they can be effectively used for the development of a transgenic plant in which a heterologous gene whose pollen-specific gene expression is needed is introduced and contributes to the development of a male-sterile transgenic plant.

Description

ANTHER-SPECIFIC GENES DERIVED FROM MALUS X

DOMESTICA, PROMOTERS THEREOF AND A METHOD FOR

PREPARING TRANSFORMED PLANT USING THE SAME

Technical Field

The present invention relates to novel anther- specific genes and promoters thereof.

Background Art Since human started cultivating, man has been continuously improving the crops. For thousands of years, the selection of a useful genetic characteristic from crossbreeding has been a key point in the improvement. That is, a target characteristic has to be searched among hybrids produced by crossbreeding, which requires time-consuming numbers of trial and error. By a collaboration of Chilton, M. D., Van Montagu, M. and Schell, J. with Monsanto Co., USA in 1983, it was proved that a direct insertion of a heterogeneous gene is possible in tobacco using Agrobacterium tumefaciens without crossing. Since then, methods for transformation of useful crops ranging from dicotyledons to monocotyledons have been developed.

With the advance of technologies for inserting heterogeneous gene, a promoter that can strongly induce gene expression in a plant has been required. Since Chua, N. H. et al. confirmed the usefulness of 35S promoter of cauliflower mosaic virus (CaMV) (Odell, J. T. et al., Nature, 313(6005): 810-2, 1985) for transforming a plant, in fact 80% of plant transformation have been achieved by using the 35S promoter at present. However, since 35S promoter is derived from a virus, it has a possibility to induce over-expression of a normal gene through producing a new variety of virus by a conjugation or a recombination with another virus residing in a plant (Cummins, J., Ho, M. W. and Ryan A., Nat. Biotechnol., 18(4): 363, 2000). Further, non-tissue specific nature of 35S promoter is another problem, which means the promoter is always expressed in every tissue, far from being efficient. Therefore, a tissue specific promoter has been searched since 1980s. As a result, starting with TA29, tissue specific promoters derived from the root, the anther, the fruit and the leaf have been developed. In 1994, a fruit specific promoter was used to develop a tomato having a phenotype of delayed afterripening (Product name; Flavr-Savr) , which is a genetically modified crop.

Since 1930, seed companies have sold Fl hybrid seeds produced by the crossbreeding of inbred ancestors. The Fl hybrid seeds are widely used for the seed production of cereals, feed grains and garden plants including fruits and vegetables owing to their excellent properties such as disease tolerance, yield potential and good quality, which are peculiar to hybrid. However, most plants are self-pollinated naturally. Thus, in order to produce the hybrid seeds, a stamen, a male reproduction organ, has to be removed and then pollen of inbreeded species has to be controlled-pollinated. The controlled pollination has problems of increased production costs and questioning in purify of produced hybrid seeds. Therefore, other approaches have been made to produce a genetically engineered male-sterile plant by inserting a specific gene or knocking out it. Anyway, searching pollen- or anther-specific promoter is the first consideration. In 1990, TA29 promoter, specifically expressed in tapetum of anther, was developed (Koltunow, A. M. et al., Plant Cell, 2(12): 1201-1224, 1990; Sa, G. et al., Transgenic Res., 11(3): 269-78, 2002). And, a male- sterile system activated by treating a ribosome inactivating protein or N-acetyl-L-phosphinothricin has been developed. Yet, no other effective anther- specific promoter excepting TA29 has been reported, although the development of effective anther-specific promoters is unquenchable.

Thus, the present inventors have studied to develop an anther-specific gene and promoter thereof. As a result, the present inventors completed the present invention by confirming that novel genes MdASGl, MdASG2 and MdASG3 are expressed specifically in anther of an apple {Malus x domestica cv. Fuji) and promoters PMdASGl, PMdASG2 and PMdASG3 induce pollen-specific expression of a heterogeneous gene. Furthermore, a transgenic male-sterile plant can be produced by transforming a self-pollinated plant with a recombinant gene cassette generated by using the promoters and a gene encoding a cytotoxic protein.

Technical Problem

It is an object of the present invention to provide anther-specific genes derived from Malus x domestica cv. Fuji.

It is another object of the present invention to provide pollen-specific promoters derived from the above genes .

It is a further object of the present invention to provide recombinant vectors containing the anther- specific genes or the pollen-specific promoters, transformants transfected with those recombinant vectors and a method for preparing the same.

It is also an object of the present invention to provide a transformant to produce a male-sterile plant by using the recombinant vector of the invention and a method for preparing the same.

Technical Solution The terms and arts described in the present invention are used in general sense understood by those in the art, if not defined otherwise. And articles cited in the present invention are incorporated herein by references.

In order to achieve the above objects, the present invention provides an isolated or recombinant gene comprising a nucleotide sequence selected from the group consisting of SEQ. ID. NO: 1 (MdASGl), SEQ. ID. NO: 2 (MdASG2) and SEQ. ID. NO: 3 (MdASG3) or a homologue thereof having 70 - 99% homology and being functionally equal thereto. The homologue of the present invention preferably has 80 - 99% homology with the isolated genes. The present invention also provides an isolated or recombinant polynucleotide having pollen-specific promoter activity comprising a nucleotide sequence selected from the group consisting of SEQ. ID. NO: 4 (PMdASGl), SEQ. ID. NO: 5 (PMdASG2) and SEQ. ID. NO: 6

(PMdASG3) or an active partial fragment thereof having transcriptional activity. At this time, the active partial fragment is not limited to specific ones, but a polynucleotide comprising from -55^th to +89^th nucleotide on the basis of the 2786^th nucleotide of SEQ. ID. NO: 5, which is transcription start site of PMdASG2, or a homologue having 70 - 90% homology with the polynucleotide and being functionally equal thereto is preferred. In particular, a partial fragment comprising from -255^th to +l^st nucleotides on the basis of the 2786^th nucleotide of SEQ. ID. NO: 5 is more preferred.

The present invention provides a recombinant vector comprising an isolated or recombinant gene comprising a nucleotide sequence selected from the group consisting of SEQ. ID. NO: 1 [MdASGl), SEQ. ID. NO: 2 (MdASG2) and SEQ. ID. NO: 3 (MdASG3) , or a homologue thereof having 70 - 99% homology and being functionally equal thereto or an antisense sequence thereof. The recombinant vector comprising said gene or said anti-sense sequence of the present invention is not limited to, but a recombinant vector selected from the group consisting of MdASGs/pBluescript, MdASGs/pRTL2 and MdASGs/pCAMBIA2301 shown in the cleavage maps of Fig. 3 is preferred, and the MdASGs/pRTL2 is more preferred. Herein, 's' of 'MdASGs¹ indicates one of the digits from 1 to 3.

The present invention also provides a recombinant expression vector containing an isolated or recombinant polynucleotide having pollen-specific promoter activity comprising a nucleotide sequence selected from the group consisting of SEQ. ID. NO: 4 (PMdASGl), SEQ. ID. NO: 5 (PMdASG2) and SEQ. ID. NO: 6 (PMdASG3) or an active partial fragment thereof having transcriptional activity. At this time, an Agrobacterium binary vector for plant transformation is preferred as a recombinant expression vector but not always limited thereto. And also the active partial fragment having transcriptional activity is not limited to a specific one but a polynucleotide comprising from -55^th to +89^th nucleotides on the basis of the 2786^th nucleotide of SEQ. ID. NO: 5, which is a transcription start site of PMdASG3, or a homologue thereof having 70 - 99% homology and being functionally equal thereto is preferred, and an active partial fragment comprising from -255^th to +l^st nucleotides on the basis of the 2786^th nucleotide of SEQ. ID. NO: 5 is more preferred. It is also preferred that the recombinant vector containing said polynucleotide having pollen-specific promoter activity further comprises at least one of genes encoding a heterologous genes operably linked to said promoter. There is no limitation in heterologous genes, however, a gene selected from the group consisting of a gene encoding ribosome inactivating protein (RIP), MdASGl₁ MdASG2 and MdASG3 of the invention or anti-sense sequence thereof is preferred. The recombinant expression vector is not limited to a specific one, either, but an Agrobacterium binary recombinant vector containing T-DNA of Agrobacterium is preferred to transform a plant effectively, and pMd2proRIP shown in the cleavage map of Fig. 19 or pMd2proASMd2 presented by cleavage map of Fig. 20 is more preferred.

