Patent Application: US-46840799-A

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:
as shown in fig1 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 fig3 . 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 fig2 . 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 fig4 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 . fig5 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 . fig6 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 ( 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 fig7 . 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 . & lt ; 120 & gt ; title of invention : a method to improve productivity of higher plants transgenic plant e i d i a v d p c e g t n l c a y g q p g s m a v l a i s e a t e n l k i l s e c l d r a g v i s a a a m r c l g g h f q g q l i y d p e v v k t g l g i t d p d r v y d a n e l a s g q e v l f a a c g i t p g & lt ; 120 & gt ; title of invention : a method to improve productivity of higher plants transgenic plant met leu tyr ile gly glu glu val gly ile tyr arg asp ala asp lys asp pro cys glu gly thr asn leu cys ala tyr gly gln pro gly ser met ala val leu ala ile ser glu lys gly gly leu phe 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