WO1997020936A1

WO1997020936A1 - Modification of starch synthesis in plants

Info

Publication number: WO1997020936A1
Application number: PCT/GB1996/002990
Authority: WO
Inventors: Peter Lewis Keeling; Mary Elizabeth Knight
Original assignee: Zeneca Limited
Priority date: 1995-12-06
Filing date: 1996-12-04
Publication date: 1997-06-12
Also published as: GB9524938D0; EP0865494A1; JP2000500982A; AU1037197A; AU725926B2; CA2238399A1

Abstract

Plants, particularly cereal plants which have modifications to their starch synthesising pathway contain a DNA specifying the enzyme soluble starch synthase which has the sequence SEQ ID NO 1 or SEQ ID NO 2 or SEQ ID NO 3. The inserted gene may be inserted in a sense or anti-sense construct. The alteration introduced by the inserted genes may be a greater or reduced ability to produce starch or starch which has a different fine structure such as a different pattern of branching.

Description

MODIFICATION OF STARCH SYNTHESIS LN PLANTS

This invention relates to the alteration of the biosynthetic pathway which leads to production of starch in plants. By the term "alteration" we mean a change from normal of the amount or quality of the starch which the plant produces. More particularly, the invention relates to the isolation, purification and characterisation of the DNAs encoding several forms of the enzyme soluble starch synthase and the use of those DNAs through genetic modification of the plant genome to alter the starch production. The invention also relates to novel plants having an improved ability to produce starch including an improved ability to produce structurally-altered starch.

Our previous studies have led to a new understanding of the metabolic pathway of starch synthesis in developing starch storing tissues (Keeling et al, 1988, Plant Physiology, 87:311-319; Keeling, 1989, ed. CD. Boyer, J.C. Shannon and R.C. Harrison; pp.63-78, being a presentation at the 4th Annual Penn State

Symposium in Plant Physiology).

Starch is an important end-product of carbon fixation during photosynthesis in leaves and is an important storage product in seeds and fruits. In economic terms, the starch produced by the edible portions of three grain crops, wheat, rice and maize, provide approximately two-thirds of the world's food calculated as calories.

Starch is synthesised in the plastid compartment, the chloroplast, in photosynthetic cells or the amyloplast in non- photosynthetic cells. The biochemical pathway of starch biosynthesis in leaves has been well-characterised (Figure 1 ). In contrast, little is known of the pathway of starch biosynthesis in storage organs.

Two principal methods for the control of gene expression are known. These are referred to in the art as "antisense downregulation" and "sense downregulation" or "cosuppression". Both of these methods lead to an inhibition of expression of the target gene. Overexpression is achieved by insertion of one or more than one extra copies of the selected gene. Other lesser used methods involve modification of the genetic control elements, the promoter and control sequences, to achieve greater or lesser expression of an inserted gene.

In antisense downregulation, a DNA which is complementary to all or part of the target gene is inserted into the genome in reverse orientation and without its translation initiation signal. The simplest theory is that such an antisense gene, which is transcribable but not translatable, produces mRNA which is complementary in sequence to mRNA product transcribed from the endogenous gene: that antisense mRNA then binds with the naturally produced "sense" mRNA to form a duplex which inhibits translation of the natural mRNA to protein. It is not necessary that the inserted antisense gene be equal in length to the endogenous gene sequence: a fragment is sufficient. The size of the fragment does not appear to be particularly important. Fragments as small as 40 or so nucleotides have been reported to be effective. Generally somewhere in the region of 50 nucleotides is accepted as sufficient to obtain the inhibitory effect. However, it has to be said that fewer nucleotides may very well work: a greater number, up to the equivalent of full length, will certainly work. It is usual simply to use a fragment length for which there is a convenient restriction enzyme cleavage site somewhere downstream of fifty nucleotides. The fact that only a fragment of the gene is required means that not all ofthe gene need be sequenced. It also means that commonly a cDNA will suffice, obviating the need to isolate the full genomic sequence.

The antisense fragment does not have to be precisely the same as the endogenous complementary strand of the target gene. There simply has to be sufficient sequence similarity to achieve inhibition of the target gene. This is an important feature of antisense technology as it permits the use of a sequence which has been derived from one plant species to be effective in another and obviates the need to construct antisense vectors for each individual species of interest. Although sequences isolated from one species may be effective in another, it is not infrequent to find exceptions where the degree of sequence similarity between one species and the other is insufficient for the effect to be obtained. In such cases, it may be necessary to isolate the species-specific homologue. Antisense downregulation technology is well-established in the art. It is the subject of several textbooks and many hundreds of journal publications. The principal patent reference is European Patent No. 240,208 in the name of Calgene Inc. There is no reason to doubt the operability of antisense technology. It is well-established, used routinely in laboratories around the world and products in which it has been used are on the market.

Both overexpression and downregulation are achieved by "sense" technology. If a full length copy of the target gene is inserted into the genome then a range of phenotypes is obtained, some overexpressing the target gene, some underexpressing. A population of plants produces by this method may then be screened and individual phenotypes isolated. As with antisense, the inserted sequence is lacking in a translation initiation signal. Another similarity with antisense is that the inserted sequence need not be a full length copy. Indeed, it has been found that the distribution of over- and under- expressing phenotypes is skewed in favour of underexpression and this is advantageous when gene inhibition is the desired effect. For overexpression, it is preferable that the inserted copy gene retain its translation initiation codon. The principal patent reference on cosuppression is European Patent 465,572 in the name of DNA Plant Technology Inc. There is no reason to doubt the operability of this technology. It is well- established, used routinely in laboratories around the world and products in which it has been used are on the market.

Sense and antisense gene regulation is reviewed by Bird and Ray in Biotechnology and Genetic Engineering Reviews 9: 207-227 (1991). The use of these techniques to control selected genes in tomato has been described by Gray et.al., Plant Molecular Biology, 19: 69-87 (1992).

