Patent Application: US-67349200-A

Abstract:
a method is disclosed for producing a transgenic plant with a modified inulin producing profile comprising in its genome a combination of one or more expressible 1 - sst enzyme encoding genes and one or more expressible 1 - fft enzyme encoding genes , wherein either of these genes or both of them comprise one or more recombinant genes containing one or more 1 - sst , respectively 1 - fft , enzyme encoding dna sequences of plant origin or an expressible homologous sequence thereof . the invention also relates to a method for modifying and controlling the inulin profile of plants and to a method for producing inulin from said transgenic plants . furthermore , a novel cdna sequence of a 1 - sst enzyme encoding gene of helianthus tuberosus and a novel cdna sequence of a 1 - fft enzyme encoding gene of cichorium intybus are disclosed , novel recombinant dna constructs and genes derived thereof , as well as novel combinations of expressible 1 - sst and 1 - fft enzyme encoding genes . moreover , the invention also relates to novel polypeptides , homologues thereof and fragments thereof , which have 1 - sst activity of 1 - fft activity , and to antibodies capable of specifically binding one or more of them .

Description:
the present invention results in significant technical and economical advantages . at first , the invention provides novel plant sources of inulin thus helping to solve the problem of sufficiency of inulin supply . said plant sources may comprise known inulin producing plants which as a result of a genetically modification according to the invention produce more inulin and / or inulin of a preferred modified profile entailing improved functional properties , as well as non - inulin producing plants which have been transformed to produce inulin , particularly inulin having a desired profile . secondly , the method according to the present invention enables to obtain directly , i . e . without any additional process steps such as e . g . size fractioning or partial hydrolysis , inulin with a certain desirable inulin profile , such as inulin essentially composed of inulin chains with a dp ≦ 10 . this obviously has considerable advantages with respect to manufacturing and manufacturing costs . furthermore , the invention provides the possibility of producing many transgenic plant species and variants thereof within the scope of the present invention , resulting from the flexibility of the method of the present invention for producing a transgenic plant with a modified inulin producing profile . indeed , the possibility to produce many different transgenic plants , starting from different host plant species and by using various different combinations of the 1 - sst and / or 1 - fft enzyme encoding sequences according to the invention , i . e . including different 1 - sst , respectively 1 - fft enzyme encoding dna sequences originating from various different plant species or homologous sequences thereof , as defined above , and different ratio &# 39 ; s of said 1 - sst , respectively 1 - fft enzyme encoding dna sequences , enables to produce various inulin compositions with a different , desired profile . said desired inulin profile is often translated in improved functional properties of the inulin and commonly in improved physico - chemical and / or organoleptic properties of end products containing said inulin , and of hydrolysates and of derivatives of said inulin . the invention enables furthermore to produce transgenic plant species like for example cultural crops and ornamental plants with an increased resistance against abiotic stresses like e . g . drought and / or cold , which also results in economically important advantages . the polypeptides , homologues and fragments thereof , as defined above , may be used to catalyse specific in vitro chemical reactions , such as , for example , the use of polypeptide of seq id no : 2 in the synthesis of 1 - kestose from sucrose by a fructosyl transfer reaction ; the use of polypeptide of seq id no : 4 in the synthesis of fructo - oligosaccharides or inulin by a fructosyl transfer reaction from fructo - oligosaccharides ; and the use of a combination of the polypeptides of seq id no : 2 and seq id no : 4 , or respective homologues or fragments thereof as defined above , for the synthesis of fructo - oligosaccharides and inulin from one or more carbohydrates selected from the group consisting of sucrose , 1 - kestose and oligofructoses . the invention is further described in detail in the experimental part below , comprising the methodology and examples . it is emphasised that the examples are given as merely illustrative , non - limitative examples of the invention . dna and rna isolation , subcloning , restriction analysis and sequencing were performed using standard methods described in molecular biology manuals ( e . g . sambrook et al . 1989 ; ausubel et al . 1994 ). dna and protein alignments were performed using the dna analysis software genworks 2 . 5 . 1 . ( intelligenetics , inc ., ca ). this program uses a progressive alignment method , building the alignment in approximate phylogenetic order using an algorithm similar to fasta . protein alignments use a pam - 250 scoring matrix . construction and screening of a cdna library from h . tuberosus ten μg of poly ( a ) + rna isolated from tubers of helianthus tuberosus ‘ colombia ’, harvested in july , was used as starting material for the construction of an uni - zap xr cdna library ( stratagene , la jolla , calif ., usa ). construction , plating and screening of the library were performed according to the protocols developed by stratagene ( usa , cat . no . 237211 ). about 100 . 000 plaques of the unamplified cdna library of h . tuberosus were blotted onto hybond n + ( amersham ) membranes and screened with a probe comprising two dna fragments . a 840 bp dna fragment was synthesised by pcr using primers 5 ′- tacgctgtcaactcgtcgg - 3 ′ and 5 ′- ttgaatagaacatcgggctctagcg - 3 ′ and plasmid psst103 ( sst103 cdna in pbluescript phagemid , van der meer et al . 1998 ) as a template . a 860 bp dna fragment was obtained by pcr using primers 5 ′- gttcaacgctgcttgatccacc - 3 ′ and 5 ′- accacggtccttccaaacgg - 3 ′ and plasmid pfft111 ( fft111 cdna in pbluescript phagemid van de meer et al ., 1998 ) as a template . the two pcr fragments were radiolabelled by the radprime dna labelling system ( life technologies ). hybridisation was at 50 ° c . in 500 mm nap - buffer , ph 7 . 