Patent Application: US-46323800-A

Abstract:
the invention relates to isolated nucleic acids obtainable from potato which encode isoamylases , a type of starch branching enzyme , particularly seq id nos : 1 - 3 or variants encoding seq id nos : 4 - 6 ; vectors comprising the nucleic acids ; plant cells and plants transformed therewith ; and methods for making plants which produce starches with modified branching characteristics .

Description:
briefly , cdna clones from potato were isolated from cdna libraries synthesised from mrna from both developing tubers and from in vitro grown minitubers . the probe was an est from arabidopsis ( at69012 . new_est ) which was identified by the present inventors as showing significant homology to the su1 gene from maize . a ) from mrna from developing tubers from greenhouse cultivated potato plants ( solanum tuberosum var desiree ) b ) from mrna from minitubers indued on stem explants of potato ( solanum tuberosum var desiree ) cultured in vitro according to the method of visser et al ( 1994 ) physiol plantarum 90 : 285 - 292 . minitubers were used in addition to tubers in order to assess starch synthesising organ which has a different gene express ion profile to tubers . the cdna was synthesized by reverse transcription using as a template poly ( a ) rna passed twice over oligo ( dt ) cellulose . the poly ( a ) rna was reverse transcribed to form the first strand cdna and the second strand was prepared using dna polymerase i ( large subunit ), t4 dna polymerase and rnaase h as described in the manufacturer &# 39 ; s instructions for the cdna synthesis kit ( amersham plc , uk ). cdna was ligated to ecori adaptors as described in the rapid adapter ligation kit ( amersham plc , uk ) and then cloned into the ecori of the λ cloning vector λgt10 according to the manufacturers instructions for the λgt10 cloning kit ( amersham plc , uk ). the probe used was a fragment of an arabidopsis est ( embl id no . at69012 . new_est ; accession no . h36690 ). this est was identified initially using a blast search of est databases . in order to determine the extent of the homology between the arabidopsis est and the su1 gene product , the est was further sequenced using an applied biosystems taq cycle sequencing kit ( perkin elmer ) and an abi automated sequencer . this generated a further 780 bp sequence data . it showed significant homology to the deduced amino acid sequence for the product of the su1 gene from maize that encodes an isoamylase type of debranching enzyme . the probe was prepared using pcr amplification of a miniprep of the plasmid . the pcr used the m13 reverse primer : and also a primer specific for the 3 ′ end of the est , at a point before the polya tail . this was designated g3712 : the amplified fragment was cut with psti to remove the sequences from the vector . the fragment was purified and then labelled using an oligonucleotide random priming labelling kit to provide the probe . approximately 60 , 000 plaques from the tuber library ( unamplified ) and 60 , 000 plaques from the minituber library ( unamplified ) were used to infect e . coli ( strain nm514 ) and the resultant plaques were screened using a 1 . 2 kb fragment of est cdna clone ( at69012 . new_est ) which lacked the poly ( a ) tail . filters were subsequently washed at low stringency ( 2 × ssc , 0 . 5 % sds , 55 ° c . for two washes ). 16 independent phages ( 5 from tuber and 11 from minituber ) that showed different levels of hybridization to the est probe were selected . dna from 9 independent clones was subcloned into either pcr2 . 1 ( invitrogen ) or pbluescript ( stratagene ) in e . coli . those in pcr2 . 