Gene control by any of the methods described requires insertion of the sense or antisense sequence, with appropriate promoters and termination sequences containing polyadenylation signals, into the genome of the target plant species by transformation, followed by regeneration of the transformants into whole plants. It is probably fair to say that transformation methods exist for most plant species or can be obtained by adaptation of available methods. For dicotyledonous plants the most widely used method is Agrobacterium- mediated transformation. This is the best known, most widely studied and, therefore, best understood of all transformation methods. The rhizobacterium Agrobacterium tumefaciens, or the related Agrobacterium rhizogenes, contain certain plasmids which, in nature, cause the formation of disease symptoms, crown gall or hairy root tumours, in plants which are infected by the bacterium. Part of the mechanism employed by Agrobacterium in pathogenesis is that a section of plasmid DNA which is bounded by right and left border regions is transferred stably into the genome of the infected plant. Therefore, if foreign DNA is inserted into the so-called "transfer" region (T-region) in substitution for the genes normally present therein, that foreign gene will be transferred into the plant genome. Thers are many hundreds of references in the journal literature, in textbooks and in patents and the methodology is well-established.

The effectiveness of Agrobacterium is restricted to the host range of the microorganism and is thus restricted more or less to dicotyledonous plant species

In general monocotyledonous species, which include the important cereal crops, are not amenable to transformation by the Agrobacterium method. Various methods for the direct insertion of DNA into the nucleus of monocot cells are known. In the ballistic method, microparticles of dense material, usually gold or tungsten, are fired at high velocity at the target cells where they penetrate the cells, opening an aperture in the cell wall through which DNA may enter. The DNA may be coated on to the microparticles or may be added to the culture medium.

In microinjection, the DNA is inserted by injection into individual cells via an ultrafine hollow needle.

Another method, applicable to both monocots and dicots, involves creating a suspension of the target cells in a liquid, adding microscopic needle-like material, such as silicon carbide or silicon nitride "whiskers", and agitating so that the cells and whiskers collide and DNA present in the liquid enters the cell. In summary, then, the requirements for both sense and antisense technology are known and the methods by which the required sequences may be introduced are known. What remains, then is to identify genes whose regulation will be expected to have a desired effect, isolate them or isolate a fragment of sufficiently effective length, construct a chimeric gene in which the effective fragment is inserted between promoter and termination signals, and insert the construct into cells of the target plant species by transformation. Whole plants may then be regenerated from the transformed cells.

An object of the present invention is to provide DNAs encoding soluble starch synthases.

An further object of the invention is to provide novel plants having an increased capacity to produce starch and a capacity to produce starch with an altered fine structure.

According to the present invention there is provided cDNAs having the sequences of the inserts in plasmids pSSS6, pSSSlO. l and pSSS6.31 and sequences having sufficient similarity such that when inserted into the genome of an organism which produces starch, the synthesis of starch is altered.

The plasmid pSSS6 was deposited under the terms of the Budapest Treaty, with the National Collections of Industrial and Marine Bacteria Limited, 23 St Machar Drive, Aberdeen AB1 2RY, on 13th June 1994, under the Accession Number 40651. The plasmids pSSS6.31 and pSSSlO.l were deposited under the terms of the Budapest Treaty, with the National Collections of Industrial and Marine Bacteria Limited, 23 St Machar Drive, Aberdeen AB1 2RY, on 22nd August 1994, under the Accession Numbers NCIMB 40679 and 40680 respectively.

The invention also provides the cDNAs, encoding soluble starch synthases which have the sequences SEQ-ID-NO- 1 , SEQ-ID-NO-2 AND SEQ-ID-NO-3.

The invention also provides transformed plants containing one or more copies of one or more of the said cDNAs in sense or antisense orientation. The description which follows will describe a method for the isolation of the genes encoding soluble starch synthases from maize. These DNAs can be used for the isolation of the corresponding genomic sequences. Either the cDNAs or the genes can then be used in studies leading to the increase in starch yield. One possible application could be the use of these sequences to increase gene dosage of SSS in transformed crop plants to determine the contribution of SSS to the net regulation of starch biosynthesis, and to modify the levels of starch synthesised by the plant. The introduction of additional copies of SSS genes should produce greater levels of the enzyme in the amyloplasts.

Increased gene expression may also be elicited by introducing multiple copies of enhancer sequences into the 5'-untranscribed region of SSS gene. If the enzyme is rate-limiting to starch biosynthesis, then the rate of starch biosynthesis would be expected to increase in the transformed plants. By virtue of this invention it will also be possible to alter the kinetic properties of the endopserm enzyme through protein engineering. Obviously a number of other parameters could also be improved. The present invention will now be described, by way of illustration, by the following Example and with reference to the accompanying drawings of which: Figure 1 shows the reactions involved in the biosynthetic pathways of starch and glucose in leaves. The abbreviations used are: G-3-P,glyceraldehyde-3- phosphate; DHAP, dihydroxyacetone phosphate; Pi, orthophosphate; PPi, inorganic pyrophosphate. The reactions are catalysed by the following enzymes:

I) phosphoglycerate kinase/glyceraldehyde- 3 -phosphate dehydrogenase 2) triose-phosphate isomerase

3) aldolase

4) fructose- 1 ,6-bisphosphatase

5) hexose phosphate isomerase

6) phosphoglucomutase 7) ADP-glucose pyrophosphorylase

8) starch synthase

9) UDP-glucose pyrophosphorylase

10) sucrose phosphate synthase

I I ) sucrose phosphatase 12) orthophosphate/triose phosphate translocator

13 ) inorganic pyrophosphatase Figure 2 shows the proposed metabolic pathway of starch biosynthesis in wheat endosperm (Keeling et. al. 1988). The abbreviations used are the same as in Figure 1. The reactions are catalysed by the following enzymes:

1 ) sucrose synthase

2) UDP-glucose pyrophosphorylase

3) hexokinase

4) phosphoglucomutase 5) hexose -phosphate isomerase

6) ATP-dependent phosphofructokinase

7) PPi-dependent phosphofructokinase

8) aldolase

9) triose-phosphate isomerase 10) hexose-phosphate translocator

11 ) ADP-glucose pyrophosphorylase

12) starch synthase

13) sucrose phosphate synthase

14) sucrose phophatase

USEOFSOLUBLESTARCHSYNTHASEORBRANCHINGENZYME

Using standard cloning techniques, the SSS genes may be isolated. The source of the genes was a US yellow-dent com line of Zea mays, from which the enzyme protein was purified. Endosperms from the maize line were homogenised in a buffer which maintains the SSS in active form.