2 , 7 % sds , 1 % bsa , 1 mm edta . filters were washed to a final stringency of 2 × ssc , 0 . 1 % sds at 50 ° c . ( 2 × 30 min ) and finally exposed to x - omat ar ( kodak ). positive plaques were purified in 34 rounds of plaque hybridisation . the pbluescript phagemids were excised from the uni - zap vector using the exassist / solr system ( stratagene ). the inserts were analysed by restriction enzyme analysis and sequencing . construction and screening of a cdna library from cichorium intybus ten μg of poly ( a ) + rna isolated from tap roots of cichorium intybus ‘ cassel ’, which was harvested in july , was used as starting material for the construction of a lambda triplex cdna library ( clontech laboratories ). construction , plating and screening of the library were performed according to the protocols developed by clontech ( palo alto , calif ., cat . no . cs1010t ). about 60 . 000 plaques of the unamplified chicory library were screened with a mixture of the two 32 p - labelled dna probes obtained by pcr and radprime labelling as described above . hybridisation and washing of hybond - n + membranes were performed under low stringency conditions ( hybridisation at 50 ° c ., final wash step with 2 × ssc , 0 . 1 % sds , 50 ° c .). positive plaques were purified in 3 - 4 rounds of plaque hybridisation . the lambda trip1ex clones were converted into ptriplex clones using the cre - recombinase mediated site - specific recombination at the loxp sites flanking the embedded ptriplex ( clontech ). the ptriplex clones were analysed by restriction enzyme analysis and dna sequencing . pcr was performed in 50 μl pcr buffer ( life technologies ), containing 100 pmol plasmid as a template , and 100 pmol of gene specific primers ( specific for sst103 , fft111 , a33 , c33 , or c86b , depending on the experiment ). amplification involved 30 cycles of denaturing ( 0 . 5 min , 92 ° c . ), annealing ( 1 min , 55 ° c .) and amplification ( 1 min , 72 ° c .). the resulting fragments were checked by dna sequencing and restriction digestion to confirm the identity . transformation of potato was performed according to visser ( 1991 ), with the following modifications . stem internodes , cut from in vitro grown solanum tuberosum , were placed in r 3 b agar plates , on top of filter paper , which was soaked with 2 ml pacm medium . the internodes were incubated 24 h at 21 ° c . the binary plasmid pa33 . 236 was introduced into the agrobacterium tumefaciens strain ‘ agl0 ’ ( lazo et al . 1991 ) by adding 0 . 5 microgram of plasmid dna to 200 microliter of competent agrobacterium cells . competent cells were prepared according to sambrook ( 1989 ). the plasmid dna - agrobacterium mixture was incubated on ice , for 30 min , then frozen in liquid nitrogen and thawed in a water bath at 37 ° c . for 5 min . after addition of 1 ml yep medium , the bacteria were incubated at 28 ° c . for 2 hours with gentle shaking . cells were pelleted and resuspended in 100 μl yep - medium . finally , transformed bacteria were selected on yep - agar plates containing 25 mg / l kanamycin . the presence of pa33 . 236 was tested by restriction enzyme analysis . a . tumefaciens cells , transformed with pa33 . 236 were grown at 30 ° c . in 5 ml lb medium , containing 50 μg / ml kanamycin and 100 μg / ml rifampicin . after 48 h of growth , the cells were washed twice in 5 ml 2 mm mgso 4 , then suspended in 5 ml ms medium . stem intemodes were incubated in 20 ml of a diluted ( 100 × in ms ) a . tumefaciens suspension , and gently shaken for 30 min . after incubation , the stem internodes were blotted dry on filter paper and placed on top of pcm - agar plates ( 48 h at 21 ° c .). callus formation was induced by transferring the internodes to pcm agar plates , containing 200 mg / l cefotaxime and 100 mg / l kanamycine ( 96 h at 21 ° c .). shoot formation was induced at 21 ° c . by transferring the stem internodes to psm - agar , containing 200 mg / l cefotaxime and 100 mg / l kanamycin . regenerating explants were transferred to fresh medium every three weeks . root formation was induced by transferring the transgenic shoots to ms 30 medium , containing 200 mg / l cefotaxime and 50 mg / l kanamycine . rooted plantlets of 5 - 10 cm were transferred to the greenhouse . total dna was isolated from mature young mature leaves which were harvested 3 months after transfer of the plants to the greenhouse . aliquots of the dna was digested with a number of restriction enzymes . total rna was isolated from stolons , tubers of various ages , stems , leaves and flower tissues . after electrophoresis on 1 % agarose , dna or rna was blotted onto hybond - n + ( amersham ) and uv cross - linked . filters were hybridised with either an fft111 , sst103 ( see construction and screening of a cdna library from h . tuberosus ) or an a33 probe . a 1140 bp dna fragment specific for a33 was synthesised by pcr using primers 5 ′- caacccaattctcttccctcctccg - 3 ′ and 5 ′- acaaacactitgggcggc - 3 ′ and plasmid pa33 ( a33 cdna in pbluescript ) as a template . the gene specific dna fragments were radio labelled with alpha 32 p - atp using the radprime kit ( life technologies ). hybridisation was at 65 ° c . in 500 mm nap - buffer , ph 7 . 2 , 7 % sds , 1 % bsa , 1 mm edta . filters were washed to a final stringency of 0 . 1 × ssc , 0 . 1 % sds at 65 ° c . ( 2 × 15 min ) and finally exposed to x - omat ar ( kodak ). transgenic sugar beets ( beta vulgaris ) were generated by a stomatal guard cell based transformation system ( hall et . al . 1996 ). guard cell protoplasts were obtained from shoot cultures of the diploid breeding line bv - nf ( hall et . al . 1996 ). one million guard cell protoplasts were transformed with 50 μg of the plasmid in the presence of peg . regeneration and selection of transformants was as described ( hall et . al . 1996 ). plants were grown in a greenhouse at 18 / 15 ° c . ( day / night ) under a 16 h photoperiod . two months old leaves were cut to prevent spread with powdery mildew . total dna was isolated from young mature leaves which were harvested 3 months after transfer of the plants to the greenhouse . dna was digested with specific restriction enzymes . total rna was isolated from 2 - 5 month old tap roots or tubers . after electrophoresis on 1 % agarose , dna or rna was blotted onto hybond - n +( amersham ) and uv cross - linked . filters were hybridised with a gene specific probe . the dna fragment was radio labelled with alpha 32 p - atp using the radprime kit ( life technologies ). hybridisation was at 65 ° c . in 500 mm nap - buffer , ph 7 . 