CA2365279A1 - Genetically modified plants with altered starch - Google Patents

Abstract

Starch yield of wheat and maize plants grown under higher temperatures than control plants is increased by the introduction of a chimaeric gene comprising a glycogen synthase coding sequence under the control of a promoter directing expression and a terminator. A transit peptide for translocation of the glycogen synthase to the plant plastid may also be included in the chimaeric gene. The starch may also have altered processing characteristics, in particular an increased chain length.

Description

Genetically Modified Plants with altered Starch This invention relates to genetically modified plants, and in particular to genetically modified maize and wheat. The genetically modified plants have an altered starch synthesising ability following the introduction, by recombinant DNA
techniques, of one or more gene sequences coding for enzymes in the starch or glycogen biosynthetic pathway into the plant.
Starch is a complex polymer of glucosyl residues. It is the major form in which carbohydrate is stored in the tissues and cells of most species of higher plants. It is accumulated in the leaves of plants during the day as a result of photosynthesis and is used to supply the needs of the plant for energy and biosynthesis during the night. Starch is also accumulated in non-photosynthetic cells, especially those involved in reproduction such as in seeds, fruits and tubers.
Therefore, starch is of great importance to the productivity of the plant and its survival.
Starch is also highly significant to man. Firstly, it forms a major component of animal diets, supplying man and his domestic animals with a large portion of their carbohydrate intake. Secondly, the type of starch in a plant affects the quality of the processed plant product. Thirdly, starch is used industrially in the production of paper, textiles, plastics and adhesives, as well as providing the raw material for some bio-reactors. Starch from different species have preferred uses.
On a world scale, starch producing crops are agriculturally and economically by far the most important, and these crops include wheat, maize, rice and potatoes. The type of starch will affect the quality of a processed product and the profitability of the processed crop. In addition, the quantity and quality of starch present in the harvested organ of a plant will affect the gross yield and the processing efficiency. It is also known that some starch producing crops produce a lower yield of starch when grown under higher temperature conditions. This reduction in yield is undesirable in terms of gross yield and processing efficiency of the crop. Starch yield may be measured in terms of the number of seeds harvested or the weight of the seeds harvested.
In plants, i.e. vascular plants, the starch consists of linear chain and branched chain glucans known as amylose and amylopectin respectively. Starch with various amounts of amylose and amylopectin are found in different plants.
Typically, plant starch contains 10-25o amylose, the remainder being amylopectin, the branched chain glucan. Amylopectin contains short chains and long chains, the short chains ranging from 5-30 glucose units and the long chains ranging from 30-100 glucose units, or more. It is thought that the ratio of amylose to amylopectin and the distribution of short to long chains in the amylopectin fraction affect the physical properties of starch, e.g. thermal stabilisation, retrogradation and viscosity. These properties also affect the utility of starch, as mentioned above. Starches from different plants have different properties, which also affects their suitability for processing under certain conditions and for certain uses. It can be seen, therefore, that modifying the starch generated in a plant can have particular utility in the downstream processing or the yield of the starch in the plant storage organ. It can also be seen that providing a plant having an improved starch yield when grown under higher temperature conditions compared with unmodified plants is also desirable.
Waxy corn starch lacks amylose and this starch has unique properties. Also, most mutations in the waxy locus of maize, which encodes starch granule bound synthase I (GBSSI), result in plants which produce much reduced amylose. When no functioning GBSSI is synthesised in the homozygous waxy mutant it also lacks amylose (Echt & Schwartz, 1981).
The genetic modifications of the present invention produce altered starch composition and properties, which properties are ideally beneficial in terms of starch processing. The genetic modifications surprisingly also affected starch yields under more stringent growing conditions.
In the last few years this concept of modifying starch properties has been postulated and put into practice in varying degrees. In the patent literature Ir~ternational Patent Application, Publication No. WO 94/11520 (Zeneca) described constructs having a target gene which encodes an enzyme involved in the starch or glycogen biosynthetic pathway under control of a gene switch, for example, a chemical or temperature controlled on-off mechanism. Various crops were postulated as being suitable for use in the method but no plant transformation was actually carried out. Some constructs were made but no examples or results were given. International Patent Application, Publication No. WO 94/09144 (Zeneca) was very similar to the just described application. Only the first steps in the transformation process were demonstrated. No results are given for any plant, and only the transformation of tomato is described with reference to the exemplary methodology, although other plants are mentioned. International Patent Application, Publication No. WO 92/11376 (Amylogene) described introducing antisense genes for GBSSI in to potatoes to down-regulate amylose production with the intention of producing a potato with practically no amylose-type starch. Whilst great detail is given of methodology, no actual results from transformed plants are given and no plant transformations other than potato are postulated. Only a small number of constructs are actually produced to enable one to carry out the invention. The results for potato were eventually published in the scientific literature by Visser et al in 1991. Increases in the amylopectin content of the starch was seen. Further scientific papers on altering GBSSI in potato using antisense GBSSI
constructs, e.g. Visser et al (1991a) and Kuipers et al (1994), have shown actual transformation and alteration of starch composition.
In terms of successful transformation using non-plant derived starch-related genes, in International Patent Application, Publication No. WO 92/11382 (Calgene) and their later publication (Shewmaker et al, 1994) potato was actually transformed with E.coli glgA (Glycogen synthase) and E.coli glgC
(ADPG pyrophosphorylase). Higher specific gravity measurements were obtained from transformed potato plants compared with two control events, as well as altered starch characteristics.
It can be seen, therefore, that work to date has involved introducing certain genes involved in glycogen biosynthesis specifically into potato. The effects and their potential usefulness for other plants and other non-plant derived starch-related genes has only been postulated.
This invention seeks to transform cereal crops and specifically wheat and maize with an enzyme involved in the synthesis of microbial glycogen, namely glycogen synthase (E. C.
2.4.1.21).
This invention also seeks to identify properties of the starch in these transformed plants which are particularly useful and/or advantageous in the downstream processing of starch or the plant itself.
The present invention provides transgenic wheat or maize plants, said plants having therein a chimaeric gene comprising a promoter, a coding sequence for glycogen synthase, which coding sequence is derived from a microorganism, and a terminator.
As used herein, the term chimaeric gene refers to a combination of nucleic acid sequences for each part of the chimaeric gene, which sequences have been engineered into relationship by recombinant DNA techniques, which sequences may also be in their separate parts endogenous or exogenous to the plant into which the chimaeric gene is to be introduced.
A construct and a chimaeric gene comprising nucleic acid causing the expression of the sequences above mentioned are also aspects of the invention.
Plant cells containing a chimaeric gene comprising a nucleic acid sequence encoding glycogen synthase are also an aspect of this invention, as are other plant parts, such as for example, seed of the transformed plant containing a chimaeric gene according to the invention. Seed of the transformed plants grown on average at more than 20°C can exhibit a higher weight than seed of the control plants grown on average at more than 20°C. Seed of the transformed plants can in addition or alternatively exhibit less of a loss in yield compared with control plants when both are grown on average at a higher temperature of more than 20°C.
The present invention also provides a method of altering the starch in maize or wheat plants, the method comprising the steps of stably introducing into the plant genome a nucleic acid sequence encoding glycogen synthase under the direction of a suitable promoter and a suitable terminator, and regenerating a plant having an altered genome.
The present invention also provides a starch obtained from transformed wheat or maize, said starch having an altered chain length and/or processing property compared with control starch from a non-transformed plant.
The present invention also provides a method of reducing the loss of starch yield in wheat or maize grown under high temperature conditions, the method comprising the steps of stably introducing into the plant genome a nucleic acid sequence encoding glycogen synthase under the direction of a suitable promoter and a suitable terminator, and regenerating a plant having an altered genome.
The chain length and/or branching of the starch may be increased or decreased. Other parameters which may be altered include the degree of retrogradation, the viscosity, the pasting temperature, the gelling temperature, each of which may be increased or decreased. The starch may also have modified properties for chemical derivitisation. The yield of starch in seed may also be less affected under more stringent growing conditions, and in particular under growing conditions where the temperature on average is greater than 23°C, and more preferably on average greater than 25°C and even more preferably is on average about 27°C, 30°C, 33°C or 36°C, or more, or incrementally in full degrees from 23°C upwards to 36°C. Using the inventive method a greater than normal starch yield in seeds can be achieved at higher temperatures during seed growth compared with control seed grown at 20°C.
The turnover of starch in leaves is of central importance to the growth of the plant. A change in the structure of the starch in the granule without a complementary change in other enzymes of starch breakdown might be expected to restrict the export of carbon from the leaf at night. This might be expected to cause an altered ratio of source to sink with a subsequent effect on yield.
Preferably the promoter is capable of directing expression in a particular tissue of the plant and/or at particular stages of development of the plant. The promoter may be heterologous or homologous to the plant. Preferably the promoter directs expression to the endosperm of the plant seed. A preferred promoter is the high molecular weight glutenin (HMWG) gene of wheat. Other suitable promoters will be known to the skilled man, such as the promoters of gliadin, branching enzyme, ADPG
pyrophosphorylase, starch synthase and actin, for example.