The present invention further provides a transformed microorganism prepared by transforming a host with a recombinant vector containing said gene or said promoter. The host microorganism is not limited to a specific one, but Agrobacterium is preferred for transformation of a plant. The above transformed microorganism is not limited to a specific one, either, but the recombinant Agrobacterium LBA4404/pMd2proASMd2 which is deposited under Accession No: KTCT 10769BP is more preferred.

Further, the present invention provides a method for preparing a transgenic plant showing anther- or pollen-specific gene expression comprising the following steps: i ) Construction of a recombinant expression vector containing a gene comprising a nucleotide sequence selected from the group consisting of SEQ. ID. NO: 1 (MdASGl), SEQ. ID. NO: 2 (MdASG2) and SEQ. ID. NO: 3 (MdASG3) , a homologue thereof having 70 - 99% homology and having functionally equal thereto, or an anti-sense sequence thereof, or a recombinant expression vector containing a polynucleotide having pollen-specific promoter activity comprising a nucleotide sequence selected from the group consisting of SEQ. ID. NO: 4 (PMdASGl), SEQ. ID. NO: 5 (PMdASG2) and SEQ. ID. NO: 6 (PMdASG3) or an active partial fragment thereof having transcriptional activity and a heterologous gene operably linked to said polynucleotide having pollen-specific promoter activity; ii ) preparing a transformed Agrobacterium by introducing the recombinant expression vector into

Agrobacterium; iii) transforming plant cells by co-culture of the plant cells and the transformed Agrobacterium; and iv) re-differentiation of the transformed plant cells by tissue-culture.

The present invention also provides a method for preparing a male-sterile plant comprising the following steps : i ) Constructing a recombinant expression vector containing a polynucleotide having pollen-specific promoter activity comprising a nucleotide sequence selected from the group consisting of SEQ. ID. NO: 4

(PMdASGl), SEQ. ID. NO: 5 (PMdASG2) and SEQ. ID. NO: 6 (PMdASG3) or an active partial fragment thereof having transcriptional activity and a heterologous gene encoding a cytotoxic protein operably linked to said polynucleotide or an anti-sense sequence of a gene necessary for pollen formation; ii ) preparing a transformed Agrobacterium by introducing the recombinant expression vector into

Agrobacterium; iii) transforming plant cells by co-culture of the plant cells and the transformed Agrobacterium} and iv ) re-differentiating the transformed plant cells by tissue-culture.

At this time, the cytotoxic protein is not limited to a specific one, but a ribosome inactivating protein is preferred. The above gene necessary for pollen formation is not limited to a specific one, either, but

MdASGl, MdASG2 or MdASG3 of the invention is preferred.

Description of Drawings

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Fig. 1 is a photograph showing the results of PCR with cDNAs extracted from a leaf, a flower bud, a mature flower, a receptacle after fertilization, and a young fruit of Malus x domestica cv. Fuji using random primers .

Fig. 2 is a photograph showing the results of RT-

PCR with the novel gene MdASGl (GenBank Accession No: AF403122) of the present invention using cDNAs obtained from each genital organ of a flower of Malus x domestica cv. Fuji as a template.

Fig. 3 is a photograph showing the organ-dependent expressions of MdASG2 (GenBank Accession No: AF403123) and MdASG3 (GenBank Accession No: AF403124) cloned by using MdASGl as a probe for RT-PCR analysis.

Fig. 4 is a diagram showing the comparison of amino acid sequences deduced from MdASGl, MdASG2 and MdASG3. Fig. 5 is a photograph showing that the expression pattern of MdASGl of the present invention confirmed by mRNA in situ hybridization.

Fig. 6 is a schematic diagram showing the construction process of a recombinant expression vector for generating transgenic plants in which mRNA expressions of MdASGl and MdASG2 are suppressed.

Fig. 7 is a photograph showing the comparisons of flowers (A) , anthers (B) and fruits (C) between a transgenic tobacco plant in which MdASGl expression is suppressed and a wild type tobacco plant.

Fig. 8 is a photograph showing the comparisons of flowers (A) , stamens and pistils (B) , anthers (C) and pollen grains (D) between a transgenic tobacco plant in which MdASG2 expression is suppressed and a wild type tobacco plant.

Fig. 9 is a diagram showing the nucleotide sequence of a genomic gene encoding MdASGl of Malus x domestica cv. Fuji and the nucleotide sequence of its promoter . Fig. 10 is a diagram showing the nucleotide sequence of a genomic gene encoding MdASG2 having homology with MdASGl of Malus x domestica cv. Fuji and the nucleotide sequence of its promoter.

Fig. 11 is a diagram showing the nucleotide sequence of a genomic gene encoding MdASG3 having homology with MdASGl of Malus x domestica cv. Fuji and the nucleotide sequence of its promoter.

Fig. 12 is a diagram showing the homology and locations of potential regulatory sites of the nucleotide sequences of promoters of MdASGl, MdASG2 and MdASG3 derived from Malus x domestica cv. Fuji.

Fig. 13 is a schematic diagram showing the preparation process of a deletion mutant of PMdASG2 promoter region. Fig. 14 is a graph showing the GUS enzyme activity in the anther of a transgenic tobacco plant introduced with a mutant gene construct having serial deletion of MdASG2 promoter in the direction from 5' to 3'.

Fig. 15 is a photograph illustrating that the developmental process of a flower of a transgenic tobacco plant with the insertion of promoter deletion mutant MdASG2 04 is classified into 10 stages.

Fig. 16 is a graph illustrating that the GUS enzyme expression was investigated according to each developmental stage of a flower by a biochemical method. Fig. 17 is a photograph showing the GUS enzyme expression profiles of each differentiation and developmental stage of a genital organ.

Fig. 18 is a fluorescent photograph taken by confocal microscopy which illustrates the pollen grains of a transgenic tobacco plant with the insertion of promoter deletion mutant MdASG2 04.

Fig. 19 is a schematic diagram showing the construction process of a recombinant expression vector in which MdASG2 04 promoter is operably linked to a gene encoding RIP (ribosome inactivating protein) .

Fig. 20 is a schematic diagram showing the construction process of a recombinant vector in which MdASG2 cDNA is reversely linked to MdASG2 04 promoter. Fig. 21 is a photograph showing the floral organ of a transgenic tobacco plant with the insertion of a vector constructed by operably linking a RIP coding gene to MdASG2 04 promoter of the invention.

Fig. 22 is a photograph showing the floral organs of a transgenic tobacco plant introduced with a vector constructed by linking MdASG2 cDNA reversely to MdASG2 04 promoter and a wild type tobacco plant.

Fig. 23 is a set of photographs showing the morphologies of pollen grains of a transgenic tobacco plant with the introduction of a vector constructed by operably linking a RIP coding gene to MdASG2 04 promoter of the invention and a wild type tobacco plant, which are stained with cotton blue (A) , and the activities of pollen of a transgenic tobacco plant and a wild type, which are stained with fluorescein diacetate (B) .

