MXPA01000503A - Modification of starch biosynthetic enzyme gene expression to produce starches in grain crops - Google Patents

Abstract

The instant invention discloses utilization of a cDNA clone to construct sense and antisense genes for inhibition of starch synthase enzymatic activity in corn. More specifically, this invention concerns a method of controlling the starch fine structure of starch derived from the grain of cereal crops comprising:(1) preparing a chimeric gene comprising a nucleic acid fragment encoding a starch synthase structural gene or a fragment thereof, operably linked in either sense or antisense orientation on the upstream side to a nucleic acid fragment encoding a promoter that directs gene expression in corn endosperm tissue, and operably linked on the downstream side to a nucleic acid fragment encoding a suitable regulatory sequence for transcriptional termination;and (2) transforming cereal crops with said chimeric gene, wherein expression of said chimeric gene results in alteration of the fine structure of starch derived from the grain of said transformed cereal crops compared to the fine structure of starch derived from cereal crops not possessing said chimeric gene.

Description

MODIFICATION OF THE GENETIC EXPRESSION OF BIOSINTETIC STARCH ENZYME TO PRODUCE CEREAL CROPS FIELD OF THE INVENTION This invention is in the field of plant molecular biology. More specifically, this invention pertains to the modification of the biosynthetic genetic expression of starch to produce starches in plants and seeds.

BACKGROUND OF THE INVENTION Starch is a mixture of two polysaccharides, amylose and amylopectin. Amylose is a debranched chain of up to several thousand α-D-glucopyranose units linked by α-1,4-glucosidic bonds. Amylopectin is a highly branched molecule made up of 50,000 residues of α-D-glucopyranose bound to α1,4 and α1,6 glucosidic bonds. Approximately 5% of the glycosidic linkages in the amylopectins are a-1, 6 bonds, which lead to the branched structure of the polymer.

The molecules of amylose and amylopectin are organized in granules that are stored in plastids. The granules of REF.125794 starch produced by most plants are 15-30% amylose and 70-85% amylopectin. The ratio of amylose to amylopectin and the degree of branching of amylopectin affects the physical and functional properties of starch. The functional properties, such as viscosity and stability of the gelatinized starch determine the utility and therefore the value of the starches in industrial and food applications. When a specific functional property is needed, starches obtained from various crops such as corn, rice, potato or wheat can satisfy the functionality requirements. If a starch does not satisfy a required functional property, such as the need for a stable viscosity under conditions of high temperatures and acids, the functionality can usually be achieved by chemically modifying the starch. Various types and grades of chemical modifications are used in the food industry, and the labeling and use of chemically modified starches must meet government regulations.

In the production of starch in plant organs, the ratio of amylose to amylopectin and the degree of branching of amylopectin are under genetic control. For example, homozygous corn plants for the recessive serosity (wx) mutation lacks the enzyme starch synthase binding to the granule and produces almost 100 % amylopectin. Corn plants homozygous for mutations of amilose modifier of recessive viscosity (ae) and decharacterized modifier genes can produce, according to the information received, starch granules that are approximately 80% to 90% amylose (see US Patent No. 5,300,145). The inactivated maize mutant lacks the starch synthase different from the one missing in the waxy lines and has a starch characterized by more amylose and a higher proportion of shorter branches in the amylopectin molecule than the normal starches.

Most cereal crops are managed as products, and many of the animal and industrial feed requirements for these crops can be found for common varieties that are widely developed and produced in volume. However, there is currently a growing market for crops with special end-use properties which are not found in cereals of standard compositions. More commonly, special corn differs from "normal" corn by altered endosperm properties, such as a total change in the ratio of amylose to amylopectin as in amylose-rich or waxy-rich maize, an increased accumulation of sugars as in sweet corn, or an alteration in the degree of hardness of endosperm such as food-grade or poplar corn (Glover, DV and E- T. Mertz (1987) in Corn: Nutri ti onal Quali ty of Cereal Grains, Genetic and Agronomic Improvement, RA Olson and KJ Frey, eds., American Society of Agronomy, Madison Wisconsin, pp. 183-336; Rooney, L. W. and S. 0. Serna-Saldivar (1987) Food Uses of Whole Corn and Dry-milled Fractions, in Corn: Chemistry and Technology, S. Watson and P. E. Ramsted, eds. American Association of Cereal Chemists, Inc., St. Paul, Minnesota, pp. 399-429). Currently the invention offers buyers of specialty cereals a source of starch that has properties other than waxy starch and offers farmers the opportunity to develop a higher value-added crop than normal or waxy corn.

The purified starches are obtained from plants by grinding process. Corn starch is extracted from the grains by the use of a wet milling process. Wet milling is a multi-stage process that involves the maceration and crushing of the grains and separation of the fractions of starch, protein, oil and fiber. A review of the wet corn grinding process is given in S.R. Eckhoff (1992) in the Proceeding of Fourth Corn Utilization Conference, June 24-26, St. Louis, MO., Printed by the National Corn Growers Association, CIBA-GEIGY Seed Division and the United States Department of Agriculture. Wheat is also an important source of purified starch. The production of wheat starch was reviewed in J. W. Knight and R. M. (1984) Olson in Starch: Chemi stry and Technology 2a. Ed., Academic Press. Eds. Whisler et al.

Starch is used in numerous industrial and food applications and is the largest source of carbohydrates in the human diet. Typically, the starch is mixed with water and cooked to form a thick gel. This process is called gelatinization. Three important properties of a starch are the temperature at which gelatinization takes place, the gel viscosity achieved, and the stability of the viscosity of the gel over time. The physical properties of unmodified starches during heating and cooling limits have utility in many applications. As a result, considerable effort and cost was required for chemically modified starches in order to overcome these starch limitations and expand the utility of starch to industrial applications.

Some limitations of unmodified starches and properties of modified starches are given in Modifi ed Starches: Properti es and Uses, 0. B. Wurzburg, ed., (1986) CRC Press Inc., Boca Raton, FL. Unmodified starches have very limited use in foodstuffs because of granule swelling and rupture easily, thus forming undesirable, bodily weak gels. Chemical modifications are used to stabilize starch granules thus making the starch suitable for thousands of industrial and food applications including baby food, coffee powder, medical powders for surgery, calibration of yarn and paper and adhesives Common chemical modifications include crosslinking, in which the chemical bond is introduced to act as stabilizing bridges between the starch molecules, and substitution in which substituent groups such as hydroxyethyl, hydroxypropyl or acetyl groups are introduced into the starch molecules.

The use of chemically modified starches in the United States is regulated by the Food and Drug Administration (FDA). Starches "modified for food starches" starches that can be used in food but must meet the specified treatment limits, and starches "modified for industrial starch" can be used in products such as containers that come in contact with food and must also satisfy requirements specified by the regulations; Code of Federal Regulation, Title 21, Chapter 1, art 172, Food additives allowed in Food for Human Consumption, Section 172, 892, Modified Starch for Foods, U.S. Government Printing Office, Washington, D.C. 1981; (a) Part 178, Indirect Food Additives, Sec. 178.3520, Modified Industrial Starch. These regulations limit the degree of chemical modification by definition of the maximum amount of chemical reagent that can be used in the modification stages. The levels of by-products in starch that result from the modification process are also regulated. For example, residues of propylene chlorohydrin in hydroxypropyl starch are especially related (Tuschhoff, JV (1986) hydroxypropylated starches, in Modified Starches: Properties and Uses, OB Wurzburg, ed. CRC Press, Boca Raton, FL, pp. 55-57) .

In addition to its use as a purified ingredient, starch is an important component of whole flours, such as wheat flour, used in the production of breads, bakery products and pasta. The starches comprise between 50 and 70% by weight of a wheat grain and their importance in the results of wheat flours are well known in the art. Although wheat genetic complexes have limited variations in the structure of fine starch that is adequate in whole flours, the production of new starch structures in wheat or other flours can result in an improved result of these whole flours in product applications. food. The structure of starch is also an important component of the quality of whole grain grains consumed such as rice. Differences in amylopectin of fine structure have been related to the texture of cooked rice (Reddy et al. (1993) Carbohydr, Polymers 22: 267-275).

Differences in the degree of branching or polymerization of starch are known to result in a change in the physical-chemical properties of starch. It has been suggested that starches, made on demand for specific applications, can be generated by alterations of the chain distribution in branches of the amylopectin molecule, the relative ratio of amylose to amylopectin or the degree of polymerization of amylose.

Some authors (Shi and Seib (1992) Carbohydr Res. 227: 131-145; Jane et al. (1999) Cereal Chemistry In Press), it has been published that the tendency to retrograde is reduced in starches from different botanical sources that contain increased proportions of very short chains (DP 6-9) in their amylopectin, but it is not suggested that this be done, as it was achieved in corn. However, it has been problematic to achieve phenotypic alterations of the starch composition; While key enzymes in starch biosynthesis have been identified, their exact role remains uncertain. Therefore, the correlation of activities of particular enzymes with particular molecular characteristics of starch structure and, in turn, with the function of starch in industrial and food products has become difficult. Although desirable functional properties that an ideal starch should need may be contemplated, there is only a limited understanding that the molecular structure of the starch should achieve this and little understanding of how particular starch biosynthetic enzymes specifically affect these parameters. For example, the role of individual enzymes in determining the branching and extension patterns of the branches is not yet clear and consists of the lack of understanding of how branched enzymes and starch synthases interact. In addition, while the role of the granule-binding starch synthase encoded by the waxy gene is carefully well understood; see Denyer et al. (1996) Plant J. 10: 1135-1143), the exact number and functions of other starch synthases, soluble or granular bond, are well understood. (Smith et al. (1996) Ann. Rev. Plant Phys. And Mol. Biol. 48: 67-87).

WO 94/09144 discusses the generation of corn plants with improved capacity to synthesize starch at elevated temperatures. This publication proposes that the limiting factor in completing the grain at high temperature is the lability of certain starch biosynthetic enzymes, particularly starch synthase (SS) and starch branching enzyme (SBE). The introduction of genes that encode enzymes that have a higher optimal activity temperature or that have a higher tolerance to heating in plants that can result in an increase in the amount of starch deposited in the corn kernel. Furthermore, it is claimed that this strategy can be used to generate altered fine structure starch as a result of the introduction of donor genes whose expression can alter the balance of the different starch biosynthetic enzymes. It is suggested that donor genes include those that encode enzymes that exhibit improved kinetic or allosteric properties in relation to the endogenous enzyme or an extra copy of the endogenous gene that compensate for losses in enzymatic activity incurred due to thermal lability. As a means of altering the structure of starch, WO 94/09144 also suggests the use of genes in sense and antisense to alter the natural proportions of the different synthases and branching enzymes of starch in the recipient plant. This publication describes the effect of temperature on the catalytic activity and enzymatic stability for certain biosynthetic starch enzymes. However, no data are presented that prove the proposed molecular strategies. Indeed, while this publication suggests the use of altered starch synthase expression to alter the fine structure of starch, both the amylose / amylopectin ratio and the degree of amylopectin branching, other before and after publications suggest that branching starch enzymes , not exactly the starch synthases, would be required or that still other factors should be considered. For example, Smith and co-workers (1995, Plant Phys. 107: 673-677) suggest two different views about the determination of the amylopectin branching pattern: first, that the pattern represents a balance between the activities of branching enzymes and debranching, and second, that the pattern can be explained extensively by the properties of branching enzymes. No paper is provided for the starch synthases. Guan and Preiss (1993, Plant Phys. 102: 1269-1271) suggest a study of the interactions between the multiple forms of branching enzymes and starch synthase. The specificity and function of the individual isoenzymes and the mechanism of biosynthesis can be understood. amylopectin. Therefore, Guan and Preiss imply the need to alter both enzymes at the same time. Lately, Van den Koornhuyse et al (1996, J. Biol. Chem. 271: 16281-16287) propose that sugar concentrations in lower nucleotides are either directly or indirectly responsible for the greatest differences observed in the composition or structure of starch during storage. In sum, it is clear from the different points of view that there is no consensus on exactly what factors affect the structure of the starch and therefore how to alter them. In addition, no work, including WO 94/09144, presents evidence demonstrating that the soluble starch synthases limit the polymerization rate and therefore either increase or decrease its level will currently alter the fine structure of the starch. WO 94/09144 further does not demonstrate how to differentiate between genes encoding isoforms that make a minimal contribution to starch biosynthesis and more active forms. Reducing the expression pattern of a relatively inactive enzyme (at the enzymatic level, not necessarily at the transcriptional level) is indistinct to have an effect. In sum, WO 94/09144 suggests but does not show in sufficient detail for those skilled in the art to currently produce an altered starch in fine structure.

There have been several reports of alteration of starch structure due to modification of SBE expression in both potatoes (Virgin et al., (June 1994) in the 4th International Congress of Molecular Biology of Plants, and Chritensen et al., And Kossman et al. (July, 1994) at the Symposium on Plant Polysaccharides) and maiz (Broglie et al., WO 97/22703). None of these works is directed towards the alteration potential of the expression of starch synthases. Several authors have speculated that altering the expression of the synthase, starch synthase I non-binding with the granule (non-GBSSI) could alter the structure or composition of starch, but this was not clearly demonstrated in cereals (Block et al, WO 9745545A, Frohberg and Koss ann, WO 9744472, Frohberg and Kossmann, WO 972362).

Although the enzymatic steps are known, the molecular details of starch biosynthesis are not well understood. It is not clear if the different SS isoforms contribute equally throughout the starch biosynthesis or if each isoform plays a different role in assembling the amylopectin molecule in discrete stages over a mandatory cycle. In consideration of the possible inter. -between starch branching enzymes and multiple starch synthases that function in the elongation of the glucan chain, it is possible to ensure predictions concerning the structure of starch based on the catalytic properties of each enzyme.

In addition to the clear role of GBSSI in amylose biosynthesis, the exact roles of individual starch synthases are unclear. There is evidence of some, but not all, species whose individual SS isoforms contribute qualitatively differently to amylopectin biosynthesis and whose GBSSI may also contribute to the biosynthesis of amylopectin as well as amylose (Smith et al. (1996) Ann. Rev. Plant Phys. And Mol. Biol. 48: 67-87). Numerous starch synthases have been cloned from different species, but Edwards et al. (1960, Plant Phys. 112: 89-97), Mu-Foster et al. (1996, Plant Phys. 111: 821-829) demonstrated that the distinctions previously made between soluble link-granule and starch synthases can not reflect the situation in vi and will not be used in the present with the exception of the waxy protein, GBSSI. Since its original isolation from corn (Kldsgen et al. (1986) Mol. Gen. Genet. 203: 237-244) this gene has been cloned from many species. Numerous other SSs have been cloned from a variety of species, but appear to be less closely related through species than GBSSI. The potato contains at least two other starch synthases, SSII and SSIII (Marshall and colleagues (1996) Plant celld: 1121-1135). The pea contains a designated SSII synthase that appears to be present in two forms, one derived from the process of the other (Edwards et al. (1995) Plant Phys. 112: 89-97). Block and collaborators (WO 9745545a) isolated two clones of wheat starch synthase cDNA. Three forms of soluble starch synthase were purified from rice. This was shown to be derived from a primary form by the isolation of the corresponding gene referred to as soluble starch synthase I (SSSI) (Baba et al. (1993) Plant Phys, 103: 565-573; Tanaka et al. (1995) Plant Phys. 108: 677-683). The Expressed Rice Sequence (ESTs) has been identified that shows homology with the pea and potato starch synthase II sequences (AA752475, AA753266, AA751702, AA751557, AA751512A; Nahm, B. H. and collaborators). A sequence related to rice SSI was isolated from corn (Harn et al. (1995) Plant Phys. 108: S-50; Keeling et al, WO 9720936) and SSI of corn was designated. Two clones of starch synthase cDNA were isolated by Keeling et al (WO 9720936). The Expression of the genes encoding these starch synthases has been studied and their representation in the maize genome has been published (Harn et al. (1998) Plant Mol. Biol. 37: 639-649). Frohberg and Kossmann (WO 97/44472 and WO 97/26362) have also published the isolation of two of these sequences of corn starch synthases. The locus responsible for the mutation was recently shown to encode another starch synthases (Gao et al. (1998) Plant Cell 10: 399-412). In a study that characterizes the activities of soluble corn starch synthase in maize endosperama Cao et al. (1999), Plant Physiol. 120: 205-215) published that DU1 and SSSI are similarly responsible for all the soluble starch synthase activity in the development of the endosperm. Single-celled organisms also contain multiple starch synthases (Fontaine et al. 81993) J. Biol. Chem. 268: 16223-16230; Buleon et al. (1997) Plant Physiol. 115: 949-957). While for some of these enzyme authors have speculated about their particular role, in no case has it been elucidated as the total complement of starch synthase isoforms working together to elongate the amylopectin branches or as the complete set of biosynthetic enzymes Starch in particular species interact and work together to produce the starch structure observed in the mature seed or tuber. In particular, the role of low abundant starch synthases in endosperm is unclear.