Purification ofthe SSS from maize has been achieved by a combination of ammonium sulphate precipitation, DEAE-cellulose chromatography, gel- filtration, phenyl Superose and FPLC using a Mono-Q column. This results in several hundred- fold purification with yields up to 5%. The SSS polypeptide was a single subunit of molecular weight 76kDa. Other SSS polypeptides were present in a US dent inbred line at around 60kDa, 70kDa and 105kDa molecular weight. Ammonium sulphate precipitation of SSS I is best achieved using 10-35% ammonium sulphate which produces a translucent SSS-enriched pellet which is next dialysed and further fractionated using DEAE-cellulose ion-exchange chromatography (2.5 x 5cm column). SSS was eluted with a 150 ml gradient of KCI (0-0.6M) and fractions collected. These steps increase specific activities by up to 12-fold. The DEAE peak fractions were concentrated by precipitation with ammonium sulphate (40%) and the resulting pellet dissolved in buffer and fractionated on a Sephacryl S-200 column (2.5 x 100 cm) equilibrated with buffer and fractions collected. These steps increase specific activities by up to 8-fold. A Phenyl-Superose column was equilibrated with buffer containing ammonium sulphate. SSSI did not bind and was present in the pass-through fraction. These steps increase specific activities by up to 2-fold. Finally, a Mono-Q column was equilibrated with buffer and charged with the Phenyl-Superose pass-through fraction. The errzymes were eluted from the column using a 12 ml linear gradient of 0-0.5 M KCI and fractions collected. These steps increase specific activities by up to 5-fold.

In the final purification step the SSS preparations were loaded on to SDS PAGE gels. The bands corresponding to the SSS polypeptides were cut out and eluted. The polypeptide was sequenced using standard amino acid sequencing techniques.

In order to produce a pure antigen for antibody production, we decided to use starch granules as our starting-point for isolation of SSS proteins. Kernels were homogenised in buffer by grinding in a Waring blender. The homogenate filtered through miracloth and centrifuged. After discarding the supematant and the discoloured material that overlays the white starch pellet, the pellet was washed twice with buffer and centrifuged. Starch was washed a final time with chilled acetone and following centrifugation, dried under a stream of air before storing at -20C. Granule protein was extracted by boiling 1.4 g starch for 10 minutes in 50ml SDS-PAGE sample buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol and 62.5 mM Tris/HCI, pH 6.8) which lacked bromophenol blue. After cooling and centrifugation at 25,000 g at 4C for 15 minutes, the supernatant was mixed with an equal volume of 30% TCA and allowed to stand at 4C for 1 hour. The solution was centrifuged again and pellet washed twice with 10 ml acetone before resuspension in 1.4 ml SDS-PAGE sample buffer. Following separation of granule-derived proteins by SDS-PAGE, the SSS proteins (eg 60kDa, 76kDa etc) bands were electroeluted and used as antigen (three 50ug doses at 4-week intervals, in New Zealand white rabbits) to generate polyclonal antibodies in a rabbit. The antibodies were then tested for specificity to the SSS polypeptides. Antibodies were monospecific and have enabled a thorough analysis of enzyme activities and expression studies.

N-terminal amino acid sequences were also obtained from the polypeptides. These proteins were shown to be identical with soluble proteins on the basis of (i) N-terminal sequences to the SSSs as purified by conventional means and sequenced were identical to the granule derived proteins, and (ii) protease digests gave peptide maps which were also identical.

Amino acid sequencing ofthe maize SSS polypeptide has yielded the following partial sequences:

N-terminal... C V A E L S R E G P A P R

Intemal sequences: KNYANAFYTETHI

ELGGYIYGQNDMFVVNNDHASLVPVLLAAKYIR

EVTTAEGGSGLNELL

GKIDNTVVVASEQDSY

The antibodies may be used to screen a maize endosperm cDNA library for clones derived from the mRNAs for SSS in an in vitro transcription/ translation system. Synthetic oligos may be constructed and used to screen maize endosperm cDNA library. The SSS sequence may be compared to the amino acid sequence of pea SSS I and SSS II published by Dry et al (1991, Plant Journal 2: 193-202) or rice SSS published by Baba et al (1993, Plant Physiology 103, 565-573). Interestingly, the clone obtained from rice SSS is not correctly identified. The N-terminal sequence AELSREG is stated to be part of the transit peptide sequence ofthe rice clone. This error must have occurred because of protein isolation problems from rice kernels: presumably a portion ofthe protein was cleaved prior to isolation. Using our N-terminal sequence, the corrected molecular weight of the rice clone is around 69kDa and not 55 or 57kDa as suggested by Baba et al. cDNA LIBRARY SCREENING AND ISOLATION OF SSS cDNA CLONES

RNA was extracted from from 21 DAP endosperm (obtained from the inbred line B73) after removal of pericarp and embryo. The library consisted of -900,000 recombinant clones. A probe for granule bound starch synthase was generated using PCR and used to screen an aliquot of the library, -500,000 recombinants. This screening yielded approximately 200 positive signals. Isolation and sequencing of a number showed them to be full length GBSS cDNA clones.

An oligonucleotide was synthesised to N-terminal sequence obtained from the purified SSS protein and used to screen the same aliquot of library as that used for the GBSS screening. No positive signals were obtained. A long oligonucleotide probe was then synthesised to the

ADP-ADPG binding region and following sequence, based on a comparison of the sequences published for pea SSS, rice SSS and maize GBSS.

The sequence of the oligonucleotide was GGT/C GGA/G CTA/T GGAGATGTTTGTGGA/T

TCACTCCCAATTGCTCTT/G GCTCTTCGTGGA T CATCGTGTG ATGGTTGT. Fifteen strong signals were obtained,all were picked, of these ten plaque purified after two rounds of purification. Restriction analysis of all ten showed them to fall into two classes.