Preferably the chimaeric gene also contains a sequence that encodes a transit peptide which provides for translocation of the glycogen synthase and/or a marker gene to the plant plastid.
Suitable transit peptides include those from the small sub-unit of the ribulose bisphosphate carboxylase enzyme (ssu of Rubisco) from pea, maize or sunflower, for example. Combinations of transit peptides may also be used. Other suitable transit peptides for transporting to the amyloplast will be known to those skilled in the art, such as the transit peptide for the plant plastid acyl carrier protein (ACP) or for GBSSI.
The coding sequence encoding glycogen synthase is a sequence obtained from a microorganism, such as a unicellular organism, for example, bacteria, which sequence has the necessary ability to encode glycogen synthase.
Suitably the glycogen synthase is derived from a bacterial source such as E.coli (for example, Baecker, P.A. et al, 1983 or Kumar, A. et al 1986), Agrobacterium (Uttaro, A.D., & Ugalde, R.A. 1994), Salmonella (Leung, P.S.C. & Preiss, J. 1987), or Bacillus (Kiel, J.A. et al 1994). Standard methods of cloning by hybridisation or polymerase chain reaction (PCR) techniques may be used to isolate the sequences from such organisms: for example, molecular cloning techniques such as those described by Sambrook, J. et al 1989 and the PCR techniques described by Innis, M.A., et al 1990. Other microbial sequences may be obtained in a similar manner.

Depending on the homology of the nucleotide sequences encoding glycogen synthases, different conditions of stringencies may be used in the hybridisation procedures. By way of example and not limitation, hybridisation procedures using such conditions of high stringency are as follows:
hybridisation to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in O.IxSSC/0.1%SDS at 68°C (Ausubel F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley and Sons, Inc., New York, at p.
2 . 10 . 3 ) . Other conditions of high stringency which may be used are well known in the art. Hybridisation procedures using conditions of moderate stringency that may be used are as follows: hybridisation to filter-bound DNA in 0.5 M NaHP04, 7%
sodium dodecyl sulfate (SDS) , 1 mM EDTA at 65°C, and washing in 0.2xSSC/0.1% SDS at 42°C (Ausubel et al, 1989, supra). Other conditions of moderate stringency which may be used are well-known in the art.
The chimaeric gene may comprise one or more additional coding sequences from the starch or glycogen biosynthetic pathway, such as, for example, branching enzyme (EC 2.4.1.18).
The transformation techniques for the method of the invention are advantageously direct DNA transfer techniques, such as electroporation, microinjection or DNA bombardment (the biolistic approach). Alternatively, plant cell transformation using plant vectors introduced into plant pathogenic bacteria, such as Agrobacterium-mediated transfer (Cheng, M. et al (1997)), may be used. In any transformation method positive or negative selectable markers may be used, at least initially, in order to determine whether transformation has actually occurred.
Useful negative selectable markers include enzymes which confer resistance to an antibiotic, such as gentamycin, hygromycin, kanamycin and the like, or resistance to a herbicide, such as asulam or basta. Alternatively, markers which provide a compound identifiable by a colour change, such as GUS, or luminescence, such as luciferase, may be used.
The chimaeric gene may also comprise a gene switch mechanism which determines under what conditions or when the coding sequence is to be expressed. The gene switch may be a chemically induced promoter or a temperature controlled promoter, for example.
In order that the invention may be easily understood and readily carried into effect, reference will now be had, by way of example, to the following diagrammatic drawings in which:
Figure 1 shows a map of the plasmid pJIT117 used in the preparation of the plamid of Figure 2;
Figure 2 shows a map of the plasmid pBSl7R used in the sticky-feet polymerase chain reaction;
Figure 3 shows a diagrammatic representation of the steps in the sticky-feet polymerase chain reaction;

Figure 4 shows a map of the plasmid pBSHMVJGP used in the preparation of the plasmid of Figure 6;
Figure 5 shows a map of the plasmid pDV02000 used in the preparation of the plasmid of Figure 6;
Figure 6 shows a map of the plasmid pDV03000 used in the preparation of the plasmid of Figure 7;
Figure 7 shows a map of the plasmid pDV03191 according to one aspect of the invention and used in the transformation process of the invention;
Figure 8 shows a standard chromatogram of glucose at 1mM
concentration;
Figure 9 shows a standard chromatogram of maltose at 1mM
concentration;
Figure 10 shows a standard chromatogram of maltotriose at 1mM concentration;
Figure 11 shows a standard chromatogram of maltohexaose at 1mM concentration;
Figure 12 shows a standard chromatogram of a mixture of maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose each at 1mM concentration;
Figure 13 shows a chromatogram of an isoamylase digest of wheat starch from wheat plants according to the invention;
Figure 14 shows a graph of starch branch chain lengths for starch from the seed of a single transgenic wheat plant compared with starch from the seed of a control wheat plant;

Figure 15 shows a graph of starch branch chain lengths for starch from the seed of a further single transgenic wheat plant compared with starch from the seed from a control wheat plant;
Figure 16 shows a comparison of branch chain length for a family of starches from the seed of transgenic lines against a family of starches from the seed of control wheat plants;
Figure 17 shows a western blot of proteins extracted from the seed of transgenic maize plants according to the invention.
Figure 18 shows the differences in two lines of wheat in a dry weight of seed at two different temperatures; and Figure 19 shows the effect of temperature on the rate of starch synthesis in two transgenic lines of wheat compared to a control.
The invention will now be described, by way of example, with reference to an embodiment for incorporating glgA from E.coli strain LCB618 into wheat and maize.

Construction of crlgA plasmids used for particle bombardment of wheat embryos.
Isolation of E.coli chromosomal DNA
The coding sequence for glgA was originally isolated by PCR
using chromosomal DNA from the E.coli strain LCB618 as template. E.coli LCB618 was obtained from E.coli Genetic Stock Center, Yale University, U.S.A.
E.coli LCB618 was grown up in 100m1 LB o/n at 37°C. Cells were pelleted and re-suspended in 9.5m1 lOmM Tris-HCl, 1mM EDTA
(TE) pH8.0 and 0.5m1 10% w/v Sodium dodecyl sulphate (SDS) and 50,1 proteinase K 20mg/ml were added. The mixture was incubated at 37°C for 1 hour to lyse cells. 1.8m1 of 5M NaCl followed by l.5ml of CTAB (cetyl trimethyl ammonium bromide)/NaCl solution (10% w/v CTAB in 0.7M NaCl) were added and the mixture incubated at 65°C for 20 minutes. The lysate was extracted with an equal volume of chloroform and centrifuged at 6000g to separate the layers. The upper layer was removed to a fresh tube and DNA was precipitated by the addition of 0.6 volumes isopropanol. The DNA was removed from the solution with a sealed pasteur pipette, placed into a fresh tube and washed with 70 % ethanol . The DNA
was dried in vacuo and re-suspended in TE pH8Ø The DNA was purified on a CsCl gradient.
Sticky-~eet PCR
In order for the E.coli glycogen synthase to function in plants the protein has to be transported into the amyloplast.
This transport can be facilitated by attachment of a plastid transit peptide to the amino terminus of the E.coli polypeptide.