It is well known that the waxy mutation in corn results from the lack of a functional GBSSI enzyme and an altered starch composition. Similarly in wheat, poor amylose varieties lacking GBSS have been selected. Dominant forms of analogous mutations in potato have been elaborated by expression of GBSSI antisense genes in transgenic potato plants (Visser et al. (1989) Plant Mol Biol. 17: 691-699). She maker and collaborators (1994, Plant Physiol., 104: 1159-1166) the structure of altered starch in potato has been published through the expression of glycogen synthase of E. coli in tubers.

The modified expression of non-GBSSI starch synthases have been published in potato. The reduction of SSII expression in transgenic potato tubers has been achieved using antisense technology. The decreased levels of SSII protein was not correlated with any detectable change in starch content or composition and the morphology of the starch granule appears normal (Edwards and co-workers (1995) Plant J. 8: 283-294). Recently, small changes in the distribution of the amylopectin branched chain (dp 6-35) have been published in antisense SSII from potato plants (Edwards et al. (1999) Plant J. 17: 251-261). Different results were observed when the highest synthase activity of 1 soluble starch of potato tubers, SSIII was inhibited by an antisense approximation. In these transgenic plants, the content and composition of starch was not changed, however, the morphology of the starch granule was markedly affected (Marshall et al. (1996) Plant Cell 8: 1121-1135). Changes in the amylopectin branched chain distribution were observed but this was different from those found in plants with antisense SSII (Edward et al. (1999) Plant J. 17: 251-261). A pea mutant, rug 5, was found to be lacking in a starch synthase isoform that is highly homologous to potato SSII. Although the two starch synthases share homology in the amino acid sequence, different results were produced when these starch isoforms were inhibited in peas. They were apparent, notable changes in short branched chains (dp <15), medium (dp 15-45) and very long (dp ~ 1000) amylopectin. In addition, these structural changes were associated with large changes in the morphology of the starch granule. Therefore, while the transgenic potato results suggest that in a specific organ, different isoforms of starch synthase play different roles in starch biosynthesis, results obtained from the mutant rug 5 of pea indicate that the homologous isoforms can not necessarily disprove the same function in different organs that store starch. It becomes difficult to generalize about the role of specific isoforms and the prediction of phenotypic changes in starch that accompany modified expression, due to differences in the number of isoforms represented in different organs as well as differences in the relative amount of activity contributed by the different isoforms. While the proposed transgenic works in the modification of the expression of the starch synthase have been published in potato, no similar experiment has been described in corn.

US5824790 reports the isolation and sequencing of 3 cDNA clones of non-waxy corn sytases. Suggests the so of these sequences to generate constructs indicated to modify the expression of these starch synthases in transgenic plants. It is further suggested that the expression of the modified protein may increase to a change in the fine structure of the starch. While the protein and nucleotide sequences for the three starch synthases were provided, no data was given in relation to the generation and characterization of transgenic plants that carry DNA construction derived from these sequences; similarly, no data regarding the composition and structure of transgenic plants was reported. In the absence of specific roles for the different isoforms of starch synthase soluble in cereal endosperm, and with the presence of activities for the enzymes of class SSIIa and SSIIb in the endosperms in question, (Cao et al. (1999) Plant Physiol. : 205-215) it is clear that genetic sequences alone do not provide clear indications of what type of change, if any, in the structure of starch can be achieved by altering the expression of a particular soluble starch synthase gene. And in the absence of this predictive power or the current production of starch, the utility of any given change is not clear. In fact, in terms of functional attributes such as the tendency to retrograde it is clear that some structural changes of starch are currently detrimental to utility.Qiange and Thompson (1998, Carbohydr, Res. 314: 221-235) examined the retrogradation of three double mutants of maize, duwx, aewx and su2wx, compared to normal starch and demonstrated the tendency for increased retrogradation in two of the three types of amylopectins. Therefore, it is clear that the change is only insufficient to improve the usefulness of cereal starches, and that some changes can be improved while others are neutral or equally detrimental. In the absence of the ability to significantly predict the structural changes that may occur with a given genetic modification, the only way to identify useful changes is to currently produce the modified starch.

Molecular genetic solutions to the generation of starches from cereal crops with altered fine structures have an advantage over attempts to inseminate more traditional plants. Changes in the fine structure of starch can be produced by specifically inhibiting the expression of one or more of the SS or SBE isoforms by antisense inhibition or co-suppression (WO 94/09144). An antisense construct or cosuppression could act as a dominant negative regulator of genetic activity. While conventional mutations can produce negative regulations of genetic activity these effects are more likely to be recessive. The appropriate dominant negative regulation with a transgenic approach can be advantageous for certain grain production methods. In addition, the ability to restrict the expression of the altered starch phenotype in the reproductive tissues of the plant by the use of specific promoters may confer agronomic advantages over conventional mutations that will have an effect on all tissues in which the mutant gene it is expressed ordinarily. Finally, the variable levels of antisense inhibition or cosuppression that comes from the effects of the chromosomal position could produce a wider range of starch phenotypes than those resulting from the dosage effects of a mutant allele in the cereal endosperm.

Incomplete understanding of the role of different starch synthase enzymes in cereal crops makes difficult attempts to manipulate the fine structure of starch by inhibiting the genetic expression of starch synthase. However, manipulation of the genetic expression of the starch synthase enzyme by cosuppression and antisense technology is possible and would probably produce a desirable phenotype. Accordingly, one skilled in the art would only have to screen multiple transgenic plants for the desired alteration in the fine structure of starch.

BRIEF DESCRIPTION OF THE INVENTION The present invention describes the use of cDNA clones for chimeric constructions of genes in the sense and antisense for alterations in the enzymatic activity of starch synthase in grains or endosperm of aiz and in the grain or endosperm of other cereal crops. More specifically, this invention concerns a method of producing a transformed cereal crop in which the fine structure of the starch derived from a grain of the cereal crop is altered in comparison with the fine structure of starch derived from a non-cereal crop. -transformation comprising: (1) preparing a chimeric gene comprising a nucleic acid fragment encoding a structural gene of non-GBSSI starch synthase enzyme or a fragment thereof, operably linked either in sense orientation or antisense on the 5 'side of a nucleic acid fragment encoding a promoter that directs genetic expression in endosperm tissue, and operably linked on the 3' side to a nucleic acid fragment encoding a regulatory sequence suitable for transcriptional termination , and (2) transform the cereal crop with said chimeric gene, wherein the expression of the chimeric gene results in the alteration of the fine structure of starch derived from the grain of the transformed cereal crop in comparison with the fine structure of starch derived from the cereal crop that does not possess said chimeric gene . The invention also concerns a method of producing a transformed cereal crop in which the fine structure of starch derived from a grain of the cereal crop has a change in the relative proportions of amylose to amylopectin relative to that of the starch derived from the crop of cereal that does not possess the previous chimeric gene, or a change in the degree of polymerization of the amylose component of the starch derived from the transformed cereal crop in relation to the degree of starch amylose polymerization derived from cereal crops that do not possess the previous chimeric gene. To date, no publication has demonstrated an alteration in the molecular structure of the starch created by altering the expression level of non-GBSSI starch synthase in a transgenic plant. This invention describes specific alterations in starch structures, changes in the amylose to amylopectin ratio, changes in the fine structure of amylopectin, increased abundance of very short amylopectin chains (DP 6-9), and change in the degree of polymerization of amylose, which can be created by controlling the expression of non-GBSSI starch synthases in transgenic plants.

This invention also concerns varieties of cereal crops prepared by transformation using said method, the starch isolated from the grain of a variety of cereal crop prepared using the above method, and a method of preparing a thick comestible comprising combining an edible, water , and an effective amount of a starch isolated from the grain of a variety of cereal crop prepared using the method, and cooking the resulting composition as necessary to produce a thick comestible.

This invention also concerns varieties of cereal crops prepared by transformation using the above method, prepared grain meals of said varieties of cereal crops, and the preparation of bread, baked goods, and pastes by combination of water, food ingredients, and an effective amount of grain meal of cereal crop varieties prepared using the method, and cooking the resulting composition as necessary to produce a bread, baked product, or paste product.

BRIEF DESCRIPTION OF THE DRAWINGS AND DESCRIPTIONS OF SEQUENCES The invention may be more carefully understood from the following detailed description and the accompanying drawings and sequence descriptions that form a part of this application.

Figure 1 presents a restriction map of plasmid pSS43.

Figure 2 presents a restriction map of plasmid pSS64-C5.

Figure 3 presents a restriction map of the plasmid pSS65-Cll.

Figure 4 presents a restriction map of plasmid pSPB40.

Figure 5 presents a restriction map of plasmid pSPB47.

Jki¡ & Figure 6 shows the molecular weight distributions of debranched starch from Rl grains of altered segregating corn plants 944-1 and normal segregation 944-7.

Figure 7 shows the molecular weight distributions of debranched starches from single grains of line S064.1.2.1 altered segregates XGB1717-2 and normal segregates XGB01717-9.

Figure 8 shows the distribution of the relative mol percentage of the chain extension DP7 and DP30 for starch derived from an altered segregating grain and from starch derived from a non-altered segregating grain.

SEQ ID NO: 1 discloses the nucleotide sequence of the PCR primer BE62.

SEQ ID NO: 2 discloses the nucleotide sequence of the BE61 PCR primer.

SEQ ID NO: 3 discloses the nucleotide sequence of the SS7 PCR primer.

SEQ ID NO: 4 discloses the nucleotide sequence of the SS8 PCR primer.

SEQ ID NO: 5 discloses the nucleotide sequence of the composite gene sequence SS1.

SEQ ID NO: 6 discloses the nucleotide sequence of the SS1 DNA sequence inserted in pSS43.

SEQ ID NO: 7 discloses the nucleotide sequence of the MM50 PCR primer.

SEQ ID NO: 8 discloses the nucleotide sequence of the BE56 PCR primer.

SEQ ID NO: 9 discloses the nucleotide sequence of the MM62 PCR primer.

SEQ ID NO: 10 discloses the nucleotide sequence of the MM60 PCR primer.

SEQ ID NO: 11 discloses the nucleotide sequence of the SS1 DNA sequence inserted in pSS64-C5.

SEQ ID NO: 12 discloses the nucleotide sequence of the SS1 DNA sequence inserted in pSS65-cll.

SEQ ID NO: 13 discloses the nucleotide sequence of the SS9 PCR primer.

SEQ ID NO: 14 discloses the nucleotide sequence of the SS10 PCR primer.

SEQ ID NO: 15 discloses the nucleotide sequence of the inserted SSb sequence in pSPB37.

SEQ ID NO: 16 discloses the nucleotide sequence of the SSb DNA sequence inserted into pSPB40.

SEQ ID NO: 17 discloses the nucleotide sequence of the PCR primer OSPB104.

SEQ ID NO: 18 discloses the nucleotide sequence of the PCR primer OSPB105.

SEQ ID NO: 19 discloses the nucleotide sequence of the PCR primer OSPB106.

SEQ ID NO: 20 discloses the nucleotide sequence of the SSb DNA sequence inserted in pSPB47.

Sequence Descriptions contain the one-letter code for nucleotide sequence characters and the three-letter code for amino acids as defined in accordance with IUPAC-IUB standards (1985, Nucleic Acids, Res. 13: 3021-3030 , and 1984, Biochem J. 219: 345-373) which are incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION In the context of this description, a number of terms will be used. As used herein, the term "starch" refers to a polysaccharide consisting of an α-D- (1, 4) glucan which may contain a variable proportion of a-D- (1, 6) branches. As used herein, the term "fine structure of starch" refers to the molecular structure of a starch polymer, the presence, abundance, and distribution of aD- (1, 6) bonds and the presence, abundance and extent of both branched and debranched glucans aD- (1, 4) in the polymer. The fine structure of starch is described by the distribution of branched chains of amylopectin, or by the relative ratio of amylose to amylopectin, or by the degree of polymerization of amylose. Altering any of these structural molecular components results in a fine structure of starch. One, two or all three of these parameters can be altered independently of one another. The term "degree of polymerization" refers to the number of α-D-glucopyranose units in a designated molecule or portion of a molecule such as a branched chain of amylopectin. As used herein, the term "branched chain distribution" refers to the distribution of glucan chains attached to α-1,4 which are detected following the digestion of amylopectin isoamylase and subsequent activation of the branches released by chromatography. of exclusion of size.

As used herein, "cereal harvest" means a plant that produces a seed containing starch suitable for food or industrial use, as exemplified by corn, rice, sorghum, wheat and barley. As used herein, an "isolated nucleic acid fragment" is an RNA or DNA polymer that is single or double stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a DNA polymer can be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

As used herein, "essentially similar," refers to nucleic acid fragments in which changes in one or more nucleotide bases result in the substitution of one or more amino acids, but does not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to nucleic acid fragments in which changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate the alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the present invention such as deletions or insertions of one or more nucleotide bases that do not affect the functional properties of the resulting transcript as compared to the ability to mediate the alteration. of gene expression by antisense technology or co-suppression or alteration of the functional properties of the resulting protein molecule. It is understood, therefore, that the invention encompasses more than the specific exemplary sequences. For example, it is well known in the art that antisense suppression and co-suppression of gene expression can be achieved by using nucleic acid fragments that represent less than the entire coding region of a gene, and by fragments of nucleic acid that do not share 100 % identity with the gene to be deleted (US Patent No. 5, 107, 065).

In addition, alterations in a gene that result in the production of an amino acid chemically equivalent at a given site, but do not affect the functional properties of the encoded protein, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, can be replaced by a codon encoding another less hydrophobic residue, such as glycine. Similarly, a codon for the amino acid alanine can be replaced by a codon encoding a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes that result in a substitution of a negatively charged residue for another, such as aspartic acid for glutamic acid, or a positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes that result in alteration of the N-terminal and C-terminal portions of the protein molecule would not be expected to alter the activity of the protein either. Each of the proposed modifications belong to the routine of experts in the field, such as the determination of the retention of the biological activity of the coded products. In addition, those skilled in the art recognize that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under severe conditions (0.1X SCC, 0.1% SDS, 65 ° C), with the sequences exemplified herein. Preferred substantially similar nucleic acid fragments of the present invention are fragments of nucleic acid whose DNA sequences are 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are 90% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are those which are identical to the DNA sequence of the nucleic acid fragments reported herein. The percent identity used herein can be precisely determined by the DNASTAR protein alignment protocol using the Jotun-Hein algorithm (Hein, J. J. (1990) Meth. Enz. 183: 626-645). Defective parameters by the Jotun-Hein method for multiple alignment are GAP PENALIZED = 11, GAP PENALIZED IN EXTENSION = 3; by alignments in pairs KTUPLE 6.

