Abstract:
The present invention relates to an anther-specific cotton gene (CoFS), and active promoter fragments thereof. These promoters show strong anther-specific activity.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Technical Field.  
           [0002]    The present invention relates to the field of plant molecular biology. In particular, the invention pertains to cotton promoters and their uses in creating transgenic plants, and more specifically to cotton anther-specific promoters.  
           [0003]    2. Description of the Background Art.  
           [0004]    Cotton is the most extensively used natural fiber in the textile industry. Annual production of cotton worldwide is over 100 million bales valued at 45 billion U.S. dollars. Although significant improvements have been made in quality and yield by means of classical breeding in the past decades, the potential for further improving the properties of cotton through classical breeding is limited due to requirements for species compatibility and available traits. Genetic engineering provides novel approaches for further improving cotton by introducing genes to create new germplasms with highly desirable characteristics, for example, insect pest resistance.  
           [0005]    The anther is the male reproductive organ in flowering plants. Anther development can be divided into two general phases. During phase 1, most of specialized cells and tissues differentiate, microspore mother cells undergo meiosis and tetrads of microspores are formed. During phase 2, microspores are released from tetrads followed by pollen grain maturation, tissue degeneration, dehiscence and pollen release. Genes specifically expressed during anther and pollen development have been studied in a few plant species. Allen and Lonsdale,  Plant J . 3:261-271, 1993; Bird, et al.,  Plant Mol. Biol . 11:651-662, 1988; Brown and Crouch,  Plant Cell  2:263-274, 1990; Grierson et al.,  Nucl. Acids Res . 14:8595-8603, 1986; Hanson, et al.,  Plant Cell  1:173-179, 1989; Ursin et al.,  Plant Cell  1:727-736, 1989; John and Petersen,  Plant Mol. Biol . 26(6):1989-1993, 1994; Atanassov et al.,  Plant Mol. Biol . 38:1169-1178 1998; Liu et al.,  Plant Mol. Biol . 33:291-300, 1997; Treacy et al.,  Plant Mol. Biol . 34:603-611, 1997; Agnes et al.,  Plant Mol. Biol . 40:857-872, 1999. Among the 20,000 to 25,000 expressed genes in tobacco anther, only 10,000 genes are anther-specific. Kamalay and Goldberg,  Proc. Natl. Acad. Sci. USA  81:2801-2805, 1984; Koltunow, et al.,  Plant Cell  2:1201-1224, 1990.  
           [0006]    A promoter is a DNA fragment which determines the temporal and spatial specificity of gene expression during plant and animal development. Many tissue-specific genes and their promoters have been identified and isolated from a wide variety of plants and animals over the past decade, including cotton tissue-specific genes and promoters. Loguerico et al.,  Mol. Gen. Genet . 261(4/5):660-671, 1999; Kawai et al.,  Plant Cell Physiol . 39(12):1380-1383, 1998; Song and Allen,  Biochem. Biophys. Acta  1351(1):305-312, 1997; Ma et al,  Biochim. Biophys. Acta  1344(2):111-114, 1997 ; John, Plant Mol. Biol . 30(2):297-306, 1996; Rinehart et al.,  Plant Physiol . 112(3):1331-1341, 1996; Hasenfratz et al.,  Plant Physiol . 108(4):1395-1404, 1995; John and Peterson,  Plant Mol. Biol . 26(6): 1989-1993, 1994; John and Crow,  Proc. Natl. Acad. Sci. USA  89(13):5769-5773, 1992. These plant tissue-specific promoters can be used to control the expression of foreign genes in transgenic plants in a tissue-specific manner that will dominate the majority of the second generation of transgenic crops. Some plant tissues do not express high levels of the transgene in all desired tissues or the particular desired tissue. In transgenic Bt cotton, for example, Bt gene expression level is extremely low in the flower, including in the anther, resulting in little protection from pest insects in these tissues. To achieve better control of pest insects of cotton, it would be highly advantageous to identify anther-specific promoters which can produce higher levels of gene expression in these tissues.  
         SUMMARY OF THE INVENTION  
         [0007]    Accordingly, the present invention provides a promoter that is cotton another-specific, comprising the promoter of the cotton CoFS gene. The invention also provides a cotton anther-specific promoter comprising SEQ ID NO:2. In yet a further embodiment, the invention provides a transgenic plant expressing a transgene under control of a cotton anther-specific promoter of the cotton CoFS gene. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0008]    [0008]FIG. 1 shows the results of a CoFS cDNA differential display assay.  
         [0009]    [0009]FIG. 2 provides a Northern blot showing CoFS gene expression.  
         [0010]    [0010]FIG. 3 is a schematic diagram of constructs of CoFS promoter vector constructs and the cotton CoFS gene.  
         [0011]    [0011]FIG. 4 provides the results of an assay of the expression of the GUS gene under the control of the CoFS gene promoter in transgenic tobacco plants.  
         [0012]    [0012]FIG. 5 shows the results of an expression assay of the GUS gene under the control of the CoFS promoter in transgenic tobacco plants. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0013]    An anther-specific gene (CoFS) and its corresponding promoter were isolated from cotton by differential display assay. The activity and tissue specificity of the isolated promoter was confirmed in transgenic tobacco plants using the CoFS promoter to control the expression of the GUS reporter gene.  
         [0014]    Northern blot analysis of cDNAs from a variety of cotton tissues showed that a cDNA clone comprising the CoFS gene was strongly expressed in anthers, and also expressed in petal tissue, but less or not at all in other tissues. See FIG. 1.  