Sequence analysis showed both classes to be starch synthases.

Screening of a maize seedling library (Clontech) gave positive signals using 5' probes from one class of clones only. A cDNA library from the inbred line W64A was screened and full length clones were isolated as judged by comparison with N-terminal sequence.

CHARACTERISATION OF cDNA CLONES

The isolated cDNAs were sequenced and are given herewith as SEQ-ID-NO-1, NO-2 and NO- 3.

For comparison, the deduced amino acid sequences are shown here with the sequences obtained directly from the protein :-

CVAELSREGPAPR peptide derived

CVAELSREGPAPR deduced cDNA 6.4

KXYANAFYTETΗI peptide derived

KNYANAFYSEKHI deduced cDNA

10.52 EVTTAEGGSGLNELL peptide derived

EVTTAEGGQGLNELL deduced cDNA 10.52

ELGGYIYGANXMFWNXXHASLVPVLLAAKY peptide derived

ELGGYIYGQNCMLWNDWHASLEPVLLAAKY deduced cDNA 10.52

GKIDNTVVVASEQDSY peptide derived

GSIDNTVVVASEQDSE deduced cDNA 10.52

Isolated from soluble 76kDa protein. GLWTRDRDRIQ-VASNR peptide derived

GAVVTADRIVTVSKGYS deduced cDNA 10.52

Clone SSS6.31 contained none of these intemal sequences. The motif for the binding-site of ADPG and ADP, thought to be part of the active site of starch synthases is found in all clones near to the 5' end and is followed by the highly conserved sequence on which the oligonucleotide probe was based. The highly conserved domain SRFEPCGLNQLYAMXYGTXXXXXXXGGLRDTV is present in SSS 10.52 but is slightly modified in SSS6.31 in that the EPC motif is replaced with an AG motif.

Expression of maize starch synthases in Escherichia coli BL21(DE3).

These SSS clones have been transfected into E.coli. The SSS activity was measured and are reported in the Table below.

Plasmids Maize starch N-terminus Protein Specific synthase genes (mg/mL) Activities*

(units/mg Protein) pET21a Native plasmid <no insert> 1.8 .009

pEXS-3a MSSSII GENVMNVrV 2.8 0.069 (MSSS631) V pEXS-8 1.9 0.097

MSSSI (MSSS6- CVAELSREGP pEXS-9 4) 1.8 0.515

GSVGAALRSY pEXS-wx MSSSIII 2.0 0.033 (MSSS5.6) ASAGMNVVF V

MGBSS (waxy)

• One unit activity is defined as one mmol glucose incoφorated into a- 1,4 glucan per minute at 25°C using 5 mg/mL glycogen as primer. GENE CONSTRUCTS FOR TRANSFORMATION The gene constructs require the presence of an amyloplast transit peptide to ensure its correct localisation in the amyloplast. It is believed that chloroplast transit peptides have similar sequences but other potential sources are available such as that attached to ADPG pyrophosphorylase (Plant Mol. Biol. Reporter (1991) 9, 104-126). Other potential transit peptides are those of small subunit RUBISCO, acetolactate synthase, glyceraldehyde-3P- dehydrogenase and nitrite reductase. For example,

Consensus sequence of the transit peptide of small subunit RUBISCO from many genotypes has the sequence:

MASSMLSSAAV³/ATRTNPAQAS MVAPFTGLKSAAFPVSRK QNLDITSIA

SNGGRVQC and the com small subunit RUBISCO has the sequence:

MAPTVMMASSAT-ATRTNPAQAS AVAPFQGLKSTASLPVARR SSRSLGNVA

SNGGRIRC

The transit peptide of leaf starch synthase from corn has the sequence:

MA ALATSQLVAT RAGLGVPDAS TFRRGAAQGL RGARASAAAD TLSMRTASARA APRHQQQARR GGRFPSLVVC

The transit peptide of leaf glyceraldehyde-3P- dehydrogenase from com has the sequence: MAQILAPS TQWQMRITKT SPCATPITSK MWSSLVMKQT KKVAHSAKFR

VMAVNSENGT

The putative transit peptide from ADPG pyrophosphorylase from wheat has the sequence:

RASPPSESRA PLRAPQRSAT RQHQARQGPR RMC

It is possible however to express the genes constitutively using one of the well-known constitutive promoters such as CaMV35S but there may be biochemical penalties in the plant resulting from increased starch deposition throughout the entire plant. Deposition in the endosperm is much preferred.

Possible promoters for use in the invention include the promoters ofthe starch synthase gene, bound starch synthase gene, endopserm hsp70 gene, ADPG pyrophosphorylase gene, and the sucrose synthase gene.

FOR TESTING GENE EXPRESSION IN ENDOSPERM TISSUE:

Plasmid name Promoter Intron Targetting Gene pHKHl CaMV35S adhl WxTrPep GUS pShlPIGN CaMV35S adhl WxTrPep GUS pSh2PIGN CaMV35S adhl WxTrPep GUS

FOR TESTING IN SUSPENSION CELL CULTURES:

Plasmid name Promoter Intron Targetting Gene p*** l CaMV35S Shl WxTrPep GUS p***2 CaMV35S adhl WxTrPep GUS

FULL VECTORS FOR PLANT TRANSFORMATION

Plasmid name Promoter Intron Targetting Gene p***21 Waxy Shl WxTrPep SSS and/or BE p***22 Waxy Adhl WxTrPep SSS and/or BE p***23 Shl Shl WxTrPep SSS and/or BE p***24 Shl Adhl WxTrPep SSS and or BE p***25 Sh2 Shl WxTrPep SSS and/or BE p***26 Sh2 Adhl WxTrPep SSS and or BE p***27 hsp70 Shl WxTrPep SSS and/or BE p***28 hsp70 Adhl WxTrPep SSS and/or BE TRANSFORMATION

(i) Insertion of extra copies of the gene

Maize genomic DNAs isolated as above may subsequently be transformed into either protoplasts or other tissues of a maize inbred line or population. The existing gene promoters ensure that the extra genes are expressed only in the developing endosperm at the correct developmental time. The protein sequences likewise ensure that the enzymes are inserted into the amyloplast.