Fig. 24 is a set of photographs showing the morphologies of pollen grains of a transgenic tobacco plant with the introduction of a vector in which MdASG2 cDNA is reversely linked to MdASG2 04 promoter and a wild type tobacco plant, which are stained with cotton blue (A) , and the activites of pollen of the above tobacco plants which are stained with fluorescein diacetate (B) . Fig. 25 is a photograph showing the result of the observation under scanning electron microscope on the morphologies of pollen grains of a transgenic tobacco plant introduced with a vector in which a RIP coding gene is operably linked to MdASG2 04 promoter and a wild type tobacco plant.

Fig. 26 is a photograph showing the result of the observation under scanning electron microscope on the morphologies of pollen grains of a transgenic tobacco plant introduced with a vector in which MdASG2 cDNA is reversely linked to MdASG2 04 promoter and a wild type tobacco plant.

Fig. 27 is a set of photographs showing the result of investigation on the fructification by self- pollination (A) in a transgenic tobacco plant with the insertion of a RIP coding gene operably linked to MdASG2 04 promoter and the fructification by cross- pollination (B) .

Fig. 28 is a set of photographs showing the result of investigation on the fructification by self- pollination (A) in a transgenic tobacco plant with the insertion of MdASG2 cDNA reversely linked to MdASG2 04 promoter and the fructification by cross-pollination

(B) .

Best Mode

Hereinafter, the present invention is described in detail .

The present inventors performed PCR (differential display PCR) with cDNAs obtained from a leaf, a flower bud, a flower, a receptacle and a fruit of Malus x domestica cv. Fuji to clone a novel gene expressed specifically in mature flowers of Malus x domestica cv. Fuji. After sequencing the nucleotide sequence, the novel gene was confirmed to have a nucleotide sequence represented by SEQ. ID. NO: 1, and then named MdASGl

{Malus x domestics anther specific gene 1) . After searching more genes that have homology with the nucleotide sequence of the novel gene, two other novel genes were additionally cloned, which were named

'MdASG2' and 'MdASG3' respectively. From the RT-PCR with those genes, it was confirmed that those genes were expressed specifically in flowers containing the anther (see Fig. 3 - 6) . MdASGl, MdASG2 and MdASG3 of the present invention include open reading frame (ORF) each encoding a potential protein composed of 75, 88 and 75 amino acids, respectively, and the amino acid sequences thereof have high homology with one another. The homology was further investigated by using BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) provided by National Center for Biotechnology Information (NCBI, USA) . And as a result, no other genes were confirmed to have homology with the above three novel genes.

The functions of those genes were also investigated, for which a tobacco plant was transformed with a recombinant vector constructed by linking MdASGl or MdASG2 reversely to 35S promoter (see Fig. 7 and 8) . As a result, the transgenic plant displayed abnormal phenotype particularly in the development of a stamen. As shown in Fig. 8 , in a transgenic plant, a stamen was shorter, pollen grains were less formed and pollen was less developed, resulting in poor fructification. The above results indicate that MdASGl and MdASG2 genes derived from Malus x domestica cv. Fuji play an important role in the development of the anther.

The present inventors further cloned genomic DNAs of MdASGl, MdASG2 and MdASG3 as well as their promoter regions having a transcriptional activity. The cloned promoters were named 'pMdASGl', 'pMdASG2' and 'pMdASG3' respectively. Those promoters contain the whole or a partial fragment having a transcriptional activity, each represented by SEQ. ID. N0:0: 4, 5 and 6. The promoters of the invention express heterologous genes pollen-specifically. From the sequencing of the nucleotide sequences of the promoters, it was confirmed that the promoters showed high sequence homology in the region from the transcription start site to -255 - +1 and include TATA box and CAAT box for transcription start, a transcription factor binding site 56/59 box harboring a key factor of a pollen-specific gene (Eyal et al., Plant Cell, 7:373-384, 1995), GTGA motif found in a pollen gene of a tobacco plant (Rogers et al., Plant MoI. Biol., 45: 577-585, 2001) and a pollen- specific transcription factor POLLEN1LELAT52 comprising the nucleotide sequence of AGAAA (Bate et al., Plant MoI. Biol., 37: 859-869, 1998). Particularly, TCCACCATA, along with POLLEN1LELAT52 which is a transcription factor sequence regulating anther- specific protein expression, is closely located to TATA box therein (see Fig. 12) . Hereinafter, the phrase 'pMdASG promoters' is used for meaning nucleotide sequences represented by SEQ. ID. N0:0: 4, NO: 5 and No: 6 and active partial fragments thereof having a transcriptional activity, if not described otherwise.

The present inventors prepared a fusion gene construct consisting of said promoter and a reporter gene GUS (β-glucuronidase) operably linked to the promoter, in order to investigate the activity of the promoter of the invention (see Fig. 13) . As a result, the promoter of the invention was strongly expressed at the stage of tetrad of pollen development in the anther of a plant. Based on the founding, the present inventors prepared a fusion gene construct consisting of an active fragment having the region from 1 to 2874 of the nucleotide sequence represented by SEQ. ID. NO: 5 or a partial fragment particularly having a promoter activity and a heterologous gene operably linked thereto. The fusion gene construct which has a structure consisting of a PMdASG2 promoter gene and a heterologous gene encoding a cytotoxic protein or a specific structural gene operably linked thereto interrupts normal pollen development. Therefore, the fusion gene construct of the present invention can be effectively used for the preparation of a male-sterile plant (see Fig. 25 and 26) .

The present inventors further prepared a gene construct for transformation in which a gene encoding cytotoxic RIP (ribosome inactivating protein) or an antisense sequence of MdASG2 gene is operably linked to the downstream of PMdASGl, PMdASG2 or PMdASG3 promoter and prepared a recombinant vector by cloning said gene construct into an Agrobacterium binary vector for plant transformation. Then, an Agrobacterium was transformed with the recombinant vector. Among Agrobacterium transformants in which the gene construct for transformation is introduced, the Agrobacterium LBA4404/pMd2proASMd2, transformed with the recombinant gene construct in which MdASG2 antisense sequence is operably linked to PMdASG2 04 promoter, was deposited at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) (52 Oun-Dong, Yusong-Gu, Taejon, Korea) under accession No: KTCT 10769BP. At last, a transgenic plant in which the recombinant gene construct is itegrated was prepared by infecting a plant with the Agrobacterium transformant . When the flower development of the transgenic plant was investigated, a typical male-sterility was observed, i.e., a fruit was not produced by self-pollination, while a fruit was normally produced by cross-pollination with the pollen of a wild type plant (see Fig. 27 and 28) . It is definitely understood by those in the art that RIP or the antisense of MdASG2 used in the present invention is an example to illustrate the invention and the present invention is not limited thereto. In fact, any heterologous gene that can be operably linked to the promoter of the invention can be used for the construction of a pollen-specific recombinant gene construct and the production of a transgenic plant using the same. The preparation method of a transgenic plant of the present invention can be effectively used for the preparation of a male-sterile plant in which unwanted self-pollination is inhibited and the development of a genetically modified (GM) plant in which a pollen-specific gene expression is needed.

Mode for Invention

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

The percentage unit used in the following examples indicates the ratio of weight-to-volume (w/v) , if not described otherwise.