         [0015]    An anther-specific gene (named CoFS)was isolated from cotton. The isolated complete CoFS cDNA is 8.4 kb in length. See Table I. Based on the CoFS cDNA sequence, a CoFS promoter fragment (2.6 kb) was isolated. See Table II. Comparing the nucleotide and predicted polypeptide sequences of the cotton CoFS gene with published sequences revealed that the gene was about 54-58% identical at the amino acid level with acyl-CoA synthetase (probable long-chain-acid cCoA ligase, EC 6.2.1.3) genes from some plants such as  Brassica napus  (X94624) and Arabidopsis (AL078468, AL161560). Less homology was found at the nucleotide level, indicating that CoFS is a novel gene found in cotton. Analyzing the CoFS gene sequence revealed that it may contain 9-10 exons and 8-9 introns in its open reading frame, based on amino acid sequences of the known acyl-CoA synthetases.  
         [0016]    The CoFS promoter fragment was fused with the GUS gene to construct gene expression vectors for analyzing the function of the promoter. Transgenic tobacco plants with the CoFS promoter/GUS fusion genes were identified by Southern blot hybridization. In the transgenic plants studied, GUS activity was detected in anther, and weakly in ovaries, styles and stigmas, but not in petals or other tissues. This result, together with Northern blot analysis, indicates that the CoFS promoter is anther-specific in cotton. The promoter controls specific gene expression at the transcriptional level in cotton anthers. The isolated promoter may be used in improving expression of desired genes in anther and related tissues of the plant sexual organs to create new plant varieties, thereby enhancing quality and yield of the plant by gene manipulation.  
         [0017]    The promoters of the present invention are useful in creating transgenic plants, especially including cotton, having improved expression of the transgene in anther tissue. Better expression of protective genes, such as the Bt gene, in anther tissue results in a plant with increased resistance to Bt-sensitive pests. Genes which may be expressed under the control of this promoter include any gene suitable for the purpose.  
                         TABLE I                       Sequence of the CoFS Gene From Cotton (SEQ ID NO:1).                                CCTCACATTTAAGCGGAAAAAAATATTAACTAATTACTAATTACTAAGGTCATGGGT                   TGCGCATTAAAGTTCACTGACGATTGTGCAAATGATGTTCCATAGAGCTTAATTGAT               GAAATGGGAACTCATGACCCGCTTGAAGTAACTCGACTTGTAGAACTCATGAAGAA               GCTTATCTACTTGAAGTTTTGGTAGCCCAATGAAATACTCTCGTAAATCTAGAGTTAT               TAGTGTAAACCCTAAAGGGATCAAATTGTATAAATTTAAATCCCTTATGACTTTCAA               TTGTAGATAGACTCTAATCTCGATCATGGATGTAACTCAATCTATTTGTTGGGTTTGG               GGTGATTACTTCAATTCATTCCATTCATAGTTGTGAATATATTTGAGAGTATTTACGC               AAACATTTGGTGTGTGCTATTTTTCCTTTGGTCTTTTGTTCTTCGTTGCCCATTCGTTC               GAGTTTGCTTTCGCTATATTTTAATGCCTTAGAAAATTTTTGCGAGAATTCTCATTTT               GTGAGAGTTAAGCGAACTTAGAATTATTTTTTTTAAAATCGCTTAAGGCTGTATGGT               CTGTGAGACTAAAATTCTAGTCTCGTAACACTAATACAATCACAAGTAATTTACATT               GTTCAAGTTCTTATTCACATAAGCGGTTGGATAAAGAAAATTAAAAAAAAACAATC               GGATATAATTACAAAAAAATAAATTGAAATGTGCAATAATACAAATAATAATTATT               GCTAAAGGTAAATAAAAAATGTAAATAATATCAATGAAGTTTGAAACCTTAAATG               GTGAAGTTTGTGTCAACTAATAGAAGAAAAAATAAATTATTTATATAACTCTACTAA               TGTATTATTTTATTTTGTAAAATTGATTTATTTATATTATTTCTACTAAATTGATGTGG               AATTAGTGATATCTACTTAATTAACTATATATAATTATAATGAATCTCCGGGACTGT               GACTGGTCAAAGATCATAAAGTGGTATCCAATAAATTTAAAATGCACTTGTAAAATA               TTAGACTCATGATGGCACTGAGGCGGAGGTGAAGAGGCGGCAAAGCACATGGAGA               AGCTATATAGAAAATTCTTTCACGAAAAAGGCAACTCTTGGCTTGTGTGTTGGGAAT               TGTGTTAAGAACTGGATTATATAAAAACAATTATATGGGGAAAGGAAATGGTCCAC               TGTCAATAGTTTACTATAAGCAAGTTGGAGATATAAAATTAAATATATATTCAGTAC               ATATACGAGTTTGAGCAACAAAATTAGAGATCTTTTTTGTCAAGTTGATATCTTCAA               TTTTATAACGTAAATGTTCTTTTGAAGGCAACAGTAATGATATATATATATGTAGAA               GAAATTTAACTAAAAATAGATAATTAGGCTTAATTTAATTAATTCAAGTGCAATTGT               TTTATCATAATATATATTACATTACAAGGCTTGAATTATTCATATTTTAAATTTATTT               ATTAGTTAACAAAGTAATTATTGGTGCAAAAATAAATAAATTATTACCCCATTATCT               ATTTTCTTAAATAAAAAAATATATTATATATGCCATTTACTCTCTTTAAAAAAATTTA               ATTTACAAATAAACTAATAAATTTGTATATGATGATTTCGAATGAGGGTTTTAATAC               AGTTATCATGATGATTTCAATACAATGGTTCCAAATGAATAAGGATTCCACTACAAC               ATTAAACTCACCATAATGGTGATTCCAATTGAGTGTTCCTACATAATTATCATCATA               ATTCTTACTTGGCAGGATACAAAAAATAATAATGGGTAAGGTAATTAAAAATTAAA               ATAATTATCAATAAAGTTTTTATTATGGTGACAAAGTTTGATAATCATTATTAAATTA               TTAATTGAGTAAAATATTTAAATATAGTAATGTATATATAATGAATCTCCGGCGATG               TTGGCTGTTCAAAGATCATAAAATCCAATACATTTAAAATGCAGCTGTAAAATATGA               TGTAAAGGGCGGATAAAGCACATGGAGAAGCCATATAGAAATTTCTTCCATGAAAA               AGGCGACTCTTAGCTTGTGTGTTGGGAATTGTGTTAAGTTATATGGGAAAGGAAATA               TTCCACTGTCAATAGTTTATTATAAGCAAGTGGGAGAAACAAAATTAAATATGTTAT               TTTGAAGGCAACAGTAATAATATAATTATAAATTAAAATAACATAATTTAGGCTTAA               TTTAATTAATGGTAAACTATAAAAAAAAGTCATTTTTGTTTGCTTCAGATTACATTTT               AGTCACTTATGTTTGAAATGTTACGTTTTAGTCACTTACATTACCGTTTTGTTACGAA               GTGGTCACTTTACCATTAAACTCTATTACCTCCCTAACGACAGTCCTACGTGGCAGT               CAAAATGAATTTTAAATGCTAACTTGGACGTCCAGTTGCTGGGACATTTTCCGGTTC               ACCTACAGCCACCTAATACTTAGGCGCTATATATTTTCCCAAAATTATTCTCCACATT               TCACTCCCAGAGCCCTCCATCTATGTAGAGAGCTGTAAGAGAGAATATAAAAAAGG               GAAAGCTAGCTGAGGATCGTTTGATTTTGGACATTTTGATGAAACGGCCTGGAAATA               ATTTTGTAATAGAGGTAGAGAAAGGTAAAGACCCCAGCGATGGACAGCCGTCGATC               GGTCCTGTTTATCGCAGTTCTTTTGCTGCTAATGGATTCCCTGCTCCGATTCCTGGAA               TGGAGAGTTGCTGGGACATTTTCCGGTTCGTTTTTAGCTTTCTCTTTTTTACTTTACGC               TTCTTGCTTGGCTGCTAAGAAAATAAGGATACTAGGAGAAGATTTGACTTCTTCTAT               TCTTTGCTTTGATTTCAGATGGCTTTCGCAATAGTTGCCATTTTTTTTGAATTTTTACT               TCCCCTTTTTTAAGTTGAGTAGATTTTTCTTCTAATTTGTTGGCTTTGTTATTTTTTTAT               TTCGCGACGAGGTGGCGATGAAATCGAGAAACGTACTTTTAAGGATCCTATGAGAA               AGTTATATGCAAGATCGAAACGCCTAATATTTGAAACTATTGAATTTTAACGCTCAC               ACAGAGCAAGAATCGAGTTACTGGTATTTCCATTCTTATAGCTGAAAGATTGATGGC               TTTCATTCAACTCAATGTAAAACTGTGAAATAAATTGTTTAATAGTAGTAATTATTTT               GGTTTTGATGCTTATGTGATGTGGAGATTAAAATATTGCCTCCTTATAACTTAGCTGA               ACCGTAGATATGGGCTGATTGAACTTGCTAACTAACTGTATGACAGCTCCTATTTAC               GAAAAAGTAATATTTTATTTGAGGATGATGATTTCGATTTCATTTTCTCCTTGCAGTA               