"Codon degeneracy" refers to the divergence in the genetic code that allows variation of the nucleotide sequence without effecting the amino acid sequence of a coded polypeptide. Accordingly, the present invention concerns any nucleic acid fragment that all or a substantial portion of the amino acid sequence encoding the SS1 or SSb proteins as set forth in SEQ ID NOS: 5, 11, 12, 15, 16 and 20. Those skilled in the art are well aware of the "codon-derivative" exhibited by a specific host cell in use of nucleotide codons to specify a given amino acid. Accordingly, when synthesizing a gene for enhanced expression in a host cell, it is desirable to design the gene so that its codon usage frequency approaches the preferred codon usage frequency of the host cell.

"Gene" refers to a fragment of nucleic acid that expresses a specific protein, including the regulatory sequences that precede (non-coding sequences at 5 ') and following (non-coding sequences at 3') the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a different way than they are in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not as it is normally found in the host organism, but which is introduced into the host organism by genetic transfer. Foreign genes may comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that was introduced into the genome by a transformation procedure.

"Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Start codon" and "stop codon" refers to a unit of three adjacent nucleotides in a coding sequence that specifies the start and end of the chain, respectively, of protein synthesis (mRNA translation). "Open reading frame" refers to the encoded amino acid sequence between the codons of initiation and termination of the translation of a coding sequence. "Regulatory sequences" refers to nucleotide sequences located at the 5 'end (sequences that do not encode at 5'), at, or at the 3 'end (sequences that do not encode at 3') of a coding sequence, and it influences transcription, processing RNA or stability, or translation of the associated coding sequence. Regulatory sequences may include promoter sequences, translation leaders, introns, and polyadenylation recognition sequences.

"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3 'in a promoter sequence. The promoter sequence consists of proximal and more distal 5 'elements, the posterior elements are often mentioned as enhancers. Accordingly, an "enhancer" is a dNA sequence that can stimulate the activity of the promoter and can be an innate element of the promoter or a heterologous element inserted to improve the level of tissue specificity of a promoter. The promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or likewise comprise segments of synthetic DNA. Those skilled in the art understand that different promoters can direct the expression of a gene in different types of tissues or cells, or at different stages of development, or in response to different environmental conditions. Promoters that cause a gene to be expressed in most types of cells in more times are commonly referred to as "constitutive promoters". New promoters of various useful types in plant cells have been constantly discovered; numerous examples can be found in the compilation of Okamuro and Goldberg, (1989) Biochem Plants 15: 1-82. It is further recognized that since in most cases the exact links of the regulatory sequences have not been fully defined, DNA fragments of different extensions may have identical promoter activity.

The "leader translation sequence" refers to the DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the mRNA processed completely 5 'of the translation initiation sequence. The leader sequence of translation can affect the process of the primary transcript to mRNA, the stability of mRNA or efficiency of translation. Examples of leader translation sequence are described in Turner, R. and Foster, G. D. (1995) Molecular Biotechnology 3: 225.

The term "3 'non-coding sequences" refers to DNA sequences located 3' of a coding sequence and includes polyadenylation recognition sequences and other sequences encoding regulatory indications capable of affecting the mRNA process or gene expression. The indication for polyadenylation is usually characterized by affecting the addition of polyadenylic acid towards the 3 'end of the mRNA precursor. The use of different 3 'non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1: 671-680.

"RNA transcript" refers to a product that results from transcription catalyzed by RNA polymerase from a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, is referred to as the primary transcript or can be an RNA sequence derived from the post-transcriptional process of the primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)" refers to an RNA that is without introns and that can be transferred to a protein by the cell. "CDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "RNA" in the sense "refers to a transcribed RNA that includes mRNA and can also be translated into protein by the cell." Antisense RNA " refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (US Patent No. 5,107,065) The complementarity of an antisense RNA can be with any part of the transcript of the specific gene, for example, in the 5 'non-coding sequence, 3' non-coding sequence, introns, or the coding sequence "Functional RNA" refers to antisense RNA, ribosome RNA, or other RNA that is not transferred still that has an effect on the cellular process.

The term "operably linked" refers to the association of nucleic acid sequences in a nucleic acid fragment alone so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (eg, that that coding sequence is under the transcriptional control of the promoter). The coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term "expression", as used herein, refers to the transcription and stable accumulation of RNA in the sense (mRNA) or antisense derived from the nucleic acid fragment of the invention. The expression can also refer to translation of mRNA in a polypeptide. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Over-expression" refers to the production of a gene product in transgenic organisms that exceed production levels in normal or non-transformed organisms. "Co-suppression" refers to a phenomenon in plants with which foreign or endogenous genes are silenced by the introduction of sufficiently homologous transgenes. The mechanisms of genetic activation are not well understood but can take place either by blocking the transcription or by inhibiting the accumulation of mRNA. For example, US Pat. No. 5,231,020 describes the production of RNA transcripts in the sense capable of suppressing the expression of identical or essentially similar foreign or endogenous genes.

"Altered levels" refers to the production of gene products in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

"Transformation" refers to the transfer of a nucleic acid fragment in the genome of a host organism resulting from genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as organisms "Transgenic: Examples of plant transformation methods include transformation mediated by Agrobacterium (De Blaere et al. (1987) Meth. Enzymol 143: 277) and accelerated particle or "shotgun gene" transformation technology (Klein et al. 81987) Nature (London) 327: 70-73; US Patent No. 4,945,050).

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, e. F. and Maniatis, T. Molecular Cloning: a Laboratory Manual; Cold Spring harbor laboratory Press; Cold Spring Harbor, 1989 (hereinafter "Maniatis").

The term "filling" refers to an irreversible physical change in starch granules or a suspension of starch granules characterized by the swelling and hydration of the granules, a rapid increase in the viscosity of a suspension, and the formation of a sol of the suspension. This change is also known as cooking or gelatinization. The abbreviation "SNU" refers to the number of units of agitation, approximately equal to 10 centipoises, which is a measure of viscosity. For conversion to SI units (pascals per second), multiply the centipoise by 1000, for example 1 PaSec = 1000 cp. Therefore, 1 SNU = 0.01 PaSec. The term "sun" refers to a fluid colloidal system. The term "viscosity" is a measure of the internal friction of a fluid that can be, however, the consistency or thickened of a fluid.

This invention concerns the construction of plants carrying transgenic grains in which the expression of the genes encoding the enzymes involved in the synthesis of starch, specifically the formation of starch polymers (starch synthases) are modulated to effect a change in the distribution of the branched chain of amylopectin, the relative proportion of amylose to amylopectin, or the degree of polymerization of amylose component of starch. Such modification of the fine structure of starch results in the alteration of the physical properties of isolated starch from the transgenic grain harvest. This alteration in the fine structure of starch will lead to the generation of new starches that possess properties that are beneficial in food and industrial applications.

Among these preferred genes are the genes encoding the monocotyledonous starch synthase of different GB? SI, the cloning of which was discussed above. These genes can be isolated by techniques routinely employed by those skilled in the art for gene isolation when the nucleotide sequence of the desired gene is known, or when the sequence of a homologous gene of another organism is known. The sequence information about the desired gene can be used to prepare oligonucleotide probes for identification and isolation of the complete starch synthase enzyme gene from an appropriate gene pool. This pool can be a genomic pool, in which the coding region can be contained in a single DNA fragment or it can be contained in several different DNA fragments. In addition, two or more exons encoding the enzyme complete starch synthase can be separated by one or more introns. Alternatively, the pool may be a cDNA pool in which the probability of isolating a cDNA clone comprising the entire coding region as a contiguous sequence is greater. In either case, the appropriate clones can be identified by DNA-DNA hybridization with probes corresponding to one or more portions of the desired genes. Alternatively, oligonucleotide primers can be prepared and used as PCR primers in order to amplify and subsequently isolate all or part of the starch synthase enzyme encoding the genomic DNA region, or from the cDNA or genomic assemblages described above.

Several different assays can be used to measure the activity of the starch synthase enzyme. The activity can be verified using a variety of methods that evaluate the incorporation of radiolabeled ADP-glucose (14C-glucose) into alcohol-insoluble polymer (Pollock and Priess (1980) Arch. Biochem. Biophys., 204, 578-588; Keeling et al. 1994) Aust. J. Plant Physiol., 21: 807-827; Fontaine et al. 1993 J. Biol. Chem. 268: 16223-16230). The method of Keeling and collaborators is typical. The endosperm tissue from developed corn grains is dissected, lyophilized and cultivated in liquid nitrogen. An extract is prepared by suspension of 100 mg of tissue grown in 2 ml of regulator (50 mM Hepes, pH 7.5, 5 mM MgC12, 1 mM DTT) and homogenized with a mechanical homogenizer. The homogenate is centrifuged at 30,000 x g and the supernatant is verified for soluble starch synthase activity. Briefly, the soluble synthase activity is verified in 1.5 ml tubes with 25 ml of rabbit liver glycogen (2 mg) and 100 ml of regulator (200 mM Vicien, 9 mM EDTA, 50 mM KCl and 20 mM of reduced glutathione, pH 8.3). 50 ml of the soluble extract is added and pre-incubated for 2 minutes. The assay is started with the addition of 25 ml of 8.0 mM ADP-glucose (14C, 444 dpm nmol-1) and was maintained to proceed for 10 minutes before the addition of 100 ml of 0.25M NaOH. glucan by adding 1.0 ml of methanol, cooling on ice for 5 minutes, and centrifuging. The glucan is resolubilized in 0.1 M NaOH and precipitated a second time with methanol. The precipitate is then gelatinized by heating before the addition of the scintillation mixture and measured to the radioactivity in a scintillation counter.

In order to alter the fine structure of starch in corn, a chimeric gene is constructed in which the expression of the gene encoding the enzyme starch synthase is under the control of the appropriate regulatory elements for the expression of gene 1) in tissues of desired plants, 2) in stages of development that provides the maximum desired effect, and 3) to levels of gene expression that result in the alteration of the function of the enzyme starch synthase so that the expression affects a measurable change and significant in the fine structure of starch. The expression of foreign genes in plants is well established (Klein et al. (1987) Nature (London) 327: 70-73, and De Blaere et al. (1987) Meth. Enzymol. 143: 277-291). The proper level of gene expression of sense synthase enzyme or antisense in corn may require the use of different chimeric genes that use different regulatory elements. In addition, effective modulation of the genetic expression of endogenous starch synthase by co-suppression or antisense suppression may require the construction of chimeric genes that comprise different regions of the sense or antisense sequences of the starch synthase. The well-known variability of cosuppression and antisense techniques indicates that even while using different genetic constructs, multiple plants can be screened in order to identify them with the desired phenotype.

The promoters used to direct gene expression in transgenic plants can be derived from many sources, while the selected promoters have sufficient transcriptional activity to achieve the invention by expression of translatable mRNA, mRNA suitable for co-suppression, or antisense RNA in the tissue desired guest. For example, promoters for expression in a broad array of plant organs include those that direct 19S and 35S transcripts in cauliflower mosaic virus (Odell et al. (1985) Nature 313: 810-812; Hull et al. (1987) Virology 86 : 482-493), small subunits of ribulose 1, 5-bisphosphate carboxylase (Morelli et al. (1985) Nature 315: 200-204; Broglie et al. (1984) Science 224: 838-843; Herrera-Estrella y collaborators (1984) Nature 310: 11-120; Coruzzi et al. (1984) EMBO J 3: 1671-1679; faciotti et al. (1985) Bio / Technology 3: 241) and chlorophyll a / b binding protein (Lamppa et al. (1986) Nature 316: 750-752).

Depending on the application, it may be desirable to select promoters that are specific for expression in one or more organs of the plant. Examples include inducible promoters in light of the small ribulose 1,5-bisphosphate carboxylase subunit, if expression is desired in photosynthetic organs, or promoters specifically active in seeds.

Preferred promoters are those that allow expression specifically in seeds. These can be especially useful, since seeds are the primary location for long-term starch accumulation. In addition, seed-specific expression can avoid any potential deleterious effects that modulation of the enzyme starch synthase may have on non-seed organs. Examples of seed-specific promoters include, but are not limited to, the promoters of proteins stored in seeds. The expression of proteins stored in seeds is strictly regulated in the plant, which is expressed almost exclusively in seeds in a highly specific organ and in a specific stage (Higgins et al. (1984) Ann Rev Plant Physiol. 35: 191-221; Goldberg et al (1989) Cell 56: 149-160; Thompson et al. (1989) Bioessays 10: 108-113). In addition, different proteins stored in seeds can be expressed in different stages of seed development. There are currently numerous examples for seed-specific expression of protein genes stored in seeds in transgenic plants. This includes genes from monocotyledonous plants such as barley ß-hordein (Marris et al. (1988) Plant Mol Biol. 10: 359-366) and wheat glutenin (Colot et al. (1987) EMBO J 6: 3559-3554). In addition, promoters of seed-specific genes, operably linked to heterologous encoding sequences in chimeric genetic constructs, also maintain their spatial and temporal expression pattern in transgenic plants (Goldberg et al. (1989) Cell 56: 149-160). Such examples include binding to the albumin promoters either phaseolin or 2S Arabidopsis to the coding sequence of albumin nut 2S and expressing such combination in tobacco, Arabidopsis, or Brassi ca napus (Altenbach et al. (1989) Plant Mol. Biol .. 13_513-522; Altenbach et al 81992) Plant Mol. Biol. 18: 235-245; De Clercq et al 81990) Plant Physiol 94: 970-979), the use of bean lecithin and b-phaseolin bean promoters to express luciferase (Riggs et al. 81989) Plant Sci. 63; 47-57), and wheat glutenin promoters for expressing chloramphenicol acetyl transferase (Colot et al. (1987) EMBO J. 6: 3559-3564).

Of particular use in the expression of nucleic acid fragments of the invention will be the promoters of several protein genes stored in extensively characterized maize seeds such as endosperm specific promoters from the 10 kD ceina or maize gene (Kiribara et al. 1988) Gene 71: 359-370), the 15 kD ceina or maize gene (Hoffman et al. (1987) EMBO J. 6: 3213-3221; Schernthaner et al. (1988) EMBO J. 7: 1249-1253; Williamson et al. (1988) Plant Physiol. 88: 1002-1007), the 27 kD ceina or maize gene (Prat et al. (1987) gene 52: 51-49; Gallardo et al. (1988) Plant Sci. 54: 211-281), and the 19 kD ceina or corn (Marks et al. 81985) J. Biol. Chem. 260: 16451-16459). The relative transcriptional activities of these promoters in maize have been published (Kodrzyck et al. 81989) Plant Cell 1: 105-114) which provides a basis for selecting a promoter for use in chimeric genetic constructs for maize. In addition, promoters that drive the expression of genes encoding enzymes involved in starch biosynthesis can be used in the practice of this invention. This includes but is not limited to the 5 'regulatory sequences of the sucrose synthase (Yang, NS and Russell, D. (1990) Proc. Nati, Acad. Sci. USA 87: 4144-4148), the genes of the starch synthase I that binds to the granule or waxy (Unger et al. (1991) Plant Physiol 96: 124), the sh2 genes (Bhave et al. (1990) Plant Cell 2: 581-588) and bt2 (Bae et al., ( 1990) Maydica 35: 317-322) whose products constitute the enzyme ADP-glucose pyrophosphorylase. Those skilled in the art recognize that the easiest examples can now be supplemented by the plethora of genes specific to other isolated starch biosynthetics and seeds using modern genomic science techniques, which provide an almost unlimited source of seed-specific promoters that can be used for the practice purposes of the present invention.

When necessary, cDNA clones can be used to isolate genomic clones containing the regulatory sequences of interest. The expression of any of these promoters could be increased by the use of enhancer sequences, which include those found in introns sequences (see, for example, "Callis et al. (1987) Genes Dev. 1: 1183-1200; Maas et al. 1991) Plant Mol. Biol .. 16: 199-207; Luehrsen, KR and Walbot, V. (1991) Mol. Gen. Genet 225: 81-93; Orad et al. (1989) Plant Cell Rep 8: 156- 160).