2 , 7 % sds , 1 % bsa , 1 mm edta . filters were washed to a final stringency of 0 . 1 × ssc , 0 . 1 % sds at 65 ° c . ( 2 × 15 min ) and finally exposed to x - omat ar ( kodak ). leaf and root tissues were ground in liquid nitrogen to a fine powder . five hundred mg of the powdered tissue were extracted in 1 ml 50 mm bistris ph 5 . 5 , 1 mm edta , 1 mm mgso 4 , 1 mm dtt , 1 mm pmsf , 20 mm na - metabisulfite and 15 g 1 − 1 pvpp . the extract was centrifuged ( 20 min at 10000 × g ) and the supernatant desalted and concentrated by centrifugation through centriprep 30 ultrafiltration devices ( amicon , breda , the netherlands ). a 240 μl aliquot was mixed with 240 μl assay mixture , containing either 100 mm sucrose , gf 2 or gf 3 in 20 mm bistris ph 5 . 5 , 2 mm dtt and 0 . 01 % na - azide t ( w / v ). after 8 , 16 and 24h of incubation , an aliquot of the reaction mixture was boiled for 5 min and store at − 20 ° c . the assay mixtures were then analysed by hpaec as described below . the inulin composition of transgenic plants beet was measured by tlc ( thin layer chromatography ) and hpaec ( high performance anion exchange chromatography ). two to five months after transfer of the plants to the greenhouse , fresh plant material ( 500 mg ) was collected and cut into 2 mm thick slices with a sterile razor blade . a 20 mm phosphate buffer , ph 7 . 0 , of 100 ° c . was added to the slices to a final volume of 3 ml , and kept at 85 ° c . for 30 min , with occasional vortexing . the extract was centrifuged at 14000 rpm and the supernatant collected . tlc analysis was performed on silica gel g ( schleicher and schuell nederland bv , the netherlands ) developed two times in a mixture of 1 - butanol : 2 - propanol : water 3 : 12 : 4 . carbohydrates were stained with a fructose - specific urea phosphoric spray ( wise et al ., 1955 ). for hpaec analysis , the extracts were deionized with a mixture of equal amounts of q - sepharose and s - sepharose fast flow ( pharmacia , upssala , sweden ), which were pre - equilibrated in 20 mm phosphate buffer , ph 7 . 0 . the ion exchanger was added to 50 % of the total extract volume , mixed for 5 min at 600 rpm , then centrifuged at 14000 rpm for 2 min . the supernatant was analysed by high pressure anion exchange chromatography / pulsed amperometric detection ( hpaec - pad , dionex , the netherlands ) equipped with 250 × 4 mm carbopac pa1 anion exchange column and a 25 × 3 mm carbopac pa guard column . inulins were separated with a 80 min linear gradient of 0 to 0 . 4 mol m − 3 naac in 0 . 1 mol m − 3 naoh at a flow rate of 1 ml min − 1 , or over 85 minutes with an aqueous gradient ( a : naoh 0 . 1 mol m − 3 ; b : ( 0 . 1 mol m − 3 naoh + 0 . 4 mol m − 3 naac ); c : naoh 1 mol m − 3 ) as follows : min 0 - 5 : a : b 96 : 4 ; min 5 - 15 linear gradient a : b from 96 : 4 to 60 : 40 ; min 15 - 35 : linear gradient a : b from 60 : 40 to 30 : 70 ; min 35 - 50 linear gradient a : b from 30 : 70 to 10 : 90 ; min 50 - 60 linear gradient a : b from 10 : 90 to 0 : 100 ; min 60 - 85 a : b 0 : 100 ; followed by regeneration min 0 - 5 with a : b : c : 0 : 0 : 100 and min 5 - 30 with a : b 96 : 4 , at a flow rate of 1 ml min − 1 . standard grade inulin was used as a standard . cgc analysis was made according to the method described by l . de leenheer et al ., starch - stärke , ( 1994 ), vol . 46 , p . 193 - 196 . isolation of a new cdna from the cdna library of helianthus tuberosus homologous to sst 103 and fft111 from helianthus tuberosus an uni - zap cdna library , constructed from mrna isolated from h . tuberosus tubers , was screened with a mixture of a 840 bp sst103 and a 860 bp fft111 fragment . the sst103 specific fragment was obtained by pcr using primers 5 ′- tgtcagcccatcccttggaaagg - 3 ′ and 5 ′- tacgctgtcaactcgtcgg - 3 ′ and psst103 ( van der meer et al ., 1998 ) as template . the fft111 specific fragment was obtained by pcr using 5 ′- gttcaacgctgcttgatccacc - 3 ′ and 5 ′- accacggtccttccaaacgg - 3 ′ and pfft111 ( van der meer et al , 1998 ) as template . screening of about 100 . 000 cdna clones yielded about 1200 positive clones , most of them most probably representing either a sst103 or a fft111 cdna . only the lambda zap clones giving a weak positive signal ( 84 ) were picked from the primary screening . after 34 rounds of purification , 11 clones were left . from these clones , the pbluescript phagemids were excised from the uni - zap vector and the cloned insert characterised by restriction enzyme analysis and sequencing . one clone with an about 2 kb insert , designated a33 , was fully sequenced . the dna sequence of a33 ( sequence id . no . 1 ) and the corresponding amino acid sequence ( sequence id . no . 2 ) is presented in fig1 . sequence id . no . 1 , designated a33 has an open reading frame of 1845 base pairs and encodes a protein of 615 amino acid residues ( sequence id . no . 2 ). on dna level , a33 shows a 70 % identity with the 1 - sst encoding sst103 cdna sequence from h . tuberosus and 54 % identity with the 1 - fft encoding , fft111 cdna sequence from h . tuberosus . at the amino acid level , a33 shows a 75 % similarity with sst103 from h . tuberosus and a 58 % similarity with fft111 from h . tuberosus . isolation of a new cdna from the cdna library of cichorium intybus homologous to sst103 and fft111 from helianthus tuberosus a lambda triplex cdna library , constructed from mrna isolated from the tap roots of c . intybus , was screened with a mixture of a 840 bp sst103 and a 860 bp fft111 fragment ( see example 1 ). the primary screening of about 60 . 000 cdna clones yielded about 700 positive clones , of which 96 clones were picked . after 3 - 4 rounds of purification 81 clones were left . from 55 clones , the ptriplex phagemids were excised from lambda trip1ex vector and the cloned insert characterised by restriction enzyme analysis and sequencing . the dna sequence of one of the clones , designated c86b ( sequence id . no . 3 ) and the corresponding amino acid sequence ( sequence id . no . 4 ) is presented in fig2 a . sequence id . no . 3 , designated c86b , has an open reading frame of 1851 base pairs and encodes a protein of 617 amino acid residues ( sequence id . no . 4 ). on dna level , c86b shows a 61 % identity with sst103 from h . tuberosus and 77 % identity with fft111 from h . tuberosus . at the amino acid level , c86b shows a 53 % similarity with sst103 from h . tuberosus and a 78 % similarity with fft111 from h . tuberosus . the dna sequence of another clone , designated c33 ( sequence id . no . 