Patent Application: US-7045593-A

Abstract:
genetically engineered modification of potato for suppressing the formation of amylose - type starch is described . three fragments for insertion in the antisense direction into the potato genome are also described . moreover , antisense constructs , genes and vectors comprising said antisense fragments are described . further a promoter for the gene coding for formation of granule - bound starch synthase and also the gene itself are described . also cells , plants , tubers , microtubers and seeds of potato comprising said antisense fragments are described . finally , amylopectin - type starch , both native and derivatised , derived from the potato that is modified in a genetically engineered manner , as well as a method of suppressing amylose formation in potato are described .

Description:
in the practical carrying out of the invention the following materials were used : e . coli dh5alfa and dh5alfaf ′ iq ( brl ). e . coli jm105 ( pharmacia ). a . tumefaciens lba4404 ( clontech ). m13mpl8 and mp19 ( pharmacia ). pbi101 and pbi121 ( clontech ). pbi240 . 7 ( m . w . bevan ). puc plasmids ( pharmacia ). restriction enzymes and ecori linker ( brl ). union ™ dna ligation kit ( clontech ). sequenase • dna sequencing kit ( usb ). t 4 - dna ligase ( pharmacia ). the above - mentioned materials are used according to specifications stated by the manufacturers . a genomic library in embl3 has been produced by clontech on the applicant &# 39 ; s account , while using leaves of the potato bintje as starting material . the genomic library has been screened for the potato gbss gene by means of cdna clones for both the 5 ′ and 3 ′ end of the gene ( said cdna clones being obtained from m hergersberger , max plank institute in cologne ) according to a protocol from clontech . a full - length clone of the potato cbss gene , wx311 , has been identified and isolated from the genomic library . the start of the gbss gone has been determined at an ecori fragment which in called fragment w ( 3 . 95 kb ). the end of the gbss gene has also been datrind at an ecori fragment which in called fragment x ( 5 . 0 kb ). bgiii - spei fragment which is called fragment m ( 3 . 9 kb ) has also been isolated and shares sequences both from fragment w and from fragment x . the fragments w , m and x have been subcloned in puc13 ( viera , 1982 , yanisch - paron at el , 1985 ) and are called psw , psn and psx , respectively ( fig2 ). the gbss gene in potato has been characterised by restriction analysis and cdna probes , where the 5 ′ and 3 ′ end of the gbss gene has been determined more accurately ( fig2 ). seuence determination according to sangar et al , 1977 of the gbss gene has been made on subclones from psw and psx in m13mpl8 and mpl9 as well as puc19 starting eround the 5 ′ end ( see seq id no . 5 ). the promoter region has beon determined at a bgiii - nsii fr nt ( see seq id no . 4 ). transcription and translation start has been determined at an overlapping bgiii - hindiii fragment . the terminator region has in turn been determined at a spei - hindiii fragment . the hbss gene fragments according to the invention ( see seq id nos 1 , 2 and 3 . and fig2 ) have been determined in the following manner . the restriction of psw with nsii and hindiii gives fragment i ( seq id no . 1 ) which subcloned in puc19 is called 19nh35 . further restriction of 19 nh35 with hpai - ssti gives a fragment containing 342 bp of the gbss gene according to the invention . this fragment comprises leader sequence , translation start and the first 125 bp of the coding region . the restriction of psm with hpai and nsii gives fragment ii ( seq id no . 2 ) which subcloned in pjrd184 ( heusterspreute et al , 1987 ) is called pjrdmitt . further restriction of pjrdmitt with hpai - ssti gives a fragment containing 2549 bp of the gbss gene according to the invention . this fragment comprises exons and introns from the middle of the gene . the restriction of psx with sstl and spei gives fragment iii ( seq id no . 3 ) which subcloned in pbluescript ( melton et al , 1984 ) is called pblue3 ′. further restriction of pblue3 ′ with bamhi - ssti gives a fragment containing ing 492 bp of the gbss gene according to the invention . this fragment comprises the last intron and exon , translation end and 278 bp of trailer sequence . for the antisense construct phoxwa , the hpai - ssti fragment from 19nh35 has been inserted in the antisense direction into the binary vector pbi121 ( jefferson et al , 1987 ) cleaved with smai - ssti . the transcription of the antisense fragment is then initiated by the camv 35s promoter and is terminated by the nos terminator ( nos = nopaline synthase ). for the antisense construct phoxwb , the hpai - ssti fragment from 19nh35 has been inserted in the antisense direction into the binary vector pho4 ( fig3 ) cleaved with smai - ssti . the patatin i promoter which is tuber specific in potato comes from the vector pbi240 . 