Any 3 'non-coding region capable of providing an indication for polyadenylation and other regulatory sequences that may be required for self-expression may be used to achieve the invention. This will include the 3 'end of any stored protein such as the 3' end of the alpha and 10 kD, 15 kD, 27 kD ceina or maize genes, the 3 'end of the bean phaseolin gene, the 3' end of the ß-soybean conglicin gene, the 3 'end from viral genes such as the 3' end of 35S or 19S cauliflower mosaic virus transcripts, the 3 'end from optimal gene synthesis, the 3' end of genes encoding ribulose 1, 5-bisphosphate carboxylase or protein binding to chlorophyll a / b, or sequences at the 3 'end of any gene so that the sequence employed provides the necessary regulatory information in its nucleic acid sequence to give as a result the expression itself of the promoter / encode the combination of regions to which it is operably linked. There are numerous examples in the art that demonstrate the utility of the different 3 'non-coding regions (for example, see Ingelbretch et al. (1989) Plant Cell 1: 671-680). Various methods of introducing a DNA sequence (for example transforming) into eukaryotic cells of higher plants are available to those skilled in the art (see EPO publications 0 295 959 A2 and 0 138 341 Al). Such methods include high-velocity ballistic bombardment with metal particles coated with nucleic acid constructs (see Klein et al. (1987) Nature (London) 327: 70-73, and see US Patent No. 4,945,050), as well as those based on transformation vectors based on Ti and Ri plasmids of Agrobacterium um spp. , particularly the binary type of these vectors. The vectors derived from Ti transform a wide variety of higher plants, which include dicotyledonous plants, such as soy, cotton and rapeseed (Pacciotti et al. (1985) Bio / Technology 3: 241; Byrne et al. (1987) Plant Cell, Tissue and Organ Culture 8: 3; Sukhapinda et al. (1987) Plant Mol. Biol .. 8: 209-216; Lorz et al. (1985) Mol.

Gen. Genet. 199: 178-182; Potrykus et al. (1985) Mol. Gen. Genet. 199: 183-188); Qu, R. And collaborators (1996) Dev. Bio-Plant 32: 233-240; Vasil, V. et al. (1993) Bio / Technology 11: 1553-1558 and more recently monocotyledons such as rice and corn Hiei Y. Y collaborators (1994) Plant J. "6: 271-282.

Other transformation methods are available to those skilled in the art, such as capturing foreign DNA constructs (EPO publication 0 295 959 A2), and electroporation techniques (Fromm et al. (1986) Nature (London) 319: 791-793). Once transformed, the cells can be regenerated in mature plants by those skilled in the art. Also relevant are several recently described methods of introducing nucleic acid fragments into commercially important crops, such as rapeseed (De Block et al. (1989) Plant Physiol. 91: 694-701), sunflower (Everett et al. (1987) Bio / technology 5: 1201-1204), soybean (McCabe et al. (1988) Bio / technology 6: 923-926; Hinche et al. (1988) Bio / technology 6: 915-922; Chee et al. (1989) Plant Physiol. 91: 1212-1218; Christou et al. (1989) proc. Nati Acad. Sci USA 86: 7500-7504; EPO publication 0 301 749 A2), rice (Qu R. Y collaborators (1996) Dev. Bio-Plant 32: 233-240; Hie Y. et al. (1994) Plant J. 6: 271-282), wheat (Vasel V. et al. (1993) Bio / technology 11: 1553-1558), and maize (Gordon-Kamm et al. (1990) Plant Cell 2: 603-618; Fromm et al. (1990) Bio / technology 8: 833-839 .

One skilled in the art is familiar with other means for the production of transgenic maize plants which include the introduction of DNA into protoplasts and regeneration of plants from said protoplasts.

(Omirulleh et al. (1993) Plant Mol. Biol. 21: 415-42), electroporation of intact tissues (D'Halluin et al. (1992) Plant Cell 4: 1495-1505; laursen et al. (1994) Plant Mol Biol 24: 51-61), the transformation of fiber from corn cells mediated by silica carbide (Keppker et al. (1992) Theor, Appl. Genet, 84: 560-566; Frame et al. (1994) Plant J. 6: 941-948). In addition to the method of bombardment of woody woody woody cell particles described above, a person skilled in the art is familiar with the bombardment of hand-picked corn particles or suspension cultures to produce fertile transgenic plants (Koziel et al. (1993). ) Bio / Tecnology 11: 194-200; Walters et al. (1992) Plant Mol. Biol. 18: 189-200).

Those skilled in the art know that special considerations are associated with the use of cosuppression or antisense technologies in order to reduce the expression of particular genes. US Patents Nos. 5,190,931, 5,107,065 and 5,283,323 describe the feasibility of these techniques. Once the transgenic plants are obtained by one of the methods described above it will be necessary to screen the individual transgenics by which they mostly present the desired phenotype. It is well known to those skilled in the art that individual transgenic plants carry the same construction but differ in expression levels; This phenomenon is commonly referred to as "position effect". For example, when the construction in question is indicated to express higher levels of the of interest, individual plants will vary in the amount of the protein produced and consequently in enzymatic activity; this in turn will effect the phenotype. Thus in the use of these techniques their efficiency in an individual transgenic plant is unpredictable, but a large individual transgenic population with suppressed gene expression will be obtained. In both cases in order to save time, the expert in the matter will elaborate multiple genetic constructions containing one or more different parts of the gene to be deleted., since the specialty does not demonstrate a method to predict which should be more effective for a particular gene. In addition, equally the most effective construction will give an effective suppression phenotype in only a fraction of the individual isolated transgenic lines. For example, WO 93/11245 and WO 94/11516 disclose that when it was attempted to suppress the expression of the fatty acid desaturase genes in rapeseed, the current suppression was obtained in less than 1% of the verified lines. In other species the percentage is somewhat higher, but in no case does the percentage reach 100. This would not seem like a limit in the present invention, but preferably as a practical matter that is appreciated and anticipated by the person skilled in the art. Accordingly, the person skilled in the art will develop methods for screening a large number of transformants. The nature of these screens will generally be selected on practical crops, and is not an inherent part of the invention. In the present case, for example, one can screen for changes in the starch phenotype using chromatography to determine the relative proportions of the starch. amylose to amylopectin, distribution of the amylopectin branched chain, degree of polymerization, Quick Visco Analysis, a standard industry technique for measuring the functionality of food hydrocolloids, particularly starches (as given in the examples), or other means. One could also use antibodies specific for the protein encoded by the gene that is deleted, or one could establish assays that specifically measure the enzymatic activity. A preferred method would be one that allows a large number of samples to be processed quickly, since it would be expected that most of the samples would be negative.

The plants that were identified as having the fine structure of altered starch in the grain present unique genetic material that provides advantages over the traditional cereal crop lines and known starch mutants. The use of lines of the present invention with inhibited expression of SS isoforms in fertile cereal crops provides a dominant characteristic that can simplify and expedite the reproduction process. Known starch mutants can be used but are often recessive and present more complications. Additionally for cereal crops such as wheat, there are a limited number of known mutations. Additionally, the use of antisense or cosuppression to inhibit SS isoforms leads to varying levels of inhibition due to the effect of the chromosomal position. The variable levels of SS activities that result would lead to a wide range of phenotypes that it is not possible to use traditional mutants that can result in a series of limited doses of a mutant allele in endosperm of cereal crops. Additionally, potentially valuable single starch structures of the crossbreeding of newly developed corn lines with altered SS activities with each of the other and / or known starch mutants such as wx or ae will result.

EXAMPLES The present invention is further defined in the following examples. It will be understood that the examples are given to illustrate only and the present invention is not limited to the uses described in the examples. Temperature values are presented in degrees Celsius and percent values are weight by volume, unless stated otherwise. The present invention can be used to generate transgenic cereal crops whose altered starches can be used for any purpose in which their properties are useful such as in, but not limited to, food, paper, plastics, adhesives, or paints. From the above discussion and the following examples, a person skilled in the art can verify, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions. All such modifications are intended to fall within the scope of the claims.

EXAMPLE 1 PREPARATION OF TRANSGENIC CORN EXPRESSING AN ANTISENTIAL CONSTRUCTION OF MAIZE STARCH I SYNTHESIS Cloning of Synthase I Clusters of Maize Starch The cDNA sequence of a soluble rice starch synthase (Baba T. et al. (1993) Plant Physiol. 103: 565-573) was used to generate DNA probes for the detection of homologous starch synthase sequences in maize. Oligonucleotides BE62 (SEQ ID NO: 1) and BE61 (SEQ ID NO: 2) were synthesized in a synthesized Oligonucleotide Beckman Oligo 1000 ™. These initiators include nucleotides (nt) 1600-1619 and 1826-1808 respectively of the published rice sequence.

'-AAGCTTGAATTCCACAGAATCAGGGTACAGG-3 '[SEQ ID NO: l] '-GAAGGACTGGCACTAGACTGG-3 '[SEQ ID NO: 2] The first pair of primers was used to amplify a 429 bp DNA fragment from rice genomic DNA using standard PCR conditions specified in the GeneAmp® PCR kit (Perkin-Elmer). The amplification was carried out for 30 cycles consisting of 1 minute at 94 °, 2 minutes at 55 ° and 3 minutes at 72 °, followed by an extension of 7 minutes at 72 ° after the last cycle. Analysis of the nucleotide sequence showed that the amplified fragment (SSI) contained the expected cDNA sequence as well as an intron of 124 bp following nt 1678 and an intron of 81 bp following nt 1788 of the published sequence. The DNA fragment was labeled by translation of a break and used for probe for Northern blots of total RNA from the development of the corn kernels. A 2.6 kb corn transcript was detected that was present so precociously as 10 days after pollination (DAP) and reached a maximum level of 22 DAP. The rice SSI fragment was designed to contain two regions of sequence homology found to be shared by glycogen synthases or bacterial and plant starch (Baba T. et al. (1993) Plant Physiol. 103: 565-573) . A second fragment of soluble starch synthase (SS2) which lacked these amino acid conservation regions was obtained by PCR amplification of rice DNA using a pair of primers spanning nt 1083-1103 (SS7; SEQ ID NO: 3) and nt 1440-1459 (SS8; SEQ ID NO: 4) of the cDNA.

'-GGATCCGAATTCTCCTTTCTCAGCAAACGG-3 '[SEQ ID NO: 3] '-AAGCTTGAATTCCTGGGATTGCCACCTGAATTG-3 '[SEQ ID NO: 4] A 900 bp DNA fragment containing one or more introns was obtained. When used as a probe for hybridization on total corn RNA stains, this fragment detected a transcript of similar size (2.7 kb) whose expression profile corresponds to that observed with the SSI probe. The SS2 fragment was then used to screen a stock of 19 DAP maize endosperm cDNA by sequences homologous to those of soluble rice starch synthase.

Clontech constructed a corn cDNA library using polyA + RNA from harvested endosperm tissue 19 DAP. The cDNAs were cloned as EcoRI-Xhol inserts in the lambda-ZAPII vector (Stratagene). Approximately 120,000 plate-forming units of the unamplified stock were plated onto NZY agar plates and transferred in duplicate to nitrocellulose membranes (Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning, Cold Spring Harbor Laboratiry Press, New York, hereinafter "Maniatis"). The immobilized DNA was hybridized for 16 hours at 51 ° to the translated SS2 slot (2X105 dp / ml) in 6 X SSPE, 5X Denhardt 0.5% SDS, 100 mg / ml denatured salmon sperm DNA (Maniatis) . The filters were washed twice in 2 X SSC, 0.1% SDS at room temperature for 30 minutes each time and once in 1 X SSC, 0.1% SDS for 15 minutes at 50 ° (Maniatis). A total of 38 putative positive plaques were identified from this initial screening. Of these 24 were purified and subjected to additional characterization by restriction enzyme digestion and partial nucleotide sequence analysis. Two clones, designated pSS23 and pSS31 contained the longest cDNA insert and were selected for more detailed characterization. The plasmid pSS31 was found to contain a 2.2 kb cDNA insert which is comprised of 144 bp of DNA not translated in 5 ', an open reading frame of 1923 bp, and 168 bp of DNA not translated in 3'. Plasmid pSS31 thus encodes a complete copy of the corn starch synthase polypeptide. The comparison of the deduced amino acid sequence with that of the soluble rice starch synthase shows that the two proteins are 80% identical throughout their entire extension. PSS23 contains a 1954 bp insert whose sequence on the first 1715 nucleotides is identical from nt 521 to 2235 of pSS31. However, the pSS23 cDNA insert extends 239 bp beyond the 3 'end of pSS31. PSS23 thus contains an incomplete copy of the starch synthase polypeptide, which lacks 126 amino acids at the terminal end amine. The consensus sequence of SSI cDNA was obtained by comparing the sequences of pSS23 and pSS31 and is set forth in SEQ ID NO: 5. Both pSS23 and pSS31 were used to generate DNA constructs by modifying the expression of that starch synthase. in corn plants.

Preparation of an Expression Vector that encodes Corn SSI antisense transcripts The pSS23 clone of the starch synthase was used to generate an antisense construct by suppression of SSI expression in maize. PSS23 was digested first with the restriction enzyme Pvu I and the recessed 5 'end was truncated by reaction with the T4 DNA polymerase. To 10 mg of pSS23 digested with Pvu I (in 40 ml of 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgC12, 1 mM DTT) 20 units of T4 DNA polymerase and deoxynucleotide triphosphates ( dNTPs) were added to a final concentration of 0.1 mM. The reaction mixture was incubated for 15 minutes at 12 °, 10 minutes at 75 ° and DNA purified by extraction with phenol: chloroform: isoamyl alcohol (25: 24: 1) followed by ethanol precipitation. The DNA of the repaired plasmid was then incubated with the restriction enzyme Xhol in the regulator specified above. After digestion, dNTPs were added to a final concentration of 50 μM and the Xho I end was filled by reaction with the Klenow fragment of E DNA polymerase I. Coli (10 units) and dNTPs for 15 minutes at room temperature. The enzyme was inactivated by incubation at 75 ° for 10 minutes and the blunt end DNA was fractionated by electrophoresis on 0.7% low melting agarose gel in 40 mM Tris-acetate, pH 8.5, 1 mM EDTA. The 1.55 kb band (insert) was excised from the gel and combined with a 4.9 kb fragment from plasmid pML103 (ATCC 97366). The pML 103 plasmid contains a 1.05 kb Sall-Ncol promoter fragment of the 27 kD maize gene and a 3 'S to I-Sal I fragment of 0.96 kb of the 10 kD maize gene in a pGem9Zf vector (+) (Promega). Plasmid pML103 was digested with Neo I and Sma I, digested DNA was treated with Klenow and dNTPs to insert into the left projection by the enzyme, and the desired 4.9 kb vector fragment was electrophoresed and isolated as described. The combined vector and insert fragments were melted at 68 ° and ligated overnight, essentially as described (Maniatis). The ligated DNA was used to transform E. coli XLl-Blue cells (Epicurean Coli XL-1 Blue ™, Stratagene). Bacterial transformants were screened for the presence of and orientation of the insert DNA by digestion with the restriction enzyme HindIII. Plasmid pSS42 was identified from this analysis. PSS42 contains the 1.55 kb segment of pSS23 (SEQ ID NO: 6) in antisense orientation with respect to the 27 kD cein promoter fragment and the 10 kD cein at the 3 'end. To generate a construct for plant transformation, the chimeric gene of pSS42 was released by digestion with BamHl and the 3.6 kb fragment was cloned into the BamH1 site of the pKS17 vector. Plasmid pKS17 contains the hygromycin B phosphotransferase (HPT) gene that confers resistance to the hygromycin antibiotic. To generate a construct for plant transformation, the chimeric gene of pSS42 was cloned into the pKS17 vector. A derivative of the pSP72 vector (Promega), pKS17 contains the hygromycin B phosphotransferase (HPT) gene which confers resistance to the antibiotic hygromycin. PKS17 was conjuncted by the addition of a T7-promoter-HPT-T7 gene to a modified pSP72 plasmid from which the β-lactamase gene has been deleted. The chimeric gene of pSS42 was released by digestion with BamHl and the 3.6 kb fragment was cloned into the BamH1 site of the pKS17 vector. The resulting plasmid containing the kin promoter of 27 kD-SSI antisense-end 3 'of 10 kD cein in pKS17 is pSS43 terminated (Figure 1).

