Patent Application: US-201314026269-A

Abstract:
the present invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for flavonoid biosynthetic enzymes in plants , and the use thereof for the modification of flavonoid biosynthesis in plants . more particularly , the flavonoid biosynthetic enzyme is selected from the group consisting of chalcone isomerase , chalcone synthase , chalcone reductase , dihydroflavonol 4 - reductase , leucoanthocyanidin reductase , flavonoid 3 ′, 5 ′ hydrolase , flavanone 3 - hydroxylase , flavonoid 3 ′- hydroxylase , phenylalanine ammonia - olyase and vestitone reductase , and functionally active fragments and variants thereof .

Description:
preparation of cdna libraries , isolation and sequencing of cdnas coding for chi , chi - like , chs , chs - like , chr , chr - like , dfr , dfr - like , lcr , lcr - like , f3 ′ 5 ′ h , f3 ′ 5 ′ h - like , f3h , f3h - like , f3 ′ h , f3 ′ h - like , pal , pal - like , vr and vr - like proteins from white clover ( trifolium repens ) and perennial ryegrass ( lolium perenne ) cdna libraries representing mrnas from various organs and tissues of white clover ( trifolium repens ) and perennial ryegrass ( lolium perenne ) were prepared . the characteristics of the white clover and perennial ryegrass libraries , respectively , are described below ( tables 1 and 2 ). the cdna libraries may be prepared by any of many methods available . for example , total rna may be isolated using the trizol method ( gibco - brl ; usa ) or the rneasy plant mini kit ( qiagen ; germany ), following the manufacturers &# 39 ; instructions . cdnas may be generated using the smart pcr cdna synthesis kit ( clontech ; usa ), cdnas may be amplified by long distance polymerase chain reaction using the advantage 2 pcr enzyme system ( clontech ; usa ), cdnas may be cleaned using the geneclean spin column ( bio 101 ; usa ), tailed and size fractionated , according to the protocol provided by clontech . the cdnas may be introduced into the pgem - t easy vector system 1 ( promega ; usa ) according to the protocol provided by promega . the cdnas in the pgem - t easy plasmid vector are transfected into escherichia coli epicurian coli xl10 - gold ultra competent cells ( stratagene , usa ) according to the protocol provided by stratagene . alternatively , the cdnas may be introduced into plasmid vectors for first preparing the cdna libraries in uni - zap xr vectors according to the manufacturer &# 39 ; s protocol ( stratagene cloning systems ; la jolla , calif ., usa ). the uni - zap xr libraries are converted into plasmid libraries according to the protocol provided by stratagene . upon conversion , cdna inserts will be contained in the plasmid vector pbluescript . in addition , the cdnas may be introduced directly into precut pbluescript ii sk (+) vectors ( stratagene ) using t4 dna ligase ( new england biolabs ), followed by transfection into e . coli dh1ob cells according to the manufacturer &# 39 ; s protocol ( gibco brl products ). once the cdna inserts are in plasmid vectors , plasmid dnas were prepared from randomly picked bacterial colonies containing recombinant plasmids , or the insert cdna sequences were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cdna sequences . plasmid dna preparation was performed robotically using the qiagen qiaprep turbo kit ( qiagen ; germany ) according to the protocol provided by qiagen . amplified insert dnas were sequenced in dye - terminator sequencing reactions to generate partial cdna sequences ( expressed sequence tags or “ ests ”). the resulting ests were analyzed using an applied biosystems abi 3700 sequence analyser . the cdna clones encoding chi , chi - like , chs , chs - like , chr , chr - like , dfr , dfr - like , lcr , lcr - like , f3 ′ 5 ′ h , f3 ′ 5 ′ h - like , f3h , f3h - like , f3 ′ h , f3 ′ h - like , pal , pal - like , vr and vr - like proteins were identified by conducting blast ( basic local alignment search tool ; altschul et al . ( 1993 ) j . mol . biol . 215 : 403 - 410 ) searches . the cdna sequences obtained were analyzed for similarity to all publicly available dna sequences contained in the ebioinformatics nucleotide database using the blastn algorithm provided by the national center for biotechnology information ( ncbi ). the dna sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the swiss - prot protein sequence database using blastx algorithm ( v 2 . 