The coding sequence for the transit peptide (TP) from the small subunit of the ribulose bisphosphate carboxylase enzyme (ssu of Rubisco) pea has been cloned and the TP shown to target (3-glucuronidase (GUS) protein to chloroplasts (Guerineau et al, 1988) .
The plasmid pJIT117 (Guerineau et a1, 1988), the map of which is shown in Figure l, has several restriction sites downstream of the ssuTP which can be used for subcloning of coding sequences, however, the subcloning must create a translational fusion between the transit peptide and the coding sequence, and the Cys-Met amino acid sequence at the junction must be maintained.
We have previously used pJIT117 to attach the ssu transit peptide to the coding sequence for E.coli ADPG PPase g1gC16 using restriction digestion and PCR. The TP-g1gC16 DNA, herein known as SEQ.ID. No.l, was subsequently transferred to the vector pBluescript (Stratagene Ltd., Cambridge, UK) to create pBSl7R (the map for which is shown in Figure 2) and this plasmid was useful in generating a similar construct for glgA.
The glgA coding sequence has no convenient restriction sites at the 5' end. Therefore, to ensure that the open reading frame was in a translational fusion with the ssu transit peptide and to maintain the integrity of the Cys-Met cleavage site, plasmid pBSl7R was used to substitute the glgA sequence for the g1gC16 sequence with a technique called sticky-feet PCR
(Clackson and Winter, 1989).
This technique is explained diagrammatically with reference to Figure 3. In this technique, PCR primers are designed to the 5' and 3' ends of the acceptor sequence of chromosomal or genomic DNA and the sequences which are to be attached to the acceptor from a donator plasmid. In Step A, PCR is used to amplify the sequences which are to be inserted in the donator.
In Step B the amplified acceptor DNA fragment is annealed to the donator plasmid which has been made single-stranded and carries uracil residues instead of thymidine residues by using a specific type of E.coli host. In Step C, a new strand is synthesised, using the donator plasmid as template and the acceptor fragment as primer, with a combination of Taq polymerase, T7 DNA polymerase (Sequenase) and T4 DNA ligase.
The new double-stranded plasmid is a hybrid with one strand of the uracil-containing donator and one strand incorporating the acceptor fragment.
This hybrid plasmid is then transferred into a normal E.coli host where the uracil-containing strand is degraded and the acceptor strand replicated. A double-stranded plasmid incorporating the acceptor DNA can then be recovered. As an alternative, in Step D (not shown), the hybrid plasmid can be used in a PCR reaction with primers which will amplify out the acceptor DNA with the required fragments from the donator attached.
In this particular example, glgA sticky-feet primers were designed as follows:
SEQ. ID. No. 4 GLGASF5 (Pl) TGGTGGAAGAGTAAAGTGCATGCAGGTTTTACATGTATGTTCA
ssu TP 3'end ~ glgA 5' end --SEQ. ID. No. 5 GLGASF3 (P2) TCGCTCCTGTTTATGCCCTAGATCTCTATTTCGAGCGATAGTAAAGCTCACGGT
~glgC 3 ' end ~ glgA 3 ' end-The PCR primers are designed to the 5' and 3' ends of the glgA cDNA sequence.
The 5' end primer (SEQ. ID. No. 4) also has sequences which are homologous to the ssu-TP.
The 3' end primer (SEQ. ID. No. 5) also incorporates sequences which are homologous to the 3' end of the glgC coding sequence. These primers are used in a PCR process to amplify a glgA fragment with extensions which will overlap onto the sequences in pBSl7R. This is represented by Step A of Figure 3.
Plasmid pBSl7R is made into a template for sticky-feet PCR
by transferring the plasmid into the E.coli host CJ236 (Raleigh et al, 1989). This host is deficient in the enzyme dUTPase, (i.e. dut-) which results in deoxyuridine being incorporated into the DNA instead of thymidine. The absence of another enzyme uracyl N-glycosylase (ung-) means that the deoxyuridines cannot then be removed from the DNA.
In Step B of Figure 3, the extended glgA DNA (2) is annealed to the uracil-containing template which has been isolated as single-stranded DNA (3), and a new strand is synthesised as per Step C above. The new double-stranded plasmid is a hybrid (5) with one strand of the uracil-containing template (3) and the other strand consisting of the plasmid backbone and the glgA fragment now with ssu-TP and a 3' glgC
fragment attached at 5' and 3' ends respectively (4).
In Step D (not shown) , the hybrid plasmid is used in a PCR
reaction with primers (SEQ. ID. No. 6) (P3)(see below) and SEQ.
ID. No. 5 (P2) which will amplify out the extended glgA.
With reference to Figure 3, the experimental details are as follows:
The primers GLGASFS (P1) (SEQ. ID. No. 4) vs GLGASF3 (P2) ( SEQ . ID . No . 5 ) were kinased and used to ampl i fy the glgA open reading frame with extension sequences using E.coli LCB618 genomic DNA (1) as template. The DNA (2) was purified with GeneClean (BIO 101, Ltd.). The sticky-feet template DNA, single-stranded uracil pBSl7R DNA (3), was isolated from 5m1 overnight cultures of the dut- ung- E.coli strain CJ236.
The sticky-feet PCR reaction was carried out in 10,1 volume containing 20ng ss uracil pBSl7R (3); 200ng glgA DNA (2), 1~1 x Taq polymerase buffer, 1.0,1 2mM mixture of dATP, dTTP, dCTP, dGTP (2mM dNTPs); 2.5 units Taq polymerase. The mix was overlaid with 30,1 mineral oil and cycled once at 94°C, 3 min;
72°C, 2 min; 40°C, 2 min. and then cooled to room temperature.
10.1 of a solution containing 2.0,1 x5 Sequenase buffer (200mM
Tris-HC1 pH 7.5; 100mM MgCl2, 250mM NaCl), 1.51 O.lmM
Dithiothreitol (DTT); 2.0,1 lOmM Adenosine 5' triphosphate (ATP); 4 units T4 DNA lipase; 6.5 units Sequenase was then added and the reaction incubated at room temperature for 30 minutes.
Generation of TP-glgA DNA
1Ø1 of the reaction containing the hybrid plasmid (3 + 4) was taken and diluted to 101 with lOmM TE at pH8Ø 1.0,1 of the diluted sample was used in a PCR reaction in order to obtain the TP-glgA coding sequence (Step C of Figure 3). The primers used were TPSSU5 (P3) (SEQ. ID. No. 6) vs GLGASF3 (P2) (SEQ. ID.
No. 5) .
SEQ. ID. No. 6 TPSSU5 (P3) ACGTAGATCTATGGCTTCTATGATATCCTCTTC
The primers both have restriction sites for BglII, therefore after purification, the amplified DNA was digested with BglII and subcloned into the BamHI site of pDV03000 (see below) .

Construction of pDV03000 vector Transgenic wheat and maize plants are generated by particle bombardment of embryos and it is not necessary to use binary vectors. For expression of the glgA protein the coding sequence is placed under the control of an endosperm-specific promoter.
One such suitable promoter is that from the High Molecular Weight Glutenin (HMWG) gene of wheat (Bartels and Thompson, 1986). Primers (P4) and (P5) (SEQ. ID. Nos. 7 and 8 respectively) were designed so that the 430bp HMWG promoter, (the nucleotide sequence of which is given in SEQ. ID. No. 3) could be isolated by PCR and subcloned via EcoRI and Clal restriction sites into pBluescript to generate the plasmid pBSHMWGP (Figure 4).
A second set of PCR primers were designed to obtain the nopaline synthase terminator from plasmid pDV02000, the map of which is shown in Figure 5. This plasmid was previously constructed in our laboratory as an intermediate vector for the sub-cloning of coding sequences. The 5' primer, NTPRIMES (P6) (SEQ. ID. No. 9), has a BamHI restriction site, while the 3' primer NTP3NXS2 (P7) (SEQ. ID. No. 10), has restriction sites for Notl, Xhol and SacII. The amplified DNA was digested with BamHI and SacII and ligated into the pBSHMWGP plasmid to generate pDV03000 (the map of which is shown in Figure 6).