Transgenic maize plants are regenerated and the endosperms of these plants are tested foi increased SSS enzyme activity. The kernels are also tested for enhanced rate of starch synthesis ,ιt different temperatures. The plants are then included in a breeding programme to produce new maize hybrids with higher rates of starch synthesis at temperatures above the normal optimum.

(ii) Insertion of genes specifying SSS

This is also achieved by standard cloning techniques. The source ofthe temperature-stable forms ofthe SSS genes is any organism that can make starch or glycogen. Potential donor organisms are screened and identified as described above. Thereafter there are two approaches:

(a) via enzyme purification and antibody/sequence generation using the protocol described above.

(b) using SSS cDNAs as heterologous probes to identify the genomic DNAs for SSS in libraries from the organism concerned. The gene transformation, plant regeneration and testing protocols are as described above. In this instancs it is necessary to make gene constructs for transformation which contain the regulatory sequences from maize endosperm SSS or another maize endosperm starch synthesis pathway enzyme to ensure expression in endosperm at the correct developmental time (eg, ADPG pyrophosphorylase). Gene constructs used to transform plants requires the regulatory sequences from maize endosperm SSS or another maize endosperm starch synthesis pathway enzyme to ensure expression in endosperm at the correct development time (eg, ADPG pyrophosphorylase). Furthermore the gene constructs also requires a suitable amyloplast transit -peptide sequence such as from maize endosperm SSS or another maize endosperm starch synthesis pathway enzyme to censure expression ofthe amyloplast at the correct developmental time (eg, ADPG pyrophosphorylase) .

Genetic protein engineering techniques may also be used to alter the amino acid sequence ofthe SSS enzymes to impart higher temperature optima for activity. The genes for SSS may be cloned into a bacteria which relies on these enzymes for survival. Selection for bacteria surviving at evaluated temperatures enables the isolation of mutated thermostable enzyme forms. Transformation of maize with the altered genes is carried out as described above.

(iii) Changing the ratios of activities of the isoforms of enzymes SSS

This is also achieved by standard cloning techniques. The source of the SSS genes is maize using the protocol described above. Plants are then transformed by insertion of extra gene copies ofthe isoforms of SSS enzymes and/or by insertion of anti- sense gene constructs. The gene promoters and other regulatory sequences may also be altered to achieve increased amounts ofthe enzyme in the recipient plant.

(iv) Insertion of a gene or genes specifying SSS with activities which effect a change in the fine structure ofthe starch.

This is also achieved by standard cloning techniques. The source ofthe special forms of the SSS is any organism that can make starch. Potential donor organisms are screened and identified as described above. Thereafter there are two approaches:

(b) using SSS cDNAs as heterologous probes to identify the genomic DNAs for SSS in libraries from the organism concerned. The gene transformation, plant regeneration and testing protocols are as described above. In this instance it is necessary to make gene constructs for transformation which contain the regulatory sequences from maize endosperm SSS or another maize endosperm starch synthesis pathway enzyme to ensure expression in endosperm at the correct developmental time (eg, ADPG pyrophosphorylase) .

Full length clone sequences

SEQ-ID-NO1; DNA; 2992 BP.

CC NOTE: ORIGINAL SEQUENCE NAME WAS SSS 1052 and SSS64

SQ SEQUENCE 2992 BP; 758 A; 655 C; 801 G; 776 T; 2 OTHER; GAATTCGCGG CCGCCTTATT TCTGGTTGGC CACATACATC ATCCAAAAAA

CTTTATTATT

GAATTACAAC TAATAAGCAA TCTAAAAGAG GGCACCACCA ATGATGTGTT GTTGGTAGGA

GGCCGCTGGG TCTGTCAAAG CAAGTTGGAC AAAGGGCAAC AATTGTTGTA GTTGTAAGAG

GGTTGCGGGG TTAGCCGCAA ACTGCTGGTA GAAAGGCAGC AACTGTTGCT GTGTCAAGAA

GGAAGCACGG TTTGCTGCAG CTGTTGTGCC CTGATGGTTT GTACCAATGA CTGCACCAAA GATAGGGCTG GCGATTGTTG AAACAACAAG GGCGATAAAG GTATGTTGCT

TGCTGCGATT

GCTTGTTGAA GCCTATATGG TTGAAGAGCT GGGTTTTCAC ATATTGAAGC TATAATTGAT

GGAAGGTATG GGGGAAGAAG GGAAGCTATA GGAGCTTGTG AGCATTGAGG GAAAATTGTC

GCGTTAGCAA CACATGTAGA AAGAGCAAGG AGCATAAGGA GGGAAAATAT CTTGGTCGCC

ATTGTTGCGC GCGATCCACG GCCCCCCCCC CCCGCGCTCC TGTCTGCTCT CCCTCTCCGC AATGGCGACG CCCTCGGCCG TGGGCGCCGC GTGCCTCCTC CTCGCGCGGG

NCG CCTGGCC

GGCCGCCGTC GGCGACCGGG CGCGCCCGCG GAGGCTCCAG CGCGTGCTGC GCCGCCGGTG CGTCGCGGAG CTGAGCAGGG AGGGGCCCGC GCCGCGCCCG CTGCCACCCG CGCTGCTGGC