Example 1 : Searching of an anther-specific gene from Malus x domestica cv. Fuji cDNA <!-!> Gene searching

To detect an anther-specific gene derived from Malus x domestica cv. Fuji, PCR was performed using the first strand cDNAs (Sung et al . , MoI. Cells, 8(5): 565- 77, 1998) obtained from a leaf, a flower bud, a mature flower, a receptacle after pollination and an early fruit as a template. The first strand cDNA was 50-fold diluted. PCR was performed with 1 μi of the diluted cDNA by using a random 10-mer (SK020, Operon Technology Inc., USA) as a forward primer and 5'-T12VN-3' (SK030, Operon Technology Inc., USA, V=mixture of equal molarity of each dA, dC, dG; N=one of dA, dC, dG and dT) as a reverse primer. A DNA fragment detected only in a mature flower was used for cloning and the nucleotide sequence thereof was identified (Fig. 1) . Fig. 1 is a photograph showing the results of PCR with cDNAs separated from a leaf, a flower bud, a mature flower, a receptacle after pollination and a young fruit by using a random primer. Herein, L indicates a leaf, FB indicates a flower bud, MF indicates a mature flower, Fl indicates a receptacle after pollination and F2 indicates a young fruit. The red arrow indicates the location of a band of the anther-specific gene of the present invention.

A gene corresponding to the DNA fragment was obtained from the flower cDNA library and then cloned into a cloning vector (pBluescript SK (-) , Stratagene, USA) by using the DNA fragment as a probe. Two other genes showing high homology with the cloned gene were additionally cloned, which were named each MdASGl, MdASG2 and MdASG3.

To examine whether the above genes were expressed only in a mature flower of Malus x domestics cv. Fuji, PCR was performed by using the first strand cDNAs separated from a calyx, a petal, a stamen and a pistil of a plant genital organ as a template and using 5'- TCTAGTCGGAGCTTCAGTCT-3 ' (SEQ. ID. NO: 7), a part of the nucleotide sequence of MdASGl, as a forward primer and 5 '-AATTAGCTCTCGGACAACAC-3' (SEQ. ID. NO: 8), a part of the nucleotide sequence of MdASGl, as a reverse primer (Fig. 2) . Fig. 2 is a photograph showing the results of RT-PCR performed by using cDNAs separated from each genital organ as a template to examine the novel gene MdASGl (GenBank Accession No: AF403122) of the present invention. Herein, M indicates a size marker, C indicates a cDNA of a control, ¹Se' indicates a cDNA of a calyx, 'Pe' indicates a cDNA of a petal, ¹St' indicates a cDNA of a stamen and a 'Ca' indicates cDNA of a pistil. As shown in Fig. 3, it was confirmed that the anther-specific gene is expressed only in a stamen of a mature flower. In the meantime, PCR was also performed to examine MdASG2 and MdASG3, respectively, by using the above-mentioned cDNAs as a template with primers designed by the nucleotide sequence of the gene

(Fig. 3) . Fig. 3 is a photograph showing the results of RT-PCR examining expressions of MdASG2 (GenBank

Accession No: AF403123) and MdASG3 (GenBank Accession

No: AF403124), homologous genes cloned by using MdASGl as a probe, in each organ. As shown in Fig. 3, MdASG2 and MdASG3 are expressed specifically in a flower, like MdASGl .

Nucleotide sequences of MdASGl, MdASG2 and MdASG3 were analyzed. AS a result, the genes of the invention, MdASGl, MdASG2 and MdASG3, were confirmed to contain open reading frames (ORF) each encoding a different potential protein composed of 75, 88 and 75 amino acids respectively and have high homology each other with the amino acid sequence and nucleotide sequence as well. The homology was further investigated by using BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) provided by National Center for Biotechnology Information (NCBI, USA) . As a result, no other genes having homology with those genes were detected. The amino acid sequence of a hypothetical protein encoded by the ORF was also analyzed (Fig. 4) . Fig. 4 is a diagram showing the comparison of the amino acid sequences of assumed hypothetical proteins encoded by MdASGl, MdASG2 and MdASG3. As shown in Fig. 4, those potential proteins encoded by those three genes have extremely high homology in amino acid sequences, in particular amino- terminal (N-terminal) and carboxyl-terminal (C- terminal) sequences, among them, and also contains an alanine rich region, an N-glycosyl group binding site and a miristoyl group binding site.

<l-2> In situ hybridization

The expression pattern of MdASGl mRNA, during the development of the floral organ of Malus x domestica cv. Fuji, was investigated by mRNA in situ hybridization (Sung et al., Plant Physiology 120: 969-978, 1999). First, an anti-sense RNA was prepared by using sense strand DNA of MdASGl as a template. After samples were taken from each stage of flower development from an immature flower to a mature flower, they were dissected with appropriated size and were fixed with a fixing solution (PIPES 50 mM and 4% p-formaldehyde) , then embedded in paraffin. The paraffin embedded blocks were cut into 10 (M thick sections and they were placed on a glass slide. Then, the expression pattern of MdASGl mRNA was investigated by using antisense mRNA as a probe (Fig. 5) . Fig. 5 is a photograph showing the expression profile of MdASGl mRNA investigated by mRNA in situ hybridization. Herein, A indicates a flower primordial, B indicates a flower at the stage of floral organ primordial and C indicates a flower having a fully developed stamen and pistil. D-F indicate the morphologies of the anthers during the floral development. Particularly, D indicates the anther of immature stage, E indicates the anther at the stage of pollen development and F indicates the anther at the stage of aging after completion of pollen development. The blue region indicates the region where MdASGl gene is expressed. As shown in Fig. 5, the expression of MdASGl is observed from the anther development stage (B, C and D) . From the observation on the inside of the anther was confirmed that the expression, of the gene localizes to tapetum of the anther (E) , while the expression was hardly observed in the aging anther which had its wall broken (F) .

Example 2 : Preparation of a transforπtant in which the expression of mRNA of MdASGl or MdASG2 is suppressed <2-l> Preparation of an Agrobacterium transformant for the expression of MdASGl antisense pBluescript SK(-) vector harboring MdASGl separated from Malus x domestica cv. Fuji, prepared in Example 1, was digested with EcoR I and Xho I. The DNA fragments harboring MdASGl were blunted in their ends using Klenow enzyme, followed by cloning into Sma I site of pRTL-2 vector (Oregon State Univ., USA) containing a multiple cloning site in between CaMV 35S promoter and 35S terminator. Among those prepared recombinant vectors, a vector in which MdASGl was linked reversely to 35S promoter was selected. Both ends of the vector were digested with Hind III, and thus DNA fragments harboring MdASGl were prepared. The fragments were blunted in their ends using Klenow enzyme, followed by cloning into Sma I site of pCAMBIA2301 (Hajdukiewicz, P. et al . , Plant MoI. Biol., 25(6): 989-94, 1994). As a result, a plant expression vector for the suppression of gene expression was constructed (Fig. 6) . Fig. 6 is a schematic diagram illustrating the construction process of a recombinant expression vector for preparing a transformant in which mRNA expressions of MdASGl and MdASG2 were suppressed, respectively.

As shown in Fig. 6, pRTL-2 vector contains double 35S promoter and enhancer, and 35S terminator, and pCAMBIA2301, an Agrobacterium binary vector for plant transformation, contains npt II (neomycin phosphotransferase) gene as a selection marker, endowing kanamycin resistance {Plant molecular Biology Manual, 1988, A3: 1-19). The recombinant Agrobacterium binary vector was introduced into Agrobacterium tumefaciens LB4404

(Hoekema et al., Nature, 303: 179-181, 1983), and then strains harboring the recombinant Agrobacterium binary vector were selected from kanamycin containing medium.

