Abstract:
The present invention relates to a method to improve the primary metabolism of a higher plant having chloroplasts by expressing fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase derived from  Cyanobacterium synechococcus  in the chloroplasts. The invention further relates to transgenic plants comprising DNA encoding fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase derived from  Cyanobacterium synechococcus.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a method for enhancing the photosynthetic activity and growth of a higher plant to increase its harvest yield and/or to enable its earlier harvest. 
     2. Description of Related Art 
     The development of a recombinant DNA technique has realized the incorporation of a certain exogenous gene into a higher plant and expression regulation of an existing gene therein. Only few experiments has been attempted to improve the characteristics concerning food production, such as the production yield of serials and crops. Recently, incorporation of a gene, coding enzyme participating in photosynthesis or carbohydrate metabolism, was achieved. Such gene was incorporated in anti-sense direction to inhibit the expression of the gene. The results indicated functional importance of the enzyme as a rate-determining factor of photosynthesis or carbohydrate metabolism. A Researcher in Germany have played a major role in the research. 
     SUMMARY OF THE INVENTION 
     Despite of it, no attempts have been performed on phenotypic expression of a certain gene in a higher plant using recombinant DNA technique to enhance the photosynthesis, which is a primary metabolism of a higher plant, and to improve its growth. 
     The object of this invention is to achieve phenotypic expression of a certain gene in a higher plant using recombinant DNA technique to enhance the photosynthesis, which is primary metabolism of a higher plant, to improve the crop productivity and yield potentiality and/or to enable the earlier harvest of the crop. 
     This invention provides a method for improving the productivity of a higher plant having chloroplasts by the phenotypic expression of cyanobacterial fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase in the chloroplasts. 
     Moreover, this invention provides a transgenic plant comprising a higher plant with a DNA fragment incorporated therein, the DNA fragment containing a base sequence coding cyanobacterial fructose-1,6-bisphosphatase/sedoheptulose- 1,7-bisphosphatase. 
     The fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) in the chloroplasts of a higher plant are the key (rate determining) enzymes of a photosynthetic reductive carbon system. The activities of these enzymes are regulated by photoreduction-potentiality. On the other hand, fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (FBPase/SBPase), derived from  Cyanobacterium synechococcus  PCC 7942 gene, if found widely in a specific type of a prokaryotic algae—a Cyanobacterium. The primary structure and enzyme properties of cyanobacterial FBPase/SBPase are different from those of the FBPase or SBPase found in the chloroplasts of a higher plant. In addition, cyanobacterial FBPase/SBPase is composed of one protein, that is, a bi-functional enzyme exhibiting two kinds of enzyme activities, FBPase and SBPase. 
     FBPase-I, derived from  Cyanobacterium synechococcus  PCC 7942 gene, is a tetramer consisting of four subunits of 40 kDa identical with each other. After the treatment by 1 mM H 2 O 2 , the purified enzyme retained more than 80 % of native enzyme activity. The enzyme activity of FBPase-I was inhibited by AMP (Ki=0.26 mM), which is a specific inhibitor of cytoplasm-type FBPase. However, it was not inhibited by fructose-2,6-P 2 . The optimum pH for the enzyme activity was 8.0 and pI value of the enzyme was 4.8. FBPase-I hydrolyzed not only fructose-1,6-bisphosphate (Fru1,6-P 2 ), but also sedoheptulose-1,7-bisphosphate (Sed1,7-P 2 ). The activities of the purified enzyme for Fru1,6-P 2  and Sed1,7-P 2  were 11.7 μmol/min/mg protein and 12.1 μmol/min/mg protein, respectively. The Km values for Fru1,6-P 2  and Sed1,7-P 2  were 52 μM and 118 μM, respectively. The enzyme activity was proved to be dependent on Mg 2+  concentration, also equally to typical FBPase. The dose-response curve showed sigmoidal curve equally to plastid FBPase, and the S 0.5  value was shown to be 1.4±0.1 mM. This enzyme itself was described in “Archives of Biochemistry and Biophysics, Vol. 334, No. 1, pp. 27 to 36, 1996: Molecular characterization and resistance to hydrogen peroxide of two fructose-1,6-bisphosphatase from synechococcus PCC 7942”. 
     The inventor incorporated fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, isolated from  Cyanobacterium synechococcus  PCC 7942, into a tobacco plant so that the expressed protein was transferred to its chloroplasts. The FBPase activity, the SBPase activity and the photosynthetic ability of the transgenic plant were compared to those of the wild type strain. The results measured 7 weeks after seeding showed significant increase of these activities in the transgenic plant. Furthermore, after certain period of cultivation, the plant bodies of the transgenic plant proved to be taller than those of the wild type strain. In the transgenic plant, the areas of the blades, the diameters of the stalks, and the numbers and lengths of the roots were larger than those in the wild type strain. In addition, the contents of hexose, sucrose and starch were proved to be increased in blades, stalks and roots of the transgenic plant, compared with those of the wild type strain. 