TGTCAGTTGAGAAATATCCTGACAACCATATGCTTGGTCGCCGACAGATTGTGGATG               GGAAAGTATGTTGCTGCTGCAGTTTCCTTTTTTCTTGTTACTTTTGCGTTCTGTTTGTA               GTGCGGCCTTTGACTTTTAGTTCATGTATCTAAATTGACATGCTTTGATTGCAGGCTG               GAAAATACGTGTGGCAAACTTACAGAGAAGTTTATGACATTGTAACAAAAGTTGGG               AATTCCATCCGAAGTTGTGATGTTGTGGAAGTAATGCTTTAACCTCCTTTTTTCCTTT               TAATTGTAAAATTATTGTCAATTTTTTTTATAACAAATATCCTATTTCTGGGGATCAA               TATCCACCCACAATTGATGCTAATAAAAAAAAAAATTAAGCTTTTTATTCTTGCTTA               CAGGGAGGAAAGTGTGGTATTTATGGTGCCAATTGCCCAGAATGGATAATTAGCAT               GGAGGTATGATCATTCTGGCATGTTTCATCTGATTTGCACAGTGGACATCCCAAGTT               ACTTAGATGTCGCTATAACTTGTTTCTTTGGATCATACTATTTGCTACCAAATTGCTT               GTTGCCCGAAATGTTTACTAATGTTGCAAGATTGATACAGGCCTGCAACGCTCATGG               ACTCTATTGTGTTCCTTTATATGACACTTTGGGTATTTCTTCTTGAGATCCAACAAAA               GCATTCTTTCAGTTTTTGCCAAACAACTACCTTTCTCTAACAACCATCTTATGTGTAT               TATGTACTCTTCATATAGTCTGTGATCATAATATCCTTAAACTCTTTAAATTATTCTT               GCATTCCAACGCGCCTGCTACTCTTTTTGAATGTTTGATACGTTGCTACATATTTGTA               GGTGCCGGTGCTGTGGAGTTTATCATATGCCATGCAGAAATTTCTATTGCTTTTGTAG               AGGAAAAGAAGATTAATGAGGTATGCCTGTTTACATCTATATTTGAAACCCTAGTAG               TGATATGGCAACTACTGGGAAAGATACTTCTAGATACTTGCAAAAATAAGATATCTA               TCTACAAAATAGATTCGATGTTTTATTTTTATTATCAGCATTTGCTTCTATGCTTGCTG               CTCACTTATGCATAGTTGATATTGATACAAGAATCTGTAATTTCACAAATTTTCTGTT               CTTTTTTCTTTTGGGTTTCTCATGTAAGGTTTTTCTTTTTCCTTTCAGCTGTTCAAAAC               ATTTCCAGCCTCAACAGAACACTTGAAAAGTAAGCTATCTGATTATTTAGGGGGATT               CTTGAAATAGTACGTTACAAATTATTTATGTCCATGTATTTTTGCTATGCAGCAATTG               TTAGCTTCGGGAAGGTAACACCTGAGCAGAAGGCAGAAGCTGAGAAGCATGGTTTG               AAGATATATCCTTGGGAGGAATTTTTGCAACTGGTAAGCTCTTTGCTCTGTTATTTTC               CACTTCAATTTATCAGAAATAAAATTTATTCTGCTGAACTAATTGTGTCTATTTTAGC               AGACTGCCATCCTGTTAGTTACGAGTTCAGATGAAGCCATATGGCTGTAGAAAACAT               GCTTCTGGCAGTGCTTAATATGAACTAGGCTTCATTACATTTTCATGCATGCGCCTAT               ATCTTTTTTCCTTAGCTAACCGTTATTGGATTGATGGATTTTAACCTGTAGATGACTT               ATGTTCTGATTTAAGCTCTTATGTCTGTTTCTTGCAGGGAGAAAATAAGAATTATGGT               CTTCCGGTGAAGAAGAAAACTGATATCTGCACGATAATGTATACTAGTGGAACAAC               TGGTGATCCAAAGGGAGTATTGATTTCAAATGATAGTATTGTTACTCTTATAGCAGG               GGTGAAACGCCTGCTGGGGAGTGTAAATGAACAGGTGACCTTTTCATTTTATTTTTG               ACCATTTCACCAGTCACTTGGTTTGATCTGTCGTTTCTTTTCTTTCCTCCACCAAATTT               GACATGATTGTTCCCTTGTTTTCCCTTTCTCATTTGTTTGTTTCTTGTGAAATTTACAG               TTGACTATGAAGGATGTATATATTTCTTATCTTCCTCTTGCTCATATCTTTGACCGGG               TGATTGAGGAATTATTTATTTCGCATGGTGCTTCAATAGGATTTTGGCGTGGGGTAA               GCATGATTAGTTAGTACTCTGACAACAAATACGGGTTCATTCAAATCAGCAAGTGCT               TATTTGTTTCATCTTCAGGATGTGAAACTATTGGTCGAAGATATTGGAGAGCTAAAG               CCAAGTATCTTCTGTGCTGTTCCTCGTGTCTTAGATAGAATTTATTCAGGTAAACTTT               CTTATATTCGTGGATTAGGCAATGTCATTTTTGGGTTGTTTGTGGAGGTTATACTAAG               CAACCTGGAACATGTACTAGCTGGAAAACTTGTCTTAATTTACTTATTTTTAGTATTT               ATAAATGAAACAAAACTAGATTGATTCACTTTTCTGTTAAATACAATGAATATATAC               TCAGCTTTTTTCAGAAGATGCATGTTCTCAGCTGTGAGATTGTCATAACCTTTGTACA               TTATCAGGTTTACTACAGAAGATTTCTTGCGGGCGGCTTATTGAAAAAGAAGATGTT               TGATTTAGCATACACATAGTAAGTTACTCTCATATTTTCAGTTTCTTATGTGAAGCTG               TTCATTTTATCTGCTGGCCGCCCAAAAATATTGATTGGAAATAGAGTTAAATTGCTCT               ATTAGTTCTGCCACTGCAGACTCACCGGAGTAAAGGAAAATAAAAGATATTTGGGC               ATTCTCTAACAAGCAACAGGGTCAAAAGCATATTTTTCCTTGTAGACAAATATAGAA               TTTGTTAGAGTTGTGTGACCCAAATTCTAGTTAAAAAAAAGTGGCAAGATAGGGGG               ATTTGTGGGGGCATCGGAGGCCCCCACGGTACGGTACAGACTGCACAAGTGGAATT               CGTATAAAAGTACACTTCTTCTATTTGATATTGATTTGAATAAGGTGTTTCAACCTTA               TTGCATTGCTTCTATTAGGTTTTGATTAGAATAAGGTTTATAGGTCGTCGTCTCTCTC               TGCCCGTGGTTTTGTGTGTTATATTTTTACCCTCTTTCTTTACGATTCATTGTCATTAT               CGAGGTTTGTTTTTCACAGAATTGTCTCAATCCCTTTGGGTTTATGAGCTTTTGCATT               AGTAGAGATCCATTTGCAGTCTGTGATTGCACTTTTCGTGAATATGTTTAACAGAGTT               ACTGAATCAGGATTACGGTTTCTTGGCTTTGATTTTACTAATATCTGACATCTGTGAT               AACCTACAGCAAATACTACAACATGAAGAAGGGCCGCAAACATGGAGAAGCATCTC               CAATTTGTGACAAAATTGTATTTAGTAAGGTGATGAAAGTCTTCATTGATACATTAT               ATGCACGAGGCTCCTTGAATATTGGCCAAAAGCCCATTAAATCACATTTACCTGCAA               CATAATCCTCTTGACTACTCAAATCTCATGTTGAGTTGTAATTTTTCTCAGGTAAAGC               AAGGATTGGGAGGGAATGTGCGGCTTATTCTATCTGGTGCAGCACCTCTTTCAGCTC               ATGTGGAAGAGTTCTTGCGAGTTGTGGCATGTTGTCATGTTCTGCAAGGATATGGTA               TAGTTGAAGTCAGCCTTTGTGCTTGTGATAAGTTCTTTTTTTCCCTTTACCAGCTGTG               CACACTGGCTGCAACATGAACATTTATTATTATGTTGATCCAAATGTAGGTCTGACG               GAGAGTTGTGCGGGGAGTTTTGTCTCTTTACCTAATGAATTGTCAATGCTTGGTACTG               TGGGGCCTCCCAGTACCAAACATAGATGTACGCCTGGAATCTGTTCCCGAAATGAAT               TATGATGCCCTTGCTAGCACACCACGGGGGGAAATTTGTATCAAAGGAAATACATTA               TTCTCAGGATACTACAAACGTGAAGACCTCACCCGTGAAGTATTGATTGATGGATGG               TTCCACACAGGTCTTCCAACTTTTGTTTCTTTTAAGGTTCTATGCATTATTAGTTTTTA               TCTATAAGTTGAAGACCTTGAATCTTTGTGCATTAGGGGATATTGGAGAGTGGCAAC               CTAATGGAAGCATGAAGATTATTGATCGAAAGAAGAACATTTTTAAGCTTTCACAAG               GTGAATATGTTGCTGTTGAGAACCTGGAGAACATTTACGGTCTCGTGTCAGCTATTG               ATTCGGTACATCTCTTATGCTCTCTTTGATACATTAACATACACTGCTTCTCGGATAT               GTAGCCATGCACTGAATGTTGGTCAAACGTAAAATTGATTTTGAAATGATTGGCAAA               TTAAACATTTTCTTTCTTATGTTACCTTATGATTGCATTCCTTTTTTAGCACTAGGTTT               CAACCCATTGCCATTGATGGTTGCTTGATTGAACAAAAATAAACATAATAATCGAAA               TATGCATGTCATGTTACAGTGTTTTTATCGTATCAGTTGTGTAAACATGTGTCAAAAT               CCTTTAACAGAAATATGACAAATGTACTAAATATGTTAAATCATGCTTAAGCGCATC               ATATGGTATCTAAATTTGTCATACATATATGTCATGGAAGTGATGTAAAATAAACTA               TAGTTTATGTCAGATTTGTAATTTACTTGCTGGAGATTGGCATTCTTTTAAACTTTTC               AGTTTCATGTCTTTATCAATTTCAGATATGGATTTACGGAAACAGCTTTGAGTCGTTC               CTTGTTGCGGTTGTTAACCCCAATAAGGAAGCACTTGAAAGCTGGGGTGCCGACAAT               AACGTAAGTGGTGACTTCGAGTCCCTCTGTCAAAACCCCAAGGCCAAAGAGTTCATA               CTTGGGGAGCTCGCAAAGACTGGCAAAGAGAAAAAGGTTAGTTATTCATGCTTTTTG               CCCCCTTTTTTATTTTTCAAAATTTATGAAATATGGGTTTATCAATTCATACTGAAAT               ATTATAATCTTTACTCAGCTAAAAGGTTTTGAATTCTTTGGATCC                  
 