SEQ. ID. No. 7 HMWGPR05 (P4) GACATCGATCCCAGCTTTGAGTGGCCGTAGATTTGC
SEQ. ID. No. 8 HMWGPR03 (P5) GACGAATTCGGATCTCTAGTTTGTGGTGCTCGGTGTTGT
SEQ. ID. No. 9 NTPRIME5 I(P6) CAGGATCCGAATTTCACCCGATCGTTCAAACA
SEQ. ID. No. 10 NTP3NXS2 (P7) GACCCGCGGCTCGAGGCGGCCGCCCGATCTAGTAACATAGATGACACCGC
pDV03000 vector has the HMWG promoter-nos terminator sequences separated by unique restriction sites for EcoRI, Pstl, Smal and BamHI.
Construction of pDV03191 TP-glgA DNA amplified from the sticky-feet PCR sample with primers TPSSU5 vs GLGASF3 (Step D, Figure 3) was digested with BglII, purified and ligated into the BamHI site of pDV03000.
Plasmid pDV03191 (the map of which is shown in Figure 7) was confirmed by restriction enzyme digestion and by sequencing of the junctions between promoter and coding sequence. E.coli XL1 Blue (Stratagene Ltd., UK) harbouring pDV03191 was deposited by Advanced Technologies (Cambridge) Limited of 210 Cambridge Science Park, Cambridge CB4 OWA, under the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the purposes. of Patent Procedures at the National Collection of Industrial and Marine Bacteria (NCIMB), 23 Machar Street, Aberdeen, Scotland GB on 4 August 1998 under accession number NCIMB 40962. The micro-organism is E.coli XL1 Blue: strain LCB618 containing PDV03191. The DNA for E.coli glgA was inserted as described above into pBluescript with the ssu transit peptide, the HMWG promoter and nos terminator. The vector is useful for altering starch properties.
Transformation of wheat Methods for the transformation of wheat by particle bombardment are well known in the art, for example see Vasil et al, 1992 .
Immature embryos of wheat are used to initiate embryogenic callus. The callus is subcultured and used for particle bombardment with gold particles coated with plasmid DNA.
Two plasmids are used per bombardment, one plasmid carries the construct of interest, in this case pDV03191. The second plasmid carries the selectable marker which expresses the gene responsible for resistance to the herbicide Basta. Plants resistant to Basta are generally found to also have the recombinant gene of interest present.

Bombarded calli are grown on Basta selection media and surviving calli are transferred to regeneration medium. Rooted plants are transferred to soil and grown to maturity in a growth room.
Primary transformant wheat plants (To) are selfed to produce transgenic seed.
Seed are extracted for protein and the protein analysed by western blotting for the presence of E.coli glgA polypeptide.

Biochemical Analysis of c~rlgA transformed maize 1. Expression of glgA protein Soluble protein samples were prepared from individual maize grain derived from transformed maize plants. Each grain was pulverised in a pestle and mortar until a fine powder was obtained. A portion of this powder (100-200mg) was placed in an Eppendorf tube and 5001 of ice cold extraction buffer (50mM
HEPES, pH 8.0; lOmM DTT; lOmM EDTA) added. The powder was homogenised with a micropestle to release soluble proteins.
The extract was centrifuged at 13000 rpm for 1 minute and the supernatant decanted into a fresh Eppendorf tube and stored on ice. The total protein content in the soluble protein sample was assayed using The Bradford dye binding method (Bradford, M.
1976 ) .

An aliquot of the soluble protein sample containing 100mg total protein was placed into an Eppendorf tube and excess acetone (ca l.5ml) was added to precipitate the proteins. The proteins were collected by centrifuging the sample at 13000 rpm for 5 minutes. The acetone was decanted off and the samples were air-dried until all the residual acetone had evaporated.
SDS PAGE loading buffer (4% (w/v) SDS; 12% (w/v) glycerol;
50mM Tris-HCl pH 6.8; 20 (v/v) (3-mercaptoethanol; 0.01% Serva blue G) in an amount of 100,1 was added to the protein sample contained in the Eppendorf tube. Samples were boiled for 1 minute before loading onto a polyacrylamide gel.
Electrophoresis was carried out according to the method of Schagger and Von Jagow (1987). The resolving gel composition was loo acrylamide, 3% bis-acrylamide. Gels were run at 50 V
constant for 16 hours.
Separated proteins were transferred from the acrylamide gel onto PVDF membrane by electroblotting (Transfer buffer: 20%
methanol; 25mM Tris-HCl pH 8.3; 190mM glycine. Run in a Biorad blotting apparatus at 50 V for 3 hours).
To detect expression of glgA the membrane was challenged with a rabbit anti-glgA antiserum (raised glgA-GST fusion protein purified from E.coli). Specific cross-reacting proteins were detected using an anti-rabbit IgG-alkaline phosphatase conjugate secondary antibody and visualised by the NBT/BCIP
reaction.

NuPacreTM Electrophoresis Alternatively, an aliquot of the soluble protein sample, containing 100mg total protein was placed into an Eppendorf tube and excess acetone (ca l.5ml) was added to precipitate the proteins. The proteins were collected by centrifuging the sample at 13000 rpm for 5 minutes. The acetone was decanted off and the samples were air-dried until all the residual acetone had evaporated.
NuPageTM loading buffer (2% (w/v) SDS; 10% (w/v) sucrose; 25 mM Tris-HC1 pH 8.5; 1% (v/v) (3-mercaptoethanol; 0.5 mM EDTA;
0.02% Serva blue 6250; 0.006% Phenol Red) 100 ~.1, was added to the protein sample contained in the Eppendorf tube. Samples were heated at 100°C for 1 minute before loading onto a polyacrylamide gel. Electrophoresis was carried out on NuPageTM
precast gels according to the manufacturer's instructions (Novex, San Diego CA) . Gels were run at 200 V constant for 60 minutes using MES SDS running buffer (20 mM MES/20 mM Tris-HCl pH 7.3; 1% (w/v) SDS; 1 mM EDTA).
Separated proteins were transferred from the acrylamide gel onto PVDF membrane by electroblotting (Transfer buffer: 200 methanol; 25 mM Bis-Tris/25 mM Bicine pH 8.3; 1 mM EDTA. Run in a Novex electroblotting apparatus at 25 V for 1.5 hours).
To detect expression of glgA the membrane was challenged with a rabbit anti-glgA antiserum (raised against glgA-GST
fusion protein purified from E. coli). Specific cross-reacting proteins were detected using an anti-rabbit IgG-horse Radish Peroxidase conjugate secondary antibody and visualised using enhanced chemiluminesence (ECL) as supplied by Amersham International.
Several transformed lines were found to express a 50 kDa protein in their grain, which was not present in control grain derived from non-transformed maize plants.
2. Preparation of wheat starch Starch was extracted from grain of separate field grown samples of two of the glgA expressing lines and a control line.
Wheat grains of each sample (3-4g) were placed in a mortar, 30m1 of 1% sodium bisulphate was added and placed on ice for 30 minutes. The grains were then gently pulverised using a pestle.
The solution was filtered through a nylon filter sieve and collected in a centrifuge tube. The pulverised wheat grains were re-extracted with a further 30m1 of 1% sodium bisulphate and the filtrates were combined. The filtrate was centrifuged at 6000 rpm for 5 minutes. After decanting off the supernatant, the pellet of extracted starch was re-suspended in water and centrifuged at 6000 rpm for 5 minutes. This was repeated once.
The resulting starch pellet was re-suspended in acetone, centrifuged at 6000 rpm for 5 minutes and the supernatant decanted away. This was repeated once and the starch left to air dry. Once dried the starch was stored at -20°C.

3. Branch chain length analysis of wheat starch Portions of the starch samples were digested with isoamylase and the resulting unbranched linear glucan chains were analysed by HPLC.
75mg of isolated wheat starch was placed in a l5ml Pyrex boiling tube and suspended in 3.Om1 of water. The sample was placed in a boiling water bath for 6 minutes, occasionally removed and vortex mixed. The sample was cooled to room temperature and 250,1 of 200mM sodium acetate, pH 3.5 and 180 units of isoamylase enzyme added. The samples were made up to a final volume of 3.8m1 with water. After mixing, the sample was placed in a 37°C water bath for 4 hours. The samples were occasionally vortex mixed throughout this incubation period. At the end of the incubation the sample was placed in a boiling water bath for 2 minutes, and then allowed to cool to 4°C. The sample was centrifuged at 3,400 rpm for 20 minutes. The resulting supernatant was transferred to Eppendorf tubes and centrifuged at 13000 rpm for 15 minutes. Finally, the sample was filtered through a 0.2mm syringe filter and stored at 4°C
until required.
Separate isoamylase digest samples were normalised to a constant total glucan content by digesting a portion of the sample to glucose using a,-amylase and amyloglucosidase.