     Accordingly, the photosynthetic ability of the transgenic plant, obtained by incorporation of cyanobacterial FBPase/SBPase into a tobacco plant, was improved. As the result, the ability of the transgenic plant to synthesize carbohydrate and starch is increased, and the growth was enhanced, indicating the increase of final anabolism of the transgenic plant. Therefore, incorporation of cyanobacterial FBPase/SBPase into the chloroplasts of a higher plant was proved to be a very effective technique for producing rareripe or high-yield plants. 
     The effect might be explained as follows. Triggered by environmental stresses, light and oxygen toxicity causes various kinds of injuries to plant bodies, resulting in a critical and limiting factor of food production. Contrary to FBPase and SBPase derived from a higher plant, cyanobacterial FBPase/SBPase is resistant against oxygen injury and thus considered to function under various environmental stresses. Moreover, a gene encoding the cyanobacterial FBPase/SBPase does not exist in higher plants, thereby eliminating the possibility of adverse effects by gene silencing. 
     In this invention, a vector to produce a recombinant DNA includes plasmids pBI101, pIN19 and pMSH-1. A wide variety of useful cultivated plants and woods capable of photosynthesis can be adopted as a higher plant in which the inventive recombinant DNA is incorporated. For example, the invention may be applied to serials such as maize, rice, wheat, barley, oat wheat, millet and barnyard millet, beans such as soy bean, vegetables such as potato and tomato, useful cultivated plants such as coleseed, cotton and tobacco, and trees. 
     An amino acid sequence may be deleted from or added to the amino acid sequence of sequence number 1, or a part of the sequence of the sequence number 1 may be substituted with another amino acid sequence in the scope of this invention, so far as the resulting peptide retains its enzymatic activity properties as fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphasphatase. Preferably, not lower than 85 percent, more preferably not lower than 95 percent of the amino acid sequence may be overlapped or identical with the amino acid sequence of the sequence number 1. 
     In a base sequence of sequence number 2, a base sequence referred to as base numbers from 1 to 1068 is essential for this invention, because this base sequence corresponds to a structural gene portion, that is, an amino acid sequence of the sequence number 1. In addition, a base sequence referred to as base numbers from −180 to 1170 is the most preferred embodiment of this invention. 
     These and other features and advantages of this invention will become apparent upon a reading of the detailed description and drawings. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic view showing the structure of a plasmid incorporated into tobacco chloroplasts. 
     FIG. 2 is a graph showing the heights of the plant bodies of a wild type strain and a transgenic plant under water culture. 
     FIG. 3 is a graph showing the photosynthetic ability of a wild type strain and a transgenic plant. 
     FIG. 4 is a photograph showing appearance of plant bodies of a wild type strain and a transgenic plant, on the 112th day of cultivation under water culture, 
     FIG. 5 is a photograph showing the appearance of blades and stems of a wild type strain and a transgenic plant, on the 112th day of cultivation under water culture, 
     FIG. 6 is a photograph showing the appearance of roots of a wild type strain and a transgenic plant, on the 112th day of cultivation under water culture. 
     FIGS. 7 a ,  7   b , and  7   c  are graphs showing the contents of intermediate metabolites of a wild type strain and a transgenic plant. FIG. 7 a  is a comparison of the content of the metabolite intermediate hexose produced by photosynthesis. FIG. 7 b  is a comparison of the content of the metabolite intermediate sucrose produced by photosynthesis. FIG. 7 c  is a comparison of the content of the metabolite intermediate starch produced by photosynthesis. 
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
     As shown in FIG. 1, tomato rbcS promoter, coding region of a transit peptide and cyanobacterial fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (S.7942 FBP/SBPase) gene (fbp-I) were conjugated with pBI101 to construct a plasmid. The gene named fbp-I indicates a base sequence referred to as base numbers from −180 to 1170 of the sequence number 2, derived from  Cyanobacterial synechococcus  PCC7942. The plasmid was incorporated into agrobacterium tumefacience LBA4404, which was used for infection of leaf disk of tobacco ( Nicotiana tabacum  cv Xanthi), to incorporate fbp-1 into a tobacco nuclear gene. After isolating the genomic DNA, incorporation of fbp-1 was confirmed by PCR and immunoblotting methods. 7 strains of transformants (TFI-1 to TFI-7) were obtained. The chloroplasts were isolated from transformed strains (T2 generation), then expression of S.7942 FBPase/SBPase was confirmed by western blotting. Moreover, it was confirmed, by cell fractionation, that the protein expressed from the incorporated gene is localized in the chloroplasts (The FBPase activity, SPBase activity and photosynthetic activity). 