7 obtained from m . bevan , institute of plant science research , norwich . the transcription of the antisense fragment is then initiated by the patatin i promoter and is terminated by the nos terminator . for the antisense construct phoxwd , the hpai - ssti fragment from 19nh35 has been inserted in the antisense direction into the binary vector pho3 ( fig3 ) cleaved with smai - ssti . pho3 is a new binary vector which is constructed on the basis of pbi101 . this vector which contains the promoter according to the invention ( see seq id no . 4 ) ( gbss promoter ) of the now characterised potato gbss gene according to the invention has been restriction - cleaved with smai and ssti , the hpai - ssti fragment from 19nh35 being inserted in the antisense direction . the transcription of the antisense fragment is then initiated by its own gbss promoter and is terminated by the nos terminator . this means that the antisense fragment is transcribed only in the potato tuber , since the gbss promoter like the patatin i promoter is tuber - specific . for the antisense construct phoxwf , the hpai - ssti fragment from pjrdmitt has been inserted in the antisense direction into the binary vector pho4 cleaved with smai - ssti . the transcription of the antisense fragment is then initiated by the patatin i promoter and terminated by the nos terminator . for the antisense construct phoxwg , the hpai - ssti fragment from pjrdmitt has been inserted in the antisense direction into the binary vector pho3 cleaved with smai - ssti . the transcription of the antisense fragment is then initiated by its own gbss promoter and is terminated by the nos terminator . for the antisense construct phoxwk , the bamhi - ssti fragment from pblue3 ′ has been inserted in the antisense direction into the binary vector pho4 cleaved with bamhi - ssti . the transcription of the antisense fragment is then initiated by the patatin i promoter and is terminated by the nos terminator . for the antisense construct phoxwl , the bamhi - ssti fragment from pblue3 ′ has been inserted in the antisense direction into the binary vector pho3 cleaved with bamhi - ssti . the transcription of the antisense fragment is then initiated by its own gbss promoter and is terminated by the nos terminator . the formed antisense contructs ( fig4 , 6 ) have been transformed to agrobacterium tumefaciens strain lba4404 by direct transformation with the “ freeze - thawing ” method ( hoekema et al , 1983 , an et al , 1988 ). the antisense constructs are transferred to bacteria , suitably by the “ freeze - thawing ” method ( an et al , 1988 ). the transfer of the recombinant bacterium to potato tissue occurs by incubation of the potato tissue with the recombinant bacterium in a suitable medium after some sort of damage has been inflicted upon the potato tissue . during the incubation , t - dna from the bacterium enters the dna of the host plant . after the incubation , the bacteria are killed nd the potato tissue is transferred to a solid medium for callus induction and is incubated for growth of callus . after pausing through further suitable media , sprouts are formed which are cut away from the potato tissue . checks for testing the expression of the antisense constructs and the transfer thereof to the potato genome are carried out by e . g . southern snd northern hybridisation ( manatis et al ( 1982 )). the number of copies of the antisense construct which has been transferred is determined by southern hybridisation . the testing of the expression on protein level is suitably carried out on microtubers induced in vitro on the transformed sprouts , thus permitting the testing to be performed an quickly as possible . the effect of the antisense constructs on the function of the gbss gene with respect to the activity of the gbss protein is examined by extracting starch from the microtubers and analysing it regarding the presence of the gbss protein . in electrophoresis on polyacrylamide gel ( hovenkamp - hermelink et al , 1987 ), the gbss protein forms a distinct band at 60 kd . when the gbss gene functions . when the gbbs gene is not expressed , i . e . when the antisense gbss gene is fully expressed , thereby inhibiting the formation of gbss protein , no 60 kd band is demonstrated on the gel . the composition of the starch in microtubers is identical with that of ordinary potato tubers , and therefore the effect of the antisense constructs on the amylose production is examined in microtubers . the proportion of amylose to amylopectin can be determined by a spectrophotometric method ( e . g . according to hovenkamp - hermelink et al , 1988 ). amylopectin is extracted from the so - called