Two 1001 aliquots of isoamylase digested starch were placed in two separate Eppendorf tubes (one is to be used as a blank). To one aliquot was added: 5001 of 200mM sodium acetate pH 4.8; 50.1 of a-amylase solution containing 10 units of a-amylase; 1001 of amyloglucosidase solution containing 10 units of amyloglucosidase and water to a final volume of l.Oml. To the second (blank) aliquot was added: 500.1 of 200mM sodium acetate pH 4.8 and 400,1 of water. The samples were left to digest at 25°C for 16 hours.
The glucose content of the digest and blanks was assayed spectrophotometrically using a coupled enzyme assay. An aliquot of the total glucose digest or the blank was added to a cuvette containing in a final volume of 990 ~1 100mM HEPES, pH 8.0; 5mM
MgCl2; 4mM NAD; 1mM ATP and 1 unit of hexokinase. The optical density (OD) of the reaction mixture at 340nm was measured prior to the addition of 101 containing 1 unit of glucose-6-phosphate dehydrogenase. The OD at 340nm was monitored until there was no further change and the difference in OD after the addition of glucose-6-phosphate dehydrogenase compared to before the addition of glucose-6-phosphate dehydrogenase was determined. This figure was used to determine the total glucose amounts in the original isoamylase digests. These samples were diluted with water to a standard concentration of 8mM total glucose and stored at 4°C until required for HPLC analysis.

The samples were then analysed by Dionex HPLC using a Dionex PA 100 column and PED-Integrated Amperometric detection.
The solvent flow rate was l.Om1/min and a gradient system was developed. Solvent 1 consisted of 100mM NaOH and Solvent 2 was 100mM NaOH, 0.60M sodium acetate. The gradient profile was as shown in Table l, with the pulsed electrochemical detection (PED) parameters shown in Tables 2.1 and 2.2.
Table 1 Gradient Profile Event Start Solvent 1 (~) Solvent 2 ($) Time (min) 30.1 100 0 Table 2.1 Waveform Table Time (sec) Potential (V) 0 0.1 0.5 0.1 0.51 0.6 0.59 0.6 0.6 -0.6 0.65 -0.6 WO 00/55331 PCTlGB00/00848 Table 2.2 Integration Begin (sec) End (sec) 0.3 0.5 Three isoamylase digestions were performed for each sample and three aliquots of each isoamylase digest were analysed by the HPLC system. Separate chromatogram peaks were assigned to specific linear glucan sizes by reference to standard mixtures containing linear glucans of known numbers of glucose molecules (see Figures 8-12). Peak areas were abstracted from the primary data and averaged for the replicate chromatograms.
Figures 8 to 12 are HPLC traces of standards for various sugars. The standards in Figures 8-12 allow the peak area for each peak of the inventive sample of Figure 13 to be converted to a quantitative representation of the number of glucan chains in each peak, and the position (on the x-axis) of each peak to the number of glucose residues in each chain, i.e. the chain length. In Figures 14 and 15 this conversion has been done for wheat starch extracted from a single transgenic line and its paired control. In Figure 16, a family of starches from transgenic lines are compared with a family of controls. Figure 16 clearly shows that the transgenic starches have a different chain length distribution from the control starches. The starch has therefore been altered, which alteration affects its processing capabilities.

Maize plants transformed with glcrA recombinant ctene In the transformation step, immature maize embryos are used instead of wheat and are subject to particle bombardment with gold particles coated with plasmid DNA. Methods for the transformation of maize are well known in the art, for example see Gordon-Kamm et al (1990) and Fromm et al (1990). After rooted primary transformant plants (To) are transferred to soil and grown to maturity, maize plants are back-crossed to produce transgenic seed which can be extracted and analysed according to Example 2. Further back-crossing is performed to introgres the transgene into elite varieties and selfing of transgenic plants is performed to obtain plants and seed which are homozygous for the transgene. Seed from these generations can also be extracted and analysed according to method 2.
Seed from a number of back-crossed primary transformants were shown to be expressing the glgA protein. The plants grown up from the remaining seeds were subsequently selfed and progeny seed were extracted for protein and western blotting according to Example 2. Figure 17 shows the presence of glgA polypeptide in seed from two of these second generation lines 2-AM4-5'-2 and 2-AM4-6'-1.

Viscometry measurements of transgenic wheat seed extracts.
Flour was extracted from T2 and T3 progeny seed of primary transformant wheat line 72.11B which was shown to be expressing the glgA polypeptide by western blots. 4g of ground sample (140 moisture) was mixed with 25m1 water or with 24.5m1 water + 0.5m1 10% AgN03 solution. The presence of silver nitrate will inhibit any amylase activity in the slurry and allows the true viscosity developed by the flour to be assessed.
The slurry was subject to rapid viscometric analysis (RVA) using standard profile 1 (Table 3). Results of the RVA are tabulated in Table 4 and Table 5 below.
Standard 1: Idle temperature . 50 ~ 1°C
End Test (HH:MM:SS) . 00:13:00 Table 3 Time Type Value (HH:MM:SS) 00:00:00 Speed 960 rpm 00:01:00 Speed 160 rpm 00:01:00 Temp. 50C

00:04:45 Temp. 95C

00:07:15 Temp. 95C

00:11:00 Temp. 50C

Table 4 RVA STD without AaN03 Pasting Peak BKD Final temperature viscosity viscosity CYMMIT control 87.2 191 61 222 72.11B/62 87.2 181 57 208 72.11B/39/4 88.1 182 53 223 72.11B/49/11 86.3 184 53 230 I72.11B/41/22 88.1 I 185 I 52 I 226 I

Table 5 RVA Modified with AaN03 Pasting Peak BKD Final Peak Final temp. visc. visc. AgN03 visc.
- AgN03 Peak -standard FV std CYMMIT ctrl 86.4 251 98 267 60 45 72.11B/62 87.2 251 99 259 70 51 72.11B/39/4 87.3 238 86 265 56 42 72.11B/49/11 87.2 234 80 267 50 37 72.11B/41/22 86.5 244 87 273 59 47 The RvA method is described in Edwards et al (1999).

Differential scanning calorimetry of glgA transgenic wheat seed extracts Wheat kernels were cleaned and water was added to the sample (90mg). The sample was allowed to condition in the analysis chamber at ambient temperature for 24 hours before cycling using the following conditions:
Stabilisation: lh 25min at 25°C

Raise temperature to 110°C at 1.2°C/minute Cool to 25°C at 1.2°C/minute.
The DSC results are shown in Table 6. The DSC method is described in the book of Frazier et al (1997).
Table 6 Peak 1 Peak (amylopectin) (amylose-lipid complex) Onset Temp. Enthalpy Onset Temp. Enthalpy Point peak Point Peak CYMMIT ctrl. 52 60 6.9 80.2 92.5 1.9 72.11B/62 52 59 6.7 82 93 1.4 72.11B/39/4 52 60 6.8 80 93 1.9 72.11B/49/11 52.3 59.6 6.4 80 93 1.8 ~72.11B/41/22 51.7 59.4 6.8 I 80.2 92 1.8 ~ ~ I

Example 6 Growth of plants and plant seed under high temperature conditions Seeds were planted in 6-inch pots in M2 compost (5-6 seeds per pot). They were grown to anthesis in a greenhouse at 15-25°C under a l6hr photoperiod in daylight supplemented with sodium light (photosynthetically active radiation - 160~.mol.m-z S-1~ ~ Plants were watered regularly (every day in summer) and were fed weekly with Phostrogen (Phostrogen, Corwne, Clwyd, UK) at a concentration of 1.7 gl-1. On the day of anthesis plants were tagged. 5 days post anthesis (p. a.) plants were repotted to 1 plant per pot. Plants from each line were moved at 5 days p.a. into controlled environment cabinets set at either 20°C or 27°C for a 16 hr day length. Plants were grown in cabinets until seed maturity (approx. 70 days p.a.). Mature seeds were harvested and then weighed and average seed weight calculated.
The results are given in Table 7 below.
Table 7 Line Dry Weight Dry Weight Change in ~ loss wt in wt Control 0.0360.0028 0.0450.0097 0.0087 19 (Cimmyt 101) 72.11b 0.0370.0045 0.0400.0068 0.0034 8.4 79.42a 0.0380.0024 0.0430.009 0.0045 10.6 T~Vhere ~ loss in weight = ((wt at 20°C - wt at 27°C)/wt at 20°C) * 100 As plants were grown under identical conditions with temperatures from 5 days p.a. being the only variable, it is statistically relevant to compare changes in seed weight within lines and not just with controls. It is clear that lines 72.11b and 79.42a are markedly less sensitive to temperatures above 25°C than controls, losing 10.60 and 8.40 less dry weight respectively, than the cimmyt 101 control This reduction in seed weight loss is advantageous in countries with hotter climates and increases the starch yield, in seeds at least, of transformed plants compared with control plants.
The results were similar when the experiment was repeated (see Table 8).

