Patent Application: US-77923701-A

Abstract:
this invention relates to the regulation and manipulation of sucrose content in a sugar - storing plant , such as sugarcane , by regulating the activity of the pfp enzyme in the plant . it has been found that the down regulation of the pfp enzyme by decreasing the concentration of one of subunits , namely the β - subunit , of the enzyme increases the sucrose content of the plant . in a preferred embodiment of the invention , the activity of the pfp enzyme is down regulated by the introduction of an untranslatable form , or an antisense form of an isolated nucleotide sequence of the invention .

Description:
it has been found that the sucrose content of a sugar - storing plant can be altered by regulating the activity of the pfp enzyme in the plant . although this invention describes the regulation of the pfp enzyme in sugarcane , the regulation of the activity of the pfp enzyme in other sugar - storing plants such as sweet sorghum and sugar beet will also affect the sucrose content of the plant . the pfp enzyme is a heterotetramer with two α and two β - subunits . the β - subunit is the catalytic subunit of the enzyme , and it has been found that removal of the β - subunit increases the sugar content of a sugar - storing plant , namely sugarcane . a similar effect can be attained by altering the α - subunits of the enzyme which are believed to be involved in the regulation of enzyme activity through fructose 2 , 6 - biphosphate ( fru - 2 , 6 - p 2 ). a first step of the invention was the cloning and characterization of a sugar cane pfp - β cdna fragment . a set of degenerate primers was designed , based on the consensus of the castor bean and potato pfp - β gene sequences . these primers were used to amplify a fragment from sugarcane leafroll rna which was then used as a probe to screen a sugarcane leafroll cdna library for putative pfp - β clones . the sequence of the insert of one such clone is shown as an example in fig1 . this sequence contains a 1170 bp cdna fragment . the complete sugarcane pfp - β coding sequence , as shown in fig2 was obtained by sequencing other cdna and gdna ( genomic dna ) library clones . the pfp - β cdna fragment shown in fig1 was excised and cloned into the plant expression vector pubi 510 which confers high - level constitutive gene expression in sugarcane cells . one of the vectors , termed puspc 510 , shown in fig3 contains a fragment in the sense orientation but it lacks a translation initiation codon , and is thus untranslatable . the other vector , termed paspc 510 , shown in fig4 contains a fragment in the antisense orientation . the vectors puspc 510 and paspc 510 were used to transform cells in a sugarcane callus . transformed calli were selected and putative transgenic plants were regenerated . the plants were characterized to confirm the presence of pfp - β transgene . plants containing the pfp - β transgene were hardened off and grown under glass house conditions . subsequently the expression of the transgene was investigated using northern blot analysis . the results shown in fig6 indicated that the transgene is expressed in all the tissues analyzed . pfp protein expression was then investigated by means of protein blot analysis . the results shown in fig7 indicated that endogenous pfp levels were decreased in leafroll and internodal tissue . lastly , the influence of the abovementioned changes in pfp - β protein levels / activity in sucrose accumulation in the plants was determined . the results presented in table 1 below show a substantial increase in sucrose levels in the clones with reduced pfp - β protein . to ensure optimal antisense and / or co - suppression mediated gene silencing in sugarcane the endogenous pfp - β gene sequence was isolated . a set of degenerate primers was designed , based on the consensus sequence of the castor bean and potato pfp - β gene sequences available in the international database ; genbank accession numbers z32850 and m55191 respectively ( http :// www . ncbi . nlm . nih . gov /). these primers were used to amplify a 248bp fragment from sugarcane leafroll rna using the titan ™ rt - pcr system according to the manufacturers specifications ( boehringer mannheim ). this amplified pfp - β cdna fragment was used as a probe to screen a sugarcane leafroll cdna library ( lambda zap ii system , stratagene ) for putative pfp - β clones . the cdna inserts of the isolated clones were sequenced to verify their integrity and identity . automated dna sequencing was performed with an applied biosystem abi prism 373 genetic analyzer using an abi bigdye ™ terminator cycle sequencing ready reaction kit according to the manufacturer &# 39 ; s recommendations ( perkin - elmer , part number 430 3152 ). the sequence of the insert of one such clone , pfp # 5 , is shown as an example in fig1 . comparing this sequence to the international database confirmed its identity as pfp - β ( blastx software ; http :// www . ncbi . nlm . nih . gov / cgi - bin / blast /). it contains an 1170 bp cdna fragment which represents more than 50 % of the pfp - β coding sequence and includes a 260 bp 3 ′- untranslatable sequence . the complete sugarcane pfp - β coding sequence , as presented in fig2 was obtained by sequencing other cdna - and gdna library clones . as an example , the construction of a set of plant expression vectors for the down regulation of pfp activity in sugarcane will be described in detail . these constructs contain the pfp - β fragment from the leafroll cdna library clone pfp # 5 . the 1170 bp cdna insert was excised using the restriction enzyme ecor i and cloned into the ecor i site of the plant expression vector pubi 510 ( ecacc deposit reference number : 00042603 ) which confers high - level , constitutive gene expression in sugarcane cells . the expression vector contains two promoters , the camv 35s and the maize polyubiquitin ubi promoter . one of the vectors , puspc 510 , contains the fragment in the sense orientation but it lacks a translation initiation codon and the other vector , paspc 510 , contains the fragment in the antisense orientation . schematic representations of the two expression vectors are shown in fig3 and 4 respectively . sugarcane tissue culture and transformation was done as described by snyman et al . ( 1996 ). one of the expression vectors and a selectable marker gene , neomycin phosphotransferase ii ( npt ii ), driven by the synthetic emu promoter ( chamberlain et al ., 1994 ), was co - transformed into sugarcane callus using a particle inflow gun ( finer et al ., 1992 ). transformed calli were selected on geneticin - containing medium and putative transgenic plants regenerated . as an example , the characterization of a set of transgenic sugarcane plants transformed with the puspc 510 expression vector will be described in detail . these clones were designated opu followed by a specific three digit clone number , e . g . opu 501 . as a first characterization step genomic dna ( gdna ) was extracted from putative transgenic plants and subjected to pcr analysis to confirm the presence of the pfp - β transgene before the plants were hardened off and allowed to grow under glass house conditions . fig5 represents an example of a pcr analysis used to detect transgenes in regenerated sugarcane plants . pcr analysis to identify transgenic sugarcane plants . gdna from nine putative puspc 510 transgenic plants ( lanes 2 - 10 ) was used as template for specific pcr . the 484 bp fragment in lanes 3 - 8 confirms the presence of the pfp - β cdna transgene in these clones . gdna from an untransformed nco 310 sugarcane plant was used as negative control ( lane 11 ) and a positive control using the transformation vector , puspc 510 , was also included ( lane 12 ). in this example the amplification of a 484 bp fragment confirms the presence of the cdna derived transgene . due to the presence of introns the primers used will amplify a ˜ 1250 bp fragment from the endogenous genomic sequence ( as seen in lane 10 ). moreover , the genomic sequence will amplify at low efficiency because the up - stream primer , based on the cdna sequence , overlaps an exon / intron splice site . subsequently the expression of the transgene on transcriptional level and its possible effect on endogenous pfp - β transcript levels was investigated using northern blot analyses . total rna was isolated from leafroll and young internodal tissue as described by bugos et al . ( 1995 ). after gel electrophoresis of 20 μg total rna per sample the rna was transferred to nylon ™ membranes and probed with the 1170 bp pfp - β cdna fragment . the results , as presented in fig6 clearly indicate that the transgene is expressed in all the tissues analyzed . northern blot analysis of transgenic sugarcane plants transformed with an untranslatable form of the pfp - β gene . lane 1 : untransformed sugarcane leafroll rna . lane 2 : transgenic clone opu 503 leafroll rna . furthermore , the result shows that some of the transgenic tissues had reduced levels of the endogenous pfp - β transcript . the untranslatable transgene transcript is represented by the 1 . 2 kb band and the endogenous pfp - β transcript by the 2 . 3 kb band . levels of the pfp protein in the transgenic clones were compared to an untransformed control plant by means of protein blot analyses . crude protein extracts were prepared from leafroll and young internodal tissue as described by whittaker and botha ( 1999 ). ten microgram total protein of each sample was analyzed using sds - page , where after the proteins were transferred to nitrocellulose membranes and probed with potato pfp - β antiserum . a 63 kda polypeptide cross - reacted with the antibody and the bands obtained were quantified using spot densitometry ( alphalmager 2000 ™ gel documentation and analysis system , alpha innotech ). the results , presented in fig7 indicated that the relative level of pfp - β protein decreased in the transgenic plants . this is especially evident in the tissues with lower levels of pfp , e . g . internode 5 . finally the influence , of the above mentioned changes in pfp - β protein levels /- activity on sugar accumulation , was determined . sugars were extracted from internodes 7 to 13 of an untransformed control and the three opu clones and quantified enzymatically . the results presented in table 1 clearly shows an increase in sucrose levels in the clones with reduced pfp - β protein . table 1 . sugar content of intemodal tissue of transgenic sugarcane clones with reduced pfp activity . sugar content ( umol g − 1 fresh weight ) untransformed internode control opu 501 opu 502 opu 503 no hexoses sucrose hexoses sucrose hexoses sucrose hexoses sucrose 7 3 . 30 385 . 93 114 . 24 238 . 23 151 . 92 197 . 22 50 . 21 376 . 15 9 2 . 55 320 . 65 13 . 73 886 . 64 154 . 60 228 . 93 8 . 70 796 . 22 11 2 . 40 344 . 25 5 . 58 750 . 94 63 . 64 412 . 27 6 . 14 894 . 22 13 4 . 86 295 . 69 5 . 07 862 . 60 20 . 69 372 . 98 5 . 58 881 . 95 in conclusion , it was found that the isolated pfp - β gene fragments could be used to down regulate the level of active pfp in the cells and thereby manipulating sucrose metabolism in the cells . ausubel f a , brent r , kingston r e , moore d d , seidman j g , smith j a , struhl k ( eds ) ( 1998 ) current protocols in molecular biology . john wiley and sons , new york . black c c , loboda t , chen j - q , sung s - j s ( 1995 ) can sucrose cleavage enzymes serve as markers for sink strength and is sucrose a signal molecule during plant sink development ? in : pontis h g , salemo g l , echeverria e j ( eds ) sucrose metabolism : biochemistry , physiology and molecular biology . waverley press , baltimore , md ., pp 49 - 64 . isbn 0 - 943088 - 32 - 3 . botha f c , small j g c , de vries c . 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