         [0018]    [0018]                         TABLE II                       Sequence of the Cotton CoFS Promoter (SEQ ID NO:2).                                GGGAAGCTTATCTACTTGAAGTTTTGGTAGCCCAATGAAATACTCTCGTAAATCTAG                   AGTTATTAGTGTAAACCCTAAAGGGATCAAATTGTATAAATTTAAATCCCTTATGAC               TTTCAATTGTAGATAGACTCTAATCTCGATCATGGATGTAACTCAATCTATTTGTTGG               GTTTGGGGTGATTACTTCAATTCATTCCATTCATAGTTGTGAATATATTTGAGAGTAT               TTACGCAAACATTTGGTGTGTGCTATTTTTCCTTTGGTCTTTTGTTCTTCGTTGCCCAT               TCGTTCGAGTTTGCTTTCGCTATATTTTAATGCCTTAGAAAATTTTTGCGAGAATTCT               CATTTTGTGAGAGTTAAGCGAACTTAGAATTATTTTTTTTAAAATCGCTTAAGGCTGT               ATGGTCTGTGAGACTAAAATTCTAGTCTCGTAACACTAATACAATCACAAGTAATTT               ACATTGTTCAAGTTCTTATTCACATAAGCGGTTGGATAAAGAAAATTAAAAAAAAAC               AATCGGATATAATTACAAAAAAATAAATTGAAATGTGCAATAATACAAATAATAAT               TATTGCTAAAGGTAAATAAAAAATGTAAATAATTATCAATGAAGTTTGAAACCTTAA               ATGGTGAAGTTTGTGTCAACTAATAGAAGAAAAAATAAATTATTTATATAACTCTAC               TAATGTATTATTTTATTTTGTAAAATTGATTTATTTATATTATTTCTACTAAATTGATG               TGGAATTAGTGATATCTACTTAATTAACTATATATAATTATAATGAATCTCCGGGAC               TGTGACTGGTCAAAGATCATAAAGTGGTATCCAATAAATTTAAAATGCACTTGTAAA               ATATTAGACTCATGATGGCACTGAGGCGGAGGTGAAGAGGCGGCAAAGCACATGGA               GAAGCTATATAGAAAATTCTTTCACGAAAAAGGCAACTCTTGGCTTGTGTGTTGGGA               ATTGTGTTAAGAACTGGKATTATATAAAAACAATTATATGGGGAAAGGAAATGGTCC               ACTGTCAATAGTTTACTATAAGCAAGTTGGAGATATAAAATTAAATATATATTCAGT               ACATATACGAGTTTGAGCAACAAAATTAGAGATCTTTTTTGTCAAGTTGATATCTTC               AATTTTATAACGTAAATGTTCTTTTGAAGGCAACAGTAATGATATATATATATGTAG               AAGAAATTTAACTAAAAATAGATAATTAGGCTTAATTTAATTAATTCAAGTGCAATT               GTTTTATCATAATATATATTACATTACAAGGCTTGAATTATTCATATTTTAAATTTAT               TTATTAGTTAACAAAGTAATTATTGGTGCAAAAATAAATAAATTATTACCCCATTAT               CTATTTTCTTAAATAAAAAAATATATTATATATGCCATTTACTCTCTTTAAAAAAATT               TAATTTACAAATAAACTAATAAATTTGTATATGATGATTTCGAATGAGGGTTTTAAT               ACAGTTATCATGATGATTTCAATACAATGGTTCCAAATGAATAAGGATTCCACTACA               ACATTAAACTCACCATAATGGTGATTCCAATTGAGTGTTCCTACATAATTATCATCAT               AATTCTTACTTGGCAGGATACAAAAAATAATAATGGGTAAGGTAATTAAAAATTAA               AATAATTATCAATAAAGTTTTTATTATGGTGACAAAGTTTGATAATCATTATTAAATT               ATTAATTGAGTAAAATATTTAAATATAGTAATGTATATATAATGAATCTCCGGCGAT               GTTGGCTGTTCAAAGATCATAAAATCCAATACATTTAAAATGCAGCTGTAAAATATG               ATGTGAAGGGCGGATACCACACATGGAGAAGCCATATAGAAATTTCTTCCGGTACC               ATGAAAAAGGCGACTCTTAGCTTGTGTGTTGGGAATTGTGTTAAGTTATATGGGAAA               GGAAATATTCCACTGTCAATAGTTTATTATAAGCAAGTGGGAGAAACAAAATTAAAT               ATGTTATTTTGAAGGCAACAGTAATAATATAATTATAAATTAAAATAACATAATTTA               GGCTTAATTTAATTAATGGTAAACTATAAAAAAAAGTCATTTTTGTTTGCTTCAGATT               ACATTTTAGTCACTTATGTTTGAAATGTTACGTTTTAGTCACTTACATTACCGTTTTGT               TACGAAGTGGTCACTTTACCATTAAACTCTATTACCTCCCTAACGACAGTCCTACGT               GGCAGTCAAAATGAATTTTAAATGCTAACTTGGACGTCCAGTTGCTGGGACATTTTC               CGGTTCACCTACAGCCACCTAATACTTAGGCGCTATATATTTTCCCAAAATTATTCTC               CACATTTCACTCCCAGAGCCCTCCATCTATGTAGAGAGCTGTAAGAGAGAATATAAA               AAAGGGAAAGCTAGCTGAGGATCGTTTGATTTTGGACATTTTGGGATCC                    
         [0019]    [0019]                         TABLE III                       Sequence of a CoFS 275 bp cDNA Fragment (SEQ ID NO:3).                                GGTCACTGTGACGTGCCGTGGCTACTGTGAAACGAGCCGTGGCTACTGTGAACGTGC                   CGTGGCTACTGTGAACGAGCCGTGGCTACTGTGAACGTGCCGTGGCTACTGTGAACG               TGCCGTGGCTACTGTGAACGAGCCGTGGTCACTGTGATACGTGCCGGGAGTTTTGTC               TCTTTACCTAATGAATTGTCAATGCTTGGTACTGTGGGGCCCCATACCAAACATAGA               TGTACGCCTGGAATCTGTTCCGAAAAAAAAAAAACTGAATTCCGAGT                    
         [0020]    The following non-limiting examples are included to illustrate the invention.  
       EXAMPLE 1  
     Isolation of an Anther-specific cDNA Fragment Encoding a CoFS Sequence Expressed in Cotton Anthers  
       [0021]    Cotton seeds were surface-sterilised with 70% ethanol for 30-60 seconds and 10% H 2 O 2  for 30-60 minutes, followed by washing with sterile water. The seeds were germinated on ½ MS medium at 28 C with 16 hr lighting. Cotyledons and hypocotyls were cut from sterile seedlings as transformation explant material. Cotton plants were grown in pots for DNA and RNA extraction. Total RNA was extracted from young fibres, ovaries, anthers, petals, sepals, leaves and roots of cotton using the guanidinium thiocyanate method or SV Total RNA Isolation System (Promega). Poly (A) + RNA was purified using oligo(dT)-cellulose spin columns from an mRNA purification kit (Pharmacia Biotech). Total RNAs from different tissues of cotton were used to reverse-transcribe first-strand cDNAs. These cDNAs were used as templates in differential display PCR.  
         [0022]    Differential display analysis was carried out with the Differential Display Kit (Clontech). First-strand cDNA was synthesised with 2 μg total RNA as starting materials of reverse transcription and oligo(dT) as primers at 42 C for 1 hour. Reactions of differential display PCR were carried out with an initial cycle consisting of 94 C for 5 minutes, 40 C for 5 minutes and 68 C for 5 minutes, followed by two cycles consisting of 94 C for 2 minutes and 40 C for 5 minutes and 68 C for 5 minutes, and then 25 cycles consisting of 94 C for 1 minute, 60 C for 1 minute and 68 C for 2 minutes, and a final extension at 68 C for 7 minutes. Target differential display bands were excised and re-amplified for further analysis. PCR fragments, DNA and cDNA fragments were sub cloned into vectors, and plasmid DNA and phagemid DNA prepared with a Qiagen Plasmid Kit were used as templates in PCR reactions. The PCR products were sequenced by autosequencer.  
         [0023]    Cotton cDNA was synthesised using a cDNA synthesis kit (Stratagene). Cotton cDNA libraries were constructed by inserting the cDNA fragments into the ZAP express vector (Stratagene). Reproducible anther- and petal-specific differential display products (see FIG. 1) were targeted for further analysis. The cDNAs in each target band were harvested and regenerated by PCR amplification. The isolated cDNAs were subsequently sub cloned into a vector and sequenced.  
         [0024]    To confirm which cDNA transcripts specifically accumulated in cotton anthers, cDNA expression patterns were analyzed by Northern blot hybridization with total RNA isolated from cotton fibers, ovules, anthers, petals, sepals, stems, leaves and roots, using probes from the cDNA clones. For Northern blot analysis, RNA samples from different cotton tissues were separated on agarose-formaldehyde gels, and transferred onto Hybond-N nylon membranes by capillary blotting. RNA Northern blots were hybridised in ExpressHyb solution (Clontech) at 68 C with  32 P-cDNA probes prepared by random labelling (Prime-a-Gene Labelling System, Promega). After hybridisation, the blots were washed at 68 C in 0.1×SSC, 0.5% SDS for 30-60 minutes.  
         [0025]    One clone was identified as a 275 bp CoFS cDNA fragment (see Table III). The cDNA fragment was found to share 73% homology with the acyl-CoA synthetase gene (X94624) of  Brassica napus  in a region of 33 amino acids of the open reading frame. Northern blot hybridization revealed the CoFS cDNA transcripts accumulated largely in cotton anthers, and also accumulated more or less in petals, but these transcripts were not detected in RNA from fibers, ovules, stems, leaves and roots (see FIG. 2). This result shows that CoFS cDNA expression is anther-specific in cotton.  
       EXAMPLE 2  
     Isolation and Structure Analysis of CoFS Gene  
       [0026]    Plant materials from cotton were prepared as in Example 1. Tobacco seeds were surface-sterilized with 70% ethanol for 30-60 seconds and 0.1% HgCl 2  for 15 minutes, followed by washing with sterile water. The seeds were germinated on ½ MS medium in light at 28 C, and leaves cut from sterile seedlings were used as experimental materials.  
         [0027]    Total DNA was extracted and purified from leaves of cotton and tobacco plants according to the following method. Leaf tissues (z-4 g) were thoroughly homogenised in liquid N 2 . The homogenized tissues were placed in a 50 ml tube with 20 ml ice-cold extraction buffer and sedimented at 2500 rpm for 15 minutes. After removing the supernatant, each pellet was resuspended in 10 ml lysis buffer and incubated at 65 C for 30 minutes. Ten milliliters chloroform was added to each tube and mixed with the samples. The samples then were sedimented at 3500 rpm for 10 minutes. The supernatant was transferred to a clean tube, and chloroform extraction was repeated once more. The supernatant was transferred to a clean tube, and 0.6 volume isopropanol was added to each tube for DNA precipitation. After centrifuging at 3500 rpm for 30 minutes, the DNA was washed with 70% ethanol. The isolated genomic DNA was then dissolved in sterile water for use.  