Table 8 Line Seed weight (g) Seed weight(g) Change in change weight (g) Control 0.032 0.0002 0.039 0.0004 0.007 17.9 Cimmyt 101 72.11b 0.033 0.0015 0.038 0.0003 0.005 13.2 79.42a 0.028 0.0009 0.031 0.0004 0.003 9.7 Example 7 Measurement of rate of starch synthesis Starch synthesis The rate of starch synthesis was measured by following incorporation of [U-14C] sucrose into starch. Sixty wheat endosperm were placed in a manometer flask containing a centre well and sidearm. The centre well contained 10% KOH and a piece of fluted filter paper to aid absorbtion of CO2. The endosperm were placed in incubation medium (lOmM Mes-NaOH, 319mM sorbitol, 60mM KCl, 6mM MgCl2, pH5.6). After equilibration for 30 minutes 20mM (U-14C] sucrose 37KBq (final concentration) was added from the sidearm and the flasks incubated for a further 3 hours. At the end of the incubation the medium and KOH paper were removed from the flask and the radioactivity determined.
The endosperm were removed from the flask and placed into a screw top eppendorf. The tissue was washed 5 times with 1 ml incubation medium (as above) per wash and then frozen in liquid nitrogen. TCA (lml) was added to a final concentration of 10%
and the tissue allowed to thaw. The endosperm were ground using a micropestle and then centrifuged for 5 minutes at 10,0008.
The supernatant was removed and the pellet washed in lml methanol:KC1 (75%:1% v:w) by re-suspension and centrifuged (10,0008, 5 minutes) (MSE microcentaur) five times. The radioactivity was determined in all of the washes from the above steps.
0.5m1 50mM acetic acid-NaOH buffer (pH4.8) was added to the tissue and boiled for 30 minutes. After cooling, starch present in the sample was digested by adding 40 units amyloglucosidase and 40 units a-amylase and incubating at 37°C for 12-16 hours.
Digests were centrifuged for 10 minutes at 10,0008. The supernatant (degraded starch) was then added to 4m1 scintillation fluid (Ecoscint A) and the radioactive counts were determined using a liquid scintillation counter (Tri-carb 300C).
Controls contained a) boiled tissue and b) tissue stopped at zero time. The amount of 14C label present in the degraded starch was calculated by subtracting the values for the control samples from the digests. The nmoles of hexose incorporated into starch was then derived from the amount of 14C label incorporated into starch. The rate of starch synthesis may therefore be derived from the time of incubation. The results are shown in Figure 19.

References:
Baecker, P.A., Preston, A., Furlong, C.E. and Preiss J. (1983) Biosynthesis of bacterial glycogen. Primary Structure of E.coli ADPG glucose synthetase as deduced from the nucleotide sequence of the glgC gene. J. Biol. Chem. 258 (8), 5084-5088.
Bartels, D. and Thompson, R.D. (1986). Synthesis of messenger-RNAs coding for abundant endosperm proteins during wheat-grain development. Plant Sci., 46 (2) 117-125.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, (1-2), 248-254.
Cheng, M., Fry, J.E., Pan, S.Z., Zhou H.P., Hironaka C.M., Duncan D.R., Conner, T.W., and Wan, Y.C. (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens.
Plant Physiology, 115 (3), 971-980.
Clackson, T. and Winter, G. (1989). "Sticky-Feet"-directed mutagenesis and its application to swapping antibody domains.
Nucl. Acids Res., 17, 10163-10170.
Echt, C.S. and Schwarz, D. (1981) Evidence for the inclusion of controlling elements within the structural gene at the waxy locus in maize. Genetics, 99, 275-284.
Edwards, E., Fulton, D.C., Hylton, C.M., Jobling, S.A., Gidley, M., Rossner, U., Martin, C. and Smith, A.M. (1999). A combined reduction in activity of starch synthases II and III of potato has novel effects on the starch of tubers. Plant J. 17:251-161.
Frazier, P.J., Donal, A.M. and Richmond, P. Starch: Structure and Functionality (1997). Royal Society of Chemistry, Cambridge, UK.
Fromm, M.E., Morrish, F., Armstrong, C., Williams, R., Thomas, J. and Klein, T.M. (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants.
Bio/Technology, 8 (9), 833-839.
Geurineau, F., Woolston, S., Brooks, L. and Mullineaux, P.
(1988). An expression cassette for targeting foreign proteins into chloroplasts. Nucl. Acids Res., 16 (23), 11380.
Gordon-Kamm, W.J., Spencer, T.M., Mangans, M.L., Adams, R.T., Dames, R.J. , Start, W.G. , O'Brien, J.V. , Chambers, S.A. , Adams, W.J. et a1. (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell, 2 (7), 603-618.
Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J.
(1990). PCR Protocols. A Guide to Methods and Applications.
Published Academic Press.
Kiel, J.A., Boels, J.M., Beldman, G. and Venema, G. (1994) Glycogen in Bacillus subtilis: molecular characterisation of an operon encoding enzymes involved in glycogen biosynthesis and degradation. Mol. Microbiol., 11(1), 203-218.

Kuipers, A.G.J; Jacobsen, E; Visser, R.G.F._, (1994). Formation and deposition of amylose in the potato tuber starch granule are affected by the reduction of granule-bound starch synthase gene expression. Plant Cell, 6 (1), 43-52.
Kumar, A., Larsen, C.E., Preiss, J. (1986) Biosynthesis of bacterial glycogen primary structure of E. coli ADP-glucose a-1,4-glucan, 4-glucosyltransferase as deduced from the nucleotide sequence of the glgA gene. J. Biol. Chem., 261 (34), 16256-16259.
Leung, P., and Preiss J. (1987) Cloning ADP glucose pyrophosphorylase glgC with glycogen synthase glgA structural genes from Salmonella-typhimurium. J. Bacteriol., 169 (9), 4349-4354.
Raleigh, E.A., Lech, K., and Brent, R. (1989) Current Protocols in Molecular Biology, Eds. Ausubel F.M. et al. Publishing Associates and Wiley Interscience, New York, Unit 1.4 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Publd. Cold Spring Harbor, U.S.A.
Schagger and Von Jagow (1987). Tricine-SDS-Polyacrylamide gel electrophoresis for the separation of proteins in the range from 1-100 kDA. Analy. Biochem., 166(2), 368-379.
Shewmaker, C.K; Boyer, C.D; Wiesenborn, D.P; Thompson, D.B;
Boersig, M.R; Oakes, J.V. (1994). Expression of Escherichia coli glycogen synthase in the tubers of transgenic potatoes (Solanum tuberosum) results in a highly branched starch. Pl.
Physiol, 104(4), 1159-1166.
Uttaro, A.D. and Ugalde, R.A. (1994) A chromosomal cluster of genes encoding ADP-glucose synthetase, glycogen synthase and phosphoglucomutase in Agrobacterium tumefaciens. Gene, 150 (1), 117-122.
Vasil, V., Castillo, A.M., Fromm, M.E. and Vasil, I.K. (1992).
Herbicide-resistant transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus.
Bio/Technology, 10(6), 667-674 Visser, G.F.; Stolte, A; Jacobsen, E, (1991) Expression of a chimaeric granule bound starch synthase-GUS gene in transgenic potato plants. P1. Mol. Biol, 17 (4), 691-699.
Visser, R.G.F.; Somhorst, I.; Kuipers, G.J.; Ruys, N.J.;
Feenstra, W.J.; Jacobsen, (1991a). Inhibition of the expression of the gene for granule bound starch synthase in potato by antisense constructs. Mol. Gen Genet., 225 (2), 289-296.
Materials Abbreviations LB - Luria broth TF - Tris-HC1, 1mM EDTA
SDS - sodium dodecyl sulphate CTAB - cetyl trimethyl ammonium bromide dATP - 2' - deoxy adenosine 5' triphosphate dTTP - 2' - deoxy thymidine 5' triphosphate dCTP - 2' - deoxy cytosine 5' triphosphate dGTP - 2' - deoxy guanosine DTT - dithiothreitol ATP - adenosine 5' triphosphate HEPES N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]
NBT - nitroblue tetrazolium BCIP - 5-bromo-4-chloro-3-indolyl phosphate GST - glutathione S transferase NAD - nicotinamide adenine dinucleotide IgG - immunoglobulin G
Mes - 2-[N-morpholino]ethane sulfonic acid ~~ ~~.4PEST TREATY ON THE INTERNATIONAL
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<W~.> Auvanced Technologies (Cambridge) Limited <120> Genetically Modified Plants with altered Starch <130> RD-ATC-20 <140>
<141>
<160> 10 <170> PatentIn Ver. 2.1 <210> 1 <211> 1467 <212> DNA