<2-2> Preparation of a transgenic tobacco plant expressing MdASGl antisense

A transgenic tobacco plant in which a recombinant gene construct for the suppression of MdASGl expression was introduced was prepared by using the above Agrobacterium transformant for the expression MdASGl antisense prepared in Example 2-1. First, a mature tobacco seed was dipped in 70%(v/v) ethanol for one minute, followed by washing three times with sterile water. Then, the seed was dipped again in 2% sodium hypochloride (NaOCl) solution for 15 minutes, and then the surface of the seed was washed with sterile water more than 7 times . The seed was placed on germination medium (1/2 MS salts, 3% sucrose, 0.8% agar) . A young leaf was taken from a plant which was growing for 5 weeks from the germination, and was cut into 2 cm² fragments, resulting in the preparation of target tissues. The transformed Agrobacterium tumefaciens LBA4404, prepared in the above Example 2-1, was cultured in YEP (1% yeast extract, 1% peptone, 0.5% NaCl) medium supplemented with 50 mg/L of kanamycin until OD₆₀₀ reached 0.8. The cultured transformed Agrobacterium was co-cultured in 1/2 MS medium containing 50 μM of acetosyringone at 22 ^°C with 150 rpm for 2 hours, then the tobacco leaf fragments selected as a target tissue were added thereto, followed by culture at 22 ^°C for 1 more minute. The fragments were transferred onto coculture medium (MS salts, 3% sucrose, 0.7% agar), followed by further culture for two more days. The cotyledon fragments were transferred onto regeneration medium (MS salts, NAA 0.1 mg/L, BA 0.5 mg/L, sucrose 3%, cefotaxime 250 mg/L, kanamycin 200 mg /L, 0.7% agar) and sub-cultured twice every three weeks. During the culture, transformed shoots generated from the sections were transferred onto rooting medium (MS salts, 3% sucrose, kanamycin 200 mg/L, 0.7% agar) supplemented with kanamycin and then a transformant was selected therefrom. Non-transformed shoots displayed color change and necrosis, while transformed shoots were growing normally with taking roots in the medium. The shoot with roots induced was transferred on soil after acclimating. To confirm whether a recombinant gene construct for the suppression of MdASGl expression was introduced in the transgenic plant, PCR was performed with an nptll primer set represented by SEQ. ID. NO: 9 (nptll Forward, 5 ' -gaggctattcggctatgactg-3 ' ) and SEQ. ID. NO: 10 (nptll Reverse, 5'- atcgggagcggcgataccgta-3 ' ) as follows; at 95^°C for 1 minute, at 55^°C for 1 minute and at 72 ^°C for 1 minute (30 cycles) . From the result of PCR using the nptll primer set, DNA fragments were detected in the transgenic plant, indicating that a heterologous gene was successfully introduced in the transgenic plant.

A recombinant vector for the suppression of MdASG2 expression and a transformant thereby were prepared by the same manner as described above (Fig. 6 - 8) . Fig. 7 is a photograph showing the comparisons of flowers (A) , anthers (B) and fruits (C) between a transgenic tobacco plant in which MdASGl expression was suppressed and a wild type tobacco plant, and Fig. 8 is a photograph showing the comparisons of flowers (A) , stamens and pistils (B) , anthers (C) and pollen grains (D) between a transgenic tobacco plant in which MdASG2 expression was suppressed and a wild type tobacco plant. As shown in Fig. 7, the pollen formation and the fructification were inhibited in the transgenic plant in which MdASGl expression was suppressed. As shown in Fig. 8, the elongation of stamen was rather inhibited and the pollen development and the fructification were inhibited in the transgenic plant in which MdASG2 expression was suppressed. Pollen grains were stained with cotton blue. The decrease of the amount of pollen grains and the broken cell wall were observed.

Example 3: Isolation of MdASGl, MdASG2 and MdASG3 from genomic DNA

Following experiments were performed to isolate genomic DNA containing MdASGl, MdASG2 or MdASG3 of the present invention. First, chromosomal DNA was extracted from a leaf of Malus x domestica cv. Fuji. The extracted DNA was digested with a restriction enzyme Dra I or EcoR V. The chromosomal DNA fragments were ligated to an adaptor primer included in Gene Walker™ kit (Clonetech, USA) to separate a promoter. PCR was performed with an MdASGl specific primer (5'- gacgccaacagcaccattg-3 ' ) represented by SEQ. ID. NO: 11 recognizing specifically the nucleotide sequence of MdASGl by using the above kit. Then, 0.85 kb genomic DNA fragment amplified from the PCR was cloned (Fig. 9) . Fig. 9 is a diagram showing the nucleotide sequence of the genomic gene encoding MdASGl of Malus x domestica cv. Fuji and the nucleotide sequence of the promoter.

To isolate MdASG2 and MdASG3, PCR was also performed respectively by using MdASG2 specific primer

(5 ' -gagtaagtagcgttatagcttc-3 ' ) represented by SEQ. ID.

NO: 12 and MdASG3 specific primer (5¹- aaatcatgaagaataaatttaaata-3 ' ) represented by SEQ. ID. NO: 13. And then, 3.2 kb and 1.9 kb genomic DNA fragments were cloned (Fig. 10 and Fig. 11) . Fig. 10 is a diagram showing the nucleotide sequence of a genomic gene encoding MdASG2 having homology with MdASGl of Malus x domestica cv. Fuji and the nucleotide sequence of the promoter. Fig. 11 is a diagram showing the nucleotide sequence of a genomic DNA encoding MdASG3 having homology with MdASGl of Malus x domestica cv. Fuji and the nucleotide sequence of the promoter.

Example 4: Analysis of MdASGl₁ MdASG2 and MdASG3 promoters

The promoter of MdASG2, obtained from genomic DNA fragments of MdASG2 cloned in the above Example 3, consists of the nucleotide sequence represented by SEQ. ID. NO: 5 corresponding to the region from the upstream of translation starting point to -2874 bp (Fig. 10) . The present inventors named the promoter 'pMdASG2' and investigated the characteristics of the nucleotide sequence of the promoter by using promoter analyzing programs (The Markov Chain Promoter Prediction Server, http://genes.mit.edu/McPromoter.html; Neural Network Promoter Prediction, http: //www. fruitfly.org/seq_tools/promoter .html; PLACE, http: //www. dna.affrc.go.jp/htdocs/PLACE/signalscan. html ; GENSCAN, http://genes.mit.edu/GENSCAN.html; and TRANSFAC, http://transfac.gbf- braunschweig.de/TRANSFAC/index.html) . Likewise, the promoter regions of MdASGl and MdASG3 identified from the nucleotide sequences of MdASGl and MdASG3 isolated in the above Example 3 were named 'pMdASGl' and 'pMdASG3' respectively (Fig. 12).

Fig. 12 is a diagram showing the homology of the nucleotide sequences among MdASGl, MdASG2 and MdASG3 of Malus x domestica cv. Fuji and the location of each potential transcription regulatory region. As shown in Fig. 12, PMdASG2 promoter was confirmed to have eukaryotic promoter regulatory elements, in addition to TATA box (-35) for transcription starting and CAAT box (-241) . The nucleotide sequence of PMdASG2 was compared with those of PMdASGl promoter (Fig. 9) and PMdASG3 promoter (Fig. 11) . As a result, high homology of nucleotide sequence was observed in the region from -255 to +1 nucleotides on the basis of transcription start site. In the meantime, PMdASG2 promoter was confirmed to have constituents of pollen-specific genes, such as 56/59 box (Eyal, Y. et al . , Plant Cell, 7: 373- 384, 1995) which is a transcription factor binding site, GTGA motif (Rogers, H. J. et al . , Plant MoI. Biol., 45: 577-585, 2001) which is a nucleotide sequence found in pollen-specific genes of tobaccos and POLLENlLELAT52 (Bate, N. and Twell, D., Plant MoI. Biol., 37: 859-869, 1998) which is a pollen-specific transcription factor having the nucleotide sequence of AGAAA. In particular, along with TCCACCATA, numbers of POLLEN1LELAT52 which is a transcription factor inducing anther-specific expression are observed in the promoters, and the locations of them are very close to TATA box (Fig. 12) .

As described above, the 'pMdASG2' promoter of the present invention includes factors regulating the pollen-specific expression, so the promoter of the invention can be effectively used for the expression of an anther-specific protein and the development of male- sterile plants.

