Patent Application: US-13450002-A

Abstract:
the present invention includes modified phytochrome a nucleic acid molecules in which dna sequences coding for “ active site ” amino acid residues have been mutated to generate hyperactive phytochromes . in particular ; a serine / threonine residue at the hinge between the n - and c - terminal domains as well as at the n - terminal serine / threonine cluster of phytochromes for pr / pfr - dependent phosphorylation and dephosphorylation by a phytochrome phosphatase was substituted with alanine . in addition , amino acid residues within the phytochrome chromophore pocket are mutated to generate the bathchromic shift of the pr - absorption band of both wild type and above - mentioned mutant phytochromes . the plants with the bathchromically shifted absorption spectrum are expected to respond to the canopy and shade conditions for growth and greening responses to far - red light with greater efficiency than are the wild type plants with normal absorption band maxima . these mutative modifications confer hyperactivity to the far - red light responsive phytochromes a . thus , the biological activity of the modified oat phya was shown to be hyperactive compared to wild type phya , characterized by its ability to reduce internode elongation of adult plants . overexpression of the phytochrome phosphatase exhibits a suppressed growth with shorter internodes and belated flowering , qualitatively consistent with the phenotype of a ser598ala mutant oat phytochrome . the invention also includes plants having at least one cell expressing the modified phya , vectors comprising at least one portion of the modified phya nucleic acids , and methods using such vectors for producing plants with reduced stature .

Description:
phytochromes are the best characterized photoreceptor that regulate diverse aspects of growth and development in higher plants . upon irradiation , it exhibits interconvertible photo - conversion between biologically inactive pr ( red absorbing phytochrome ) form and biologically active pfr ( far - red absorbing phytochrome ) form that enables it to act as a molecular light switch ( butler et al ., 1959 ). the activated pfr triggers downstream signaling that result in diverse photo - responses . upon pfr formation after red light absorption , phytochrome undergoes several conformational changes . the pfr - chromophore is more exposed than the pr - chromophore ( park et al ., 2000 ). the n - terminal domain is more exposed in the pr form than in the pfr form . the hinge region is preferentially exposed in the pfr form . these conformational changes would trigger downstream signaling events . phosphorylation is a primary mechanism that transduces signaling in eukaryotes . phytochrome signaling involves several phosphorylation events . phytochrome itself exhibited ser / thr kinase activity ( yeh and lagarias , 1997 ). pks1 , one of the phytochrome interacting factors including pif3 and ndpk2 have been phosphorylated by phytochrome ( fankhauser , et al ., 1999 ). interestingly phytochrome is also phosphorylated in a pfr - dependent manner . the 598 th serine residue is preferentially phosphorylated in the pfr form in vivo ( lapko et al ., 1999 ). in vitro kinase assay showed that the 598 th serine was shown to be important for the light - regulation of autophosphorylation / phosphotransfer activity of phytochrome . as an effort to characterize the biological role of phosphorylation at 598 th serine of phytochrome in vivo , we performed site - directed mutagenesis and generated mutant phya of which 598 th serine was substituted by alanine . after generation of transgenic plants that overexpress wildtype phya or mutant phya using phya - null arabidopsis mutant , the phenotypes of transgenic plants were examined . using immunoblot analysis , we identified transgenic lines that overexpress foreign gene , phya or mutant phya ( fig3 a and fig3 b ). two lines of wildtype phya overexpressing lines , designated as wt # 4 and wt # 6 , and several lines of s598a phya overexpressing lines were chosen for further analysis . to test whether introduced phya is biologically functional in arabidopsis , we grew seedlings under fr light or in the dark . as shown in fig4 , ler wild type showed typical fr - responses , including shortened hypocotyl , expanded cotyledons , while phya - null mutant exhibited skotomorphogenic development , such as long hypocotyl , closed cotyledons . the wt # 4 and wt # 6 transgenic seedlings showed typical light - dependent photomorphogenic development . under the same condition , s598a phya transgenic lines complemented phya null mutant , exhibiting fr - dependent photomorphogenic development . these results indicate that s598a phya is functional , complementing phya - deficiency of phya - 201 mutant in arabidopsis . previously oat phya was shown to be active in several dicot plants ( boylan and quail , 1989 ; boylan and quail , 1991 ), mediating fr - hir . in arabidopsis , transgenic lines that overexpressing phya did not show any effects on adult morphology , while