GCCCCCGCTC GTGCCCGGCT TCCTCGCGCC GCCGGCCGAG CCCACGGGTG AGCCGGCATC GACGCCGCCG CCCGTGCCCG ACGCCGGCCT GGGGGACCTC GGTCTCGAAC

CTGAAGGGAT

TGCTGAAGGT TCCATCGATA ACACAGTAGT TGTGGCAAGT GAGCAAGATT CTGAGATTGT

GGTTGGAAAG GAGCAAGCTC GAGCTAAAGT AACACAAAGC ATTGTCTTTG TAACCGGCGA

AGCTTCTCCT TAATCGAAAG TCTGGGGGTC TAGGAGATGT TTGTGGTTCA TTGCCAGTTG

CTCTTGCTGC TCGCGGTCAC CGTGTGATGG TTGTAATGCC CAGACATTTA AATGGTACCT CCGATAAGAA TTATGCAAAT GCATTTTACT CAGAAAAACA CATTCGGATT

CCATTCTTTG

GCGGTGAACA TGAAGTTACC TTCTTCCATG AGTATAGAGA TTCAGTTGAC TGGGTGTTTG

TTGATCATCC CTCATATCAC AGACCTGGAA ATTTATATGG AGATAAGTTT GGTGCTTTTG

GTGATAATCA GTTCAGATAC ACACTCCTTT GCTATGCTGC ATGTGAGGCT CCTTTGGTCC

TTGAATTGGG AGGATATATT TATGGACAGA ATTGCATGTT GGTTGTCAAT GATTGGCATG CCAGTCTAGA GCCAGTCCTT CTTGCTGCAA AATATAGACC ATATGGTGTT

TATAAAGACT

CCCGCAGCAT TCTTGTAATA CATAATTTAG CACATCAGGG TGTAGAGCCT GCAAGCACAT

ATCCTGACCT TGGGTTGCCA CCTGAATGGT ATGGAGCTCT GGAGTGGGTA TTCCCTGAAT GGGCGAGGAG GCATGCCCTT GACAAGGGTG AGGCAGTTAA TTTTTTGAAA GGTGCAGTTG

TGACAGCAGA TCGAATCGTG ACTGTCAGTA AGGGTTATTC ATGGGAGGTC ACAACTGCTG AAGGTGGACA GGGCCTCAAT GAGCTCTTAA GCTCCAGAAA GAGTGTATTA

AACGGAATTG

TAAATGGAAT TGACATTAAT GATTGGAACC CTGCCACAGA CAAATGTATC CCCTGTCATT

ATTCTGTTGA TGACCTCTCT TGAAAGGCTA AATGTAAAGG TGCATTGCAG AAGGAGCTGG

GTTTACCTAT AAGGCCTGAT GTTCCTCTGA TTGGCTTTAT TGGAAGATTG GATTATCAGA

AAGGCATTGA TCTCATTCAA CTTATCATAC CAGATCTCAT GCGGAAGAAT GTTCAA TTTG TCATGCTTGG ATCTGGTGAC CCAGAGCTTG AAGATTGGAT GAGATCTACA

GAGTCGATCT

TCAAGGATAA ATTTCGTGGA TGGGTTGGAT TTAGTGTTCC AGTTTCCCAC CGAATAACTG

CGGCTGGCGA TATATTGTTA ATGCCATCCA GATTCGAACC TTGTGGTCTC AATCAGCTAT

ATGCTATGCA GTATGGCACA GTTCCTGTTG TCCATGCAAC TGGGGGCCTT AGAGATACCG

TGGAGAACTT CAACCCTTTC GGTGAGAATG GAGAGCAGGG TACAGGGTGG GCATTCGCAC CCCTAACCAC AGAAAACATG TTTGTGGACA TTGCGAACTG CAATATCTAC

ATACAGGGAA

CACAAGTAAT AATGGGAAGG GCTAATGAAG CCAGGCATGT CAAAAGAGTT CACGTGGGAC

CATGCCGCTG AACAATACGA ACAAATCTTC CAGTGGGCCT TCATCGGATC GACCCGATGT TCAATGGAAA AAAGGGACCA AAGTTGGTTG GTTCCTTGAA GATTATCAGT TCATCATCCT

ATAGTAAGCT GAATGATGAA AGAAAACCCC TGTACATTAC ATGGAAGGCA GACCGGCTAT TGGCTCCATT GCTCCAATGT CTGCTTTGGC TGCCTTGCCT CGATGGACCG

GATGCAGTGA

GGAATCCAGN CGAACGACAG TTTTGAAGGA TAGGAAGGGG AGCTGGAAGC AGTCACGCAG

GCAGGCAAGC CTTCGCCGTT AATTCATATG GAACAAGCTG GAGTCAGTTT CTGCTGTGCC

ACTCACTGTT TACCTTAAGA TTATTACCTG TGTTGTTCTC CTTTGCTCGT TAGGGCTGAT

AACATAATGA CTCATTAAGA ATATAATTCA CTCTGCCTCG TTTTTAATCT TAAGTGAAGT CGAGATCTAC TTCGTCATTC CTTCCCCGTT TAAAAAAAAA AAAAAAAAAA AA

SEQ-ID-NO2; DNA; 2085 BP.

CC NOTE: ORIGINAL SEQUENCE NAME WAS SSS CLONE 6.31 SQ SEQUENCE 2085 BP; 456 A; 521 C; 629 G; 479 T; 0 OTHER;

AACGCCGCAT TGGCACGTTG AGATCAAGTC CATCGTCGCC GCGCCGCCGA CGAGCATAGT

GAAGTTCCCA GGGCGCGGGC TACAGGATGA TCCTTCCCTC TGGGACATAG CGCCGGAGAC TGTCCTCCCA GCCCCGAAGC CACTGCATGA ATCGCCTGCG GTTGACGGAG

ATTCAAATGG

AATTGCACCT CCTACAGTTG AGCCATTAGT ACAGGAGGCC ACTTGGGATT TCAAGAAATA CATCGGTTTT GACGAGCCTG ACGAAGCGAA GGATGATTCC AGGGTTGGTG CAGATGATGC