0 . 1 ) ( gish and states ( 1993 ) nature genetics 3 : 266 - 272 ) provided by the ncbi . the cdna sequences obtained and identified were then used to identify additional identical and / or overlapping cdna sequences generated using the blastn algorithm . the identical and / or overlapping sequences were subjected to a multiple alignment using the clustalw algorithm , and to generate a consensus contig sequence derived from this multiple sequence alignment . the consensus contig sequence was then used as a query for a search against the swiss - prot protein sequence database using the blastx algorithm to confirm the initial identification . identification and full - length sequencing of cdnas encoding perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra proteins to fully characterize for the purposes of the generation of probes for hybridization experiments and the generation of transformation vectors , a set of cdnas encoding perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra proteins was identified and fully sequenced . full - length cdnas were identified from our est sequence database using relevant published sequences ( ncbi databank ) as queries for blast searches . full - length cdnas were identified by alignment of the query and hit sequences using sequencher ( gene codes corp . ; ann arbor , mich .). the original plasmid was then used to transform chemically competent xl - 1 cells ( prepared in - house , cacl 2 protocol ). after colony pcr ( using hotstartaq , qiagen ), a minimum of three pcr - positive colonies per transformation were picked for initial sequencing with m13f and m13r primers . the resulting sequences were aligned with the original est sequence using sequencher to confirm the identity . one of the three clones having the best initial sequencing result was picked for full - length sequencing . sequencing was completed by primer walking , i . e . oligonucleotide primers were designed to the initial sequence and used for further sequencing . in most cases , the sequencing could be done from both 5 ′ and 3 ′ end . the sequences of the oligonucleotide primers are shown in table 3 . in some instances , however , an extended poly - a tail necessitated the sequencing of the cdna to be completed from the 5 ′ end . contigs were then assembled in sequencher . the contigs include the sequences of the smart primers used to generate the initial cdna library as well as pgem - t easy vector sequence up to the ecori cut site both at the 5 ′ and 3 ′ end . plasmid maps and the full cdna sequences of perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra proteins were obtained ( fig1 , 116 , 121 , 126 , 131 , 136 , 141 , 146 , 151 , 156 , 161 , 166 , 171 , 176 , 181 , 186 and 191 ). development of transformation vectors containing chimeric genes with cdna sequences from perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chshh , dfrd , f3ha , pala , palb , palf and vra to alter the expression of the proteins involved in flavonoid biosynthesis , protein binding , metal chelation , anti - oxidation , uv - light absorption , tolerance to biotic stresses such as viruses , micro - organisms , insects and fungal pathogens ; pigmentation in for example flowers and leaves ; herbage quality and bloat - safety and isoflavonoid content leading to health benefits , perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra , through antisense and / or sense suppression technology and for over - expression of these key enzymes in transgenic plants , a set of sense and antisense transformation vectors was produced . cdna fragments were generated by high fidelity pcr using the original pgem - t easy plasmid cdna as a template . the primers used ( table 4 ) contained recognition sites for appropriate restriction enzymes , for example ecori and xbai , for directional and non - directional cloning into the target vector , after pcr amplification and restriction digest with the appropriate restriction enzyme ( usually xbai ), the cdna fragments were cloned into the corresponding site in pdh51 , a puc18 - based transformation vector containing a camv 35s expression cassette . the orientation of the constructs ( sense or antisense ) was checked by dna sequencing through the multi - cloning site of the vector . transformation vectors containing chimeric genes using full - length open reading frame cdnas encoding