transgenic lines of tobacco and tomato exhibited several agronomic important traits such as dwarfism . when we grew the transgenic arabidopsis plants that overexpress s598a phya , the transgenic lines showed dwarfism , while transgenic lines of phya were normal , compared to wild type ( fig5 ). the results suggest that s598a phya is hyperactive to mediate adult dwarfism in arabidopsis , compared to wildtype phya . this trait is a potent agronomical target that can be applied to flowering plants to reduce cell / organ elongation resulting in improved agronomic values seeds of pea plant was germinated and grown under sterile condition on the murashige and skoog ( ms ) media . the arabidopsis thaliana ecotype ler , phya - 201 mutants , and transgenic lines were grown on 0 . 5 × ms medium . all arabidopsis cultures were maintained in a controlled environment culture room at 26 ° c ., 70 % humidity and for the photoperiod of 16 hours . the arabidopsis transformation was performed according to the simplified floral dip method , a well known technique to the art . for fr - high irradiance response , growth chamber ( model e - 30led1 ; percival scientific , inc ., boone , iowa ) equipped with fr light - emitting diode was used . dna manipulations were carried out according to the standard procedures with some modifications whenever required . restriction enzyme digestions were routinely done in 20 μl reaction volumes with an enzyme of 1 - 5 units per microgram dna , and the mixtures were incubated at an appropriate temperature for 1 - 2 hours . restriction enzyme digestion buffers used were those supplied by the manufacturer for each particular enzyme , unless specified otherwise . for ligation reactions , dna fragments , either a digestion mixture or a pcr product , were first separated on 0 . 8 - 1 . 5 % agarose gels , depending on the sizes of the dna fragments of interest , and the desired dna fragment was purified from the gel piece using either the geneclean ii kit ( bio 101 , vista , usa ) or the gel extraction kit ( omega biotek , doraville , usa ). ligations were performed usually at the molar ratio of 1 : 1 to 1 : 3 in a 10 μl volume using the buffer supplied by the manufacturer , and the mixture was incubated at 13 - 16 ° c . for 10 minutes ( for sticky - end ligations ) or 30 minutes ( for blunt - end ligations ). t4 dna ligase and its corresponding ligase buffer ( neb , beverly , mass ., usa ) were routinely used with 5 - 10 units of ligase in a 10 μl volume reaction . polymerase chain reaction ( pcr ) was usually carried out 25 cycles , each with 1 minute denaturation at 94 ° c ., 1 minute annealing at 60 ° c ., and polymerization at 72 ° c . for 2 minutes per 1000 bases using the pfu polymerase . for quantitative analysis , pcr was run 15 - 20 cycles , depending the gene expression levels , using the taq polymerase ( promega , madison , wis .). for general cloning purpose , e . coli strain xl1 - blue was routinely used as host cells for the transformation with plasmid dnas . the competent e . coli cells were prepared in the laboratory and usually had an efficiency of 5 × 10 − 6 to 10 − 7 colonies per μg control vector dna . three to five microliter of the ligation mixture was usually used to transform 100 μl of the competent e . coli cells . after incubation on ice for 20 minutes , the cell - dna mixture was heat - shocked at 42 ° c . for 1 minute , and 1 ml of soc medium was added . the mixture was then gently rotated at 37 ° c . for 1 hour to render the cells recovered from damage , and 50 - 300 μl was spread on lb plates containing an appropriate antibiotic . the plates were incubated at 37 ° c . overnight or until positive colonies were visible . vector dna was isolated routinely by the alkaline - sds method from e . coli culture . a 1 ml ( for high copy number plasmid ) or a 10 ml lb - ampicillin culture ( for low copy number plasmid ) was routinely prepared for the small scale purification of plasmid dna . for the large scale purification , tb medium ( terrific broth , 47 . 6 grams of tb mix per liter , difco , detroit , usa ) which gives higher plasmid dna yields , instead of lb medium , was used . to prepare plasmid dna for dna sequencing and agrobacterium transformation , those isolated by the alkaline - sds method was further purified using the plasmid miniprep kit ii ( omega biotek , seoul , korea ). after the screening of the transgenic plants , rt - pcr technique was used to confirm the transcription of the introduced gene . total rnas from the transgenic seedlings were prepared by using rneasy ® plant mini kit ( qiagen , 74903 ) and followed the standard procedure to generate cdna by mmrv - reverse transcriptase ( strategene ). 