     The FBPase activity of the blades of the plant body of the transgenic tobacco cultivated 7 weeks after seeding was compared with that of the wild type strain without the incorporated gene. The result showed that the enzyme activity of the wild type strain was 1.04±0.22 μmol/min/mg chlorophyl and that of the transgenic plant was 1.82±0.24 μmol/min/mg chlorophyl. Therefore, the activity of the transgenic plant is 1.75 times higher than that of the wild type strain. 
     The SBPase activity was also compared. The SBPase activity of the wild type strain was 1.37 μmol/min/mg chlorophyl and that of the transgenic plant was 2.40 μmol/min/mg chlorophyl. Therefore, the activity of the transgenic plant is 1.75 times higher than that of the wild type strain. 
     The photosynthetic activity under conventional condition (360 ppm CO 2 ) was compared. As the result, significant difference between the wild type strain and the transgenic plant was not observed under illumination of 0, 10, 50 and 100 μE/s/m 2 . However, the photosynthetic activity of the transgenic plant increased significantly, under illumination of 200 μE/s/m 2 , compared with that of the wild type strain. Under 1600 μE/s/m 2  of illumination, the enzyme activity of the wild type strain was 1.24 times higher than that of the wild type strain. These results are shown in FIG.  3 . 
     (Effect of Transformation Upon the Plant Growth) 
     Using Hogrant medium, water culture was performed on the wild type strain and the transgenic plant. The experiment was performed under condition of 400 μmol/m 2 /s, a relative humidity of 60% and a temperature of 25° C. On the 63rd day, 72nd day, 77th day, 82nd day, 85th day, 90th day, 97th day, 102nd day, 105th day, 109th day, 112th day of cultivation, the heights of the plant bodies were measured. The results are shown in FIG.  2 . On the 64th day of cultivation, the height of the wild type strain was 14.0±4.6 cm and that of the transgenic plant was 16.6±2.9 cm. However, on the 112th day of cultivation, the height of the wild type strain was 58.3±7.0 cm and that of the transgenic plant was 84.5±7.8 cm, indicating significant enhancement of growth in the transgenic plants (about 1.45 times). The pictures in FIG. 4 show plant body of the wild type strain (left) and that of the transgenic plant (right). 
     During the whole period of growth, the blades, stems and roots of the transgenic plant grew better than those of the wild type strain. That is, the leaves are thicker with broader surface area, the stems are thicker and the number of roots are larger with each root longer. FIG. 5 is a photograph showing the appearance of the blades and stems on the 112th day of cultivation, and the wild type strain is shown in the left and the transgenic plant is shown in the right. FIG. 6 is a photograph showing the appearance of roots on the 112th day of cultivation, and the wild type strain is shown in the left and the transgenic plant is shown in the right. 
     (The Contents of Metabolic Intermediates) 
     The contents of metabolic intermediates (hexose, sucrose, starch) were measured on upper blades (fourth blade from the top), lower blades (third blade from the bottom), stems and roots of plant bodies 12th week after seeding, for comparing the contents between the wild type strain and the transgenic plant. The results are shown in FIG.  7 . The contents of metabolic intermediates in the transgenic plant increased significantly in all parts, including the upper blades, the lower blades, the stems and the roots, compared with the wild type strain. Especially, hexose and sucrose contents in the upper blades considerably increased. The accumulation of starch was observed in the lower blades. This is considered that sucrose synthesized in the upper blades was transferred into the lower blades. 
     As shown in these results, photosynthesis in higher plants was enhanced by this invention, increasing the production of carbohydrate and starch bio-synthesized in the transgenic plant to promote the plant growth. The dry weight of the plant bodies of the wild type strain was 14.1±2.2 g at the period of flower bud production, and that of the transgenic plant was 21.0±1.9 g. The dry weight of the transgenic plant increased 1.5 times compared with that of the wild type strain, indicating increase of final anabolism. 
     As described above, photosynthetic ability was enhanced in the transgenic plant of this invention compared with the wild type strain to improve the capability of biosynthesis of carbohydrate and starch, to promote the growth and to increase the final anabolism in the transgenic plant. Accordingly, the incorporation of FBPase/SBPase into chloroplasts of a higher plant is proved to be a very effective technique to produce a rareripe and/or high-yield crop. There has been no technique which enables production of a rareripe and/or high-yield crop using recombinant DNA technique to improve the potosynthetic ability of a higher plant, which is its primary metabolism. Therefore, this invention provides an important key technique to solve the coming crisis of food shortage. 
     Sequence List 
     &lt;110&gt;Applicant name: President of Nara institute of science and technology 
     &lt;120&gt;Title of invention: A method to improve productivity of higher plants transgenic plant 
     &lt;160&gt;Total number of sequences: 2 
     &lt;210&gt;Sequence number: 1 
     &lt;211&gt;Sequence length: 356 
     &lt;212&gt;Sequence type: PRT 
     &lt;213&gt;Organism:  Cyanobacterium Synechococcus    
     &lt;220&gt;Feature of sequence 
     Topology: linear 
     Source: fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase derived from  Cyanobacterium Synechococcus  PPC 7942 gene 
     &lt;400&gt;Sequence: 
     