amylopectin potato ( potato in which the formation of amylose has been suppressed by inserting the antisense constructs according to the invention ) in a known manner . depending on the final use of the amylopectin , its physical and chemical qualities can be modified by derivatisation . by derivatisation is here meant chemical , physical and enzymatic treatment and combinations thereof ( modified starches ). the chemical derivatisation , i . e . chemical modification of the amylopectin , can be carried out in different ways , for example by oxidation , acid hydrolysis , dextrinisation , different forms of etherification , such as cationisation , hydroxy propylation and hydroxy ethylation , different forms of esterification , for example by vinyl acetate , acetic anhydride , or by monophosphatising , diphosphatising and octenyl succination , and combinations thereof . physical modification of the amylopectin can be effected by e . g . cylinder - drying or extrusion . in enzymatic derivatisation , degradation ( reduction of the viscosity ) and chemical modification of the amylopectin are effected by means of existing enzymatic systems . the derivatisation is effected at different temperatures , tures , according to the desired end product . the ordinary range of temperature which is used is 20 - 45 ° c ., but temperatures up to 180 ° c . are possible . the invention will be described in more detail in the following examples . production of microtubers with inserted antisense constructs according to the invention the antisense constructs ( see fig4 and 6 ) are transferred to agrobacterium tumefaciens lba 4404 by the “ freeze - thawing ” method ( an et al , 1988 ). the transfer to potato tissue is carried out according to a modified protocol from rocha - sosa et al ( 1989 ). leaf discs from potato plants cultured in vitro are incubated in darkness on a liquid ms - medium ( murashige & amp ; skoog ; 1962 ) with 3 % saccharose and 0 . 5 % mes together with 100 μl of a suspension of recombinant agrobacterium per 10 ml medium for two days . after these two days the bacteria are killed . the leaf discs are transferred to a solid medium for callus induction and incubated for 4 - 6 weeks , depending on the growth of callus . the solid medium is composed as follows : subsequently the leaf discs are transferred to a medium having a different composition of hormones , comprising : the leaf discs are stored on this medium for about 4 weeks , whereupon they are transferred to a medium in which the “ claforan ” concentration has been reduced to 250 mg / l . if required , the leaf discs are then moved to a fresh medium every 4 or 5 weeks . after the formation of sprouts , these are cut away from the leaf discs and transferred to an identical medium . the condition that the antisense construct has been transferred to the leaf discs is first checked by analysing leaf extracts from the regenerated sprouts in respect of glucuronidase activity by means of the substrates described by jefferson et al ( 1987 ). the activity is demonstrated by visual assessment . further tests of the expression of the antisense constructs and the transfer thereof to the potato genome are carried out by southern and northern hybridisation according ing to maniatis et al ( 1981 ). the number of copies of the antisense constructs that has been transferred is determined by southern hybridisation . when it has been established that the antisense constructs have been transferred to and expressed in the potato genome , the testing of the expression on protein level begins . the testing is carried out on microtubers which have been induced in vitro on the transformed sprouts , thereby avoiding the necessity of waiting for the development of a complete potato plant with potato tubers . stem pieces of the potato sprouts are cut off at the nodes and placed on a modified ms medium . there they form 35 microtubers after 2 - 3 weeks in incubation in darkness at 19 ° c . ( bourque et al , 1987 ). the medium is composed as follows : the effect of the antisense constructs on the functions of the gbss gene in respect of the activity of the gbss protein is analysed by means of electrophoresis on polyacrylamide gel ( hovenkamp - hermelink et al , 1987 ). starch is extracted from the microtubers and analysed regarding the presence of the gbss protein . in a polyacrylamide gel , the gbss protein forms a distinct band at 60 kd , when the gbss gene functions . if the gbss gene is not expressed , i . e . when the antisense gbss gene is fully expressed so that the formation of gbss protein is inhibited , no 60 kd band can be seen on the gel . the composition of the starch , i . e . the proportion of amylose to amylopectin , is determined by a spectrophotometric method according to hovenkamp - hermelink et al ( 1988 ), the content