<213> ArtificialSequence <220>

<221> CDS

<222> (1)..(171) <223> Pea ssu transit peptide <220>

<221> CDS

<222> (172)..(1467) <223> E. coligCl6 gl <220>

<223> Description Artifici..l Sequence:
of Pea ssu TP

linked to E. g1gC16 CDS
coli <400> 1 atg get tct atatcctct tcagetgtg actacagtc agccgtget 48 atg Met Ala Ser IleSerSer SerAlaVal ThrThrVal SerArgAla Met tct acg gtg tcggccgcg gtggetcca ttcggcggc ctcaaatcc 96 caa Ser Thr Val SerAlaAla ValAlaPro PheGlyGly LeuLysSer Gln atg act gga ccagttaag aaggtcaac actgacatt acttccatt 144 ttc Met Thr Gly ProValLys LysValAsn ThrAspIle ThrSerIle Phe aca agc aat ggaagagta aagtgcatg cttagttta gagaagaac 192 ggt Thr Ser Asn GlyArgVal LysCysMet LeuSerLeu GluLysAsn Gly gat cac tta ttggcgcgc cagctgcca ttgaaatct gttgccctg 240 atg Asp His Leu LeuAlaArg GlnLeuPro LeuLysSer ValAlaLeu Met ata ctg gcg ggacgtggt acccgcctg aaggattta accaataag 288 gga Ile Leu Ala GlyArgGly ThrArgLeu LysAspLeu ThrAsnLys Gly cga gca aaa ccg gcc gta cac ttc ggc ggt aag ttc cgc att atc gac 336 Arg Ala Lys Pro Ala Val His Phe Gly Gly Lys Phe Arg Ile i-ie Asp 100 105 110.
ttt gcg ctg tct aac tgc atc aac tcc ggg atc cgt cgt atg ggc gtg 384 Phe Ala Leu Ser Asn Cys Ile Asn Ser Gly Ile Arg Arg Met Gly Val atc acc cag tac cag tcc cac act ctg gtg cag cac att cag cgc ggc 432 Ile Thr Gln Tyr Gln Ser His Thr Leu Val Gln His Ile Gln Arg Gly tgg tca ttc ttc aat gaa gaa atg aac gag ttt gtc gat ctg ctg cca 480 Trp Ser Phe Phe Asn Glu Glu Met Asn Glu Phe Val Asp Leu Leu Pro gca cag cag aga atg aaa ggg gaa aac tgg tat cgc ggc acc gca gat 528 Ala Gln Gln Arg Met Lys Gly Glu Asn Trp Tyr Arg Gly Thr Ala Asp gcg gtc acc caa aac ctc gac att atc cgt cgt tat aaa gcg gaa tac 576 Ala Val Thr Gln Asn Leu Asp Ile Ile Arg Arg Tyr Lys Ala Glu Tyr gtg gtg atc ctg gcg ggc gac cat atc tac aag caa gac tac tcg cgt 624 Val Val Ile Leu Ala Gly Asp His Ile Tyr Lys Gln Asp Tyr Ser Arg atg ctt atc gat cac gtc gaa aaa ggt gta cgt tgt acc gtt gtt tgt 672 Met Leu Ile Asp His Val Glu Lys Gly Val Arg Cys Thr Val Val Cys atg cca gta ccg att gaa gaa gcc tcc gca ttt ggc gtt atg gcg gtt 720 Met Pro Val Pro Ile Glu Glu Ala Ser Ala Phe Gly Val Met Ala Val gat gag aac gat aaa act atc gaa ttc gtg gaa aaa cct get aac ccg 768 Asp Glu Asn Asp Lys Thr Ile Glu Phe Val Glu Lys Pro Ala Asn Pro ccg tca atg ccg aac gat ccg agc aaa tct ctg gcg agt atg ggt atc 816 Pro Ser Met Pro Asn Asp Pro Ser Lys Ser Leu Ala Ser Met Gly Ile tac gtc ttt gac gcc gac tat ctg tat gaa ctg ctg gaa gaa gac gat 864 Tyr Val Phe Asp Ala Asp Tyr Leu Tyr Glu Leu Leu Glu Glu Asp Asp cgc gat gag aac tcc agc cac gac ttt ggc aaa gat ttg att ccc aag 912 Arg Asp Glu Asn Ser Ser His Asp Phe Gly Lys Asp Leu Ile Pro Lys atc acc gaa gcc ggt ctg gcc tat gcg cac ccg ttc ccg ctc tct tgc 960 Ile Thr Glu Ala Gly.Leu Ala Tyr Ala His Pro Phe Pro Leu Ser Cys gta caa tcc gac ccg gat gcc gag ccg tac tgg cgc gat gtg ggt acg 1008 Val Gln Ser Asp Pro Asp Ala Glu Pro Tyr Trp Arg Asp Val Gly Thr ctg gaa get tac tgg aaa gcg aac ctc gat ctg gcc tct gtg gtg ccg 1056 Leu Glu Ala Tyr Trp Lys Ala Asn Leu Asp Leu A~a Ser Val Val Pro 340 345 . 350 aaa ctg gat atg tac gat cgc aat tgg cca att cgc acc tac aat gaa 1104 Lys Leu Asp Met Tyr Asp Arg Asn Trp Pro Ile Arg Thr Tyr Asn Glu tca tta ccg cca gcg aaa ttc gtg cag gat cgc tcc ggt agc cac ggg 1152 Ser Leu Pro Pro Ala Lys Phe Val Gln Asp Arg Ser Gly Ser His Gly atg acc ctt aac tca ctg gtt tcc gac ggt tgt gtg atc tcc ggt tcg 1200 Met Thr Leu Asn Ser Leu Val Ser Asp Gly Cys Val Ile Ser Gly Ser gtg gtg gtg cag tcc gtt ctg ttc tcg cgc gtt cgc gtg aat tca ttc 1248 Val Val Val Gln Ser Val Leu Phe Ser Arg Val Arg Val Asn Ser Phe tgc aac att gat tcc gcc gta ttg tta ccg gaa gta tgg gta ggt cgc 1296 Cys Asn Ile Asp Ser Ala Val Leu Leu Pro Glu Val Trp Val Gly Arg tcg tgc cgt ctg cgc cgc tgc gtc atc gat cgt get tgt gtt att ccg 1344 Ser Cys Arg Leu Arg Arg Cys Val Ile Asp Arg Ala Cys Val Ile Pro gaa ggc atg gtg att ggt gaa aac gca gag gaa gat gca cgt cgt ttc 1392 Glu Gly Met Val Ile Gly Glu Asn Ala Glu Glu Asp Ala Arg Arg Phe tat cgt tca gaa gaa ggc atc gtg ctg gta acg cgc gaa atg cta cgg 1440 Tyr Arg Ser Glu Glu Gly Ile Val Leu Val Thr Arg Glu Met Leu Arg aag tta ggg cat aaa cag gag cga taa 1467 Lys Leu Gly His Lys Gln Glu Arg <210> 2 <211> 488 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Pea ssu TP
linked to E. coli g1gC16 CDS
<400> 2 Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala Ser Thr Val Gln Ser Ala Ala Val Ala Pro Phe Gly Gly Leu Lys Ser Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser Ile Thr Ser Asn Gly Gly Arg Val Lys Cys Met Leu Ser Leu Glu Lys Asn Asp His Leu Met Leu Ala Arg Gln Leu Pro Leu Lys Ser Val Ala Leu Ile Leu Ala Gly Gly Arg Gly Thr Arg Leu Lys Asp Leu Thr Asn Lys Arg Ala Lys Pro Ala Val His Phe Gly Glr Lys Phe Arg Ile Ile Asp Phe Ala Leu Ser Asn Cys Ile Asn Ser Gly Ile Arg A~5 Met Gly Val Ile Thr Gln Tyr Gln Ser His Thr Leu Val Gln His Ile Gln Arg Gly Trp Ser Phe Phe Asn Glu Glu Met Asn Glu Phe Val Asp Leu Leu Pro Ala Gln Gln Arg Met Lys Gly Glu Asn Trp Tyr Arg Gly Thr Ala Asp Ala Val Thr Gln Asn Leu Asp Ile Ile Arg Arg Tyr Lys Ala Glu Tyr Val Val Ile Leu Ala Gly Asp His Ile Tyr Lys Gln Asp Tyr Ser Arg Met Leu Ile Asp His Val Glu Lys Gly Val Arg Cys Thr Val Val Cys Met Pro Val Pro Ile Glu Glu Ala Ser Ala Phe Gly Val Met Ala Val Asp Glu Asn Asp Lys Thr Ile Glu Phe Val Glu Lys Pro Ala Asn Pro Pro Ser Met Pro Asn Asp Pro Ser Lys Ser Leu Ala Ser Met Gly Ile Tyr Val Phe Asp Ala Asp Tyr Leu Tyr Glu Leu Leu Glu Glu Asp Asp Arg Asp Glu Asn Ser Ser His Asp Phe Gly Lys Asp Leu Ile Pro Lys Ile Thr Glu Ala Gly Leu Ala Tyr Ala His Pro Phe Pro Leu Ser Cys Val Gln Ser Asp Pro Asp Ala Glu Pro Tyr Trp Arg Asp Val Gly Thr Leu Glu Ala Tyr Trp Lys Ala Asn Leu Asp Leu Ala Ser Val Val Pro Lys Leu Asp Met Tyr Asp Arg Asn Trp Pro Ile Arg Thr Tyr Asn Glu Ser Leu Pro Pro Ala Lys Phe Val Gln Asp Arg Ser Gly Ser His Gly Met Thr Leu Asn Ser Leu Val Ser Asp Gly Cys Val Ile Ser Gly Ser Val Val Val Gln Ser Val Leu Phe Ser Arg Val Arg Val Asn Ser Phe Cys Asn Ile Asp Ser Ala Val Leu Leu Pro Glu Val Trp Val Gly Arg Ser Cys Arg Leu Arg Arg Cys Val Ile Asp Arg Ala Cys Val Ile Pro Glu Gly Met Val Ile Gly Glu Asn Ala Glu Glu Asp Ala Arg Arg Phe Tyr Arg Ser Glu Glu Gly Ile Val Leu Val Thr Arg Glu Met Leu Arg Lys Leu Gly His Lys Gln Glu Arg <210> 3 <211> 421 <212> DNA
<213> Triticum aestivum <220>
<221> promoter <222> (1) . . (421) <223> High Molecular Weight Giuc~nir_.Promoter Genomic DNA
<400> 3 cccagctttg agtggccgta gatttgcaaa agcaatggct aacagacaca tattctgcca 60 aaccccaaga aggataatca cttttcttag ataaaaaaga acagaccaat atacaaacat 120 ccacacttct gcaaacaata catcagaact aggattacgc cgattacgtg gctttagcag 180 actgtccaaa aatctgtttt gcaaagctcc aattgctcct tgcttatcca gcttcttttg 240 tgttggcaaa ctgcgctttt ccaaccgatt ttgttcttct cgcgctttct tcttagccta 300 aacaaacctc accgtgcacg cagccatggt cctgaacctt cacctcgtcc ctataaaagc 360 ctagccaacc ttcacaatct tatcatcacc cacaacaccg agcaccacaa actagagatc 420 c 421 <210> 4 <211> 43 <212> DNA
<213> Artificial Sequence <220>
<221> primer bind <222> (1) . . (19) <223> Primer to 3' end of ssu transit peptide <220>
<221> primer bind <222> (20) . . (43) <223> Primer to 5' end of glgA CDS
<220>
<223> Description of Artificial Sequence: Oligo primer <400> 4 tggtggaaga gtaaagtgca tgcaggtttt acatgtatgt tca 43 <210> 5 <211> 54 <212> DNA
<213> Artificial Sequence <220>
<221> primer bind <222> (1) . . (19) <223> Primer to 3' end of glgC CDS
<220>
<221> primer bind <222> (26) . . (54) <223> Primer to 3' end of glgA CDS
<220>
<223> Description of Artificial Sequence: Oligo primer <400> 5 tcgctcctgt ttatgcccta gatctctatt tcgagcgata gtaaagctca cggt 54 <210> 6 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Oligo primer <220>
<221> primer bind <222> (11)..(33) <223> Primer to 5' end of ssu transit peptide <400> 6 acgtagatct atggcttcta tgatatcctc ttc 33 <210> 7 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Oligo primer <220>
<221> primer bind <222> (10) . . (36) <223> Primer to 5' end of HMWG promoter <400> 7 gacatcgatc ccagctttga gtggccgtag atttgc 36 <210> 8 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Oligo primer <220>
<221> primer bind <222> (10) . . (39) <223> primer to 3' end of HMWG promoter <400> 8 gacgaattcg gatctctagt ttgtggtgct cggtgttgt 39 <210> 9 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<221> primer bind <222> (9) . . (32) <223> Primer to 5' end of nopaline synthase terminator <220>
<223> Description of Artificial Sequence: Oligo primer <400> 9 caggatccga atttcacccg atcgttcaaa ca 32 <210> 10 <211> 50 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Oligo primer <220>
<221> primer bind <222> (23)..(50) <223> Primer to 3' end of nopaline synthase terminator <400> 10 gacccgcggc tcgaggcggc cgcccgatct agtaacatag atgacaccgc 50

