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 ). 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 - 30ledi ; 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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( 1998 ). eukaryotic phytochromes : light - regulated serine / threonine protein kinases with histidine kinase ancestry . proc . natl . acad . sci . usa 95 , 13976 - 13981 . thr phe gly cys leu leu ala leu asp glu lys ser phe asn val ile ala phe ser glu asn ala pro glu met leu thr thr val ser his ala arg ser leu phe ser asp gln gly ala thr ala leu his lys ala leu gly phe ala asp val ser leu leu asn pro ile leu val gln cys lys thr ser gly lys pro phe tyr ala ile val his arg ala thr gly cys lys ile gln ser leu pro gly gly ser met glu met leu cys asn thr val val lys glu val phe asp leu thr gly tyr asp arg val met ala tyr lys phe his glu asp asp his gly glu val phe ser glu ile thr ile pro gln ala ala arg phe leu phe met lys asn lys val arg met leu pro phe asp ile ser leu cys gly ser ala leu arg ala pro his ser cys his leu gln tyr met glu asn met asn ser ile ala ser leu tyr ala cys glu phe leu ala gln val phe ala val his val asn arg gln thr met leu ser asp met leu phe arg glu ala ser pro leu thr ile val ser gly asn pro asn ile met asp leu val lys cys asp gly thr glu ser gln ile his asp ile ala phe trp leu ser asp val his lys ile asn ser lys asp ile leu phe trp phe arg ser his thr ala ala glu ile arg trp gly gly ala lys asn asp pro ser asp met asp asp ser arg arg met his pro arg leu ser phe lys ala phe leu glu val val lys met lys ser leu pro trp ser asp tyr glu met asp ala ile his ser leu gln leu ile leu arg gly thr leu asn asp ala ser lys pro lys arg glu ala ala leu asp asn gln ile gly asp leu lys leu asp gly leu ala glu leu gln ala val thr ser glu met val arg leu met glu thr ala thr val pro ile leu ala val asp gly asn gly leu val asn gly trp asn gln lys ala ala glu leu thr gly leu arg val asp asp ala ile gly arg his ile leu thr leu val glu asp ser ser val pro val val gln arg met leu tyr leu ala leu gln gly lys asp asp gly pro val ile leu val val asn ala cys ala ser arg asp leu his asp his val val gly val cys phe val ala gln asp met thr val his lys leu val met asp lys phe thr arg val glu gly asp tyr ala asp glu phe gly trp cys ser glu trp asn ala ala met thr lys leu thr gly trp asn arg asp glu val leu asp lys met leu leu gly glu val phe asp ser ser asn ala ser cys pro leu lys asn arg asp glu thr glu lys ala pro phe gly phe phe asp arg ser gly lys tyr ile thr gly val phe cys phe ile his val ala ser his glu leu gln leu lys ala phe ser tyr met arg his ala ile asn asn pro leu ser gly met leu tyr ser arg lys ala leu lys asn thr asp leu asn glu glu gln met lys gln ile his val gly asp asn cys his his gln ile arg ile ser cys asn leu pro glu arg phe met lys gln ser val tyr leu arg ile lys his gln gly leu gly val pro ala glu leu met ala his leu arg glu ala gly val ser thr phe ile ile thr ala glu leu