5 ) and the corresponding amnino acid sequence ( sequence id . no . 6 ) is presented in fig2 b . sequence id . no . 5 designated c33 , has an open reading frame of 1920 base pairs and encodes a protein of 640 amino acid residues ( sequence id . no . 6 ). on dna level , c33 shows a 97 % identity with 1 - sst from chicory ( genbank accession no u81520 ). on amino acid level c33 shows a 88 % /( similarity with 1 - sst from chicory ( genbank no u81520 ). analysis of genomic organisation and expression of a33 in helianthus tuberosus to estimate the number of a33 genes present in the jerusalem artichoke genome , southern blot analysis of digested h . tuberosus dna was performed , using a radio labelled a33 fragment of 1140 bp as a probe . ( plant journal 15 , 489 - 500 ). under stringent conditions ( hybridisation at 65 ° c . and washing at 65 ° c ., 0 . 1 × ssc , 0 . 1 % sds ), only one , maybe two fragments of the aflii digest hybridises to the a33 probe ( fig3 ), suggesting that only one , maybe two a33 genes are present in the jerusalem artichoke genome . transcript levels o a33 , sst103 and fft111 were studied in different organs and different developmental stages of the tubers of jerusalem artichoke ( fig4 ). in correspondence to earlier experiments ( van der meer 1998 ), data in fig4 a shows that sst103 is highly expressed in tubers , and to a less extent in fibrous roots , flowers and receptacle . the expression pattern of fft111 ( fig4 b ) resembles that of sst103 , although in general the expression of fft111 is 3 - 10 times lower than that of sst103 . in contrast to sst103 and fft111 , a33 is not expressed to a measurable level in any of the tested tissues ( fig4 c ). to quantify the a33 , sst103 and fft111 expressing in tubers , 100 . 000 plaques of the cdna library of jerusalem artichoke tubers were blotted onto hybond filters , hybridised to either an a33 , sst103 or fft111 probe at 65 ° c ., then washed at high stringency ( 65 ° c ., 0 . 1 × ssc , 0 . 1 % sds ). expression levels of a33 , sst103 or fft111 in tubers were 0 . 001 , 1 and 0 . 2 %, respectively . if a33 encodes a functional fructosyltransferase protein , it does not effectively contribute to inulin synthesis in tubers of jerusalem artichoke . from a functional point of view , a33 can be considered as a silent gene . construction of a chimeric a33 gene construct for transformation into potato by pcr , using primers 5 ′- atcaacatgtcttccacccc - 3 ′ and 5 ′- tggatcctcaaggccgccctc - 3 ′, an afliii site was introduced at the first atg ( start of the open reading frame ) and a bamhi site was introduced down - stream of the stop codon of the full length a33 cdna clone ( pa33 ), isolated from jerusalem artichoke . the newly obtained a33 pcr fragment was cloned into pmosblue ( amersham life science ) yielding pmosa33 . 10 . from the plasmid pfft405 ( see further , for a complete description ), which contains the enhanced cauliflower mosaic virus 35s promoter ( enh35s ), alfalfa mosaic virus rna4 leader sequence ( amv ), cdna fft111 from jerusalem artichoke and the nos terminator sequence ( tnos ), the complete fft111 sequence was replaced by the a33 pcr fragment . the complete a33 pcr fragment , resulting from a digestion of pmosa33 . 10 with afliii and bamhi ( partial ), was ligated into pfft405 , from which the fft111 was removed by a digestion with ncoi and bamhi ( partial ). replacing the fft111 cdna in pfft405 with the a33 pcr fragment yielded pa33 . 102 . the enh35s - amv - a33 - tnos - fragment was cut from pa33 . 102 with saci and sali , and ligated into the plant transformation vector pbinplus ( van engelen et al ., 1995 ) digested with saci / sali , which resulted in pa33 . 236 ( fig5 ). pfft 405 was obtained by cloning the penh35s - amv - fft111 - tnos - fragment cut from pfft209 ( van der meer et al ., 1998 ), with ecori ( partial digest ) and hindiii into pbluescript sk + ( stratagene , usa ) cut with ecori / hindiii . about 25 transgenic potato plants were generated harbouring the pa33 . 236 construct . ten potato plants were transformed with the agrobacterium strain agl0 lacking a binary vector . these plants were used as a control . southern blot analysis of genomic dna isolated from 22 transformed plants showed that 17 plants has integrated into their genome less than 3 copies of the introduced chimeric gene ( data not shown ). plant numbers 23 , 27b , 74 , 84 and 93 had integrated one copy . northern analysis showed that a33 was highly expressed in plants numbers 27b , 74 and 93 , and less , but still clearly expressed in plant numbers 23 and 84 . the carbohydrate composition of the a33 harbouring plants was analysed by two essentially different techniques : thin layer chromatography ( tlc ) and high pressure anion exchange chromatography ( hpaec ). analysis of leaf extracts from the potato harbouring the pa33 . 26 construct showed that plant numbers 23 , 27b , 50 , 83 and 93 accumulate a range of fructose containing compounds in leaves and tubers ( fig6 ). a comparison with the inulin standard extracted from jerusalem artichoke , containing inulins up to a dp of 30 , shows that the tuber extracts accumulate inulins up to a degree of polymerisation of at least 10 ( fig6 ), whereas the leaves accumulate inulins with a dp of 3 , 4 and 5 . the presence of inulins in leaf extract and tuber extracts of the a33 - harbouring plants is confirmed through hpaec analysis . hpaec indicates that plant numbers 23 , 27b , 93 ( only 93 is shown in fig7 ) accumulate inulins up to a degree of polymerisation of 11 . this clearly indicates that a33 encodes a fructosyltransferase enzyme , able to synthesise inulins up to a dp of at least 11 . since the a33 encodes an enzyme that can catalyse all steps required for inulin synthesis , including reaction ( 1 ), the conversion of sucrose into gef , the a33 encoded enzyme ( a33 ) belongs to the group of sst encoding genes . construction of a chimeric a33 gene construct for transformation into sugar the complete chimeric a33 gene ( penh35s - amv - a33 - tnos ) was cut from pa33 . 102 with noti and sali and ligated into pucm2 , digested with noti / sali , resulting in puca33 . 1 . plasmid pucm2 is derived from the cloning vector pucap ( van engelen et al . 