       
         
               
               
               
             
           
               
                 1 
                 M E K T I G L E I I E V V E Q 
                 15 
               
               
                   
               
               
                 16 
                 A A I A S A R L M G K G E K N 
                 30 
               
               
                   
               
               
                 31 
                 E A D R V A V E A M R V R M N 
                 45 
               
               
                   
               
               
                 46 
                 Q V E M L G R I V I G E G E R 
                 60 
               
               
                   
               
               
                 61 
                 D E A P M L Y I G E E V G I Y 
                 75 
               
               
                   
               
               
                 76 
                 R D A D K R A G V P A G K L V 
                 90 
               
               
                   
               
               
                 91 
                 E I D I A V D P C E G T N L C 
                 105 
               
               
                   
               
               
                 106 
                 A Y G Q P G S M A V L A I S E 
                 120 
               
               
                   
               
               
                 121 
                 K G G L F A A P D F Y M K K L 
                 135 
               
               
                   
               
               
                 136 
                 A A P P A A K G K E T S I K S 
                 150 
               
               
                   
               
               
                 151 
                 A T E N L K I L S E C L D R A 
                 165 
               
               
                   
               
               
                 166 
                 I D E L V V V V M D R P R H K 
                 180 
               
               
                   
               
               
                 181 
                 E L I Q E I R Q A G A R V R L 
                 195 
               
               
                   
               
               
                 196 
                 I S D G D V S A A I S C G F A 
                 210 
               
               
                   
               
               
                 211 
                 G T N T H A L M G I G A A P E 
                 225 
               
               
                   
               
               
                 226 
                 G V I S A A A M R C L G G H F 
                 240 
               
               
                   
               
               
                 241 
                 Q G Q L I Y D P E V V K T G L 
                 255 
               
               
                   
               
               
                 256 
                 I G E S R E S N I A R L Q E M 
                 270 
               
               
                   
               
               