Example 5 ; Construction of a recombinant vector comprising deletion mutant of pMdASG2 promoter

To prepare a pMdASG2 deletion mutant, the promoter region of pMdASG2 was amplified by PCR using Taq DNA polymerase (Takara, Japan) and sequence specific primers . For the PCR, forward primers represented by SEQ. ID. NO: 14 - 17 and a reverse primer represented by SEQ. ID. NO: 18 were used, and all the forward primers included Hind III restriction enzyme site, while the reverse primer was designed to have Sma I restriction enzyme site. The sizes of the amplified DNA fragments having deletions in pMdASG2 were 2815, 1979, 1532 and 358 bp respectively. The PCR products were digested with Hind III and Sma I and then subcloned into pBHOl plasmid vector (Clontech, USA) , a binary vector containing a gene encoding GUS (β- glucuronidase) and a NOS terminator. As a result, pMdASG2 deletion mutant plasmid vectors pBI MdASG2 28, pBI MdASG2 20, pBI MdASG2 15 and pBI MdASG2 04 having deletion construction of -2.8 kb, -2.0 kb, -1.5 kb and -0.4 kb were constructed (Fig. 13) . Fig. 13 is a schematic diagram showing the construction processes of the deletion mutant of pMdASG2 promoter of the present invention.

Example 6 : GUS expression in a transgenic tobacco plant using pMdASG2 promoter

<6-l> Measurement of GUS activity in a deletion mutant The pMdASG2 deletion mutant gene construct, prepared in the Example 5, was introduced into a tobacco plant by the same manner as described in Example 2, resulting in the preparation of a transgenic tobacco plant. The GUS activity of the transgenic tobacco plant in which the pMdASG2 deletion mutant gene construct was inserted was measured by histochemical staining. For the histochemical staining, the fragments were treated in 0.1 mM phosphate buffer (pH 7.0) solution containing 1 mM X-Gluc, 0.3 mM potassium ferricyanide, 0.3 mM potassium ferrocyanide and 0.2% Triton-X 100 at 37°C for 12 hours. The solution was eliminated, followed by destaining with 70% (v/v) ethanol . And then observation under microscope was performed. To measure the enzyme activity, the level of conversion of 4-MUG (4-methylumbelliferyl β-D- glucuronide) into 4-MU (7-hydroxy-4-methylcoumarin; 4- methylumbelliferone) was measured using fluorescence

(Jefferson, R. A., Plant MoI. Biol. Rep., 5(4): 387-405,

1987) . The promoter activity was calculated by the amount of produced GUS (Fig. 14) . Fig. 14 is a graph showing the GUS enzyme activity in a transgenic tobacco plant with the insertion of a mutant containing a recombinant promoter region in which MdASG2 promoter was deleted serially from 5 'to 3'.

As shown in Fig. 14, the GUS activity was very high in a transgenic plant in which pBI MdASG2 04 was introduced. On the contrary, the GUS activity was not much increased, compared with that of a control, in transgenic plants each transfected with pBI MdASG2 28, pBI MdASG2 20 and pBI MdASG2 15. The above results indicate that a key factor playing an important role in inducing anther- or pollen-specific gene expression exists in the region between -255 and +1 which shows high nucleotide sequence homology among pMdASGl, pMdASG2 and pMdASG3. In addition, it is believed that a regulatory element for inhibiting the activity of factors inducing the GUS expression exists in the upstream of MdASG2 04.

In the meantime, the GUS expression in the anther of a transgenic tobacco plant with pMI PMdASG2 04 insertion was also investigated according to following each stage of development: First, the development from a flower bud formation through a flower formation was divided into 10 stages according to the length of a flower, and flowers of each stage were dissected properly, followed by fixing with a fixing solution and paraffin embedding. The embedded sections were cut into 10 μm thick sections, followed by histochemical staining. The GUS activity was measured by the same manner as described above (Fig. 15 - 17) . Fig. 15 is a photograph showing the 10 divided stages of the flower development of a transgenic tobacco plant with the insertion of a deletion mutant promoter MdASG2 04. Fig. 16 is a graph illustrating the GUS enzyme levels of each developmental stage of a flower measured by a biochemical method. Fig. 17 is a photograph showing the GUS expressions of each developmental stage of a genital organ.

As shown in Fig. 16 and 17, the GUS expression was observed from the turning point of stage 5 to stage 6, which is the stage of tetrad formation from a microspore, and in the histological aspect, the GUS expression was strongly induced in the inner wall of the anther or the anther inner wall derived pollen grains .

<6-2> Observation of pollen grains under confocal microscopy

To find out the location of the GUS expression in pollen grains, 488 nm of excitation wavelength was irradiated and then emission wavelength over 505 nm was observed with a confocal microscope (Carl Zeiss LSM 510 META, Germany) (Fig. 18) . Fig. 18 is a fluorescent photograph taken by confocal microscopy which illustrates the pollen grains of a transgenic tobacco plant with the insertion of a deletion mutant promoter MdASG2 04. As shown in Fig. 18, the GUS expression was confirmed in the wall of pollen grains.

Example 7 : Preparation of a male-sterile tobacco plant using PMdASG2 promoter

<7-l> Construction of a vector for production of a male-sterile plant by using a heterologous protein RIP

A recombinant vector for plant transformation that is able to express a heterologous protein was constructed as follows by linking a gene encoding a foreign protein operably to the MdASG2 promoter of the present invention. At first, MdASG2 04 promoter (358 bp fragment amplified by using the sequences represented by SEQ. ID. NO: 17 and 18) prepared in the above Example 5 was cloned into a PCR cloning vector

(pGEM T-vector, Promega, USA) , which was then digested with a restriction enzyme Sma I. Next, PCR was performed with a forward primer represented by SEQ. ID.

NO: 19 (RIP Forward: 5'- gtttcagctgaaatgaagatatatgtagtgg-3 ' ) and a reverse primer represented by SEQ. ID. NO: 20 (RIP Reverse 5'- cctcaatattcgactttcatgcacccaaatgca-3 ' ) by using pMTA2913/RIP (Cho, H. J. et al., MoI. Cells, 10(2): 135-41, 2000) as a template to amplify a heterologous gene encoding ribosome inactivating protein (referred as 'RIP' hereinafter) which is a cytotoxic protein. The amplified polynucleotide was cloned into the PCR cloning vector. Then, the PCR cloning vector harboring the heterologous gene encoding RIP was digested with Ssp I and Pvu II. The obtained DNA fragments were ligated to pGEM T-vector having MdASG2 04 promoter, and then digested with Hind III and £coR I. The ends of the fragments were blunted by Klenow enzyme, which were cloned into pRTL-2 vector with the deletion of Hinc II and Sma I sites. Among those recombinant vectors, a vector in which the gene was cloned forward to the 35S terminator was selected. The recombinant vector was digested with Hind III again, which was cloned into Hind III restriction enzyme site of pCAMBIA3301, resulting in the construction of a recombinant vector for the heterologous gene expression (Fig. 19) . Fig. 19 is a schematic diagram showing the construction processe of a recombinant expression vector in which MdASG2 04 promoter is operably linked to the heterologous gene encoding RIP.