         [0028]    Cotton genomic DNA libraries were constructed from leaves of cotton plants. DNA was partially digested with BamH I, and the DNA fragments were cloned in the BamH I site of ZAP express vector (Stratagene). The cotton genomic DNA libraries were screened using CoFS gene fragments isolated by Genome Walk PCR as probes.  
         [0029]    Genome Walker libraries were constructed using the Universal Genome Walker Kit (Clontech). Genomic DNA from leaves of cotton plants was digested with five restriction enzymes respectively, purified by phenol/chloroform extraction and precipitated in ethanol. The digested DNA was ligated to Genome Walker adaptors.  
         [0030]    Two Genome Walker polymerase chain reactions were carried out successively: 1 μl of each Genome Walker DNA library was used as the templates in the primary PCR, and the primary PCR products were used as templates in the secondary PCR. The PCR was started at 95 C for 1 minute, followed by 35 cycles of 95 C for 15 seconds and 68 C for 4 minutes and a final extension at 68 C for 6 minutes. Target PCR bands were purified using a Geneclean Kit (Bio 101).  
         [0031]    The screens revealed two CoFS gene positive clones. One clone contained a 4.801 kb cotton CoFS gene region, and the other contained a 3.913 kb cotton DNA fragment covering part of CoFS promoter region. Three CoFS promoter fragments (0.7, 1.4 and 2.6 kb, respectively) were isolated from the cotton Genome Walker libraries. The complete CoFS gene isolated from cotton was 8.4 kb in length, including a 2.6 kb promoter region. The sequences are provided in Tables I and II.  
       EXAMPLE 3  
     Functional Analysis of CoFS Promoters  
       [0032]    To characterize the function of the CoFS promoter in anther-specific expression of the CoFS gene, a 0.7 kb fragment, a 1.4 kb fragment and a 2.5 kb fragment of the CoFS promoter were fused with the GUS coding sequence in the gene expression vector pBI121 (replacing the CaMV35S promoter), respectively. See FIG. 3.  
         [0033]    Vectors were constructed as follows. A Hind III site and a BamH I site were created by PCR at the 5′-end and 3′-end of the 0.7, 1.4 and 2.4 kb CoFS promoter fragments respectively. The Hind III/BamH I fragment was initially sub cloned into pGEM-T vector (Promega). Plasmid DNA containing the CoFS promoter fragments was digested with Hind III and BamH I, and the digested fragment was isolated by agarose gel electrophoresis. Three chimeric CoFS promoter/GUS constructs were generated by insertion of the 0.7, 1.4 or 2.4 kb fragment, respectively, replacing the CaMV 35S promoter, into the Hind III/BamH I sites of pBI121 vector.  
         [0034]    The CoFS promoter/GUS fusion gene constructs were used to transform tobacco by Agrobacterium-mediated gene transfer, using the pBI121 vector containing a CaMV35S promoter/GUS fusion protein as a positive control. The CaMV35S promoter is a constitutive promoter, active in all plant tissues. Odell et al.,  Nature  313:810-812, 1985; Ow et al.,  Proc. Natl. Acad. Sci. USA  84:4870-4874, 1987; McCabe and Martinell,  Biotechnol . 11:596-598, 1993.  
         [0035]    The binary vectors containing CoFS promoter/GUS fusion genes were transferred into Agrobacterium tumefaciens strain LBA 4404. Tobacco transformations were carried out using the leaf-disc method (Horsch, et al., 1985). Tobacco leaves were cut into pieces about 2×2 cm, and immersed in the Agrobacteria suspension for five minutes. The infected tobacco explants were cultivated on MS medium with 1 mg/L 6-BA for 48 hours at 28 C and transferred onto selection MS medium containing 100 mg/L kanamycin and 1 mg/L 6-BA for 20-30 days. Kanamycin-resistant (transformed) shoots were selected. The transformed shoots were cut from the calli and rooted on MS medium with 50-100 mg/L kanamycin. The transformed tobacco plants were transplanted to soil for growing to maturity.  
         [0036]    Transgenic tobacco plants possessing the chimeric CoFS promoter/GUS gene (or 35S/GUS gene), and negative control, non-transformed plants were analyzed by DNA Southern blot hybridization and by GUS histochemical assay. For Southern blot analysis, total genomic DNA from the transgenic tobacco leaves was digested with restriction enzymes, separated on agarose gels, and transferred onto Hybond-N nylon membranes by capillary blotting. DNA Southern blots were hybridized in ExpressHyb solution (Clontech) at 68 C with  32 P-DNA probes prepared by random labelling (Promega Prime-a-Gene Labelling System). After hybridization, the blots were washed at 68 C in 0.1×SSC, 0.5% SDS for 30-60 minutes. The  32 P-labelled nylon membranes were exposed to X-ray film at -70 C for autoradiograph. See FIG. 4 for the results.  
         [0037]    Histochemical assays for GUS activity in transgenic tobacco plants were conducted according to a protocol described previously, Jefferson,  Plant Mol. Biol. Rep . 5:387-405, 1987, with some modifications. Fresh tissues from the plants were incubated in X-gluc (5-bromo-4-chloro-3-indolylglucuronide) solution consisting of 0.1 M sodium phosphate (pH 7.0), 10 mM ethylene diaminetetraacetic acid (EDTA), 0.5 mM potassium ferrocyanide and 0.1% X-gluc (Clontech chemical) overnight. The stained plant materials were then cleared and fixed by rinsing with 100% and 70% ethanol successively, and the samples were examined and photographed directly or under a microscope. See FIG. 5.  
         [0038]    The results of Southern blot analysis demonstrated that CoFS promoter/GUS gene was integrated into the tobacco genome. More than 50 tobacco transgenic plants were obtained and transplanted in soil to grow to maturation. Consistent with the results from Northern blot analysis of cotton, the GUS gene driven by the CoFS promoter was specifically and strongly expressed in tobacco anthers. Weak activity of GUS gene under CoFS promoter was also detected in ovaries, styles and stigmas, but no GUS activity was detected in petals or other tissues in all the transgenic tobacco plants studied. This result, together with the above Northern blot analysis, indicates that the CoFS promoter is able to control specific gene expression at the transcriptional level in plant anthers.  
       References  
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         [0040]    OW D W, Jacobs J D, Howell S H, 1987. Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc. Natl. Acad. Sci. USA, 84:4870-4.  
         [0041]    McCabe D E and Martinell B J, 1993. Transformation of elite cotton cultivars via particle bombardment of meristems. Biotechnology, 11:596-8.  
         [0042]    John M E, 1996. Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene. Plant Mol. Biol., 30(2):297-306.  
         [0043]    Kawai M, Aotsuka S, Uchimiya H. 1998. Isolation of a cotton CAP gene: a homologue of adenylyl cyclase-associated protein highly expressed during fiber elongation. Plant Cell Physiol., 39(12):1380-3.  
         [0044]    Song P and Allen R D, 1997. Identification of a cotton fiber-specific acyl carrier protein cDNA by differential display. Biochim. Biophys. Acta, 1351(1):305-12.  
         [0045]    Ma dp, Liu H C, Tan H, Creech R G, Jenkins J N, Chang Y F, 1997. Cloning and characterization of a cotton lipid transfer protein gene specifically expressed in fiber cells. Biochim. Biophys. Acta, 1344(2):111-4.  
         [0046]    Rinehart J A, Peterson M W, John M E, 1996. Tissue-specific and developmental regulation of cotton gene FbL2A. Demonstration of promoter activity in transgenic plants. Plant Physiol., 112(3):1331-41.  
         [0047]    John M E and Crow L J, 1992. Gene expression in cotton fiber: cloning of the mRNAs. Proc. Natl. Acad. Sci. USA, 89(13):5769-73.  
         [0048]    Jefferson R A, 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep., 5:387-405.  
         [0049]    Jefferson R A, Kavanagh T A, Bevan M W, 1987. GUS fusion: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 6:3901-7.  
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             1  
             8367  
             DNA  
             Arabidopsis sp.  
           