                 271 
                 G I T D P D R V Y D A N E L A 
                 285 
               
               
                   
               
               
                 286 
                 S G Q E V L F A A C G I T P G 
                 300 
               
               
                   
               
               
                 301 
                 L L M E G V R F F K G G A R T 
                 315 
               
               
                   
               
               
                 316 
                 Q S L V I S S Q S R T A R F V 
                 330 
               
               
                   
               
               
                 331 
                 D T V H M F D D V K T V S L P 
                 345 
               
               
                   
               
               
                 346 
                 L I P D P K W R P E R 
                 356 
               
             
          
         
       
     
     &lt;110&gt;Applicant name: President of Nara institute of science and technology 
     &lt;120&gt;Title of invention: A method to improve productivity of higher plants transgenic plant 
     &lt;160&gt;Total number of sequences: 2 
     &lt;210&gt;Sequence number: 2 
     &lt;211&gt;Sequence length: 1350 
     &lt;212&gt;Sequence type: DNA 
     &lt;213&gt;Organism:  Cyanobacterium Synechococcus    
     &lt;220&gt;Feature of sequence 
     Topology: linear 
     Source: fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase derived from  Cyanobacterium Synechococcus  PPC 7942 gene 
     &lt;400&gt;Sequence: 
     
       
         
               
               
               
               
             
           
               
                 −180 
                 CGTCGCCCGCTCCATGCCCGCAGCTGCGCCTTTGATGCCGCGGAA 
                 −136 
                   
               
               
                   
               
               
                 −135 
                 GATATTGCCGCCAACTAACGATANNAGTCACTGCGATCGCAACTA 
                 −91 
               
               
                   
               
               
                 −90 
                 AAGCCAGAGATGTGAGGAGGGGATCCGGCCTTTGGTAGACTCAAC 
                 −46 
               
               
                   
               
               
                 −45 
                 TGTTGGAATCCCCAGAAGCAATCATCCGTAAGGAGTCAGGACGGC 
                 −1 
               
               
                   
               
               
                 1 
                 GTGGAGAAGACGATCGGTCTCGAGATTATTGAAGTTGTCGAGCAG 
                 45 
               
               
                   
               
               
                 46 
                 GCAGCGATCGCCTCGGCCCGCCTGATGGGCAAAGGCGAAAAGAAT 
                 90 
               
               
                   
               
               
                 91 
                 GAAGCCGATCGCGTCGCAGTAGAAGCGATGCGGGTGCGGATGAAC 
                 135 
               
               
                   
               
               
                 136 
                 CAAGTGGAAATGCTGGGCCGCATCGTCATCGGTGAAGGCGAGCGC 
                 180 
               
               
                   
               
               
                 181 
                 GACGAAGCACCGATGCTCTATATCGGTGAAGAAGTGGGCATCTAC 
                 225 
               
               
                   
               
               
                 226 
                 CGCGATGCAGACAAGCGGGCTGGCGTACCGGCTGGCAAGCTGGTG 
                 270 
               
               
                   
               
               
                 271 
                 GAAATCGACATCGCCGTTGACCCCTGCGAAGGCACCAACCTCTGC 
                 325 
               
               
                   
               
               
                 326 
                 GCCTACGGTCAGCCCGGCTCGATGGCAGTTTTGGCCATCTCCGAG 
                 360 
               
               
                   
               
               
                 361 
                 AAAGGCGGCCTGTTTGCAGCTCCCGACTTCTACATGAAGAAACTG 
                 405 
               
               
                   
               
               
                 406 
                 GCTGCACCCCCAGCTGCCAAAGGCAAAGAGACATCAATAAAGTCC 
                 450 
               
               
                   
               
               
                 451 
                 GCGACCGAAAACCTGAAAATTCTCTCGGAATGTCTCGATCGCGCC 
                 495 
               
               
                   
               
               
                 496 
                 ATCGATGAATTGGTGGTCGTGGTCATGGATCGTCCCCGCCACAAA 
                 540 
               
               
                   
               
               
                 541 
                 GAGCTAATCCAAGAGATCCGCCAAGCGGGTGCCCGCGTCCGTCTG 
                 585 
               
               
                   
               
               