Maí z transformation with the SSI antisense construction Immature corn embryos were dissected from caryopses developed from self-pollination of the corn germaplasma "Hi-II", which was selected from maize insemination A188 x B73 (Armstrong et al. (1991), Mai ze genetics Cooperation Newsletter 65: 92-93) . Germaplasma Hi-II has been widely used for transformation because it is characterized by a high frequency of "Type II" callus formation. This callus is derived from the decortication of immature zygotic embryos excised in vi tro. The callus type II is especially responsible for the transformation because it is friable, it proliferates rapidly and highly embryogenic. The embryos were isolated 10 to 12 days after pollination when they were 1.0 to 1.5 mm long. The embryos were placed with the lateral axis down and in contact with a solidified MS-agarose medium (Murashige, T. and Skoog, F., (1962) Physiol. Plant 15: 473) supplemented with 1 mg / L of2.4 -D. Embryos were stored in the dark at 27 0. Friable embryogenic callus consist of undifferentiated masses of cells with somatic proembryoids and embryoid terminals on suspensory structures proliferated from the decortication of these immature embryos. The embryogenic callus isolated from the primary explant was cultured in an agar-solidified N6 medium (Chu et al. (1975), Sci. Sin. Peking 18: 659-668) supplemented with 1 mg / L of 2, -D, and sub -cultivated on this medium every 2 to 3 weeks.

A segment of plasmid pML108 was used in order to provide a selectable marker in the transformation experiments. This plasmid contains the bar gene (Thompson et al. (1987) EMBO J. 6: 2519-2523), which encodes phosphinothricin acetyl transferase (PAT). The PAT enzyme confers resistance to inhibitors of the herbicide glutamine synthetase such as phosphinothricin. The bar gene in pML108 is under the control of the 35S promoter of the Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313: 810-812), and contains the 3 'region of the octopine synthase gene of the T-DNA of the Ti plasmid. Agrobacterium tumefaciens. A 2116 bp HindIII fragment containing the chimeric 35S-bar-OCS gene was isolated from pML108 and used in conjunction with the DNA characteristic of plant transformation experiments.

The particle bombardment method (Klein et al. (1987) Nature 327: 70-73) was used to transfer genes to cells grown in callus. Gold particles (1 μm in diameter) were coated with DNA using the following technique. Plasmid DNAs (1 μg of the fragment pML108 and 12 μg of pSS43) were added to 50 μl of a suspension of gold particles (60 mg per ml). Calcium chloride (50 μl of 2.5 M solution) and free base spermidine (20 μl of a 1.0 M solution) were added to the particles. The suspension was vortexed during the addition of these solutions and for 5 minutes after the addition of the last solution. After another 5 minutes, the tubes were centrifuged briefly (5 sec at 15,000 rpm) and the supernatant was removed. The particles were resuspended in 140 μl of absolute ethanol, centrifuged again and the supernatant was removed. The ethanol rinsing was carried out again and the particles were resuspended in a final volume of 55 μl of ethanol. An aliquot (6 μl) of the DNA coated gold particles was placed in the center of a Kapton ™ frisbee (Bio-Rad Labs). The particles were accelerated in the corn tissue with a Biolistic ™ PDS-1000 / He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1100 pounds per square inch, a gap distance of 0.5 cm and an air distance of 1.0 cm.

For the bombardment, the embryogenic tissue was placed on a filter paper on N6 solidified agarose medium supplemented with 1 mg / L of 2,4-D. The tissue was laid out as a thin lawn and a circular area about 5 cm in diameter was covered. The petri dish containing the tissue was placed in the PDS-1000 / He chamber approximately 8 cm from the stopping screen. The air in the chamber was then evacuated to a vacuum of 28 inches of mercury. The macrocarrier accelerated with a helium shock wave using a rupture membrane that triggers when the helium pressure in the shock tube reaches 1100 pounds per square inch.

Four days after the bombardment the tissue was transferred to N6 medium supplemented with 1 mg / L of 2,4-6 plus bialaphos (2-10 mg / l), and without casein or proline (selective medium). The tissue continued to develop in this medium. After one week, the tissue was transferred back to selective N6 medium containing 2,4-D and bialaphos. After 6-8 weeks in the selective medium, areas approximately 1 cm in diameter of the callus that grew actively were identified in some of the plates containing medium supplemented with bialaphos. This calli continued to grow when subcultured on selective medium. The callus that continued to grow vigorously on the selective medium was sampled for PCR analysis by freezing a callus mass of approximately 200-500 mg of fresh weight.

The DNA was extracted from the collected samples by frozen suspension, tissue cultured in a regulator consisting of 50 mM Tris-HCl, pH 8.0, 7 M urea, 0.35 M NaCl, 20 mM EDTA, 1% n-lauryl sarkosin and incubation at 37 ° C for 15 minutes. After this time, the samples were extracted with a sample of phenol-chloroform-isoamyl alcohol (25: 24: 1) and concentrated by precipitation with isopropanol. The DNA was resuspended in 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA (100 μl) and used as a template in PCR using primers MM50 (SEQ ID NO: 7) and BE56 (SEQ ID NO: 8).

'-AAGCTTGAATTCGGCACATCGGGCCTTATGG-3 '[SEQ ID NO: 7] '-GTCTAGTGCCAGTCCTTC-3 '[SEQ ID NO: 8] DNA (2 μl) was combined with 20 μM of each of the MM50 and BE56 primers in a standard mixture provided by the GeneAmp® PCR kit (Perkin-Elmer). The amplification was carried out by 3C cycles consisting of 1 minute at 95 °, 2 minutes at 55 ° and 3 minutes at 72 °. Samples were graded for the presence of a target band of 546 bp which encompasses the 3 'portion of the SSI fragment and the 3' end of 10 kD cein. Callus samples positive to the characteristic gene were carried forward in the transformation regime.

The plants were regenerated from the transgenic callus by first tissue transfer groups to MS medium without bialaphos or 2, 4-D and placed in the dark. After two weeks the tissue was transferred to the regeneration medium (Fromm et al. (1990) Bio / Technology 8: 833-839) in the light. A total of 35 maize plants were regenerated from a single transformation experiment using the pSS43 construct.

EXAMPLE 2 PREPARATIONS OF TRANSGENIC CORN EXPRESSING CONSTRUCTIONS IN THE SENSE OF CORN STARCH SINTASA I Plasmids pSS64-C5 and pSS65-Cll encode transcripts in the sense of the SSI gene. For both constructions, a Neo I site is introduced into the initial methionine of S? I cDNA by PCR. Oligonucleotides MM62 (SEQ ID NO: 9) and MM60 (SEQ ID NO: 10) were combined with the DNA template of pSS31 in a modified mixture by PCR (Advantage-GC ™; Clontech) designed to facilitate amplification through GC-rich regions of template DNA.

'-GAGTCACACGCGATGGC-3 '[SEQ ID NO: 9] 5' -CTCTCCGCCATGGCGACGCCCTCGGCC-3 '[SEQ ID NO: 10] The amplification was carried out using 35 cycles of 1 minute at 95 °, 1 minute at 53 ° and 1 minute at 72 °, followed by a final extension of 10 minutes at 72 °. The amplified fragment covers nucleotides 136-1003 of SSI cDNA. The DNA was digested sequentially with the restriction enzymes Kpn I and Neo I, fractionated by electrophoresis on a 1% agarose gel (Maniatis) and the 537 bp Neo I-Kpn I fragment was excised from the gel and purified by treatment with Gelase ™ (Epicenter Technologies). Plasmid pET-SSSI (PpuMI) contains a fragment spanning nucleotides 418-2235 of the SSI cDNA inserted into a truncated Neo I site of pET24d (Novagen), oriented in the direction in the direction, and at an angle to the T7 promoter. The Neo I site at the 5 'end of the SSI sequence was recreated by insertion of the SSI fragment. PET-SSSI (PpuMI) was incubated with Kpn I followed by Neo I and the digest was fractionated on a 1% agarose gel. The 7.1 kb band was excised, purified and ligated to the Neo I-Kpn I fragment of 537 bp SSI described above. The resulting plasmid, which contains the complete coding region of SSI in addition to 168 bp of untranslated 3 'DNA, is the terminated pET-SSSI @ MAT. This plasmid was used in the construction of both pSS64-C5 and PSS65-C11. To generate pSS64-C5, pET-SSSI @ MAT. It was digested with Bgl II and the 5 'overhang was inserted by reaction with Klenow and dNTPs, essentially as described above. The DNA was digested with Neo I and the fragment released from 1465 kb was cloned into the 4.53 kb Neo I-Sma I fragment from pSPB3d. This segment pSPB3d contains a 1.05 kb Sal-Incol promoter fragment of the 27 kD cein gene and a 0.96 kb Sma I-Pvu II fragment from the 3 'end of the 10 kD cein gene in the pKS17 vector, described above. The resulting plasmid, pSS64-C5 terminated (Figure 2), thereby contains the 27 kD cein promoter followed by amino acids 1-494 of the SSI coding region (SEQ ID NO: 11) and the 3 'end of the cein 10 kD. To generate pSS65-Cll, plasmid pET-SSSI @ MAT was digested with BsrGI and the 5 'overhang was inserted by reaction with Klenow and dNTPs. The DNA was digested with Neo I to release a 2.0 kb fragment which was then ligated to the 4.53 kb pSPB3d fragment described above. The derived plasmid, pSS65-Cll (Figure 3), consists of the complete SSI coding region followed by d4 bp from the 3 'untranslated DNA of SSI (SEQ ID NO: 12) surrounded by the 27 kD cein promoter and the 3 'fragment of 10 kD ceina of 0.96 kb. The DNA constructs, pSS64-C5 and pSS65-Cll were introduced into corn by the method underlined in Example 1. The callus lines with positive characteristics were identified by PCR analysis (Example 1) and carried out to regenerate transgenic plants. .

EXAMPLE 3 STARCH ANALYSIS OF TRANSFORMED CORN PLANTS CONTAINING THE ANTICIPATED CONSTRUCTION PSS43 Starch was extracted from unique seeds obtained from corn plants transformed with the antisense construct PSS43. The seeds were impregnated in a solution containing 1% lactic acid and 0.3% sodium metabisulfite, pH 3.62 and maintained at 52 ° for 22-24 h. The seeds were drained, rinsed and homogenized individually in 8-9 ml of 100 mM of a NaCl solution. Five ml of toluene was added to each tube, the tubes were shaken vigorously twice for 6 minutes and then kept to sediment for 30 minutes. Two ml of 100 mM NaCl was sprayed onto the solution, which was maintained to settle for 30 minutes, and the protein / toluene layer was removed by aspiration. The washing step with toluene was repeated. 12 ml of water was added and stirred on a paint shaker for 45 seconds. This solution was centrifuged for 10 minutes in a top plate centrifuge and the water was removed. Washing with water was repeated, followed by a final wash with 12 ml of acetone. After the agitation and centrifugation steps, the acetone was drained and allowed to evaporate for 1 hour. The starch extracts were incubated for release of the remaining acetone, overnight in a 40 ° incubator.

The extracted starches were enzymatically debranched as follows. Seven mg of each starch sample was added to a screw capped test tube with 1.1 ml of water. The tubes were heated at 120 ° for 30 minutes and then placed in a 45 ° water bath. The debranching solution was made by diluting 50 μl of isoamylase (5 × 10 units / ml, Sigma) per ml of sodium acetate buffer (50 mM, pH 4.5). Forty μl of debranching solution was added to each of the starch samples and incubated for 3 hours at 45 °. The reactions were stopped by heating at 110 ° for 5 minutes. The debranched starch samples were lyophilized and redissolved in DMSO for analysis by gel permeation chromatography (GPC). 10 μl of debranched starch was injected and run through 3 columns of narrow inner diameter (Polymer Labs. Mini-Mix C) in series at 100 ° and eluted with DMSO at a flow rate of 0.35 ml / min. The sampling interval was 30 minutes.

-, -? A refractive index detector (Waters) was used with a Waters Millenium Chromatography Manager computer System with option to GPC (version 2.15.1, Waters Corp.) for detection, calculation and analysis of data, respectively. Retention times of pullulan standards (Standard 1: 380 kD, 100 kD, 23.7 kD, 5.8 kD, 666 and 180 mw, Standard 2: 653 kD, 186 kD, 48 kD and 12.2 kD) were used to establish a linear calibration and calculate the molecular weight distributions in the Millenium® program.

As is known to those skilled in the art, the antisense phenomenon is not generally observed in each individual transgenic line. Therefore, individual grains of multiple lines were examined and as expected, some, but not all grains possessed by the lines demonstrated an altered starch phenotype in relation to the control. As is also known to those skilled in the art, transgenic maize plants produced by particle bombardment are typically heterozygous for the introduced transgene and the transgene will be segregated in a predictable Mendelian model. On the alternate ears of a plant RO (primary transformant) the triploid endosperm, which is the tissue responsible for the production of starch, will segregate * ,. - £ &• * *. 1: 1: 1: 1 for 0, 1, 2 and 3 copies of the entered transgen, respectively. In order to have a reasonable probability of observing any of these transgene dosages, 10 single grains of line S048.6.1.10, (designated XBG00944-1 to XBG00944-10) were extracted for starch, and the starch of each grain was debranched as described above. Figure 6 shows the molecular weight distribution obtained for the debranched starches of two grains representative of line S048.6.10. XGB00944-7 exposes the corresponding pattern to a normal segregator while XGB00944-1 exposes the corresponding pattern to an altered segregator.

Line S048.6.1.10 produces starches with two very different types of molecular weight distributions. The molecular weight distributions of debranched starch from seeds 944-3, 944-4, 944-7 are typical of the observed molecular weight distribution for normal indent corn starch. The molecular weight distributions of debranched starch from seeds 944-1, 944-2, 944-6, 944-8, 944-9, and 944-10 exhibits an alteration in the molecular weight distribution of debranched starch. Figure 6 sets forth the molecular weight distribution of one of each of these seed types, 944-7, is set forth as an example of a normal starch and 944-1 is set forth as an example of an altered starch. As can be seen in Figure 6 there is an increase in the amount of high molecular weight material (log MW > 4) and a decrease in the distribution of the lower molecular weight material. The proportion of occurrence of normal and altered seeds on the segregating ear was compared with the various possible modes of expected inheritance using the square x-statistic (X2). The observed frequency of 60% altered: 40% of normal seeds was a reasonable fit with the simple dominance hypothesis (in which either 1 or more doses of the transgene were sufficient to produce the altered starch structure) (X "= 1.2) or the hypothesis that 2 or more doses of the transgene were required to alter the structure of the starch / semidominance, X2 = 0.4).

EXAMPLE 4 QUANTITATIVE ANALYSIS OF STRUCTURAL ALTERATIONS OF STARCH IN SEEDS XBG00944 By quantitative comparison of the altered transgenic starch, starch XBG00944-1 was selected as representative of the most extreme alteration in the starch structure (see Figure 6) and was used for comparison of indented corn starch, starch of a dull mutant and starch of a waxy mutant. The starches from these four lines (indented, dull, wx, 944-1) were enzymatically debranched as described above and separated with a slightly modified chromatography method to provide better resolution of the branched chain distribution in the fraction of amylopectin. 10 μl of debranched starch was injected and run through 3 columns of narrow inner diameter (Polymer Labs, Mmi-Mix C, DE with a Mini-mix C safety column) in series at 90 ° and eluted with DMSO at a flow of 0.35 ml / min. The sampling interval was 35 minutes. A refractive index detector (Waters) was used with a Waters Millenium Chromatography Manager System computer with GPC option (version 2.15.1, Waters Corp.) for detection and collection and analysis of data respectively. The retention times of pullulan standards (Standard 1: 360 kD, 100 kD, 23.7 kD, 5.6 kD, 666 and 180 mw, Standard 2: 853 kD, 186 kD, and 12.2 kD) were used to establish a third-party calibration order and calculate the molecular weight distributions in the Millenium® program. The analysis was carried out in triplicate for each of the four starches that were being compared.