            1 

cctcacattt aagcggaaaa aaatattaac taattactaa ttactaaggt catgggttgc     60 

gcattaaagt tcactgacga ttgtgcaaat gatgttccat agagcttaat tgatgaaatg    120 

ggaactcatg acccgcttga agtaactcga cttgtagaac tcatgaagaa gcttatctac    180 

ttgaagtttt ggtagcccaa tgaaatactc tcgtaaatct agagttatta gtgtaaaccc    240 

taaagggatc aaattgtata aatttaaatc ccttatgact ttcaattgta gatagactct    300 

aatctcgatc atggatgtaa ctcaatctat ttgttgggtt tggggtgatt acttcaattc    360 

attccattca tagttgtgaa tatatttgag agtatttacg caaacatttg gtgtgtgcta    420 

tttttccttt ggtcttttgt tcttcgttgc ccattcgttc gagtttgctt tcgctatatt    480 

ttaatgcctt agaaaatttt tgcgagaatt ctcattttgt gagagttaag cgaacttaga    540 

attatttttt ttaaaatcgc ttaaggctgt atggtctgtg agactaaaat tctagtctcg    600 

taacactaat acaatcacaa gtaatttaca ttgttcaagt tcttattcac ataagcggtt    660 

ggataaagaa aattaaaaaa aaacaatcgg atataattac aaaaaaataa attgaaatgt    720 

gcaataatac aaataataat tattgctaaa ggtaaataaa aaatgtaaat aattatcaat    780 

gaagtttgaa accttaaatg gtgaagtttg tgtcaactaa tagaagaaaa aataaattat    840 

ttatataact ctactaatgt attattttat tttgtaaaat tgatttattt atattatttc    900 

tactaaattg atgtggaatt agtgatatct acttaattaa ctatatataa ttataatgaa    960 

tctccgggac tgtgactggt caaagatcat aaagtggtat ccaataaatt taaaatgcac   1020 

ttgtaaaata ttagactcat gatggcactg aggcggaggt gaagaggcgg caaagcacat   1080 

ggagaagcta tatagaaaat tctttcacga aaaaggcaac tcttggcttg tgtgttggga   1140 

attgtgttaa gaactggatt atataaaaac aattatatgg ggaaaggaaa tggtccactg   1200 

tcaatagttt actataagca agttggagat ataaaattaa atatatattc agtacatata   1260 

cgagtttgag caacaaaatt agagatcttt tttgtcaagt tgatatcttc aattttataa   1320 

cgtaaatgtt cttttgaagg caacagtaat gatatatata tatgtagaag aaatttaact   1380 

aaaaatagat aattaggctt aatttaatta attcaagtgc aattgtttta tcataatata   1440 

tattacatta caaggcttga attattcata ttttaaattt atttattagt taacaaagta   1500 

attattggtg caaaaataaa taaattatta ccccattatc tattttctta aataaaaaaa   1560 

tatattatat atgccattta ctctctttaa aaaaatttaa tttacaaata aactaataaa   1620 

tttgtatatg atgatttcga atgagggttt taatacagtt atcatgatga tttcaataca   1680 

atggttccaa atgaataagg attccactac aacattaaac tcaccataat ggtgattcca   1740 

attgagtgtt cctacataat tatcatcata attcttactt ggcaggatac aaaaaataat   1800 

aatgggtaag gtaattaaaa attaaaataa ttatcaataa agtttttatt atggtgacaa   1860 

agtttgataa tcattattaa attattaatt gagtaaaata tttaaatata gtaatgtata   1920 

tataatgaat ctccggcgat gttggctgtt caaagatcat aaaatccaat acatttaaaa   1980 

tgcagctgta aaatatgatg taaagggcgg ataaagcaca tggagaagcc atatagaaat   2040 

ttcttccatg aaaaaggcga ctcttagctt gtgtgttggg aattgtgtta agttatatgg   2100 

gaaaggaaat attccactgt caatagttta ttataagcaa gtgggagaaa caaaattaaa   2160 

tatgttattt tgaaggcaac agtaataata taattataaa ttaaaataac ataatttagg   2220 

cttaatttaa ttaatggtaa actataaaaa aaagtcattt ttgtttgctt cagattacat   2280 

tttagtcact tatgtttgaa atgttacgtt ttagtcactt acattaccgt tttgttacga   2340 

agtggtcact ttaccattaa actctattac ctccctaacg acagtcctac gtggcagtca   2400 

aaatgaattt taaatgctaa cttggacgtc cagttgctgg gacattttcc ggttcaccta   2460 

cagccaccta atacttaggc gctatatatt ttcccaaaat tattctccac atttcactcc   2520 

cagagccctc catctatgta gagagctgta agagagaata taaaaaaggg aaagctagct   2580 

gaggatcgtt tgattttgga cattttgatg aaacggcctg gaaataattt tgtaatagag   2640 

gtagagaaag gtaaagaccc cagcgatgga cagccgtcga tcggtcctgt ttatcgcagt   2700 

tcttttgctg ctaatggatt ccctgctccg attcctggaa tggagagttg ctgggacatt   2760 

ttccggttcg tttttagctt tctctttttt actttacgct tcttgcttgg ctgctaagaa   2820 

aataaggata ctaggagaag atttgacttc ttctattctt tgctttgatt tcagatggct   2880 

ttcgcaatag ttgccatttt ttttgaattt ttacttcccc ttttttaagt tgagtagatt   2940 

tttcttctaa tttgttggct ttgttatttt tttatttcgc gacgaggtgg cgatgaaatc   3000 

gagaaacgta cttttaagga tcctatgaga aagttatatg caagatcgaa acgcctaata   3060 

tttgaaacta ttgaatttta acgctcacac agagcaagaa tcgagttact ggtatttcca   3120 

ttcttatagc tgaaagattg atggctttca ttcaactcaa tgtaaaactg tgaaataaat   3180 

tgtttaatag tagtaattat tttggttttg atgcttatgt gatgtggaga ttaaaatatt   3240 

gcctccttat aacttagctg aaccgtagat atgggctgat tgaacttgct aactaactgt   3300 

atgacagctc ctatttacga aaaagtaata ttttatttga ggatgatgat ttcgatttca   3360 

ttttctcctt gcagtatgtc agttgagaaa tatcctgaca accatatgct tggtcgccga   3420 

cagattgtgg atgggaaagt atgttgctgc tgcagtttcc ttttttcttg ttacttttgc   3480 

gttctgtttg tagtgcggcc tttgactttt agttcatgta tctaaattga catgctttga   3540 

ttgcaggctg gaaaatacgt gtggcaaact tacagagaag tttatgacat tgtaacaaaa   3600 

gttgggaatt ccatccgaag ttgtgatgtt gtggaagtaa tgctttaacc tccttttttc   3660 

cttttaattg taaaattatt gtcaattttt tttataacaa atatcctatt tctggggatc   3720 

aatatccacc cacaattgat gctaataaaa aaaaaaatta agctttttat tcttgcttac   3780 