Claims (22)

46

1. A method of reducing the loss of starch yield in wheat or maize grown under high temperature conditions, the method comprising the steps of stably introducing into the plant genome a nucleic acid sequence encoding glycogen synthase under the direction of a suitable promoter and a suitable terminator, and regenerating a plant having an altered genome.

2. A method according to Claim 1, wherein the temperature is greater than 23°C.

3. A method according to Claim 2, wherein the temperature is greater than 25°C.

4. A method according to Claim 3, wherein the temperature is greater than 27°C.

5. A method according to any one of Claims 1 to 4, wherein a greater than normal starch yield in seeds is achieved at higher temperatures during seed growth compared with control seed grown at 20°C.

6. A method according to any one of Claims 1 to 5, wherein said nucleic acid sequence encoding glycogen synthase is a sequence obtained from a unicellular organism, an alga or bacterium, which sequence has the necessary ability to encode glycogen synthase.

7. A method according to Claim 6, wherein said glycogen synthase is derived from E.coli, Agrobacterium, Salmonella or Bacillus.

8. A method according to any one of Claims 1 to 7, wherein said promoter is capable of directing expression in a particular tissue of the plant and/or at particular stages of development of the plant.

9. A method according to any one of the preceding claims, wherein said promoter is heterologous or homologous with respect to said plant.

10. A method according to any one of the preceding claims, wherein said promoter directs expression to the endosperm of the seed.

11. A method according to Claim 10, wherein said promoter is the high molecular weight glutenin (HMWG) gene of wheat.

12. A method according to Claim 8, wherein said promoter is one or more of the group consisting of the promoters of gliadin, branching enzyme, ADPG pyrophosphorylase, starch synthase and actin.

13. A method according to any one of the preceding claims, wherein said chimaeric gene also contains a sequence that encodes a transit peptide which provides for translocation of the glycogen synthase and/or a marker gene or other coding sequence to the plant plastid.

14. A method according to Claim 13, wherein said transit peptide is one or more of the group consisting of the small subunit of the ribulose bisphosphate carboxylase enzyme (ssu of Rubisco) from pea, maize or sunflower, the transit peptide for the plant plastid aryl carrier protein (ACP) or the transit peptide for GBSSI.

15. A method according to any one of the preceding claims, wherein said chimaeric gene comprises one or more additional coding sequences from the starch or glycogen biosynthetic pathway.

16. A method according to Claim 15, wherein said additional coding sequence is the sequence for glycogen branching enzyme (EC 2.4.1.18).

17. A method according to any one of the preceding claims, wherein said chimaeric gene also comprises a gene switch mechanism which determines under what conditions or when the coding sequence is to be expressed.

18. A method according to Claim 17, wherein said gene switch is a chemically induced promoter or a temperature controlled promoter.

19. Maize or wheat plants having a higher starch yield when grown under higher temperatures compared with control seed grown at 20°C, said plants comprising cells containing a chimaeric gene comprising a promoter, a coding sequence for glycogen synthase, and a terminator.

20. Maize or wheat plants according to Claim 19, said plants having been produced by the method of any one of Claims 1-18.

21. Maize or wheat plants according to Claim 19, wherein starch obtained from said plants has an altered chain length or processing property compared with control starch from a non-transformed plant.

22. Seed of a maize or wheat plant transformed in accordance with any one of Claims 1-18, said seed exhibiting a higher weight and/or less of a loss in yield than seed of control plants grown at 20°C.