1995 ). to construct plasmid pucm2 the multiple - cloning - site of plasmid pucap was modified by the insertion of two adapters . first , adapter 5 ′- tcgaccatatcgatgcatg - 3 ′/ 5 ′- catcgctattgg - 3 ′, containing the restriction sites sali / clai / sphi , was cloned in the pucap plasmid digested with sahi / sphi . this resulted in plasmid pucm1 . next , adapter 5 ′- taagcggccgcagatctgg - 3 ′/ 5 ′- aattccagatctgcggccgcttaat - 3 ′, consisting of paci / noti / bglii / ecori restriction sites , was cloned in plasmid pucm1 digested with paci / ecori . this resulted in plasmid pucm2 . the complete chimeric a33 gene ( penh35s - amv - a33 - tnos ) was cut from puca33 . 1 with noti and asci and ligated into pucpat 34 digested with noti / asci , yielding pucpa33 . 342 ( fig8 ). pucpa33 . 342 was used to transform guard cell protoplasts of sugar beet . construction of a chimeric a33 - c86b gene for transformation into sugar beet the complete chimeric c86b gene ( penh35s - amv - c86b - tnos ), which was cut from pc86b . 2 with ecori and clai , and cloned into pucpat . 34 ( see example 8 ), was digested with ecor1 ( partial digest ) and clai . this resulted in pucpc86b . 342 . the noti / asci fragment of puca33 . 1 , containing the full length a33 - cdna ( a33 ) was ligated into pucpc86b . 342 cut with noti / asci , yielding pucpca342 . 25 . ( fig9 ). pucpca342 . 25 was used to transform guard cell protoplasts of sugar beet . construction of a chimeric sst103 - fft111 gene for transformation into sugar beet the pat gene , encoding phosphinotricin acetyl transferase ( agrevo , berlin , germany ), which confers bialaphos resistance , was cut from pigpd7 ( hall et al ., 1996 ) with ecor1 and ligated into pucm3 , digested with ecori , which yielded pucpat34 . to construct plasmid pucm3 , plasmid pucap was digested with paci / asci and the whole multiple - cloning - site was replaced by adapter 5 ′- taaggggtaccaccatcgataccgaattctacatgcatgcatggagatctcccaagcttctaagatgcggccgctaaacatgg - 3 ′/ 5 ′- cgcgccatgtttagcggccgcatcttagaagcttgggagatctccatgcatgcatgtagaattcggtatcgatggtggtaccccttaat - 3 ′ the complete chimeric sst103 gene ( penh35s - amv - sst103 - tnos ) was cut from psst403 ( van der meer et al ., 1998 ) with ecori and clai and ligated into pucm1 , digested with ecori / clai , which resulted in pucst21 . the complete chimeric fft111 gene ( penh35s - amv - fft111 - tnos ) cut from pfft405 with noti and clai was ligated into pucm2 , cut with noti / clai , resulting in pucft2l . the complete chimeric sst103 gene was cut from pucst21 with paci and clai and introduced into pucpat34 , digested with paci / clai , yielding pucps34 . 4 . the complete chimeric fft111 gene was cut from pucft21 with noti and asci and ligated into pucps34 . 4 , digested with noti / asci , yielding pucpsf344 . 18 ( fig1 ). pucpsf 344 . 18 was used to transform guard cell protoplasts of sugar beet . construction of a chimeric sst103 - c86b gene for transformation into sugar beet using pcr and primer 5 ′- cctcgaaccatggaaacagc - 3 ′ an ncoi was introduced at the first atg ( start of the open reading frame ) of the full length c86b cdna clone . a bamhi site was introduced downstream of the stop codon of c86b , using primer 5 ′- taataaaagaggatcctcatgaaacg - 3 ′. this c86b pcr fragment ( 1895 bp ) was cloned into pcr - script amp sk (+) ( stratagene , usa ) yielding p86bpcrscp . from the plasmid pfft405 , which contains the enhanced cauliflower mosaic virus 35s promoter ( penh35s ), alfalfa mosaic virus rna4 leader sequence ( amv ), fft111 cdna clone from jerusalem artichoke and the nos terminator sequence ( tnos ), the complete fft111 sequence was replaced by the c86b pcr fragment . the c86b fragment , cut from p86bpcrscp with ncoi and bamhi , was ligated into pfft405 cut with ncoi and bamhi , yielding pc86b2 . the noti / clai fragment of pc86b2 ( enh35s - amv - sst103 - tnos ) was cloned into pucm2 cut with noti / clai , yielding puc86b2 . the noti / asci fragment of puc86b2 was cloned into the pucps344 digested with noti / asci , resulting in pucpsc344 . 25 ( fig1 ). pucpsc344 . 25 was used to transform guard cell protoplasts of sugar beet . construction of a chimeric a33 - fft111 gene for transformation into sugar beet from the plasmid pucpa33 . 342 , the complete recombinant a33 gene ( penh35s - amv - sst103 - tnos ) and the complete pat cassette were excised with clai , then introduced into pucft21 cut with clai , yielding pucpaf21 . 17 ( fig1 ) two fructosyltransferase encoding cdnas from h . tuberosus ( sst 103 and fft111 ), and the pat gene conferring bialaphos resistance were cloned into the pucm3 vector . the resulting plasmid pucpsf344 . 18 ( fig1 ) was used to transform stomatal guard cell protoplasts of sugar beet . bialophos - resistent calli were obtained after bialophos selection . regeneration and selection of transformants was as described ( hall et al . 1996 ). transgenic sugar beets were analyzed by northern analysis ( fig1 ): as the sst103 probe a dna fragment was used which was prepared by pcr using primers 5 ′- ttgaatagaacatcgggctctagcg - 3 ′ and 5 ′- tacgctgtcaactcgtcgg - 3 ′ and plasmid pucft21 ( example 8 ) as a template ; as the fft111 probe a dna fragment was used which was prepared by pcr using primers 5 ′- gttcaacgctgctggatccacc - 3 ′ and 5 ′- accacggtccttccaaacgg - 3 ′ and plasmid pucft21 ( example 8 ) as a template . line 7psf22 expressed both the sst103 and the fft111 gene ( fig1 , lane 1 ). this line was further analyzed by hpaec . the inulin profile can be described as a mixture of inulin molecules up to a dp of 9 ( fig1 ). the inulin profile of sugar beet line 7psf22 is essentially different from the inulin profile of transgenic sugar beet lines comprising only the sst103 gene ( sevenier et al . 1998 ). the sugar beet comprising only the sst103 gene accumulates inulins up to a dp of 5 ( gf 2 , gf 3 and gf 4 ; sevenier et al . 1998 ). the inulin profile of line 7psf22 is also different from the standard grade inulin from chicory ( fig1 ), which comprises of a mixture of inulins up to a dp of about 55 . the plasmid prepared according to the description in example 9 , was used to transform stomatal guard cell protoplasts of sugar beet . bialophos - resistent calli were obtained after bialophos selection . regeneration and selection of transformants was as described ( hall et al . 1996 ). transgenic sugar beets were analysed by northern analysis ( fig1 ): as a c86b probe a dna fragment was used , which was prepared by pcr using primers 5 ′- gtgaccttgaggatgcatcc - 3 ′ and 5 ′- tcggttgcacccgcgctcg - 3 ′ and plasmid puc86b2 ( example 9 ) as a template . line 9psc2 , expressing both the sst103 and the c86b gene ( fig1 , lane 2 ), was selected for further analysis by hpaec . the inulin profile of sugar beet line 9psc2 can be described as a polydisperse mixture of inulin molecules with a dp ranging from 3 to 15 ( fig1 ). the inulin profile of sugar beet line 9psc2 is essentially different from transgenic sugar beet lines comprising only the sst103 gene ( sevenier et al . 