1 were subcloned following pcr amplification of the inserts using λagt10 specific oligonucleotides . those in pbluescript were isolated as ecori fragments from λdna preparations . clones were sequenced using the taq cycle sequencing kit from perkin elmer and the abi automated sequencer . to complete any incomplete sequence , primers based on the known portions of the sequence are used to ‘ walk ’ along the clones in the library to identify the remaining portions . following initial sequencing of the c9 clone , a longer cdna was obtained and sequenced . the predicted n - terminal amino acid sequences for c15 and c21 fit the criteria for plastid transit peptides . a summary of the cdna clones is presented below . this refers to the original sequences . corresponding comparisons with su1 for the new sequences are shown above . the clones encoding the isoamylases are used to construct a series of lines of antisense potato plants . the clone ( c9 , c15 and c21 ) is subcloned in antisense orientation between the camv 35s promoter and the camv terminator sequences of pjit60 ( see guerineau & amp ; mullinieaux ( 1993 ) in plant molecular biology lab fax ed . croy rrd bios scientific , oxford , uk pp 121 - 148 ). this construct has been subcloned into the primary vector pbin19 and transferred to agrobacterium tumifaciens ( lba4404 ) by transformation and from there to potato tuber discs by the method of spychalla and bevan ( 1993 ) plant tissue culture manual bii . in other transformants , full length cdna clones encoding the isoamylase type of debranching enzyme in potato are used to increase debranching enzyme activity levels in transgenic potatoes . this is achieved by cloning each of them between the 2 × camv 35s promoter and the camv terminator of pjit60 . thence into a binary vector such as pbin19 between the t - dna borders in e . coli . it is then transferred to agrobacterium tumifaciens for transformation into plants . each different cdna may be expressed in e . coli to define its activity closely and to obtain enough purified protein to produce an antiserum . this could be done using any suitable system e . g . the pstag expression vector for e . coli ( strain k38 ) ( moyano et al ( 1996 ) plant cell 8 : 1519 - 1532 ): all three cdna &# 39 ; s were inserted into a vector which permitted expression in e . coli of proteins fused to a 15 amino acid tag at the n - terminus ( pet expression system , novagen ). the amount of expressed protein in e . coli extracts was then quantified by an assay for the s - tag and the proteins were then purified on an affinity matrix specific for the s - tag , the c21 and c15 cdna &# 39 ; s were inserted into the vector after removal of the fragment encoding the putative transit peptide ( pet system manual 7th edition , novagen ). fusion proteins from the c15 and c21 cdna &# 39 ; s were successfully expressed to high levels in e . coli as determined by using sds - polyacrylamide gels ( not shown ). in both cases , single bands of protein not present in e . coli transformed with the vector alone are seen in crude extracts of both the soluble and unsoluble ( inclusion body ) fractions of the bacteria . these bands correspond closely in size to the predicted size for the expressed proteins : 87 kd for c15 and 97 kd for c21 . the recombinant isoamylase is most readily assayed when it is either purified from other hydrolases or by use of specific inhibitors to negate the contribution of interfering enzymes . it may also be visualized on non - denaturing glucan - containing polyacrylamide gels on which activities of starch hydrolozing enzymes are separated and then revealed by staining of hydrolysis products with iodine ( see kakefuda et al . 1986 planta 168 : 175 - 182 . polyclonal antibodies against c15 and c21 were produced in new zealand white rabbits using standard immunisation procedures ( harlow e . & amp ; land d . ( 1988 ) antibodies : a laboratory manual , cold spring harbor laboratory press , cold spring harbour , n . y ). the immunoblot analysis was performed according to standard procedures ( sambrook j et al ( 1989 ) molecular cloning : a laboratory manual , cold spring harbor laboratory press , cold spring harbor , n . y .). filters were incubated with the rabbit antiserum and immunoreactive bands were detected using the methods of towbin h . et al ( 1997 ) proc . natl . acad . sci . usa 76 : 4350 - 4354 . the presence of high - titer antibodies in antisera that recognised the proteins was demonstrated by immunoblot analysis of extracts from e . coli expressing eiher c15 or c21 . both c15 and c21 antisera immunoreact against c15 and c21 . met ala thr ser pro ile gln leu ala val his ser arg leu leu ser arg gly lys ile val cys ser leu arg lys leu glu leu glu asp met asn phe ser gly ile gly arg asn asn asp gln glu ala pro arg arg ser ala lys arg val pro thr tyr leu phe arg thr asp ile gly gly 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