<7-2> Construction of a vector for preparing a male- sterile plant by using the antisense mRNA of MdASG2 cDNA A recombinant vector for plant transformation that is able to express antisense mRNA of MdASG2 was constructed as follows by linking MdASG2 cDNA reversely to the MdASG2 promoter. At first, MdASG2 cDNA was amplified by PCR using a forward primer represented by SEQ. ID. NO: 21 (antisense MdASG2 cDNA Forward: 5'- ggcagctgcaaacttctacaagcctcttaacatt-3 ' ) and a reverse primer represented by SEQ. ID. NO: 22 (antisense MdASG2 cDNA Reverse: 5 ' -ttgaatatttagcgttatagcttcatttattggcg- 3'), which was then cloned into a PCR cloning vector (pGEM T-vector, Promega, USA) . The vector was digested with Ssp I and Pvu II. Then, the vector was ligated to a plasmid fragment obtained by digesting pGEM T-vector harboring MdASG2 04 promoter prepared in the above Example 7-1 with Sma I. The pGEM T-vector containing the MdASG2 04 promoter and MdASG2 antisense sequence was digested with Hind III and EcoR I and blunted by klenow enzyme in its end, which was cloned into pRTL-2 vector digested with Hinc II and Sma I. Among recombinant vectors constructed above, a vector harboring the MdASG cDNA linked reversely to the 35S terminator was selected. Both ends of the vector was digested with Hind III again, followed by cloning into Hind III site of pCAMBIA3301, resulting in the construction of a recombinant vector (Fig. 20). Fig. 20 is a schematic diagram showing the construction process of a recombinant vector harboring MdASG2 cDNA linked reversely to the MdASG2 04 promoter of the present invention. The recombinant vector for plant transformation was introduced into Agrobacterium tumefaciens LBA4404 (Hoekema et al., Nature, 303: 179- 181, 1983) . Strains containing the recombinant Agrobacterium binary vector were selected from a kanamycin containing medium. Among transformed Agrobacterium, the Agrobacterium LBA4404/pMd2proASMd2 in which a recombinant gene construct harboring MdASG2 antisense sequence operably linked to the PMdASG2 04 promoter was deposited at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) (52 Oun-Dong, Yusong-Gu, Taejon, Korea) under accession No: KTCT10769BP.

<7-3> Preparation of a male-sterile tobacco plant

The recombinant vectors for plant transformation constructed in the above Examples 7-1 and 7-2 were introduced into tobacco plants by the same manner as described in the Example 2, resulting in the preparation of transgenic tobacco plants . The developments of various floral organs of the transgenic plants were observed with the naked eye (Fig. 21 and 22) . Fig. 21 is a photograph showing floral organs of a transgenic tobacco plant with the insertion of a vector constructed by linking a heterologous gene encoding RIP operably to MdASG2 04 promoter of the present invention, and Fig. 22 is a photograph showing floral organs of a transgenic tobacco plant introduced with a vector constructed by linking MdASG2 cDNA reversely to MdASG2 04 promoter and a wild type tobacco plant. As shown in Fig. 21 and 22, those transgenic tobacco plants transformed respectively by a vector harboring a heterologous gene encoding RIP operably linked to the MdASG2 04 promoter and by a vector harboring the MdASG2 cDNA linked reversely to the MdASG2 04 promoter showed the decrease of stamen elongation. To investigate the morphologies of pollen grains of those transgenic plants, cotton blue was used for staining, and to examine the activities of pollen, fluorescein diacetate was used for staining (Fig. 23 and 24) . Fig. 23 is a set of photographs showing the morphologies of pollen grains of a transgenic tobacco plant with the introduction of a vector in which a heterologous gene encoding RIP is operably linked to MdASG2 04 promoter of the present invention and a wild type tobacco plant, which are stained with cotton blue (A) , and the activities of pollen of a transgenic tobacco plant and a wild type, which are stained with fluorescein diacetate (B) . Fig. 24 is a set of photographs showing the morphologies of pollen grains of a transgenic tobacco plant with the introduction of a vector in which MdASG2 cDNA is reversely linked to MdASG2 04 promoter and a wild type tobacco plant, which are stained with cotton blue (A) , and the activations of pollen of the above tobacco plants which are stained with fluorescein diacetate (B) . As shown in Fig. 23 and 24, the morphological changes of pollen grains and the decrease of pollen activity were observed in the transgenic plants.

Pollen grains of the transgenic plants were thoroughly examined under scanning electron microscope

(Fig. 25 and 26) . Fig. 25 is a photograph showing the result of the observation under scanning electron microscope on the morphologies of pollen grains of a transgenic tobacco plant introduced with a vector in which a heterologous gene encoding RIP gene is operably linked to MdASG2 04 promoter and a wild type tobacco plant, and Fig. 26 is a photograph showing the result of the observation under scanning electron microscope on the morphologies of pollen grains of a transgenic tobacco plant introduced with a vector in which MdASG2 cDNA is reversely linked to MdASG2 04 promoter and a wild type tobacco plant.

As shown in Fig. 25, a significant morphological change was observed in pollen grains of the transgenic tobacco plant with the insertion of a heterologous gene encoding RIP operably linked to the MdASG2 04 promoter of the present invention, compared with that of a wild type. As shown in Fig. 26, abnormal development of pollen grains was observed in the transgenic tobacco plant in which the MdASG 2 cDNA reversely linked to the MdASG2 04 promoter of the present invention, that is, microfibrils were not normally developed in the groove of the pollen grains.

Further, the present inventors performed self- pollination using the above transgenic plant and cross- pollination which is performed by covering a pistil of the transgenic tobacco plant with pollen grains of a wild type, in order to investigate whether the transgenic tobacco plant is a male-sterile in deed (Fig. 27 and 28) . Fig. 27 is a set of photographs showing the result of investigation on the fructification by self-pollination (A) in a transgenic tobacco plant in which a heterologous gene encoding RIP operably linked to MdASG2 04 promoter and the fructification by cross- pollination which is performed by covering a pistil of the transgenic tobacco plant with pollen grains of a wild type (B) . Fig. 28 is a set of photographs showing the result of investigation on the fructification by self-pollination (A) in a transgenic tobacco plant with the insertion of MdASG2 cDNA reversely linked to MdASG2 04 promoter and the fructification by cross-pollination which is performed by covering a pistil of the transgenic tobacco plant with pollen grains of a wild type (B) . As shown in Fig. 27 and 28, no fructification was observed in the case of self-pollination, while the fructification was observed in the case of cross- pollination. The results indicate that a male-sterile transgenic plant whose pistil functions normally was successfully prepared by the present invention.

As described above, the isolated genes of the present invention and promoter thereof play an important role in the development of floral organs in a plant. Particularly, the isolated genes and their promoters of the present invention can be effectively used for preparing a male-sterile plant whose organs except male-specific ones functions normally by introducing a useful pollen-specific heterologous gene and/or a recombinant gene construct designed to express a heterologous gene encoding a cytotoxic protein or an antisense mRNA of an anther-specific gene derived from Malus X Domestics cv. Fuji of the present invention.

Industrial Applicability

The present invention provides novel anther- specific genes MdASGl, MdASG2 and MdASG3 derived from Malus x domestica cv. Fuji and promoters thereof. The anther-specific genes of the present invention play an important role in the development of a stamen, a genital organ of a plant, so they can be effectively used for the examination of the development mechanism of pollen involved in plant fertilization. And the promoters PMdASGl, PMdASG2 and PMdASG3 include many recognition sites for pollen-specific expression and enable the expressions of pollen- or anther-specific genes, in addition to having a high activity, so that they can be effectively used for the development of a male-sterile plant and a transgenic plant that a pollen-specific gene expression is needed by introducing the whole or a part of the promoters into a target plant. In the meantime, a transgenic tobacco plant in which the anther-specific gene MdASGl or MdASG2 operably linked to 35S promoter displayed normal phenotypes except changes of male-specific organs. Therefore, a male-sterile plant can be generated by regulating MdASGl or MdASG2 expression.

Sequence List Text

The SEQ. ID. NO: 1 is the nucleotide sequence of anther-specific MdASGl cDNA derived from Malus x domestica cv. Fuji of the present invention.