1998 ), but also from line 7psf22 comprising a combination of sst103 and fft111 , which accumulate inulin molecules up to a dp of 9 ( fig1 ). the inulin profile of line 9psc2 is also different from the standard grade inulin from chicory ( fig1 ), which comprises of inulins up to a dp of about 55 . the plasmid prepared according to the description in example 10 , was used to transform stomatal guard cell protoplasts of sugar beet . bialophos - resistent calli were obtained after bialophos selection . regeneration and selection of transformants was as described ( hall et al . 1996 ). transgenic sugar beets were analysed by northern analysis : as the a33 probe a dna fragment was used , which was prepared by pcr using primers 5 ′- caacccaattctcttccctcctccg - 3 ′ and 5 ′- acaaacactttcggccgcc - 3 ′ and plasmid puca33 . 1 ( example 6 ) as a template . lines 8paf1 and 8paf5 which were expressing both the a33 and the fft111 gene ( fig1 , lanes 3 and 4 , respectively ), were analysed by hpaec . sugar beet line 8paf5 accumulates a inuline molecules ranging from dp 3 - 22 ( fig1 ). sugar beet line 8paf1 accumulates a mixture of inulin molecules with a dp ranging from 3 to 26 ( fig1 ). in contrast to lines 7psf22 ( example 11 ), 9psc2 ( example 12 ) and 8paf5 ( example 13 ), in which gf 2 is invariably the most abundant inulin molecule , in line 8paf1 , the concentrations of gf 2 , gf 3 , gf 4 and gf 5 are in the same range , being 2 . 5 . 2 . 5 . 2 . 4 and 2 . 2 % of total dry matter , respectively . surprisingly , line 8paf1 also accumulates significant amounts of f 2 , f 3 , f 4 , f 5 , f 6 , f 7 , f 8 and f 9 , being 0 . 4 , 0 . 4 , 0 . 5 , 0 . 4 , 0 . 4 , 0 . 3 , 0 . 2 , 0 . 1 % of total dry weight , respectively ( cgc - analysis results ). the inulin profiles of sugar beet lines 8paf1 and 8paf5 are essentially different from from sugar line 7psf22 comprising a combination of sst103 and fft11 , which accumulate inuline molecules up to a dp of only 9 . the inulin profile of lines 8paf1 and 8paf5 are also different from the standard grade inulin from chicory ( fig1 ), which comprises of inulins up to a dp of about 55 . ausubel f m , brent r , kingston r e , moore d d , seidman j g , smith j a , and struhl k . 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( 1955 ). determination of easily hydrolysable fructose units in dextran preparations . anal . chem . 27 , 33 - 6 . sevenier r ., hall r . d ., van der meer i . m ., hakkert h . j . c ., van tunen a . j . and koops a . j ., 1998 . high level fructan accumulation in a transgenic sugar beet . nature biotechnology 16 : 843 - 846 . gcaaaaatca cc atg gct tcc acc ccc acc acc cct ctt att act cac aat 51 gac ctt gaa caa cgc ccg gaa tcg acc gag tct cca ccc ggt cga tca 99 tcc atc gta aag atc ctc act gga tta ttt gtg tcc att ctt gtt ctt 147 tca tca ttg gct gca ata aca cac cgg aaa act ccc ttg cag tcc acc 195 aca gtt gat att gaa cca tcg aca agc agt ccg aag gag gtt gtg gga 243 gcg gat gat agc att gaa tgg caa cga tct gct tac cat ttt caa ccc 291 ala asp asp ser ile glu trp gln arg ser ala tyr his phe gln pro gat aaa aat ttc att agc gat cct gat ggt cca ctg tat tac aag gga 339 tgg tac cac tta ttc tac caa tac aat ccg ggg tca gcc att tgg ggc 387 trp tyr his leu phe tyr gln tyr asn pro gly ser ala ile trp gly aac ata aca tgg ggt cat gca gtc tcg aaa gac ctc atc aat tgg ttc 435 asn ile thr trp gly his ala val ser lys asp leu ile asn trp phe cac ctc cct tta gcc atg gtt ccg gat cac tgg tac gac atc cat ggt 483 his leu pro leu ala met val pro asp his trp tyr asp ile his gly gtc atg act ggg tcc gcc acc atc ctc ccc aat ggc caa atc ttc atg 531 val met thr gly ser ala thr ile leu pro asn gly gln ile phe met ctt tat agc ggc aac gcc tac gac ctc tct cag ctt caa tgc ctc gcg 579 tac ccc aaa aat gct tct gat cca ctt ctt atc gaa tgg gtc aaa tac 627 tyr pro lys asn ala ser asp pro leu leu ile glu trp val lys tyr gaa ggc aac cca att ctc ttc cct cct ccg ggc gtg ggt ctc aaa gac 675 ttt agg gac ccg tca tct ctt tgg att ggg ccc gat ggg aag tac cga 723 phe arg asp pro ser ser leu trp ile gly pro asp gly lys tyr arg atg gtt atg ggc tcc aag cac aat aat aca att ggt tgt gct tta att 771 met val met gly ser lys his asn asn thr ile gly cys ala leu ile tac cac acc act aat ttc acc cat ttt gaa ttg ttg gat gag gtg ctc 819 cat tcg gtt cag ggt acg ggt atg tgg gaa tgt gtt gat ctt tac ccc 867 his ser val gln gly thr gly met trp glu cys val asp leu tyr pro gta tcc acg acc gag aca aac ggg ttg gat atg tcg aat cat gag tcg 915 ggt gct aag tat gtg ttg aag caa agt ggg gat gag gat aga cat gat 963 gly ala lys tyr val leu lys gln ser gly asp glu asp arg his asp tgg tat gca att ggg gca tat gac gtg gtt cat gat aaa tgg tat ccg 1011 gat gat ccg gaa atg gat ttg ggt atc ggg ttg aga tat gat tat gga 1059 aag ttt tat gct tca aag acg ttt tat gac ccg agt aag aag agg cgg 1107 gtc tta tgg ggc tat gtt ggt gaa acg gat cct caa aaa gat gac ctc 1155 val leu trp gly tyr val gly glu thr asp pro gln lys asp asp leu gag aaa gga tgg gcc aat att ttg aat gtt cct aga acc gtg gtg ttg 1203 glu lys gly trp ala asn ile leu asn val pro arg thr val val leu gac acg aag acg caa agt aac ttg att caa tgg ccg gtc gag gaa aca 1251 asp thr lys thr gln ser asn leu ile gln trp pro val glu glu thr gaa act ttg aga tct gaa gag tac gat gag ttc aaa gat gtt gag ttg 1299 cgg cct gga tca ctt gtc ccg ctt gat ata ggc tca gcc aca cag ttg 1347 arg pro gly ser leu val pro leu asp ile gly ser ala thr gln leu gac ata agt gcc tca ttc gag gtt gat gaa gct ttg ctg ggt gca acc 1395 tta gaa gcc gat gtg ttg ttc aac tgc acc acg agc gag ggt tca gcc 1443 leu glu ala asp val leu phe asn cys thr thr ser glu gly ser ala atg agg ggt gtt ttg gga ccg ttt ggg ctt gtg gtt ctt gca gat tcg 1491 gca ctt tca gaa caa act cct gtt tac ttc tac att gcg aaa aac ttg 1539 ala leu ser glu gln thr pro val tyr phe tyr ile ala lys asn leu gat ggc act tca aga act tat ttc tgt gct gat gaa tca aga tca tca 1587 aag ctt tta gat gtg ggc aag atg gta tat gga agc agt gtt cct gta 1635 ctc cat ggg gaa aac tac gac atg agg tta ttg gtg gat cat tca ata 1683 leu his gly glu asn tyr asp met arg leu leu val asp his ser ile gtc gaa agc ttt gca caa gga gga aga acg gtg att aca tca aga gtg 1731 tat cct aca atg gca atc tat gat gcc gcc aaa gtg ttt gtg ttc aac 1779 tyr pro thr met ala ile tyr asp ala ala lys val phe val phe asn aat gca act gga atc act gtt aag gca tct ctc aag att tgg aag atg 