agggaggaaa gtgtggtatt tatggtgcca attgcccaga atggataatt agcatggagg   3840 

tatgatcatt ctggcatgtt tcatctgatt tgcacagtgg acatcccaag ttacttagat   3900 

gtcgctataa cttgtttctt tggatcatac tatttgctac caaattgctt gttgcccgaa   3960 

atgtttacta atgttgcaag attgatacag gcctgcaacg ctcatggact ctattgtgtt   4020 

cctttatatg acactttggg tatttcttct tgagatccaa caaaagcatt ctttcagttt   4080 

ttgccaaaca actacctttc tctaacaacc atcttatgtg tattatgtac tcttcatata   4140 

gtctgtgatc ataatatcct taaactcttt aaattattct tgcattccaa cgcgcctgct   4200 

actctttttg aatgtttgat acgttgctac atatttgtag gtgccggtgc tgtggagttt   4260 

atcatatgcc atgcagaaat ttctattgct tttgtagagg aaaagaagat taatgaggta   4320 

tgcctgttta catctatatt tgaaacccta gtagtgatat ggcaactact gggaaagata   4380 

cttctagata cttgcaaaaa taagatatct atctacaaaa tagattcgat gttttatttt   4440 

tattatcagc atttgcttct atgcttgctg ctcacttatg catagttgat attgatacaa   4500 

gaatctgtaa tttcacaaat tttctgttct tttttctttt gggtttctca tgtaaggttt   4560 

ttctttttcc tttcagctgt tcaaaacatt tccagcctca acagaacact tgaaaagtaa   4620 

gctatctgat tatttagggg gattcttgaa atagtacgtt acaaattatt tatgtccatg   4680 

tatttttgct atgcagcaat tgttagcttc gggaaggtaa cacctgagca gaaggcagaa   4740 

gctgagaagc atggtttgaa gatatatcct tgggaggaat ttttgcaact ggtaagctct   4800 

ttgctctgtt attttccact tcaatttatc agaaataaaa tttattctgc tgaactaatt   4860 

gtgtctattt tagcagactg ccatcctgtt agttacgagt tcagatgaag ccatatggct   4920 

gtagaaaaca tgcttctggc agtgcttaat atgaactagg cttcattaca ttttcatgca   4980 

tgcgcctata tcttttttcc ttagctaacc gttattggat tgatggattt taacctgtag   5040 

atgacttatg ttctgattta agctcttatg tctgtttctt gcagggagaa aataagaatt   5100 

atggtcttcc ggtgaagaag aaaactgata tctgcacgat aatgtatact agtggaacaa   5160 

ctggtgatcc aaagggagta ttgatttcaa atgatagtat tgttactctt atagcagggg   5220 

tgaaacgcct gctggggagt gtaaatgaac aggtgacctt ttcattttat ttttgaccat   5280 

ttcaccagtc acttggtttg atctgtcgtt tcttttcttt cctccaccaa atttgacatg   5340 

attgttccct tgttttccct ttctcatttg tttgtttctt gtgaaattta cagttgacta   5400 

tgaaggatgt atatatttct tatcttcctc ttgctcatat ctttgaccgg gtgattgagg   5460 

aattatttat ttcgcatggt gcttcaatag gattttggcg tggggtaagc atgattagtt   5520 

agtactctga caacaaatac gggttcattc aaatcagcaa gtgcttattt gtttcatctt   5580 

caggatgtga aactattggt cgaagatatt ggagagctaa agccaagtat cttctgtgct   5640 

gttcctcgtg tcttagatag aatttattca ggtaaacttt cttatattcg tggattaggc   5700 

aatgtcattt ttgggttgtt tgtggaggtt atactaagca acctggaaca tgtactagct   5760 

ggaaaacttg tcttaattta cttattttta gtatttataa atgaaacaaa actagattga   5820 

ttcacttttc tgttaaatac aatgaatata tactcagctt ttttcagaag atgcatgttc   5880 

tcagctgtga gattgtcata acctttgtac attatcaggt ttactacaga agatttcttg   5940 

cgggcggctt attgaaaaag aagatgtttg atttagcata cacatagtaa gttactctca   6000 

tattttcagt ttcttatgtg aagctgttca ttttatctgc tggccgccca aaaatattga   6060 

ttggaaatag agttaaattg ctctattagt tctgccactg cagactcacc ggagtaaagg   6120 

aaaataaaag atatttgggc attctctaac aagcaacagg gtcaaaagca tatttttcct   6180 

tgtagacaaa tatagaattt gttagagttg tgtgacccaa attctagtta aaaaaaagtg   6240 

gcaagatagg gggatttgtg ggggcatcgg aggcccccac ggtacggtac agactgcaca   6300 

agtggaattc gtataaaagt acacttcttc tatttgatat tgatttgaat aaggtgtttc   6360 

aaccttattg cattgcttct attaggtttt gattagaata aggtttatag gtcgtcgtct   6420 

ctctctgccc gtggttttgt gtgttatatt tttaccctct ttctttacga ttcattgtca   6480 

ttatcgaggt ttgtttttca cagaattgtc tcaatccctt tgggtttatg agcttttgca   6540 

ttagtagaga tccatttgca gtctgtgatt gcacttttcg tgaatatgtt taacagagtt   6600 

actgaatcag gattacggtt tcttggcttt gattttacta atatctgaca tctgtgataa   6660 

cctacagcaa atactacaac atgaagaagg gccgcaaaca tggagaagca tctccaattt   6720 

gtgacaaaat tgtatttagt aaggtgatga aagtcttcat tgatacatta tatgcacgag   6780 

gctccttgaa tattggccaa aagcccatta aatcacattt acctgcaaca taatcctctt   6840 

gactactcaa atctcatgtt gagttgtaat ttttctcagg taaagcaagg attgggaggg   6900 

aatgtgcggc ttattctatc tggtgcagca cctctttcag ctcatgtgga agagttcttg   6960 

cgagttgtgg catgttgtca tgttctgcaa ggatatggta tagttgaagt cagcctttgt   7020 

gcttgtgata agttcttttt ttccctttac cagctgtgca cactggctgc aacatgaaca   7080 

tttattatta tgttgatcca aatgtaggtc tgacggagag ttgtgcgggg agttttgtct   7140 

ctttacctaa tgaattgtca atgcttggta ctgtggggcc tcccagtacc aaacatagat   7200 

gtacgcctgg aatctgttcc cgaaatgaat tatgatgccc ttgctagcac accacggggg   7260 

gaaatttgta tcaaaggaaa tacattattc tcaggatact acaaacgtga agacctcacc   7320 

cgtgaagtat tgattgatgg atggttccac acaggtcttc caacttttgt ttcttttaag   7380 

gttctatgca ttattagttt ttatctataa gttgaagacc ttgaatcttt gtgcattagg   7440 

ggatattgga gagtggcaac ctaatggaag catgaagatt attgatcgaa agaagaacat   7500 

ttttaagctt tcacaaggtg aatatgttgc tgttgagaac ctggagaaca tttacggtct   7560 