5 μg of total rna was used for the cdna synthesis . after the synthesis of the cdna , pcr was performed to confirm the expression of the genes in the transgenic plants . the used primers were 5 ′- gaatgaagaacagatgaagc - 3 ′ ( seq id no : 3 ) and 5 ′- ttgtcccattgctgttggagc - 3 ′ ( seq id no : 4 ). the products are the c - terminal gene fragments of oat phya whose size is 581 base pairs . to check the expression of wt and mt proteins and the amounts , the western blot analysis was performed . the preparation of protein samples from the transgenic plants was done as follows : about 4 leaves from each plant were taken off before bolting , put the leaves between the water - soaked whatman filter papers , and incubated the leaves for at least 12 hours under dark condition . the leave samples were grinded in the microcentrifuge tubes using sea sands and plastic rods . this protein extraction procedure were performed on the ice or in the cold room under the green light condition , and the used buffer for the protein extraction composed of 70 mm tris ( ph 8 . 3 ), 35 % ethylene glycol , 98 mm ( nh 4 ) 2 so 4 , 7 mm edta , 14 mm sodium metabisulfite , 0 . 07 % polyethyleneimine and 2 . 8 mm pmsf ( all from sigma except ethylene glycol that is from fisher ). the extracted protein samples were centrifuged at 14 , 000 rpm and 4 ° c . for 15 min , and the supernatant were used as protein samples for the western blot analysis . the protein samples were quantified by using bio - rad protein assay kit ( 500 - 0001 ), and 50 ug of protein samples were loaded onto the 10 % sds - page gels for the western blot analysis . the protein bands on the sds - page gel were transferred to pvdf membrane ( hybond - p , amersham phamacia biotech ), and the membrane was incubated with oat phya - specific monoclonal antibody , oat22 and oat25 , for 2 hours and developed by using ecl ™ western blotting analysis system purchased from amersham phamacia biotech ( rpn 2108 ). for the detection of arabidopsis phya , p25 and maa7 antibodies were added to the reaction . the full size of cdna encoding avena phytochrome a ( phya ) from pfy 122 ( boylan and quail , 1989 ) was cloned to pgem ®- 11zf (+) ( promega p2411 ) by digesting with bamhi and ecori . after purifying the pgem ®- 11zf (+) plasmids containing full - length oat phya cdna , the site - directed mutagenesis in order to create ser598ala avena phya mutant was performed by using geneeditor ™ in vitro site - directed mutagenesis system ( promega q9280 ). the oligonucleotide sequence of mutagenic primer for the mutagenesis is phosphorylated - 5 ′- gcgggaagctgct ctaga taaccagattgg - 3 ′ ( seq id no : 5 ). the bold and italic bases are the mutagenized ones from the original sequence 5 ′- agtt - 3 ′ ( seq id no : 6 ) to 5 ′- gctc - 3 ′ ( seq id no : 7 ), and the underlined sequence , 5 ′- tctaga - 3 ′ ( seq id no : 8 ) is a created xbai restriction site which is used for the screening of the mutant gene . this new restriction site ( xbai ) was introduced by silent mutation near the position to be mutated , allowing rapid and efficient screening for the mutant phya ( ser598ala mutant ). after the mutagenesis , the mutagenized plasmids were purified and confirmed by xbai digestion and dna sequencing . dna sequencing was done by using sequenase version 2 . 0 dna sequencing kit ( amersham , usb , us70770 ) with 35 s - atp . all cdna and dna fragments and the junctions of the expression vector constructs were confirmed by direct dna sequencing on both strands . dna sequencing was carried out using the abi prism 310 genetic analyzer ( perkin elmer , foster city , usa ) as described in the manufacturer &# 39 ; s manual . for each sequencing run , about 500 ng of plasmid dna and 2 - 4 picomoles of 15 - 17 mer sequencing primer were used . computer - assisted sequence analysis was performed using the blast program ( ncbi , usa ). agarose gel electrophoresis of dna was usually performed using gels with a concentration range of 0 . 8 - 1 . 5 %, depending on the size of the dna fragments to be analyzed , using the tae buffer ( 40 mm tris - acetate , 1 mm edta , ph 8 . 0 ). electrophoresis was performed at a constant voltage rage of 50 - 200 , depending on the amount of dna loaded onto wells , for a desired time or until dna fragments were well separated . the gel was stained with 0 . 5 μg / ml ethidium bromide solution , visualized on an uv transilluminator , and photographed if required . the wild - type ( wt ) and ser598ala mutant ( mt ) genes were subcloned into the plant transformation vector , pbi121 ( clontech , cat no . 6018 - 1 : 13 kb , camv 35s promoter etc .). for the subcloning , the vector ( pbi121 ) was digested with bamhi and ecoicri , and the wt and mt genes in pgem ®- 11zf (+) were eluted by sequential enzyme treatment : ecori digestion , t4 polymerase treatment for making blunt ended dna and bamhi digestion . since the vector and the genes have one blunt end and one cohesive end , they can be ligated and subcloned . after the subcloning and confirmation of the genes in pbi121 , the purified plasmids were used for the transformation into phya deficient arabidopsis thaliana . since the vector has a kanamycin - resistant gene , the seeds having the transformed genes were selected by geminating on the agar plate containing 50 μg / ml kanamycin . bhoo s . h ., hirano t ., jeong h . y ., lee j . g ., furuya m . & amp ; song p . s . 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