1827 asn ala thr gly ile thr val lys ala ser leu lys ile trp lys met ggt gga gca caa ctc aac cct ttt cct ttc taattagttt agttggcttc 1877 met ala ser thr pro thr thr pro leu ile thr his asn asp leu glu ala ala ile thr his arg lys thr pro leu gln ser thr thr val asp ser ile glu trp gln arg ser ala tyr his phe gln pro asp lys asn phe ile ser asp pro asp gly pro leu tyr tyr lys gly trp tyr his leu phe tyr gln tyr asn pro gly ser ala ile trp gly asn ile thr trp gly his ala val ser lys asp leu ile asn trp phe his leu pro leu ala met val pro asp his trp tyr asp ile his gly val met thr gly ser ala thr ile leu pro asn gly gln ile phe met leu tyr ser gly asn ala tyr asp leu ser gln leu gln cys leu ala tyr pro lys asn ala ser asp pro leu leu ile glu trp val lys tyr glu gly asn pro ser ser leu trp ile gly pro asp gly lys tyr arg met val met gly ser lys his asn asn thr ile gly cys ala leu ile tyr his thr gln gly thr gly met trp glu cys val asp leu tyr pro val ser thr thr glu thr asn gly leu asp met ser asn his glu ser gly ala lys tyr val leu lys gln ser gly asp glu asp arg his asp trp tyr ala ala ser lys thr phe tyr asp pro ser lys lys arg arg val leu trp trp ala asn ile leu asn val pro arg thr val val leu asp thr lys arg ser glu glu tyr asp glu phe lys asp val glu leu arg pro gly asp val leu phe asn cys thr thr ser glu gly ser ala met arg gly glu gln thr pro val tyr phe tyr ile ala lys asn leu asp gly thr ser arg thr tyr phe cys ala asp glu ser arg ser ser lys leu leu glu asn tyr asp met arg leu leu val asp his ser ile val glu ser phe ala gln gly gly arg thr val ile thr ser arg val tyr pro thr tcgcggccgc gtcgacactt ggcccatttc cctcgaaca atg aaa aca gcc gaa 54 ccc tta agt gac ctt gag gat gca tcc aac cgc act ccc cta cta gac 102 cac cct gca cca cca ccg gcc gcc gtg aaa aag cag tcg ttc gtc agg 150 gtt ctg tcc agt atc act ttg gtg tct ctg ttc ttc gtt tta gct ttc 198 gta ctc atc gtc ctg aac cag caa gat tcc acg aac gcc act gcc aat 246 tta gca ctg ccg gag aaa tct tcg gct caa cac tat cag tcc gat cgc 294 leu ala leu pro glu lys ser ser ala gln his tyr gln ser asp arg ctg aca tgg gaa aga aca gct tac cat ttt cag cca gcc aaa aat ttc 342 leu thr trp glu arg thr ala tyr his phe gln pro ala lys asn phe atc tac gat ccc aat ggg cca ctg ttc cac atg ggt tgg tac cat ctt 390 ile tyr asp pro asn gly pro leu phe his met gly trp tyr his leu ttc tat caa tac aac ccg tac gct cca att tgg ggc aac atg tca tgg 438 phe tyr gln tyr asn pro tyr ala pro ile trp gly asn met ser trp ggt cac gcc gtg tcc aaa gac atg atc aac tgg ttc gag ctt ccc gta 486 gly his ala val ser lys asp met ile asn trp phe glu leu pro val gcc ttg aca cca acc gag tgg tac gat atc gag ggc gtc tta tcc ggg 534 ala leu thr pro thr glu trp tyr asp ile glu gly val leu ser gly tcc acc acg gcc ctc ccc aac ggt caa atc ttt gca ttg tac acc gga 582 ser thr thr ala leu pro asn gly gln ile phe ala leu tyr thr gly aat gct aat gat ttc tct caa cta caa tgc aaa gct gtt ccg tta aac 630 asn ala asn asp phe ser gln leu gln cys lys ala val pro leu asn aca tct gac cca ctc ctt ctc gag tgg gtc aaa tac gag aat aac cca 678 thr ser asp pro leu leu leu glu trp val lys tyr glu asn asn pro atc ttg ttc act cca cca ggg att gga tta aaa gac tat cgg gac ccg 726 tct aca gtt tgg acg ggt cct gat gga aaa cat cgg atg atc atg ggc 774 ser thr val trp thr gly pro asp gly lys his arg met ile met gly act aaa ata aat cgt act gga ctc gta ctt gtt tac cat act acc gac 822 thr lys ile asn arg thr gly leu val leu val tyr his thr thr asp ttc aca aac tat gta atg ttg gag gag ccg ttg cat tcg gtt ccc gat 870 phe thr asn tyr val met leu glu glu pro leu his ser val pro asp acc gat atg tgg gaa tgt gtt gac ttg tac cct gtg tca aca att aat 918 thr asp met trp glu cys val asp leu tyr pro val ser thr ile asn gac agc gca ctt gat atc gcg gct tat ggt ccc gat atg aag cat gtg 966 asp ser ala leu asp ile ala ala tyr gly pro asp met lys his val att aaa gaa agt tgg gag gga cat ggg atg gac tgg tac tcg att ggg 1014 aca tat gat gtg ata aac gat aag tgg acc ccg gat aac ccg gaa ttg 1062 thr tyr asp val ile asn asp lys trp thr pro asp asn pro glu leu gac gtg ggt att ggg tta aga gtc gat tac ggg agg ttt ttt gca tca 1110 aag agt ctt tat gac ccg ttg aag aaa cgg agg gtc act tgg ggt tat 1158 lys ser leu tyr asp pro leu lys lys arg arg val thr trp gly tyr gtt gca gaa tcg gac agt gcg gac cag gac ctt aat aga ggg tgg gct 1206 val ala glu ser asp ser ala asp gln asp leu asn arg gly trp ala act att tac aac gtt gca aga acc att gtg cta gat aga aag acc gga 1254 thr ile tyr asn val ala arg thr ile val leu asp arg lys thr gly acc cat cta ctt cat tgg cct gtt gag gaa att gag agt ttg aga tat 1302 thr his leu leu his trp pro val glu glu ile glu ser leu arg tyr gat ggt cgt gaa ttt aaa gag atc gag ctt gca ccg ggt tcg atc atg 1350 asp gly arg glu phe lys glu ile glu leu ala pro gly ser ile met cca ctc gac ata ggc ccg gct acg cag ttg gac ata gtt gcc aca ttt 1398 gag gtg gaa caa gag acg ttt atg agg aca agt gac aca aat ggt gaa 1446 glu val glu gln glu thr phe met arg thr ser asp thr asn gly glu tac ggt tgc acc acg agc gcg ggt gca acc gaa agg gga agt ttg gga 1494 ccg ttt ggg atc gcg gtt ctt gct gat gga aca ctc tcg gaa tta act 1542 pro phe gly ile ala val leu ala asp gly thr leu ser glu leu thr cct gtg tat ttc tat att tct aaa aag aca gat gga agc gtt gca aca 1590 pro val tyr phe tyr ile ser lys lys thr asp gly ser val ala thr cat ttt tgt acc gat aag cta agg tca tca ctg gat tat gac ggg gag 1638 his phe cys thr asp lys leu arg ser ser leu asp tyr asp gly glu aga gtg gta tac ggg agc act gtc cct gta ctc gat ggt gaa gaa ctc 1686 aca atg agg tta ctg gtg gat cat tca gta gtg gag ggg ttt gca atg 1734 thr met arg leu leu val asp his ser val val glu gly phe ala met gga gga agg aca gta atg aca tca cga gtg tat ccc aca aag gca ata 1782 gly gly arg thr val met thr ser arg val tyr pro thr lys ala ile tat gaa gga gcc aag atc ttc ttg ttc aac aat gcg act cat acc agt 1830 tyr glu gly ala lys ile phe leu phe asn asn ala thr his thr ser gtg aag gca tct ctc aag atc tgg caa ata gct tct gta cga atc cag 1878 met lys thr ala glu pro leu ser asp leu glu asp ala ser asn arg tyr gln ser asp arg leu thr trp glu arg thr ala tyr his phe gln pro ala lys asn phe ile tyr asp pro asn gly pro leu phe his met gly trp tyr his leu phe tyr gln tyr asn pro tyr ala pro ile trp gly asn met ser trp gly his ala val ser lys asp met ile asn trp phe glu leu pro val ala leu thr pro thr glu trp tyr asp ile glu gly val leu ser gly ser thr thr ala leu pro asn gly gln ile phe ala leu tyr thr gly asn ala asn asp phe ser gln leu gln cys lys ala val pro leu asn thr ser asp pro leu leu leu glu trp val lys