The SEQ. ID. NO: 2 is the nucleotide sequence of anther-specific MdASG2 cDNA derived from Malus x domestica cv. Fuji of the present invention.

The SEQ. ID. NO: 3 is the nucleotide sequence of anther-specific MdASG3 cDNA derived from Malus x domestica cv. Fuji of the present invention. The SEQ. ID. NO: 4 is the nucleotide sequence of anther-specific promoter PMdASGl of the present invention.

The SEQ. ID. NO: 5 is the nucleotide sequence of anther-specific promoter PMdASG2 of the present invention.

The SEQ. ID. NO: 6 is the nucleotide sequence of anther-specific promoter PMdASG3 of the present invention .

The SEQ. ID. NO: 7 is the nucleotide sequence of a forward primer used for the detection of MdASGl of the present invention.

The SEQ. ID. NO: 8 is the nucleotide sequence of a reverse primer used for the detection of MdASGl of the present invention. The SEQ. ID. NO: 9 is the nucleotide sequence of nptll forward primer used for the confirmation of the introduction of a PMdASG2 promoter deletion mutant.

The SEQ. ID. NO: 10 is the nucleotide sequence of nptll reverse primer used for the confirmation of the introduction of a PMdASG2 promoter deletion mutant. The SEQ. ID. NO: 11 is the nucleotide sequence of the MdASGl specific primer used for the cloning of genomic DNA of MdASGl.

The SEQ. ID. NO: 12 is the nucleotide sequence of the MdASG2 specific primer used for the cloning of genomic DNA of MdASG2.

The SEQ. ID. NO: 13 is the nucleotide sequence of the MdASG3 specific primer used for the cloning of genomic DNA of MdASG3. The SEQ. ID. NO: 14 is the nucleotide sequence of a forward primer used for the construction of a PMdASG2 deletion mutant.

The SEQ. ID. NO: 15 is the nucleotide sequence of a forward primer used for the construction of a PMdASG2 deletion mutant.

The SEQ. ID. NO: 16 is the nucleotide sequence of a forward primer used for the construction of a PMdASG2 deletion mutant.

The SEQ. ID. NO: 17 is the nucleotide sequence of a forward primer used for the construction of a PMdASG2 deletion mutant.

The SEQ. ID. NO: 18 is the nucleotide sequence of a reverse primer used for the construction of a PMdASG2 deletion mutant. The SEQ. ID. NO: 19 is the nucleotide sequence of a forward primer used for the cloning of a gene encoding RIP.

The SEQ. ID. NO: 20 is the nucleotide sequence of a reverse primer used for the cloning of a gene encoding RIP.

The SEQ. ID. NO: 21 is the nucleotide sequence of a forward primer used for the cloning of an antisense of MdASG2.

The SEQ. ID. NO: 22 is the nucleotide sequence of a reverse primer used for the cloning of an antisense of MdASG2.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention, Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

What is claimed is:

1. An isolated or recombinant gene comprising a nucleotide sequence selected from a group consisting of MdASGl represented by SEQ. ID. NO: 1, MdASG2 represented by SEQ. ID. NO: 2 and MdASG3 represented by SEQ. ID. NO: 3 or a homologue thereof having 70 - 99% homology and having functionally equal thereto.

2. The isolated or recombinant gene according to claim 1, wherein the homologue has 80 - 99% homology with the nucleotide sequence.

3. An isolated or recombinant polynucleotide having pollen-specific promoter comprising a nucleotide sequence selected from a group consisting of SEQ. ID. NO: 4 (PMdASGl), SEQ. ID. NO: 5 (PMdASG2) and SEQ. ID. NO: 6 (PMdASG3) or an active partial fragment thereof having transcriptional activity.

4. The isolated or recombinant polynucleotide according to claim 3, wherein the active partial fragment comprises from -55^th to +89^th nucleotide on the basis of the 2786^th nucleotide of SEQ. ID. NO: 5, which is transcription start site of PMdASG2, or a homologue having 70 - 90% homology with the polynucleotide and being functionally equal thereto.

5. The isolated or recombinant polynucleotide according to claim 3, wherein the active partial fragment comprises from -255^th to +l^st nucleotides on the basis of 2786^th nucleotide of SEQ. ID. NO: 5.

6. A recombinant gene construct containing a DNA sequence encoding a heterologous protein operably linked to the isolated or recombinant polynucleotide of claim 3.

7. A recombinant vector containing the isolated or recombinant gene of claim 1 or an antisense nucleotide thereof .

8. The recombinant vector according to claim 7, which is selected from a group consisting of MdASGs/pBluescript, MdASGs/pRTL2 and MdASGs/pCAMBIA2301, shown in the cleavage maps of Fig. 3, wherein the 's' of MdASGs indicates any number of 1 to 3.

9. The recombinant vector according to claim 8, which is MdASGs/pRTL2 shown in the cleavage map of Fig.

3 .

10. A transgenic plant transformed with the isolated or recombinant gene of claim 1 or the recombinant vector of claim 7.

11. A recombinant expression vector containing the isolated or recombinant polynucleotide of claim 3.

12. The recombinant expression vector according to claim 11, which is an Agrobacterium binary vector for plant transformation.

13. The recombinant expression vector according to claim 11, further comprising one or more genes encoding a heterologous gene or an antisense nucleotide thereof operably linked to the isolated or recombinant polynucleotide .

14. The recombinant expression vector according to claim 13, wherein the gene is selected from the group consisting of a gene encoding ribosome inactivating protein (RIP), MdASGl, MdASG2 and MdASG3 or an antisense nucleotide thereof, but not always limited thereto.

15. The recombinant expression vector according to claim 13, which is an Agrobacterium binary vector containing T-DNA of Agrobacterium.

16. The recombinant expression vector according to claim 13, which is pMd2proRIP shown in the cleavage map of Fig. 19 or pMd2proASMd2 shown in the cleavage of Fig, 20.

17. A transformed microorganism prepared by transfecting a host strain with the recombinant vector of claim 11.

18. The transformed microorganism according to claim 17, wherein the host strain is Agrobacterium.

19. The transformed microorganism according to claim 18, wherein the Agrobacterium is the recombinant Agrobacterium LBA4404/pMd2proASMd2 deposited under Accession No: KTCT 10769BP.

20. A method for the production of a transgenic plant expressing an anther- or pollen-specific gene, comprising the following steps: i ) constructing a recombinant expression vector containing the isolated or recombinant gene of claim 1 or an antisense thereof or a recombinant expression vector containing the isolated or recombinant polynucleotide of claim 3 and a heterologous gene operably linked to the polynucleotide; ii ) preparing an Agrobacterium transformant by introducing the recombinant expression vector into Agrobacterium; iii ) transforming plant cells by co-culture with the Agrobacterium transformant; and iv) re-differentiating the transformed plant cells by tissue culture.

21. The method according to claim 20, wherein the heterologous gene is an antisense nucleotide of the isolated or recombinant gene or a gene encoding ribosome inactivating protein.

22. A method for the production of a male-sterile plant comprising the following steps: i ) constructing a recombinant expression vector for plant transformation containing the isolated or recombinant polynucleotide of claim 3 and a heterologous gene encoding a cytotoxic protein operably linked to the polynucleotide or an antisense sequence of a gene necessary for pollen formation; ii ) preparing an Agrobacterium transformant by introducing the recombinant expression vector in an Agrobacterium strain; iii ) transforming plant cells by co-culture with the Agrobacterium transformant; and iv ) re-differentiating the transformed plant cells by tissue culture.

23. The method according to claim 22, wherein the cytotoxic protein is ribosome inactivating protein.

24. The method according to claim 22, wherein the gene necessary for pollen formation is selected from a group consisting of MdASGl, MdASG2 and MdASG3.