perennial ryegrass f3oh and white clover bana , chia , chid , chre , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra proteins in sense and antisense orientations under the control of the camv 35s promoter were generated ( fig1 , 119 , 124 , 129 , 134 , 139 , 144 , 149 , 154 , 159 , 164 , 169 , 174 , 179 , 184 , 189 and 194 ). development of binary transformation vectors containing chimeric genes with cdna sequences from perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsd , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra to alter the expression of the proteins involved in flavonoid biosynthesis , protein binding , metal chelation , anti - oxidation , uv - light absorption , tolerance to biotic stresses such as viruses , micro - organisms , insects and fungal pathogens ; pigmentation in for example flowers and leaves ; herbage quality and bloat - safety and isoflavonoid content leading to health benefits , perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chse , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra , through antisense and / or sense suppression technology and for over - expression of these key proteins in transgenic plants , a set of sense and antisense binary transformation vectors was produced . cdna fragments were generated by high fidelity pcr using the original pgem - t easy plasmid cdna as a template . the primers used ( table 4 ) contained recognition sites for appropriate restriction enzymes , for example ecorl and xbai , for directional and non - directional cloning into the target vector . after pcr amplification and restriction digest with the appropriate restriction enzyme ( usually xbai ), the cdna fragments were cloned into the corresponding site in a modified ppzp binary vector ( hajdukiewicz et al ., 1994 ). the ppzp221 vector was modified to contain the 35s2 cassette from pkylx71 : 35s2 as follows . pkylx71 : 35s2 was cut with clai . the 5 ′ overhang was filled in using klenow and the blunt end was a - tailed with taq polymerase . after cutting with ecori , the 2kb fragment with an ecori - compatible and a 3 ′- a tail was gel - purified . ppzp221 was cut with hindiii and the resulting 5 ′ overhang filled in and t - tailed with taq polymerase . the remainder of the original ppzp221 multi - cloning site was removed by digestion with ecori , and the expression cassette cloned into the ecori site and the 3 ′ t overhang restoring the hindiii site . this binary vector contains between the left and right border the plant selectable marker gene aaacl under the control of the 35s promoter and 35s terminator and the pkylx71 : 35s2 - derived expression cassette with a camv 35s promoter with a duplicated enhancer region and an rbcs terminator . the orientation of the constructs ( sense or antisense ) was checked by restriction enzyme digest . transformation vectors containing chimeric genes using full - length open reading frame cdnas encoding perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra proteins in sense and antisense orientations under the control of the camv 3552 promoter were generated ( fig1 , 120 , 125 , 130 , 135 , 140 , 145 , 150 , 155 , 160 , 165 , 170 , 175 , 180 , 185 , 190 and 195 ). production and analysis of transgenic arabidopsis plants carrying chimeric perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra genes involved in flavonoid biosynthesis a set of transgenic arabidopsis plants carrying chimeric perennial ryegrass and white clover genes involved in flavonoid biosynthesis , protein binding , metal chelation , anti - oxidation , uv - light absorption , tolerance to biotic stresses such as viruses , micro - organisms , insects and fungal pathogens ; pigmentation in for example flowers and leaves ; herbage quality and bloat - safety and isoflavonoid content leading to health benefits , were produced . ppzp221 - based transformation vectors with lpf3oh and trbana , trchia , trchid , trchrc , trchsa1 , trchsa3 , trchsc , trchsd2 , trchsf , trchsh , trdfrd , trf3ha , trpala , trpalb , trpalf and trvra cdnas comprising the full open reading frame sequences in sense and antisense orientations under the control of the camv 35s promoter with duplicated enhancer region ( 35s2 ) were generated as detailed in example 6 . agrobacterium - mediated gene transfer experiments were performed using these transformation vectors . the production of transgenic arabidopsis