TGGTTCTTTT GAACATTATG GGACAATGAT TCTGGGCCTT TGTGGGGAGA ATGTTATGAA CGTGATCGTG GTGGCTGCTG AATGTTCTCC ATGGTGCAAA ACAGGTGGTC

TTGGAGATGT

TGTGGGAGCT TTACCCAAGG CTTTAGCGAG AAGAGGACAT CGTGTTATGG TTGTGGTACC

AAGGTATGGG GACTATGTGG AAGCCTTTGA TATGGGAATC CGGAAATACT ACAAAGCTGC

AGGACAGGAC CTAGAAGTGA ACTATTTCCA TGCATTTATT GATGGAGTCG ACTTTGTGTT

CATTGATGCC TCTTTCCGGC ACCGTCAAGA TGACATATAT GGGGGAAGTA GGCAGGAAAT CATGAAGCGC ATGATTTTGT TTTGCAAGGT TGCTGTTGAG GTTCCTTGGC

ACGTTCCATG

CGGTGGTGTG TGCTACGGAG ATGGAAATTT GGTGTTCATT GCCATGAATT GGCACACTGC

ACTCCTGCCT GTTTATCTGA AGGCATATTA CAGAGACCAT GGGTTAATGC AGTACACTCG

CTCCGTCCTC GTCATACATA ACATCGGCCA CCAGGGCCGT GGTCCTGTAC ATGAATTCCC

GTACATGGAC TTGCTGAACA CTAACCTTCA ACATTTCGAG CTGTACGATC CCGTCGGTGG CGAGCACGCC AACATCTTTG CCGCGTGTGT TCTGAAGATG GCAGACCGGG

TGGTGACTGT

CAGCCGCGGC TACCTGTGGG AGCTGAAGAC AGTGGAAGGC GGCTGGGGCC TCCACGACAT

CATCCGTTCT AACGACTGGA AGATCAATGG CATTCGTGAA CGCATCGACC ACCAGGAGTG GAACCCCAAG GTGGACGTGC ACCTGCGGTC GGACGGCTAC ACCAACTACT CCCTCGAGAC

ACTCGACGCT GGAAAGCGGC AGTGCAAGGC GGCCCTGCAG CGGGACGTGG GCCTGGAAGT GCGCGACGAC GTGCCGCTGC TCGGCTTCAT CGGGCGTCTG GATGGACAGA

AGGGCGTGGA

CATCATCGGG GACGCGATGC CGTGGATCGC GGGGCAGGAC GTGCAGCTGG TGATGCTGGG

CACCGGCCCA CCTGACCTGG AACGAATGCT GCAGCACTTG GAGCGGGAGC ATCCCAACAA

GGTGCGCGGG TGGGTCGGGT TCTCGGTCCT AATGGTGCAT CGCATCACGC CGGGCGCCAG

CGTGCTGGTG ATGCCCTCCC GCTTCGCCGG CGGGCTGAAC CAGCTCTACG CGATGGCATA CGGCACCGTC CCTGTGGTGC ACGCCGTGGG CGGGCTCAGG GACACCGTGG

CGCCGTTCGA

CCCGTTCGGC GACGCCGGGC TCGGGTGGAC TTTTGACCGC GCCGAGGCCA ACAAGCTGAT

CGAGGTGCTC AGCCACTGCC TCGACACGTA CCGAAACTAC GAGGAGAGCT GGAAGAGTCT

CCAGGCGCGC GGCATGTCGC AGAACCTCAG CTGGGACCAC GCGGCTGAGC TCTACGAGGA

CGTCCTTGTC AAGTACCAGT GGTGAACCCT CCGCCCTCCG CATCAATATC TTCGGTTTGA TCCCATTGTA CATCGCCCTT TGACGGTCTC GGTGAAGAAC TTCATATGCA

GTGCCGTGCT

GGGGCGGTAG CAGTACTATG GGATTGCATT GAGCTGTGTC ACTATGTGCT TTCGACAGGA

CAGTAGTGAA GGTTCTATGC AAGTTTATTT TTΓTTTTCAT TACTGATATT TGGAATGTCA ACACAATAAA TAACTACTAT GTGTTTCGTA AGTAAAAAAA AAAAA

SEQ-ID-NO3: 2478 bp DNA 04-DEC-1995 CC NOTE: ORIGINAL SEQUENCE NAME WAS SSS56

SUMMARY #Molecular-weight 89141 #Length 826 #Checksum 2983

BASE COUNT 347 A 276 C 533 G 290 T

ORIGIN

1 GCNGCNGCNT GGTRRGCNYT NGTNCARGCN GARGCNGCNG TNGCNTRRGG NATHCCNATG

61 CCNGGNGCNA TΗWSNWSNWS NWSNWSNGCN TTYYTNYTNC CNGTNGCNWS NWSNWSNCCN

121 MGNMGNMGNM GNGGNWSNGT NGGNGCNGCN YTNMGNWSNT AYGGNTAYWS NGGNGCNGAR 181 YTNMGNYTNC A YTGGGCNMG NMGNGGNCCN CCNCARGA YG GNGCNGCN WS NGTNMGNGCN

241 GCNGCNGCNC CNGCNGGNGG NGARWSNGAR GARGCNGCNA ARWSNWSNWS NWSNWSNCAR

301 GCNGGNGCNG TNCARGGNWS NACNGCNAAR GCNGTNGAYW SNGCNWSNCC NCCNAAYCCN

361 YTNACNWSNG CNCCNAARCA RWSNCARWSN GCNGCNATGC ARAAYGGNAC NWSNGGNGGN

421 WSNWSNGCNW SNACNGCNGC NCCNGTNWSN GGNCCNAARG CNGAYCAYCC NWSNGCNCCN 481 GTNACNAARM GNGARATHGA YGCNWSNGCN GTNAARCCNG ARCCNGCNGG NGAYGAYGCN

541 MGNCCNGTNG ARWSNATHGG NATHGCNGAR CCNGTNGAYG CNAARGCNGA YGCNGCNCCN 601 GCNACNGAYG CNGCNGCNWS NGCNCCNTAY GAYMGNGARG AYAAYGARCC NGGNCCNYTN