asp tyr arg asp pro ser thr val trp thr gly pro asp gly lys his tyr his thr thr asp phe thr asn tyr val met leu glu glu pro leu his ser val pro asp thr asp met trp glu cys val asp leu tyr pro val ser thr ile asn asp ser ala leu asp ile ala ala tyr gly pro trp tyr ser ile gly thr tyr asp val ile asn asp lys trp thr pro val thr trp gly tyr val ala glu ser asp ser ala asp gln asp leu asp arg lys thr gly thr his leu leu his trp pro val glu glu ile glu ser leu arg tyr asp gly arg glu phe lys glu ile glu leu ala arg gly ser leu gly pro phe gly ile ala val leu ala asp gly thr leu ser glu leu thr pro val tyr phe tyr ile ser lys lys thr asp gly ser val ala thr his phe cys thr asp lys leu arg ser ser leu pro thr lys ala ile tyr glu gly ala lys ile phe leu phe asn asn cgcggccgcg tcgaccccca c atg gct tcc tct acc acc gcc acc acc cct 51 ctc atc ctc cgt gat gag act caa atc agc cca caa cta gct gga tct 99 leu ile leu arg asp glu thr gln ile ser pro gln leu ala gly ser ccg gtg ggt cgg cgt tta tcc atg gcc aat atc ctt tcc ggg atc ctc 147 gtt ttc gtc ctt gtc atc tgt gtt ctg gtt gct gtt atc cac gac caa 195 tca caa caa aca atg gcg acc aac aac cat cag gga gaa gat aaa ccc 243 ser gln gln thr met ala thr asn asn his gln gly glu asp lys pro acc tcc gcc gcc acg ttc aca gct ccg ttg cta caa gtt gat ctc aaa 291 cgg gtt ccc gga aag ttg gaa tcc aat gct gat gtt gag tgg caa cgc 339 arg val pro gly lys leu glu ser asn ala asp val glu trp gln arg tca gct tac cat ttt caa ccc gat aag aat ttc atc agc gat cct gat 387 ser ala tyr his phe gln pro asp lys asn phe ile ser asp pro asp ggt cca atg tat cac atg ggg tgg tac cat ctc ttc tac cag tac aac 435 cca gaa tca gcc ata tgg ggc aac atc aca tgg ggc cac tcc gta tca 483 pro glu ser ala ile trp gly asn ile thr trp gly his ser val ser cga gac atg atc aac tgg ttc cat ctc cca ttc gcc atg gtc ccg gac 531 arg asp met ile asn trp phe his leu pro phe ala met val pro asp cat tgg tac gac atc gaa ggg gtc atg acc gga tcc gcc acg gta ctc 579 his trp tyr asp ile glu gly val met thr gly ser ala thr val leu ccc aac ggt cag atc atc atg ctc tac act ggc aac gcg tac gat ctc 627 pro asn gly gln ile ile met leu tyr thr gly asn ala tyr asp leu tcc cag tta cag tgc tta gca tac gcc gtc aac tca tct gat cct ctc 675 ctt ctg gaa tgg aaa aag tac gaa gga aac cca att ttg ttc cca ccg 723 cct ggt gtg gga tac aaa gat ttt cga gat cca tcc aca tta tgg atg 771 pro gly val gly tyr lys asp phe arg asp pro ser thr leu trp met ggt cct gat ggg gaa tgg aga atg gta atg ggg tcc aaa cac aat gaa 819 gly pro asp gly glu trp arg met val met gly ser lys his asn glu act att ggt tgt gca ttg gtc tac cgt act act aat ttt acg cat ttt 867 thr ile gly cys ala leu val tyr arg thr thr asn phe thr his phe gaa ctg aat gag gag gta ctc cac gca gtc ccc cat act ggt atg tgg 915 glu leu asn glu glu val leu his ala val pro his thr gly met trp gaa tgt gtg gac cta tac cct gtg tcc acc acg cac acg aat ggg ttg 963 glu cys val asp leu tyr pro val ser thr thr his thr asn gly leu gac atg aag gat aat ggg ccg aat gtt aaa tat att ttg aaa caa agt 1011 asp met lys asp asn gly pro asn val lys tyr ile leu lys gln ser gga gac gaa gac cga cat gat tgg tat gcg gtt ggg act ttt gac cct 1059 gly asp glu asp arg his asp trp tyr ala val gly thr phe asp pro gag aaa gat aag tgg tac cct gac gac cct gaa aac gat gtg gga atc 1107 ggg ttg aga tac gac tac gga aag ttc tat gcg tca aag aca ttt tat 1155 gat caa cat gaa aag cgg agg gta ctt tgg ggt tat gtt ggt gaa acc 1203 asp gln his glu lys arg arg val leu trp gly tyr val gly glu thr gac ccc cct aag tcc gat ctt tta aag gga tgg gct aac atc ttg aat 1251 atc cca agg tcc gtt gtt ttg gac acg caa acc gga acc aat ttg att 1299 ile pro arg ser val val leu asp thr gln thr gly thr asn leu ile caa tgg ccg att gat gaa gtg gaa aaa ttg aga tca aca aaa tat gac 1347 gln trp pro ile asp glu val glu lys leu arg ser thr lys tyr asp gaa ttc aaa gac gtg gag ctc cga ccc gga tca ctc gtt ccc ctc gaa 1395 att ggc aca gcg aca cag ttg gac ata agt gcg aca ttt gaa atc gat 1443 caa aag aag tta caa tca acg ctt gaa gcc gat gtt ttg ttc aac tgt 1491 gln lys lys leu gln ser thr leu glu ala asp val leu phe asn cys aca act agc gaa ggt tca gtc cgg aag ggt gtg ttg gga cca ttt gga 1539 atc gtg gtt cta gcg gat gcc aac cgc tct gag caa ctt cct gtg tat 1587 ile val val leu ala asp ala asn arg ser glu gln leu pro val tyr ttc tat att gcc aaa gac acc gat gga acc tca aaa act tac ttc tgt 1635 gct gat gaa tca agg tca tcg acg gac aaa tac gtt gga aaa tgg gta 1683 ala asp glu ser arg ser ser thr asp lys tyr val gly lys trp val tac gga agc agt gtt cct gtt ctt gaa ggt gaa aat tac aac atg agg 1731 tta ctg gtg gat cat tcg ata gtg gaa ggg ttc gca caa gga gga aga 1779 leu leu val asp his ser ile val glu gly phe ala gln gly gly arg acg gtg gtg aca tca aga gtg tac ccc acg aag gcc atc tat ggc gct 1827 gct aag ata ttt ttg ttc aac aac gcc acc gga att agc gtc aag gca 1875 tct ctc aag atc tgg aaa atg gcg gaa gca caa ctc gat cca ttc cct 1923 ser leu lys ile trp lys met ala glu ala gln leu asp pro phe pro cys val leu val ala val ile his asp gln ser gln gln thr met ala thr asn asn his gln gly glu asp lys pro thr ser ala ala thr phe glu ser asn ala asp val glu trp gln arg ser ala tyr his phe gln pro asp lys asn phe ile ser asp pro asp gly pro met tyr his met gly trp tyr his leu phe tyr gln tyr asn pro glu ser ala ile trp gly asn ile thr trp gly his ser val ser arg asp met ile asn trp phe his leu pro phe ala met val pro asp his trp tyr asp ile glu gly val met thr gly ser ala thr val leu pro asn gly gln ile ile met leu tyr thr gly asn ala tyr asp leu ser gln leu gln cys leu ala tyr ala val asn ser ser asp pro leu leu leu glu trp lys lys asp phe arg asp pro ser thr leu trp met gly pro asp gly glu trp arg met val met gly ser lys his asn glu thr ile gly cys ala leu leu his ala val pro his thr gly met trp glu cys val asp leu tyr pro val ser thr thr his thr asn gly leu asp met lys asp asn gly pro asn val lys tyr ile leu lys gln ser gly asp glu asp arg his asp trp tyr ala val gly thr phe asp pro glu lys asp lys trp tyr gly lys phe tyr ala ser lys thr phe tyr asp gln his glu lys arg arg val leu trp gly tyr val gly glu thr asp pro pro lys ser asp leu leu lys gly trp ala asn ile leu asn ile pro arg ser val val leu arg pro gly ser leu val pro leu glu ile gly thr ala thr gln thr leu glu ala asp val leu phe asn cys thr thr ser glu gly ser ala asn arg ser glu gln leu pro val tyr phe tyr ile ala lys asp thr asp gly thr ser lys thr tyr phe cys ala asp glu ser arg ser val leu glu gly glu asn tyr asn met arg leu leu val asp his ser met ala glu ala gln leu asp pro phe pro leu ser gly trp ser ser