                 586 
                 ATCAGCGATGGTGACGTTTCGGCCGCGATCTCCTGCGGTTTTGCT 
                 630 
               
               
                   
               
               
                 631 
                 GGCACCAACACCCACGCCCTGATGGGCATCGGTGCAGCTCCCGAG 
                 675 
               
               
                   
               
               
                 676 
                 GGTGTGATTTCGGCAGCAGCAATGCGTTGCCTCGGCGGGCACTTC 
                 720 
               
               
                   
               
               
                 721 
                 CAAGGCCAGCTGATCTACGACCCAGAAGTGGTCAAAACCGGCCTG 
                 765 
               
               
                   
               
               
                 766 
                 ATCGGTGAAAGCCGTGAGAGCAACATCGCTCGCCTGCAAGAAATG 
                 810 
               
               
                   
               
               
                 811 
                 GGCATCACCGATCCCGATCGTGTCTACGACGCGAACGAACTGGCT 
                 855 
               
               
                   
               
               
                 856 
                 TCGGGTCAAGAAGTGCTGTTTGCGGCTTGCGGTATCACCCCGGGC 
                 900 
               
               
                   
               
               
                 901 
                 TTGCTGATGGAAGGCGTGCGCTTCTTCAAAGGCGGCGCTCGCACC 
                 945 
               
               
                   
               
               
                 946 
                 CAGAGCTTGGTGATCTCCAGCCAGTCACGGACGGCTCGCTTCGTT 
                 990 
               
               
                   
               
               
                 991 
                 GACACCGTTCACATGTTCGACGATGTCAAAACGGTTAGCCTGCCG 
                 1035 
               
               
                   
               
               
                 1036 
                 TTAATTCCTGATCCCAAATGGCGGCCGGAGCGGTAGAACGGGTAT 
                 1080 
               
               
                   
               
               
                 1081 
                 AGCTCGATCGCTTCGGTCGTTGTTTTTCAGCGAATCCATTTGCGA 
                 1125 
               
               
                   
               
               
                 1126 
                 TCGCTTTTCAAACCCTTTTTTCGTCAACCTTCTTTAAACGGCCTC 
                 1170 
               
             
          
         
       
     
     
       
         
           
             2 
           
           
             1 
             356 
             PRT 
             Cyanobacterium Synechococcus 
           
            1
Met Glu Lys Thr Ile Gly Leu Glu Ile Ile Glu Val Val Glu Gln Ala
  1               5                  10                  15
Ala Ile Ala Ser Ala Arg Leu Met Gly Lys Gly Glu Lys Asn Glu Ala
             20                  25                  30
Asp Arg Val Ala Val Glu Ala Met Arg Val Arg Met Asn Gln Val Glu
         35                  40                  45
Met Leu Gly Arg Ile Val Ile Gly Glu Gly Glu Arg Asp Glu Ala Pro
     50                  55                  60
Met Leu Tyr Ile Gly Glu Glu Val Gly Ile Tyr Arg Asp Ala Asp Lys
 65                  70                  75                  80
Arg Ala Gly Val Pro Ala Gly Lys Leu Val Glu Ile Asp Ile Ala Val
                 85                  90                  95
Asp Pro Cys Glu Gly Thr Asn Leu Cys Ala Tyr Gly Gln Pro Gly Ser
            100                 105                 110
Met Ala Val Leu Ala Ile Ser Glu Lys Gly Gly Leu Phe Ala Ala Pro
        115                 120                 125
Asp Phe Tyr Met Lys Lys Leu Ala Ala Pro Pro Ala Ala Lys Gly Lys
    130                 135                 140
Glu Thr Ser Ile Lys Ser Ala Thr Glu Asn Leu Lys Ile Leu Ser Glu
145                 150                 155                 160
Cys Leu Asp Arg Ala Ile Asp Glu Leu Val Val Val Val Met Asp Arg
                165                 170                 175
Pro Arg His Lys Glu Leu Ile Gln Glu Ile Arg Gln Ala Gly Ala Arg
            180                 185                 190
Val Arg Leu Ile Ser Asp Gly Asp Val Ser Ala Ala Ile Ser Cys Gly
        195                 200                 205
Phe Ala Gly Thr Asn Thr His Ala Leu Met Gly Ile Gly Ala Ala Pro
    210                 215                 220
Glu Gly Val Ile Ser Ala Ala Ala Met Arg Cys Leu Gly Gly His Phe
225                 230                 235                 240
Gln Gly Gln Leu Ile Tyr Asp Pro Glu Val Val Lys Thr Gly Leu Ile
                245                 250                 255
Gly Glu Ser Arg Glu Ser Asn Ile Ala Arg Leu Gln Glu Met Gly Ile
            260                 265                 270
Thr Asp Pro Asp Arg Val Tyr Asp Ala Asn Glu Leu Ala Ser Gly Gln
        275                 280                 285
Glu Val Leu Phe Ala Ala Cys Gly Ile Thr Pro Gly Leu Leu Met Glu
    290                 295                 300
Gly Val Arg Phe Phe Lys Gly Gly Ala Arg Thr Gln Ser Leu Val Ile
305                 310                 315                 320
Ser Ser Gln Ser Arg Thr Ala Arg Phe Val Asp Thr Val His Met Phe
                325                 330                 335
Asp Asp Val Lys Thr Val Ser Leu Pro Leu Ile Pro Asp Pro Lys Trp
            340                 345                 350
Arg Pro Glu Arg
        355
 