661 GCNGGNCCNA AYGTNATGAA YGTNGTNGTN GTNGCNWSNG ARTGYGCNCC NTTYTGYAAR

721 ACNGGNGGNY TNGGNGAYGT NGTNGGNGCN YTNCCNAARG CNYTNGCNMG NMGNGGNCAY

781 MGNGTNATGG TNGTNATHCC NMGNTAYGGN GARTAYGCNG ARGCNMGNGA YYTNGGNGTN

841 MGNMGNMGNT AYAARGTNGC NGGNCARGAY WSNGARGTNA CNTAYTTYCA YWSNTAYATH

901 GAYGGNGTNG AYTTYGTNTT YGTNGARGCN CCNCCNTTYM GNCAYMGNCA YAAYAAYATH

961 TAYGGNGGNG ARMGNYTNGA YATHYTNAAR MGNATGATHY T TTYTGYAA RGCNGCNGTN 1021 GARGTNCCNT GGTA YGCNCC NTG YGGNGGN ACNGTNTA YG GNGA YGGNAA YYTNGTNTTY

1081 ATHGCNAAYG AYTGGCAYAC NGCNYTNYTN CCNGTNTAYY TNAARGCNTA YTAYMGNGAY

1141 AAYGGNYTNA TGCARTAYGC NMGNWSNGTN YTNGTNATHC AYAAYATHGC NCAYCARGGN

1201 MGNGGNCCNG TNGAYGAYTT YGTNAAYTTY GAYYTNCCNG ARCAYTAYAT HGAYCAYTTY

1261 AARYTNTAYG AYAAYATHGG NGGNGAYCAY WSNAAYGTNT TYGCNGCNGG NYTNAARACN 1321 GCNGAYMGNG TNGTNACNGT NWSNAA YGGN TA YATGTGGG ARYTNAARAC NWSNGARGGN

1381 GGNTGGGGNY TNCAYGAYAT HATHAAYCAR AAYGAYTGGA ARYTNCARGG NATHGTNAAY

1441 GGNATHGAYA TGWSNGARTG GAAYCCNGCN GTNGAYGTNC AYYTNCAYWS NGAYGAYTAY 1501 ACNAAYTAYA CNTTYGARAC NYTNGAYACN GGNAARMGNC ARTGYAARGC NGCNYTNCAR

1561 MGNCARYTNG GNYTNCARGT NMGNGAYGAY GTNCCNYTNA THGGNTTYAT HGGNMGNYTN

1621 GAYCAYCARA ARGGNGTNGA YATHATHGCN GAYGCNATHC AYTGGATHGC NGGNCARGAY

1681 GTNCARYTNG TNATGYTNGG NACNGGNMGN GCNGAYYTNG ARGAYATGYT

NMGNMGNTTY

1741 GARWSNGARC AYWSNGAYAA RGTNMGNGCN TGGGTNGGNT TYWSNGTNCC NYTNGCNCAY

1801 MGNATHACNG CNGGNGCNGA YATHYTNYTN ATGCCNWSNM GNTTYGARCC NTGYGGNYTN

1861 AAYCARYTNT AYGCNATGGC NTAYGGNACN GTNCCNGTNG TNCAYGCNGT NGGNGGNYTN 1921 MGNGA YACNG TNGCNCCNTT YGA YCCNTTY AA YGA YACNG GN YTNGGNTG GACNTTYGAY

1981 MGNGCNGARG CNAAYMGNAT GATΉGAYGCN YΓNWSNCAYT GYYTNACNAC NTAYMGNAAY

2041 TAYAARGARW SNTGGMGNGC NTGYMGNGCN MGNGGNATGG CNGARGA YYT NWSNTGGGA Y

2101 CAYGCNGCNG TNYTNTAYGA RGAYGTNYTN GTNAARGCNA ARTAYCARTG GTRRGCNAAY

2161 TRRYTNGCNA CNMGNMGNMG NWSNTGYMGN MGNACNTGGA CNYTNTTYMG NMGNYTNTTY 2221 WSNYTNGCNG CNYTNATGMG NGCNWSNCAY YTNMGNMGNG CNGA YGGNMG NMGNTGGYTN

2281 GCNTAYMGNY TNMGNMGNYT NMGNGCNYTN GGNATΗTGGG CNGGNACNAT GATGCCNYTN

2341 GGNACNGGNM GNGGNGTNGT NTRRTAYGAR ACNGAYGGNG AYGGNGAYGA RGCNCAYGGN 2401 ATHTTYCCNY TNATHAAYGG NGARYTNTAY GCNACNYTNA THWSNCCNYT NYTNYTNGTN

2461 TTYATHYTNA TGGCNGCN

Claims

1. A cDNA specifying a soluble starch synthase having the sequences of the inserts in plasmids pSSSό, pSSSlO. l and pSSS6.31 and sequences having sufficient similarity such that when inserted into the genome of an organism which produces starch, the synthesis of starch is altered.

2. The cDNA of the insert of plasmid pSSSό, deposited under the terms of the Budapest Treaty, with the National Collections of Industrial and Marine Bacteria Limited, 23 St Machar Drive, Aberdeen AB1 2RY, on 13th June

1994, under the Accession Number 40651.

3. The cDNA of the insert in plasmid pSSS6.31 , deposited under the terms of the Budapest Treaty, with the National Collections of Industrial and Marine Bacteria Limited, 23 St Machar Drive, Aberdeen AB 1 2RY, on 22nd August

1994, under the Accession Number NCIMB 40679.

4. The cDNA ofthe insert in plasmid pSSSlO.l, deposited under the terms of the Budapest Treaty, with the National Collections of Industrial and Marine Bacteria Limited, 23 St Machar Drive, Aberdeen AB 1 2RY, on 22nd August

1994, under the Accession Number NCIMB 40680.

5. A cDNA, encoding soluble starch synthase which has the sequence SEQ-ID- NO-1, or SEQ-ID-NO-2 or SEQ-LD-NO-3.

6. A transformed plant containing one or more copies of one or more of the said cDNAs claimed in claim 5 in sense or antisense orientation.

7. A method of producing a plant with altered starch synthesising ability comprising stably incoφorating into the genome of a recipient plant one or more than one donor gene specifying soluble starch synthase as claimed in claim 5.

8. A method as claimed in claim 7 in which the recipient plant is of the family Gramineae.

9. A method as claimed in claim 8 in which the recipient plant is of the species Zea mays.

10. Seeds of a plant as claimed in claim 6.