To determine the content of amylose (Am) and amylopectin (Ap), the areas below the appropriate chromatography peaks were compared. The waxy mutant (which lacks amylose) was used to establish the ranges of molecular weights suitable for comparison. Table 1 shows the amylose and amylopectin content of each of the four lines.

TABLE 1 Amylose and Amylopectin Contents (Average (n = 3) and Standard Deviation) of SSSI Antisense Starch Compared to Starches du, indented, and wx % Am Deviation% Ap Standard Deviation Standard 944-1 35.40% 0.15% 64.60% 0.15% du 31.99% 0.05% 68.01% 0.05% Indented 25.65 0.14% 74.35% 0.14% wx 0.00% 0.00% 100.00% 0.00% The amylose content increases significantly (P <0.01) compared to both normal indent starch and dull mutant starch. The amylose content is similarly significantly decreased (P <0.01) compared to both lines. The suppression of the expression of starch synthase I thus resulted in the alteration of the fine structure of starch in these plants, specifically a significant change in the ratio of amylose to amylopectin.

The Millenium® program for GPC was used to produce molecular weight distributions independent of the amylose and amylopectin components of the analyzed starches and to determine the average molecular weights Mn (average molecular weight number), M ^ (average molecular weight) , M, and M + i (molecular weight of polymer sediment), peak molecular weight (MP) and polydispersity (MAMr) for the amylose and amylopectin components.

Table 2 presents the quantitative analysis of the molecular weight distributions of amylose starch 944-1, indented, and dull.

TABLE 2 Average Molecular Weight (Dalton) of the Amylose Component of Starches 944-1, du, e mdentado.

Mn MP Mw Number Weight Weight Average molecular average Molecular molecular weight peak weight 944-1 91414 ± 110 278047 ± 0.00 357427 ± 27í du 82728 ± 693 163963 ± 1322d 28320712727 addendum 90511 ± 1106 172334 ± 8444 304715 + 3460 B ^ asti »M M.- .. PD Weight Weight Polydis- molecular molecular persity, de de Mw / Mn) sedimentation-sedimentation c? Ón- +? 944-1 1123204 ± 12306 22587551464 3.910010.0074 du 894430121341 1908812191646 3.423610.0349 indented 968529136715 20953891176856 3.366710.0037 By comparing the values below column Mw in Table 2 it can be seen that the longer amylose molecules in starch of 944-1 are significantly (P <0.01) in molecular weight relative to dull and indented starch. This is reflected in the significant increase in MP, Mz and Mz + 1. The significant increase in polydispersity (P <0.01) of the amylose of 944-1 in relation to both indented and dull starches suggests that this increase in amylose molecular weight does not detract from the shorter amylose molecules but from preference for an expansion of the amylose component distribution. This is consistent with the observation of the increased relative amylose content reported in Table 1, and the increased amylose content of line 944-1 can be attributed to the occurrence of high molecular weight amylose that is not present in the indented or intermediate starches. dull The net effect, therefore, is that altering the expression of starch synthase I results in the alteration of the fine structure of starch in the seeds of these plants not only by making a significant change in the ratio of amylose to amilpectin but, because the additional amylose is larger, by altering the molecular weight distribution of the amylose component of the starch.

Table 3 presents the quantitative analysis of the molecular weight distributions of starch 944-1, indented, and dull.

TABLE 3 The Average Molecular Weights (Dalton) obtained for the Amylopectin Component of 944-1 and Control Starches.

Mn MP Mw Number Weight Weight Average molecular weight average molecular weight molecular weight 944-1 272714.5 240918.3 353415.8 du 271316.7 232018.0 3557111.2 indented 271712.3 2352114.1 373910.9 wx 2873113.0 246018.7 414418.6 Mx Mx + 1 PD Weight Weight Molecular molecular-weight persity, of M "/ Mn) sedimentation-sedimentation tionz? 944-1 4803113.9 6406126.9 1.29610.0019 du 4878120.2 6529135.4 1.31110.0009 indented 531117.1 7133118.3 1.37610.0012 wx 6196120.3 8716165.8 1.44210.0043 The amylopectin Mw of 944-1 is significantly reduced (P <0.01) in comparison to the indented such as Mz and Mz + 1, indicating a change in the distribution of amylopectin chain extension to more chain extensions. short The polydispersity of amylopectin 944-1 is also significantly reduced (P <0.01) compared to indolated amylopectin, which is dull, confirming again the visual observation in Figure 6 that the amylopectin fraction of starch 944-1 is more homogeneous in long chain than is the indented amylopectin.

Thus, the quantitative analysis confirms a significant increase in the amylose content and an increase in the molecular weight of the amylose component of starch 944-1 compared to the indented starch. The observed increase in the molecular weight of amylose is achieved by an expansion of the molecular weight distribution of the amylose chains. The observed decrease in the amylopectin content of starch 944-1 is accompanied by a change in the distribution of the branched chain in favor of shorter chains.

In summary, the quantitative analysis confirms that altering the expression of maize seed starch synthase I results in multiple changes in the fine structure of the starch in the seeds, including a significant increase in the content of anulose, an increase in the molecular weight of the amylose component of the starch, and a change to shorter chains in the reduced amylopectin component. The observed increase in the molecular weight of amylose is achieved by an expansion of the molecular weight distribution of the amylose chains.

EXAMPLE 5 FUNCTIONAL ANALYSIS OF HOMOCIGOTIC LINES STARCH FOR CONSTRUCTION ANTICIPATED IN 3 ' As is known to those skilled in the art, a homozygous line can be derived from the segregating progeny of a heterozygous plant such as line S046.6.10 by planting a sufficient number of grains from the segregating population, the resulting self-pollination of the plants of these seeds, and to sift seeds of a single progeny produced to identify an ear that has the characteristic of altered starch fixed. Once such homozygous cob is identified, a larger sample of starch can be extracted from the dried mature grains of the identified line and control lines which produce normal indented starch. For each line, 15 g of grains can be weighed in a 50 ml Erlenmeyer flask and impregnated with 50 ml of a solution (Example 3) for 18 hours at 52 °. The grains are then drained and rinsed with water. The grains are homogenized using a 20 mm Polytron probe (Kinematica GmbH, Krienz-Luzen, Switzerland) in 50 ml of cold mM NaCl. The homogenate is filtered through a 72 micron mesh screen. The filtrate is brought to a total volume of 400 ml with 50 mM NaCl and an equal volume of toluene is added. The mixture is then stirred with a magnetic stir bar for 1 hour at sufficient speed to fully emulsify the two phases. The emulsion is left overnight to separate in a covered beaker. The top layer of toluene is sucked from the beaker and discharged. The starch solid remaining at the bottom of the precipitate is re-suspended, poured into a 250 ml centrifuge bottle and centrifuged 15 minutes at 25,000 RCF. The supernatant is discharged and the starch washed sequentially with water and acetone by stirring and centrifugation as above. After washing with acetone and centrifugation the acetone is decanted and the starch is left to dry overnight in a ventilation hood at room temperature. A Visco Rapid Analyzer (Newport Scientific, Sydney, Australia) with a high sensitivity option and a Thermocline® program can be used to analyze the curve. For each line, 1.50 g of starch is weighed in the sample vessel and 25 ml of phosphate / citrate buffer (pH 6.50) containing 1% NaCl is added. The step to the analysis curve is carried out using the following temperature profile; The temperature is set at 50 °, is kept at 50 ° for 0.5 minutes, linear heating at 95 ° for 2.5 minutes, linear cooling up to 50 ° for 4 minutes, maintained at 50 ° C for four minutes.

EXAMPLE 6 PREPARATION OF TRANSGENIC CORN THAT EXPRESSES ANTICIPATED CONSTRUCTIONS AND IN THE SENSE OF THE SYNTHESIS CORN STARCH SSb From the nucleotide sequence of an Expressed Tag Sequence (EST) in corn with homology to the starch tape (T14684), the oligonucleotides SS9 (SEQ ID NO: 13) and SS10 (SEQ ID NO: 14) were designed and used for amplify a 351 bp DNA fragment by PCR using the standard conditions specified in the GeneAmp® PCR (Perkin-Elmer).

'-AAGCTTGAATTCGCAGTATGCTCGCTCTGTGC-3 '[SEQ ID NO: 13] '-GGATCCGAATTCGGTTCCACTCGCTCATGTCG-3 '[SEQ ID NO: 14] The resulting DNA fragment was labeled using a RadPrime DNA Marking System (BRL Life Technologies) and then used to screen a stock of endosperm cDNA from maize 19 DAP in lambda ZAPII. Approximately 500, 000 plaque forming units were plated on NZY agar plates and transferred in duplicate to nitrocellulose membranes (Maniatis). The immobilized DNA was hybridized to the labeled fragment and the excess probe was removed from the filters essentially as described by Maniatis. A total of 10 positive plaques were identified, purified and the DNA insert underwent additional characterization. Analysis of the DNA sequence showed that 9 out of 10 clones were related to each other and 84% were homologous over 50 bp of the probe sequence used to detect them initially. The remaining clone was distinguished from the rest and showed 95% homology with T14684. Of the group of 9 clones, one, pSPB37 contained a 2006 bp insert (SEQ ID NO: 15) and was used in the generation of an antisense construct for introduction into corn. The presence of an extra T in the sequence of the DNA insert of pSPB37 was first corrected by substituting a 431 bp Ncol fragment from another isolated SSb clone, pSPB28, for the same region in pSPB37 to give pSPB39. An SSb fragment of 1.78 kb was obtained by digestion of PSB39 with Ba nHI and BsrGI. This SSb fragment and the 4.53 kb Ncol-Smal fragment of the vector pSPB38 were blunt ended by reaction with the Klenow fragment of DNA polymerase I and ligated with each other following the standard protocols (Maniatis). The transformants - « Bacteria were screened for the presence of and orientation of the SSb-inserted DNA by restriction enzyme digestion with BamHI and XhoI. This analysis leads to the identification of pSPB40 containing the 1.8 kb SSb fragment (SEQ ID NO: 16) in antisense orientation with respect to the 27 kD cein promoter and the 3 'end of 10 kD cein. The purified pSPB40 DNA was introduced into the callus cell cultures essentially as outlined in Example 1 using 1.33 μg of pSPB40 and 0.34 μg of the marker gene fragment of pML108 by bombardment. The callus samples were examined for the presence of the characteristic genetic DNA by PCR analysis and samples positive for the characteristic of the gene were carried forward in the transformation regime.

An SSb construction in the sense of total extension was also generated and introduced into the corn callus tissue by the particle bombardment method. A complete copy of the SSB cDNA was obtained first using pSPB39 as starting material. Analysis by Northern staining of total RNA extracted from endosperm developed indicated that the SSb transcript was approximately 3.0 kb. The remaining 5 'sequence of the SSb cDNA was obtained by Rapid Amplification of the cDNA Ends (RACE) using a RACE 5' System equipment (Life Technologies) with some modifications to the instructions provided by the manufacturer. The synthesis of the first cDNA chain was carried out at 50 ° using the specific primer of the OSPB104 gene (SEQ ID NO: 17).

'-CGACTCCGTAGCACACACC-3 '[SEQ ID NO: 17] The amplification of the cDNA cut at the dl-dG ends was carried out with the AAP primer provided in the RACE kit and the specific primer of the OSPB105 gene (SEC ID NO: 18) using an Advantage GC kit for PCR (Clontech).

-GTGCCAAGGAACCTCAACAG-3 '[SEQ ID NO: 18] The re-amplification was carried out in a similar manner using the primer AAP and OSM106 (SEQ ID NO: 19).

'-GAGGGGCATCAATGAACACA-3 '[SEQ ID NO: 19] A full-length equivalent of the cDNA of SSb, pSPB45, by binding of the 1346 bp segment obtained from the digestion of the 5 'RACE product of 1485 bp with Xbal and Kpnl from the 3' SSb region of 1604 bp obtained from pSPB39 by digestion with Xbal and partial digestion with Kpnl. A Ncol site was introduced into the initiation codon of the coding region of pSPB45 by PCR to give pSPB46. PSPB46 was digested with BsrGI and the protruding 5 'end made blunt by a final insertion reaction with the Klenow fragment of DNA Polymerase I (Maniatis). Following partial digestion with Ncol, the 2248 bp SSb fragment (SEQ ID NO: 20) was isolated and cloned into the 4.53 kb Ncol-Smal segment of pSPB38 to give pSPB47 (Figure 5). Plasmid pSPB47 contains the complete SSb cDNA in orientation in the sense encircled by the 27 kD ceina promoter and the 3 'end of 10 kD cein. The purified pSPB47 DNA was introduced into callus cell cultures essentially as outlined in Example 1, using 1.43 μg of pSPB47 and 0.33 μg of the pMLlOd marker gene fragment by bombardment. The callus samples were examined for the presence of the characteristic genetic DNA by PCR analysis and the positive samples were continued in the transformation regime.

EXAMPLE 7 STARCH ANALYSIS OF TRANSFORMED CORN PLANTS CONTAINING ANTI-SENSE CONSTRUCTION SSb Starch was extracted from single seeds obtained from maize plants transformed with the SSb antisense construct as previously described. The extracted starches were enzymatically debranched as previously described and analyzed by gel permeation chromatography. 10 μl of debranched starch was injected and run through narrow inner diameter columns (Polymer Labs. Mini-Mix C, D, E with a Mini-mix C safety column) in series at 90 ° and eluted with DMSO at a flow of 0.35 ml / min. the sampling interval was 35 minutes. A refractive index detector (Waters) was used with a computer program Waters Millenium Chromatography Manager System with option for GPC (version 2.15.1, Waters Corp.) for detection and collection and analysis of data respectively.

Retention times of pullulan standards (Standard 1: 380K, 100K, 23.7K, 5.8K, 666 and 180 mw, Standard 2: 653K, 186K, 48K, and 12.2 K) were used to establish a third-order calibration and calculate the molecular weight distributions in the Millenium Program.

As is known to those skilled in the art, the antisense phenomenon is not generally observed in each individual transgenic line. Therefore, individual grains of multiple lines were examined and as expected, but not all, the grain lines demonstrated an altered starch phenotype. As is known to those skilled in the art, transgenic maize plants produced by particle bombardment are typically heterozygous for the introduced transgene and will segregate the transgene in a predictable Mendelian mode.

In an alternate ear of a RO plant the triploid endosperm, which is the tissue responsible for the production of starch, will segregate 1: 1: 1: 1 for 0, 1, 2, and 3 copies of the introduced transgene, respectively. In order to have a reasonable probability of observing any of these transgene dosages 10 unique grains of line S064.1.2.1 (designated XBG01717-1 to XBG01717-10) were extracted for starch and the starch of each grain was debranched and separated as described above. The line S064.1.2.1. produces seeds that secrete starches with different types of molecular weight distributions. Some of the starches in the seeds (XBG01717-1, 2, 3, 4, 5, 6 and 8) produced an amylopectin (the region between log MW 3 and log MW 4.2) that is more heterogeneous than amylopectin in normal indent corn , whereas normal indented corn exhibits a typical bimodal distribution (XBG01717-7, 9, and 10). Figure 5 shows the molecular weight distributions obtained by the debranched starches obtained from two representative grains, the normal segregator XBG01717-9 and the altered segregator XBG01717-2. As is typical for a normal segregator, Figure 7 shows that XBG01717-9 has a single dominant peak in log MW 3.5 and a single obvious hump in log MW 3.9. Figure 7 also shows that the unusual segregator (XBG01717-2) has a split in the main peak in log MW 3.5 and a less prominent hump in log MW 3.9. The segregadores that presented this structure of altered amylopectin also showed an increase in the abundance of the amylose fraction of the chromatogram (log MW > 4.2) although this increase was greater in some segregadores than in others. The proportion of altered and normal amylopectin occurrence that seeds contain in the segregating ear was compared to the various possible hereditary modes using the square x-statistic (X2). The observed frequency of 70% of altered seeds: 30% of normal seeds was a reasonable fit with the simple dominance hypothesis (that 1 or more doses of the transgene were sufficient to produce altered starch structures) (X2 = 0.13) or the hypothesis that 2 or more doses of the transgene were required to alter the structure of starch (semidominance, X2 = 1.6).