           
             2 
             1350 
             DNA 
             Cyanobacterium Synechococcus 
             
               misc_feature 
               (69)..(70) 
               Nucleotides at positions 69 and 70 are “n”
      wherein “n” = any nucleotide. 
             
           
            2
cgtcgcccgc tccatgcccg cagctgcgcc tttgatgccg cggaagatat tgccgccaac     60
taacgatann agtcactgcg atcgcaacta aagccagaga tgtgaggagg ggatccggcc    120
tttggtagac tcaactgttg gaatccccag aagcaatcat ccgtaaggag tcaggacggc    180
gtggagaaga cgatcggtct cgagattatt gaagttgtcg agcaggcagc gatcgcctcg    240
gcccgcctga tgggcaaagg cgaaaagaat gaagccgatc gcgtcgcagt agaagcgatg    300
cgggtgcgga tgaaccaagt ggaaatgctg ggccgcatcg tcatcggtga aggcgagcgc    360
gacgaagcac cgatgctcta tatcggtgaa gaagtgggca tctaccgcga tgcagacaag    420
cgggctggcg taccggctgg caagctggtg gaaatcgaca tcgccgttga cccctgcgaa    480
ggcaccaacc tctgcgccta cggtcagccc ggctcgatgg cagttttggc catctccgag    540
aaaggcggcc tgtttgcagc tcccgacttc tacatgaaga aactggctgc acccccagct    600
gccaaaggca aagagacatc aataaagtcc gcgaccgaaa acctgaaaat tctctcggaa    660
tgtctcgatc gcgccatcga tgaattggtg gtcgtggtca tggatcgtcc ccgccacaaa    720
gagctaatcc aagagatccg ccaagcgggt gcccgcgtcc gtctgatcag cgatggtgac    780
gtttcggccg cgatctcctg cggttttgct ggcaccaaca cccacgccct gatgggcatc    840
ggtgcagctc ccgagggtgt gatttcggca gcagcaatgc gttgcctcgg cgggcacttc    900
caaggccagc tgatctacga cccagaagtg gtcaaaaccg gcctgatcgg tgaaagccgt    960
gagagcaaca tcgctcgcct gcaagaaatg ggcatcaccg atcccgatcg tgtctacgac   1020
gcgaacgaac tggcttcggg tcaagaagtg ctgtttgcgg cttgcggtat caccccgggc   1080
ttgctgatgg aaggcgtgcg cttcttcaaa ggcggcgctc gcacccagag cttggtgatc   1140
tccagccagt cacggacggc tcgcttcgtt gacaccgttc acatgttcga cgatgtcaaa   1200
acggttagcc tgccgttaat tcctgatccc aaatggcggc cggagcggta gaacgggtat   1260
agctcgatcg cttcggtcgt tgtttttcag cgaatccatt tgcgatcgct tttcaaaccc   1320
ttttttcgtc aaccttcttt aaacggcctc                                    1350