of each starch component being determined on the basis of a standard graph . potato whose main starch component is amylopectin , below called amylopectin potato , modified in a genetically engineered manner according to the invention , is grated , thereby releasing the starch from the cell walls . the cell walls ( fibres ) are separated from fruit juice and starch in centrifugal screens ( centrisiler ). the fruit juice is separated from the starch in two steps , viz . first in hydrocyclones and subsequently in specially designed band - type vacuum filters . then a finishing refining is carried out in hydrocyclones in which the remainder of the fruit juice and fibres are separated . the product is dried in two steps , first by predrying on a vacuum filter and subsequently by final drying in a hot - air current . amylopectin is sludged in water to a concentration of 20 - 50 %. the ph is adjusted to 10 . 0 - 12 . 0 and a quatenary ammonium compound is added in such a quantity that the end product obtains a degree of substitution of 0 . 004 - 0 . 2 . the reaction temperature is set at 20 - 45 ° c . when the reaction is completed , the ph is adjusted to 4 - 8 , whereupon the product is washed and dried . in this manner the cationic starch derivative 2 - hydroxy - 3 - trimethyl ammonium propyl ether is obtained . amylopectin is sludged in water to a water content of 10 - 25 % by weight . the ph is adjusted to 10 . 0 - 12 . 0 , and a quatenary ammonium compound is added in such a quantity that the end product obtains a degree of substitution of 0 . 004 - 0 . 2 . the reaction temperature is set at 20 - 45 ° c . when the reaction is completed , the ph is adjusted to 4 - 8 . the end product is 2 - hydroxy - 3 - trimethyl ammonium propyl ether . amylopectin is sludged in water to a concentration 25 of 20 - 50 % by weight . the ph is adjusted to 5 . 0 - 12 . 0 , and sodium hypochlorite is added so that the end product obtains the desired viscosity . the reaction temperature is set at 20 - 45 ° c . when the reaction is completed , the ph is adjusted to 4 - 8 , whereupon the end product is washed and dried . in this manner , oxidised starch is obtained . amylopectin is sludged in water to a concentration of 20 - 50 % by weight , whereupon the sludge is applied to a heated cylinder where it is dried to a film . amylopectin is treated according to the process described in one of examples 3 - 5 for chemical modification and is then further treated according to example 6 for physical derivatisation . mac donald , f . d . and preiss , j ., 1985 , plant . physiol . o 78 : 849 - 852 preiss , j ., 1988 , in the biochemistry of plants 14 ( carbohydrates ). ed . j . preiss , academic press ; 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development biology 23 ( 5 ): 381 - 386 hovenkamp - hermelink , j . h . m ., jacobsen , e ., ponstein , a . s ., visser , r . g . f ., vos - scheperkeuter , g . h ., bijmolt , e . w ., de vries , j . n ., witholt , b . j . & amp ; feenstra , w . j ., 1987 , theor . appl . genet . 75 : 217 - 221 hovenkamp - hermelink , j . h . m ., de vries , j . n ., adamse , p ., jacobsen , e ., witholt , b . and feenstra , w . j ., 1988 , potato research 31 : 241 - 246 tctggtagat tccccttttt gtagaccaca catcac atg gca agc atc aca gct 234 tca cac cac ttt gtg tca aga agc caa act tca cta gac acc aaa tca 282 acc ttg tca cag ata gga ctc agg aac cat act ctg act cac aat ggt 330 gag ctc tcc tgg aag gtaagtgtga atttgataat ttgcgtaggt acttcagttt 55 aag aaa tgg gag aca ttg cta ttg ggc tta gga gct tct ggc agt gaa 157 ccc ggt gtt gaa ggg gaa gaa atc gct cca ctt gcc aag gaa aat gta 205 ile val cys gly lys gly met asn leu ile phe val gly thr glu val ala arg gly his arg val met thr ile ser pro arg tyr asp gln tyr val lys val gly asp ser ile glu ile val arg phe phe his cys tyr lys arg gly val asp arg val phe val asp his pro met phe leu glu val trp gly lys thr gly ser lys ile tyr gly pro lys ala gly leu gly glu asp val leu phe ile ala asn asp trp his thr ala leu ile pro cys tyr leu lys ser met tyr gln ser arg gly ile tyr leu asn val ala phe cys ile his asn ile ala tyr gln gly arg phe ser phe lys pro val lys gly arg lys ile asn trp met lys ala gly ile leu glu ser his arg val val thr val ser pro tyr tyr ala gln glu leu thr cys ile thr gly ile val asn gly met asp thr gln glu trp asn leu glu glu gln lys gly ser asp ile leu ala val ala ile his lys ala his met ile thr ala gly ala asp phe met leu val pro ser arg phe glu pro cys gly leu ile gln leu his ala met arg tyr gly thr val pro ile cys ala ser thr gly gly leu val asp thr val lys glu ser leu asp thr lys ser thr leu ser gln ile gly leu arg asn his glu thr lys arg pro gly cys ser ala thr ile val cys gly lys gly met asn leu ile phe val gly thr glu val gly pro trp ser lys thr ala arg ala leu ala val tyr gly thr leu ala phe ala glu met ile