Analysis of the fine structure amylopectin of corn SSb antisense segregates To extend the structural comparison of normal starch and SSb antisense, a starch from each of the two classes described above (normal vs. Altered) were compared by fluorophore labeling and electrophoresis. The starch was prepared from single corn kernels, debranched and resuspended in DMSO as described above. Four μl of diluted samples were pipetted into 0.2 ml PCR reaction tubes, and 2 μl of each fluorophore (8-amino-1,3,6-pyrenetrisulfonic acid, trisodium salt in 15% acetic acid) was added. and reducing agent (1 M sodium cyanoborohydride in water). The tubes were hermetically capped and centrifuged 2 minutes at 4000 rpm, followed by incubation at 37 ° for 16-18 hours. The standards were prepared with o.2 mg / ml maltoheptaose in water and labeled in the same manner as in the starch samples.

The gels were poured between sequential glass plates at 36 cm distance from the well for reading using 5% polyacrylamide (19: 1 acrylamide: bis) in 6M urea, IX TBE, 0.05% ammonium persulfate and 0.07% TEMED with 0.2 mm spacers. After polymerization by 3 to 4 horfíÉ ", a 36-well shark tooth collector was inserted and the wells were washed with regulator discharge (1 X TBE). The samples labeled with fluorophore were diluted 200 to 500 times in the Regulator loading (5 mM EDTA in 80% formamide with 5 mg / ml blue dextran as a visual well marker) and 1.5 μl were loaded in alternating wells Standard maltoheptaose was used to localize the DP peak, electrophoresis was performed in the Genetic Sequencer of Perkin-Elmer ABI 377 for 2 hours at 3000 volts at 51 ° and the results were analyzed using the ABI GeneScan program Figure 8 shows a graph that shows the results of ABVI as the relative% of each chain between DP7 and DP30.

The total moles with chains between DP7 and DP30 were calculated for XBG011717-2, an altered segregator, and XBG01717-7, a normal segregator, and the relative molar percent of these total chains was calculated. This distribution is shown in Figure 8 in which the altered segregator XBG01717-2 is shown to have a higher relative mole% of chains between DP7 and DP11 compared to the normal segregator XBG01717-7 which has a relative molar% of Higher strings between DP12 and DP26. The relative mole% of the altered segregator is twice that of the normal segregator for DP7 and DP8. These results demonstrate that the altered segregator is increased in very short AP chains (DP7 to DP10) and decreased in longer AP chains (DP14 to DP23). r ** eé & r LIST OF SEQUENCES < 110 > E. I. Du Pont de Nemours and Company < 120 > Modification of the genetic expression of the starch biosynthetic enzyme to produce starches in cereal crops < 130 > BB-1147-A < 140 > < 141 > < 150 > 060 / 094,436 < 151 > 1998-07-28 < 160 > twenty < 170 > Microsoft Office 97 < 210 > 1 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 1 aagcttgaat tccacagaat cagggtacag g 31 < 210 > 2 < 211 > 21 < 212 DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 2 gaaggactgg cactagactg g 21 < 210 > 3 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 3 ggatccgaat tctcctttct cagcaaacgg 30 < 210 > 4 < 211 > 33 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 4 aagcttgaat tcctgggatt gccacctgaa ttg 33 < 210 > 5 < 211 > 2491 < 212 > DNA < 213 > Zea mays < 400 > 5 agcgcgcccg aggcggcacc ccaccgtcgt agtagaagac acgggacgca cccccgcagc 60 ctcgc gcc cgctcccctc acttcctccc cgcgcgatcc acggcccccg ccccccgcgc 120 tcctgtctgc tctccctctc cgcaatggcg acgccctcgg ccgtgggcgc cgcgtgcct 180 ctcctcgcgc gggccgcctg gccggccgcc gtcggcgacc gggcgcgccc gcggcggct: 240 cagcgcgtgc tgcgccgccg gtgcgtcgcg gagctgagca gggaggggcc gcgccgcgc 300 ccgc gccac ccgcgctgct ggcgcccccg ctcgtgcccg gcttcctcgc gccgccggcc 360 gagcccacgg gtgagccggc atcgacgccg ccgcccgtgc 420 cc ccgacgccgg gggggac ctcggtctcg aacctgaagg gattgctgaa ggttccatcg ataacacagt gtggca agt 480 agtgagcaag attctgagat tgtggttgga aaggagcaag ctcgagctaa agtaacacaa 540 cc agcattgtct ttgtaaccgg cgaagcttct tatgcaa agtctggggg c aggagat 600 gtttgtggtt cattgccagt tgctcttgct gctcgtggtc accgtgtgat ggttgtaatg 660 cccagatatt taaatggtac ctccgataag atgcatttta aattatgcaa cacagaaaaa 720 cacattcgga tccatgctt tggcggtgaa catgaagtta ccttcttcca tgagtataga 780 GAIT agttg actgggtgtt tgttgatcat ccctcatatc acagacctgg aaatttatat 840 ggagataagt ttggtgcttt tggtgataat cagttcagat acacactcct ttgctatgct 900 gcatgtgagg ctcctttgat ccttgaattg ggaggatata tttatggaca gaattgcatg 960 atgattggca tttgttgtca tgccagtcta gtgccagtcc ttcttgctgc aaaatataga 1020 ccatatggtg tttataaaga ctcccgcagc attcttgtaa tacataattt agcacatcag 1080 ggtgtagagc ctgcaagcac atatcctgac cttgggttgc cacctgaatg gtatggagct 1140 ctggagtggg tattccctga atgggcgagg aggcatgccc ttgacaaggg tgaggcagtt 1200 aattttttga aaggtgcagt tgtgacagca gatcgaatcg tgactgtcag taagggttat 1260 tcgtgggagg tcacaactgc tgaaggtgga atgagctctt cagggcctca aagctccaga 1320 aagagtgtat taaacggaat tgtaaatgga attgacatta ccctgccaca atgattggaa 1380 tcccctgtca gacaaatgta ttattctgtt gatgacctct ctggaaaggc caaatgtaaa 1440 ggtgcattgc agaaggagct gggtttacct ataaggcctg atgttcctct gattggcttt 1500 attggaaggt tggattatca gaaaggcatt gatctcattc aacttatcat ac agatctc 1560 acgcgggaag atgttcaatt tgtcatgctt ggatctggtg acccagagct tgaagattgg 1620 cagagtcga atgagatcta cttcaaggat aaatttcgtg gatgggttgg atttagtgtt 1680 ccagtttccc accgaataac tgccggctgc gatatattgt taatgccatc cagattcgaa 1740 ccttgtggtc tcaatcagct atatgctatg cagtatggca tgtccatgca cagttcctgt 1800 actgggggcc ttagagatac cgtggagaac ttcaaccctt tcggtgagaa tggagagcag 1860 ggtacagggt gggcattcgc acccctaacc acagaaaaca tgttgtggac attgcgaact 1920 catacaggga gcaatatcta acacaagtcc tcctgggaag ggctaatgaa gcgaggcatg 1980 tcaaaagact tcacgtggga ccatgccgct gaacaatacg aacaaatctt ccagtgggcc 2040 ttcatcgatc gaccctatgt catgtaaaaa aaggaccaaa gtggtggttc cttgaagatc 2100 atcagttcat catcctatag taagctaaat gatgaaagaa aacccctgta cattacatgg 2160 aaggcagacc ggctattggc ccattgctc caacgtctgc tttggctggc ttgcctcgat 2220 gcagtgagga gcaccggcat atccagtcga acgacagttt tgaaggatag gaaggggagc 2280 cacgcaggca tggaagcagt gcctcgccgt gattcatatg gaacaagctg gagtcagttt 2340 ctgctatgcc actcactgtt taccttaaga ttat acctg tgttgttgtc ctttgctcgt 2400 aacataatga cagggctgat ctcattagaa aatcatgcct cgtttttatt aactgaa gtg 2460 gacacttcta cgccaaaaaa aaaaaaaaaa to 2491 < 210 > 6 < 211 > 1528 < 212 > DNA < 213 > Zea mays < 400 > 6 atcgatgaag gcccactgga agatttgttc gtattgttca gcggcatggt cccacgtgaa 60 gtcttttgac atgcctcgct tcattagccc ttcccaggag gacttgtgtt ccctgtatgt 120 agatattgca gttcgcaatg tccacaacat gttttctgtg gttaggggtg cgaatgccca 180 ccctgtaccc tgctctccat tctcaccgaa agggttgaag ttctccacgg tatctctaa? 24C gcccccagtt gcatggacaa caggaactgt gccatactgc atagcatata gctgattgag 30C accacaaggt tcgaatctgg arggcattaa caatatatcg cagccggcag ttattcggtg 36C ggaaactgga acactaaatc caacccatcc acgaaattta tccttgaaga tcgactctgt 420 agatctcatc caatcttcaa gctctgggtc accagatcca agcatgacaa attgaacatc 480 ttcccgcatg agatctggta tgataagttg aatgagatca atgcctttct gataatccaa 540 aagccaatca ccttccaata gaggaacatc aggccttata ggtaaaccca gctccttctg 600 ttacatttgg caatgcacct gaggtcatca cctttccaga acagaataat gacaggggat 660 acatttgtct gtggcagggt tccaatcatt aatgtcaatt ccatttacaa ttccgtttaa 720 tacactcttt ctggagctta agagctcatt gaggccctgt ccaccttcag cagttgtga; 780 ctcccacgaa taacccttac tgacagtcac gattcgatct gctgtcacaa ctgcaccttt 840 caaaaaatta actgcctcac ccttgtcaag ggcatgcctc ctcgcccatt cagggaatac 900 ccactccaga gctccatacc attcaggtgg caacccaagg tcaggatatg tgcttgcagg 960 ctctacaccc tgatgtgcta aattatgtat tacaagaatg ctgcgggagt ctttataaac 1020 accatatggt ctatattttg cagcaagaag gactggcact agactggcat gccaatcatt 1080 gacaacaaac atgcaattct gtccataaat atatcctccc aattcaagga tcaaaggagc 1140 gcatagcaaa ctcacatgca ggagtgtgta tctgaactga ttatcaccaa aagcaccaaa 1200 Cttatctcca tataaatttc caggtctgtg atatgaggga tgatcaacaa acacccagtc 1260 aactgaatct ctatactcat ggaagaaggt aacttcatgt tcaccgccaa agcatggaat 1320 ccgaatgtgt ttttctgtgt aaaatgcatt tgcataattc ttatcggagg taccatttaa 1380 attacaacca atatctgggc tcacacggtg accacgagca gcaagagcaa ctggcaatga 1440 accacaaaca tctcctagac c ccagactt tgcataagga gaagcttcgc cggttacaaa 1500 gacaatgctt tgtgttactt tagctcga 1528 < 210 > 7 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence; PCR INITIATOR < 400 > 7 aagcttgaat tcggcacatc gggccttatg g 31 < 210 > 8 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 8 gtctagtgcc agtccttc 1¡ < 210 > 9 < 211 > 17 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 9 GAGTCACACG CGATGGC 17 < 210 > 10 < 211 > 27 < 212 > DNA < 213 > Aritifical sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 10 ctctccgcca tggcgacgcc ctcggcc 27 < 210 > 11 < 211 > 1415 < 212 DNA < 213 > Zea mays < 400 > eleven l cS? Sl g gggccggaaC? Ccc9ggCgg? G I cSgcSgIcIccgIcIg g 9ctgggCcCttcCcCatgc c «gccggtcggccgtgggc g ccccggcccctggggctgc 12600 gtcgcggag c gagcaggg aggggcccgc gccgcgcc g ctgccaccc? c ctgctggc 180 gcccccgctc gtgcccggct tcctcgcgcc gccggccgag cccacgggtg to ccggcatc 240 gacgccgccg cccgtgcccg acgccggcct gggggacct ggtctcgaac ctgaaggga 300 tgctgaaggt tccatcgata acacagtagt tgtggcaagt gagcaagatt rtgagattgt 360 ggttggaaag gagcaagctc gagctaaagt aacacaaagc attgtc ttg aaccggcga 420 agcttctcct tatgcaaagt ctgggggtct aggagatgtt tgtggttcat t cagttgc 480 tcttgctgct cgtggtcacc gtgtgatggt tgtaatgccc agatatttaa atggtacctc 540 cgataagaat tatgcaaatg cattttacac agaaaaacae attcggat c catgc TTGG 600 cggtgaacat gaagttacct tcttccatga gtatagagat tcagttgac gggtg tgt 660 tcatatcaca tgatcatccc tttatatgga gacctggaaa gataagtttg g gcttttgg 720 ttcagataca tgataatcag cactcctttg ctatgctgca tgtgaggctc "ttgatcct 780 tgaattggga ggatatattt atggacagaa ttgcatgttt gttgtcaatg to tggcatgc 840 cagtctagtg ccagtccttc ttgctgcaaa atatagacca tatggtgttt ataaagactc 900 ccgcagcatt cttgtaatac ataatttagc acatcagggt gtagagcctg caagcacata 960 tcctgacctt gggttgccac ctgaatggta tggagctctg gagtgggtat tccctgaatg 1020 ggcgaggagg catgcccttg acaagggtga ggcagttaat tttttgaaag s gcagttgt 1080 gacagcagat ctgtcagtaa cgaatcgtga tgggaggtca gggttattcg caactgctga 1140 aggtggacag ggcctcaatg agctcttaag ctccagaaag agtgtattaa acggaattgt 1200 aaatggaatt gacattaatg attggaaccc tgccacagac aaatgtatcc cctgtcatta 1260 ttctgttgat gacctctctg gaaaggccaa atgtaaaggt gcattgcaga aggagctggg 1320 tttacctata aggcctgatg ttcctctgat tggctttatt ggaaggttgg to tatcagaa 1380 aggcattgat ctcattcaac ttatcatacc agatc 1415 < 210 > 12 < 211 > 2008 < 212 > DNA < 213 > Zea mays < 400 > 12 catggcgacg ccctcggccg tgggcgccgc gtgcctcctc ctcgcgcggg ccgcctggcc 60 ggccgccgtc ggcgaccggg cgcgcccgcg gcggctccag cgcgtgctgc gccgccggtg 120 cgtcgcggag ctgagcaggg agggscccgc gccgcgcccg ctgccacccg cgctgctggc 180 gcccccgctc gtgcccggct tcctcgcgcc gccggccgag cccacgggtg agccggcatc 240 gacgccgccg cccgtgcccg acgccggcct gggggacctc ggtctcgaac ctgaagggat 300 tgctgaaggt tccatcgata acacagtagt tgtggcaagt gagcaagatt ctgagattgt 3 < 60 ggttggaaag gagcaagctc gagctaaagt aacacaaagc attgtctttg taaccggcga 420 agcttctcct tatgcaaagt ctgggggtct aggagatgtt tgtggttcat tgccagttgc 480 tcttgctgct cgtggtcacc gtgtgatggt tgtaatgccc agatatttaa atggtacctc 540 cgataagaat tatgcaaatg cattttacac agaaaaacac attcggattc catgctttgg 600 cggtgaacat gaagttacct tcttccatga gtatagagat tcagttgact gggtgtttgt 660 tcatatcaca tgatcatccc gacctggaaa tttatatgga gataagtttg gtgcttttgg 720 ttcagataca tgataatcag ctatgctgca cactcctttg tgtgaggctc ctttgatcct 780 tgaattggga ggatatattt atggacagaa ttgcatgttt gttgtcaatg attggcatgc 840 cagtctagtg ccagtccttc ttgctgcaaa atatagacca tatggtgttt ataaagactc 900 ccgcagcatt cttgtaatac ataatttagc acatcagggt gtagagcctg caagcacata 960 , «T.». tcctgacctt gggttgccac ctgaatggta tggagctctg gagtgggtat tccctgaatg 1020 ggcgaggagg catgcccttg acaagggtga ggcagttaat tttttgaaag gtgcagttgt 1080 gacagcagat cgaatcgtga ctgtcagtaa tgggaggtca gggttattcg caactgctga 1140 aggtggacag ggcctcaatg agctcttaag ctccagaaag agtgtattaa acggaattgt 1200 aaatggaatt gacattaatg attggaaccc tgccacagac aaatgtatcc cctgtcatta 1260 ttctgttgat gacctctctg gaaaggccaa atgtaaaggt gcattgcaga aggagctggg 1320 tttacctata aggcctgatg ttcctctgat tggctttatt ggaaggttgg attatcagaa 1380 aggcattgat ctcattcaac ttatcatacc agatctcatg cgggaagatg ttcaatttgt 1440 catgcttgga tctggtgacc cagagcttga agattggatg agatctacag agtcgatctt caaggataaa 1500 tttcgtggat gggttggatt tagtgttcca gtttcccacc gaataactgc 1560 cggctgcgat atattgttaa tgccatccag attcgaacct atcagctata tgtggtctca 1620 tgctatgcag tatggcacag ttcctgttgt ccatgcaact gggggcctta gagataccgt 1680 ggagaacttc aaccctttcg gtgagaatgg agagcagggt acagggtggg cattcgcacc 1740 gaaaacatgt cctaaccaca tgtggacatt gcgaactgca atatctacat acagggaaca 1800 caagtc ctcc tgggaagggc taatgaagcg aggcatgtca aaagacttca cgtgggacca 1860 tgccgctgaa caatacgaac aaatcttcca gtgggccttc atcgatcgac cctatgtcat 1920 gtaaaaaaag gaccaaagtg gcggttcctt gaagatcatc agttcatcat cctatagtaa 1980 gctaaatgat gaaagaaaac ccctgtac 2008 < 210 > 13 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 13 aagcttgaat tcgcagtatg ctcgctctgt ge 32 < 210 > 14 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial Sequence: PCR INITIATOR < 400 > 14 ggatccgaat tcggttccac tcgctcatgt cg 32 < 210 > 15 < 211 > 2019 < 212 > DNA < 213 > Zea mays < 400 > fifteen gaattcggat ccttccctct ggggacatag cgccggagac tgtcctccca gccccgaagc 60 cactgcatga atcgcctgcg gttgacggag attcaaatgg aattgcacct cctacagttg 120 agccattagt acaggaggcc acttgggatt tcaagaaata catcggtttt gacgagcctg 180 acgaagcgaa ggatgattcc agggttggtg cagatgatgc tggttctttt gaacattatg 240 gggacaatga ttctgggcct ttggccgggg agaatgttat gaacgtgatc gtggtggctg 300 ctgaatgttc tccatggtgc aaaacaggtg tgttgtggga gtcttggaga gctttaccca 360 aggctttagc gagaagagga catcgtgtta tggttgtggt accaaggtat ggggactatg 420 tggaagcctt tgatatggga atccggaaat actacaaagc tgcaggacag gacctagaag 480 tgaactattt ccatgcattt attgatggag tcgactttgt gttcattgat gcccctcttt 540 tccggcaccg tcaagatgac atatatgggg gaagtaggca ggaaatcatg aagcgcatga 600 ttttgttttg caaggttgct gttgaggttc cttggcacgt tccatgcggt ggtgtgtgct 660 acggagatgg aaatttggtg atgattggca ttcattgcca cactgcactc ctgcctgttt 720 atattacaga atctgaaggc taatgcagta gaccatgggt gtcctcgtca cactcgctcc 780 tacataacat cgcccaccag ggccgtggtc ctgtagatga attcccgtac atggacttgc 840 ctgaacacta ccttcaacat ttcgagctgt acgatcccgt cggtggcgag cacgccaaca 900 tctttgccgc gggtctgaag atggcagacc gggtggtgac tgtcagccgc ggctacctgt 960 gacagtggaa gggagctgaa ggcggctggg gcctccacga catcatccgt tctaacgact 1020 ggaagatcaa tggcatcgtg aacggcatcg accaccagga gtggaacccc aaggtggacg 1080 tgcacctgcg gtcggacggc tacaccaact actccctcga gacactcgac gctggaaagc 1140 ggcagtgcaa ggcggccctg cagcgggagc tgggcctgga agtgcgcgac gacgtgccgc 1200 tgctcggctt catcgggcgt ctggatggac agaagggcgt ggacatcatc ggggacgcga 1260 tgccgtggat cgcggggcag gacgtgcagc tggtgatgct gggcaccggg cgcgccgacc 1320 tggaacgaat gctgcagcac ttggagcggg agcatcccaa caaggtgcgc gggtgggtcg 1380 gkttctcggt gcctatggcg catcgcatca cggcgggcgc cgacgt ct gtgatgccct 1440 cccgcttcga gccctgcggg ctgaaccagc tctacgcgat ggcatacggc accgtccctg 1500 tggtgcacgc cgtgggcggg ctcagggaca ccgtggcgcc gttcgacccg ttcagcgacg 1560 ccgggctcgg gtggactttt gaccgygccg aggccaacaa gctgatcgag gcgctcaggc 1620 actgcctcga cacgtaccgg aactacgagg agagctggaa gagtctccag gcgcgcggca 1680 tgtcgcagga cctcagctg g gaccacgcgg ctgagctcta cgaggacgtc cttgtcaagg 1740 ccaagtacca gtggtgaacc ctccgccctc cgcatcaata tcttcggttt gatcccattg 1800 tacatcgcgc gtttgacggt ctcggtgaag aacttcatat gcagtgacgc gccgctgggg 1860 tcggtagcag tactatggga ttgcattgag ctgtgtcact atgtgctttc gacaggacag 1920 tagtgaaggt tgtatgcaag tttatttttt tttcattact gatatttgga atgtcaacac 1980 aataaatgaa gctactatgt gtttcgtaaa aaactcgag 2019 < 210 > 16 < 211 > 1798 < 212 > DNA < 213 > Zea mays < 400 > 16 r gtacaatgg gatcaaaccg aagatattga tgcggagggc ggagggttca ccactggtac 60 ttggccttga caaggacgtc ctcgtagagc tcagccgcgt ggtcccagct gaggtcctgc 120 gacatgccgc gcgcctggag actcttccag ctctcctcgt agttccggta cgtgtcgagg 180 cagtgcctga gcgcctcgat cagcttgttg gcctcggcrc ggtcaaaagt ccacccgagc 240 ccggcgtcgc tgaacgggtc gaacggcgcc acggtgtccc tgagcccgcc cacggcgtgc 300 cggtgccgta accacaggga tgccatcgcg tagagctggt tcagcccgca gggctcgaag 360 cgggagggca tcaccagcac gtcggcgccc gccgtgatgc gatgcgccat aggcaccgag 420 aamccgaccc acccgcgcac cttgttggga tgctcccgct ccaagtgctg cagcattcgt 480 tccaggtcgg cgcgcccggt gcccagcatc accagctgca cgtcctgccc cgcgatccac 540 ggcatcgcgt ccccgatgat gtccacgccc ttctgtccat ccagacgccc gatgaagccg 600 agcagcggca cgtcgtcgcg cacttccagg cccagctccc gctgcagggc cgccttgcac 660 tgccgctttc cagcgtcgag tgtctcgagg gagtagttgg tgtagccgtc cgaccgcagg 720 tgcacgtcca ccttggggtt ccactcctgg tggtcgatgc cgttcacgat gccattgatc 780 ttccagtcgt tagaacggat gatgtcgtgg aggccccagc cgccttccac tgtcttcagc 840 tcccacaggt agccgcggc t gacagtcacc acccggtctg ccatcttcag acccgcggca 900 aagatgttgg cgtgctcgcc accgacggga tcgtacagct cgaaatgttg aaggtagtgt 960 tcaggcaagt ccatgtacgg gaattcatct acaggaccac ggccctggtg ggcgatgtta 1020 tgtatgacga ggacggagcg agtgtactgc attaacccat ggtctctgta atatgccttc 1080 agataaacag gcaggagtgc agtgtgccaa 'tcattggcaa tgaacaccaa atttccatct 1140 caccaccgca ccgtagcaca tggaacgtgc caaggaacct caacagcaac cttgcaaaac 1200 aaaatcatgc gcttcatgat ttcctgccta cttcccccat atatgtcatc ttgacggtgc 1260 cggaaaagag gggcatcaat gaacacaaag tcgactccat caataaatgc atggaaatag 1320 ttcacttcta ggtcctgtcc tgcagctttg tagtatttcc ggattcccat atcaaaggct 1380 tccacatagt ccccatacct tggtaccaca accataacac gatgtcctct tctcgctaaa 1440 aagctcccac gccttgggta aacatctcca agaccacctg ttttgcacca tggagaacat 1500 tcagcagcca ccacgatcac gttcataaca ttctccccgg ccaaaggccc agaatcattg 1560 gttcaaaaga tccccataat accagcatca tctgcaccaa ccctggaatc atccttcgct 1620 tcgtcaggct cgt aaaacc gatgtatttc ttgaaatccc aagtggcctc ctgtactaat 1680 ggctcaactg taggaggtgc aattc cattt gaatctccgt caaccgcagg cgattcatgc 1740 agtggcttcg gggctgggag gacagtctcc ggcgctatgt ccccagaggg aaggatcc 1798 < 210 > 17 < 211 > 20 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 17 ccatctccgt agcacacacc 20 < 210 > 18 < 211 > 20 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 18 gtgccaagga acctcaacag 20 < 210 > 19 < 211 > 20 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR INITIATOR < 400 > 19 gaggggcatc aatgaacaca 20 < 210 > 20 < 211 > 2248 < 212 > DNA < 213 > Zea mays < 400 > twenty tgactgtcag ccgcggctac ctgtgggagc tgaagacagt ggaaggcggc tggggcctcc 1440 acgacatcat ccgttctaac gactggaaga tcaatggcat cgtgaacggc atcgaccacc 1500? gagtggaa ccccaaggtg gacgtgcacc tgcggtcgga cggctacacc aactactccc 1560 tcgagacact cgacgctgga aagcggcagt gcaaggcggc cctgcagcgg gagctgggcc 1620 tggaagtgcg cgacgacgtg ccgctgctcg gcttcatcgg gcgtctggat ggacagaagg 1680 gcgtggacat catcggggac gcgatgccgt ggatcgcggg gcaggacgtg cagctggtga 1740 tgctgggcac cgggcgcgcc gacctggaac gaatgctgca gcacttggag cgggagcatc 1800 ccaacaaggt gcgcgggtgg gtcgggttct cggtgcctat ggcgcatcgc atcacggcgg 1860 gcgccgacgt gctggtgatg ccctcccgct tcgagccctg cgggctgaac cagctctacg 1920 cgatggcata cggcaccgtc cctgtggtgc acgccgtggg cgggctcagg gacaccgtgg 1980 cgccgttcga cccgttcagc gacgccgggc tcgggtggac ttttgaccgc gccgaggcca 2040 acaagctgat cgaggcgctc aggcactgcc tcgacacgta ccggaactac gaggagagct 2100 ggaagagtct ccaggcgcgc ggcatgtcgc aggacctcag ctgggaccac gcggctgagc 2160 tctacgagga cgtccttgtc aaggccaagt accagtggtg aaccctccgc cctccgcatc 2220 aatat cttcg gtttgatccc attgtaca 2248 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property in the following:

Claims (19)

1. A method of producing a transformed cereal crop that is characterized in that it comprises: (a) preparing a chimeric gene comprising a nucleic acid fragment encoding a structural gene of the non-GBSSI starch synthase enzyme or a fragment thereof, operably linked in either orientation in the sense or antisense in 5 'side of the nucleic acid fragment encoding a promoter that directs the expression of the gene in a cereal harvest tissue, and operably linked to the 3' side to a nucleic acid fragment encoding the appropriate regulatory sequence for termination transcriptional; Y (b) transforming a cereal crop with the chimeric gene of step (a), in which the expression of said chimeric gene results in the alteration of the fine structure of the starch derived from a grain of said transformed cereal crop when it is compared to the fine structure of the starch derived from a cereal crop that does not possess said chimeric gene.

2. The method of claim 1 which is characterized in that the cereal crop is a corn variety.

3. The method of claim 2 which is characterized in that the nucleic acid fragment encoding the structural gene of the starch synthase enzyme or a fragment thereof is derived from corn.

. The method of claim 3 which is characterized in that the nucleic acid fragment encoding the structural gene of the starch synthase enzyme or a fragment thereof encodes all or a portion of the enzyme starch synthase SSI.

5. The method of claim 4 which is characterized in that the nucleic acid fragment encoding the structural gene of the SSI starch synthase enzyme or a fragment thereof is substantially similar to an isolated nucleic acid fragment displayed on a member selected from the group consists of SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 11 and SEQ ID NO: 12.

6. The method of claim 1 which is characterized in that the alteration of the fine structure of 'GaPat.y * - starch comprises the alteration of the proportion of the amylose molecular component to the amylopectin molecular component of said starch.

7. The method of claim 1 which is characterized in that the alteration of the fine structure of starch comprises an alteration in the molecular weight distribution of the amylopectin component of said starch.

8. The method of claim 1 which is characterized in that the alteration of the fine structure of starch comprises an alteration in the molecular weight distribution of the amylose component of said starch.

9. The method of claim 1 which is characterized in that the nucleic acid fragment encoding the structural gene of the starch synthase enzyme or a fragment thereof is operably linked in antisense or sense orientation relative to a nucleic acid fragment that encodes a promoter that directs genetic expression in maize endosperm tissue on the 5 'side, and to a nucleic acid fragment encoding a regulatory sequence suitable for transcriptional termination on the 3' side.

10. A variety of corn that is characterized by being prepared according to the method of claim 1, or some progeny thereof.

11. The corn variety of claim 9 which is characterized in that the amylose molecular component of the starch isolated from the grain of said corn variety is significantly increased compared to the amylose molecular component of starch isolated from the untransformed corn kernel.

12. Starch isolated from the grain of a variety of corn that is characterized in that it is prepared by the method of claim 1 or some progeny thereof.

13. Prepared flour of the variety of the cereal crop characterized in that it is prepared by the method of claim 1 or some progeny thereof.

14. A method of preparing a thick comestible which is characterized in that it comprises combining an edible, water, and an effective amount of a starch derived from the flour of claim 13 and cooking the resulting composition as necessary to produce a thick comestible.

15. A corn variety transformed with a chimeric gene comprising a nucleic acid fragment encoding a structural gene of the maize SSI starch synthase enzyme or fragment thereof, operably linked in one orientation or another in the sense or antisense in the 5 'side of a nucleic acid fragment encoding a promoter that directs genetic expression in maize endosperm tissue, and operably linked on the 3' side to a nucleic acid fragment encoding a regulatory sequence suitable for transcriptional termination , or some progeny of it.

16. The method of claim 3 which is characterized in that the nucleic acid fragment encoding the structural gene of the starch synthase enzyme or a fragment thereof encodes all or a portion of a maize SSb starch synthase enzyme.

17. The method of claim 4 which is characterized in that the nucleic acid fragment encoding the structural gene of the SSb starch synthase enzyme or a fragment thereof is substantially similar to an isolated nucleic acid fragment displayed on a member selected from the group which consists of SEQ ID NO: 15 SEQ ID NO: 16 and SEQ ID NO: 20.

18. The method of claim 1 which is characterized in that said alteration of the fine structure of starch comprises the abatement of the relative moles of components amylopectin DP7 to DP10 of said starch.

19. A starch produced by the method of claim 1 which is characterized in that it has a tendency to decrease regressively.