cgtgtcagct attgattcgg tacatctctt atgctctctt tgatacatta acatacactg   7620 

cttctcggat atgtagccat gcactgaatg ttggtcaaac gtaaaattga ttttgaaatg   7680 

attggcaaat taaacatttt ctttcttatg ttaccttatg attgcattcc ttttttagca   7740 

ctaggtttca acccattgcc attgatggtt gcttgattga acaaaaataa acataataat   7800 

cgaaatatgc atgtcatgtt acagtgtttt tatcgtatca gttgtgtaaa catgtgtcaa   7860 

aatcctttaa cagaaatatg acaaatgtac taaatatgtt aaatcatgct taagcgcatc   7920 

atatggtatc taaatttgtc atacatatat gtcatggaag tgatgtaaaa taaactatag   7980 

tttatgtcag atttgtaatt tacttgctgg agattggcat tcttttaaac ttttcagttt   8040 

catgtcttta tcaatttcag atatggattt acggaaacag ctttgagtcg ttccttgttg   8100 

cggttgttaa ccccaataag gaagcacttg aaagctgggg tgccgacaat aacgtaagtg   8160 

gtgacttcga gtccctctgt caaaacccca aggccaaaga gttcatactt ggggagctcg   8220 

caaagactgg caaagagaaa aaggttagtt attcatgctt tttgccccct tttttatttt   8280 

tcaaaattta tgaaatatgg gtttatcaat tcatactgaa atattataat ctttactcag   8340 

ctaaaaggtt ttgaattctt tggatcc                                       8367 

 
           
             2  
             2454  
             DNA  
             Arabidopsis sp.  
           
            2 

gggaagctta tctacttgaa gttttggtag cccaatgaaa tactctcgta aatctagagt     60 

tattagtgta aaccctaaag ggatcaaatt gtataaattt aaatccctta tgactttcaa    120 

ttgtagatag actctaatct cgatcatgga tgtaactcaa tctatttgtt gggtttgggg    180 

tgattacttc aattcattcc attcatagtt gtgaatatat ttgagagtat ttacgcaaac    240 

atttggtgtg tgctattttt cctttggtct tttgttcttc gttgcccatt cgttcgagtt    300 

tgctttcgct atattttaat gccttagaaa atttttgcga gaattctcat tttgtgagag    360 

ttaagcgaac ttagaattat tttttttaaa atcgcttaag gctgtatggt ctgtgagact    420 

aaaattctag tctcgtaaca ctaatacaat cacaagtaat ttacattgtt caagttctta    480 

ttcacataag cggttggata aagaaaatta aaaaaaaaca atcggatata attacaaaaa    540 

aataaattga aatgtgcaat aatacaaata ataattattg ctaaaggtaa ataaaaaatg    600 

taaataatta tcaatgaagt ttgaaacctt aaatggtgaa gtttgtgtca actaatagaa    660 

gaaaaaataa attatttata taactctact aatgtattat tttattttgt aaaattgatt    720 

tatttatatt atttctacta aattgatgtg gaattagtga tatctactta attaactata    780 

tataattata atgaatctcc gggactgtga ctggtcaaag atcataaagt ggtatccaat    840 

aaatttaaaa tgcacttgta aaatattaga ctcatgatgg cactgaggcg gaggtgaaga    900 

ggcggcaaag cacatggaga agctatatag aaaattcttt cacgaaaaag gcaactcttg    960 

gcttgtgtgt tgggaattgt gttaagaact ggattatata aaaacaatta tatggggaaa   1020 

ggaaatggtc cactgtcaat agtttactat aagcaagttg gagatataaa attaaatata   1080 

tattcagtac atatacgagt ttgagcaaca aaattagaga tcttttttgt caagttgata   1140 

tcttcaattt tataacgtaa atgttctttt gaaggcaaca gtaatgatat atatatatgt   1200 

agaagaaatt taactaaaaa tagataatta ggcttaattt aattaattca agtgcaattg   1260 

ttttatcata atatatatta cattacaagg cttgaattat tcatatttta aatttattta   1320 

ttagttaaca aagtaattat tggtgcaaaa ataaataaat tattacccca ttatctattt   1380 

tcttaaataa aaaaatatat tatatatgcc atttactctc tttaaaaaaa tttaatttac   1440 

aaataaacta ataaatttgt atatgatgat ttcgaatgag ggttttaata cagttatcat   1500 

gatgatttca atacaatggt tccaaatgaa taaggattcc actacaacat taaactcacc   1560 

ataatggtga ttccaattga gtgttcctac ataattatca tcataattct tacttggcag   1620 

gatacaaaaa ataataatgg gtaaggtaat taaaaattaa aataattatc aataaagttt   1680 

ttattatggt gacaaagttt gataatcatt attaaattat taattgagta aaatatttaa   1740 

atatagtaat gtatatataa tgaatctccg gcgatgttgg ctgttcaaag atcataaaat   1800 

ccaatacatt taaaatgcag ctgtaaaata tgatgtgaag ggcggatacc acacatggag   1860 

aagccatata gaaatttctt ccggtaccat gaaaaaggcg actcttagct tgtgtgttgg   1920 

gaattgtgtt aagttatatg ggaaaggaaa tattccactg tcaatagttt attataagca   1980 

agtgggagaa acaaaattaa atatgttatt ttgaaggcaa cagtaataat ataattataa   2040 

attaaaataa cataatttag gcttaattta attaatggta aactataaaa aaaagtcatt   2100 

tttgtttgct tcagattaca ttttagtcac ttatgtttga aatgttacgt tttagtcact   2160 

tacattaccg ttttgttacg aagtggtcac tttaccatta aactctatta cctccctaac   2220 

gacagtccta cgtggcagtc aaaatgaatt ttaaatgcta acttggacgt ccagttgctg   2280 

ggacattttc cggttcacct acagccacct aatacttagg cgctatatat tttcccaaaa   2340 

ttattctcca catttcactc ccagagccct ccatctatgt agagagctgt aagagagaat   2400 

ataaaaaagg gaaagctagc tgaggatcgt ttgattttgg acattttggg atcc         2454 

 
           
             3  
             275  
             DNA  
             Arabidopsis sp.  
           
            3 

ggtcactgtg acgtgccgtg gctactgtga aacgagccgt ggctactgtg aacgtgccgt     60 

ggctactgtg aacgagccgt ggctactgtg aacgtgccgt ggctactgtg aacgtgccgt    120 

ggctactgtg aacgagccgt ggtcactgtg atacgtgccg ggagttttgt ctctttacct    180 

aatgaattgt caatgcttgg tactgtgggg ccccatacca aacatagatg tacgcctgga    240 

atctgttccg aaaaaaaaaa aactgaattc cgagt                               275