plants carrying the perennial ryegrass f3oh and white clover bana , chia , chid , chrc , chsa1 , chsa3 , chsc , chsd2 , chsf , chsh , dfrd , f3ha , pala , palb , palf and vra cdnas under the control of the camv 35s promoter with duplicated enhancer region ( 35s2 ) is described here in detail . seedling punnets were filled with debco seed raising mixture ( debco pty . ltd .) to form a mound . the mound was covered with two layers of anti - bird netting secured with rubber bands on each side . the soil was saturated with water and enough seeds ( arabidopsis thaliana ecotype columbia , lehle seeds # wt - 02 ) sown to obtain approximately 15 plants per punnet . the seeds were then vernalised by placing the punnets at 4 ° c . after 48 hours the punnets were transferred to a growth room at 22 ° c . under fluorescent light ( constant illumination , 55 μmolm - 2s - 1 ) and fed with miracle - gro ( scotts australia pty . ltd .) once a week . primary bolts were removed as soon as they appeared . after 4 - 6 days the secondary bolts were approximately 6 cm tall , and the plants were ready for vacuum infiltration . agrobacterium tumefaciens strain agl - 1 were streaked on lb medium containing 50 μg / ml rifampicin and 50 μg / ml kanamycin and grown at 27 ° c . for 48 hours . a single colony was used to inoculate 5 ml of lb medium containing 50 μg / ml rifampicin and 50 kanamycin and grown over night at 27 ° c . and 250 rpm on an orbital shaker . the overnight culture was used as an inoculum for 500 ml of lb medium containing 50 μg / ml kanamycin only . incubation was over night at 27 ° c . and 250 rpm on an orbital shaker in a 21 erlenmeyer flask . the overnight cultures were centrifuged for 15 min at 5500 × g and the supernatant discarded . the cells were resuspended in 1 l of infiltration medium [ 5 % ( w / v ) sucrose , 0 . 03 % ( v / v ) silwet - l77 ( vac - in - stuff , lehle seeds # vis - 01 )] and immediately used for infiltration . the agrobacterium suspension was poured into a container ( décor tellfresh storer , # 024 ) and the container placed inside the vacuum desiccator ( bel art , # 42020 - 0000 ). a punnet with arabidopsis plants was inverted and dipped into the agrobacterium suspension and a gentle vacuum ( 250 mm hg ) was applied for 2 min . after infiltration , the plants were returned to the growth room where they were kept away from direct light overnight . the next day the plants were returned to full direct light and allowed to grow until the siliques were fully developed . the plants were then allowed to dry out , the seed collected from the siliques and either stored at room temperature in a dry container or used for selection of transformants . prior to plating the seeds were sterilized as follows . sufficient seeds for one 150 mm petri dish ( approximately 40 mg or 2000 seeds ) were placed in a 1 . 5 ml microfuge tube . five hundred microliters ( 500 μl ) 70 % ethanol were added for 2 min and replaced by 500 μl sterilization solution ( h2o : 4 % chlorine : 5 % sds , 15 : 8 : 1 ). after vigorous shaking , the tube was left for 10 min after which time the sterilization solution was replaced with 500 μl sterile water . the tube was shaken and spun for 5 sec to sediment the seeds . the washing step was repeated 3 times and the seeds were left covered with approximately 200 μl sterile water . the seeds were then evenly spread on 150 mm petri dishes containing germination medium ( 4 . 61 g murashige & amp ; skoog salts , 10 g sucrose , 1 ml 1 m koh , 2 g phytagel , 0 . 5 g mes and 1 ml 1000 × gamborg &# 39 ; s b - 5 vitamins per litre ) supplemented with 250 μg / ml timetin and 75 μg / ml gentamycin . after vernalisation for 48 hours at 4 ° c . the plants were grown under continuous fluorescent light ( 55 μmol m - 2s - 1 ) at 22 ° c . to the 6 - 8 leaf stage and transferred to soil . three to four leaves of arabidopsis plants regenerated on selective medium were harvested and freeze - dried . the tissue was homogenized on a retsch mm300 mixer mill , then centrifuged for 10 min at 1700 × g to collect cell debris . genomic dna was isolated from the supernatant using wizard magnetic 96 dna plant system kits ( promega ) on a biomek fx ( beckman coulter ). 5 μl of the sample ( 50 μl ) were then analyzed on an agarose gel to check the yield and the quality of the genomic dna . genomic dna was analyzed for the presence of the transgene by real - time pcr using sybr green chemistry . pcr primer pairs ( table 5 ) were designed using macvector ( accelrys ). the forward primer was located within the 35s2 promoter region and the reverse primer within the transgene to amplify products of approximately 150 - 250 by as recommended . the positioning of the forward primer within the 35s2 promoter region guaranteed that homologous genes in arabidopsis were not detected . 5 μl of each genomic dna sample was run in a 50 μl pcr reaction including sybr green on an abi ( applied biosystems ) together with samples containing dna isolated from wild type arabidopsis plants ( negative control ), samples containing buffer instead of dna ( buffer control ) and samples containing the plasmid used for transformation ( positive plasmid control ). plants were obtained after transformation with all chimeric constructs and selection on medium containing gentamycin . the selection process and two representative real - time pcr analyses are shown in fig1 . genetic mapping of perennial ryegrass genes involved in flavonoid biosynthesis , protein binding , metal chelation , anti - oxidation , uv - light absorption , tolerance to biotic stresses such as viruses , micro - organisms , insects and fungal pathogens ; pigmentation in for example flowers and leaves ; herbage quality and bloat - safety and isoflavonoid content leading to health benefits the cdnas representing genes involved in flavonoid biosynthesis , protein binding , metal chelation , anti - oxidation , uv - light absorption , tolerance to biotic stresses such as viruses , micro - organisms , insects and fungal pathogens ; pigmentation in for example flowers and leaves ; herbage quality and bloat - safety and isoflavonoid content leading to health benefits , were amplified by pcr from their respective plasmids , gel - purified and radio - labelled for use as probes to detect restriction fragment length polymorphisms ( rflps ). rflps were mapped in the f1 ( first generation ) population , na6 × au6 . this population was made by crossing an individual ( na6 ) from a north african ecotype with an individual ( au6 ) from the cultivar aurora , which is derived from a swiss ecotype . genomic dna of the 2 parents and 114 progeny was extracted using the 1 × ctab method of fulton et al . ( 1995 ). probes were screened for their ability to detect polymorphism using the dna ( 10 μg ) of both parents and 5 f1 progeny restricted with the enzymes drai , ecori , ecorv or hindiii . hybridizations were carried out using the method of sharp et al . ( 1988 ). polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme ( fig1 ). rflp bands segregating within the population were scored and the data were entered into an excel spreadsheet . alleles showing the expected 1 : 1 ratio were mapped using mapmaker 3 . 0 ( lander et al . 1987 ). alleles segregating from , and unique to , each parent , were mapped separately to give two different linkage maps . markers were grouped into linkage groups at a lod of 5 . 0 and ordered within each linkage group using a lod threshold of 2 . 0 . loci representing genes involved in flavonoid biosynthesis mapped to the linkage groups as indicated in table 6 and in fig1 . these gene locations can now be used as candidate genes for quantitative trait loci associated with flavonoid biosynthesis , protein binding , metal chelation , anti - oxidation , uv - light absorption , tolerance to biotic stresses such as viruses , micro - organisms , insects and fungal pathogens ; pigmentation in for example flowers and leaves ; herbage quality and bloat - safety and isoflavonoid content leading to health benefits . finally , it is to be understood that various alterations , modifications and / or additions may be made without departing from the spirit of the present invention as outlined herein . it will also be understood that the term “ comprises ” ( or its grammatical variants ) as used in this specification is equivalent to the term “ includes ” and should not be taken as excluding the presence of other elements or features . documents cited in this specification are for reference purposes only and their inclusion is not acknowledgment that they form part of the common general knowledge in the relevant art . an , g ., watson , b . d ., stachel , s ., gordon , m . p ., nester , e . w . 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