Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
2001-09-17
2004-05-04
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S278000, C800S286000, C800S320300, C435S069100, C435S101000, C435S320100, C435S419000, C536S023600, C536S024500, C536S023200
Reexamination Certificate
active
06730825
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to plant starch compositions, and concerns novel nucleotide sequences; polypeptides encoded thereby; vectors and host cells and host organisms comprising one or more of the novel sequences; a method of altering one or more characteristics of a plant; a plant having altered characteristics; starch obtained from such plants; and uses of the starch.
BACKGROUND TO THE INVENTION
The majority of developments in cereal science in the recent past have concentrated primarily on the functionality of the gluten protein sub-units and their role in bakery systems. This has been greatly facilitated by the abundance of natural variation between cultivators for the gluten protein sub-unit components.
In contrast, although flour from commercially grown wheat varieties contains approximately 75-85% starch, the role of starch from a breeding perspective has been overlooked; this is largely due to the difficulty of measuring differences in starch structure. Of the limited amount of work that has been carried out however, there appears to be a lack of natural variation between different wheat cultivars. With the advent of recombinant DNA and gene transfer technologies it is now possible to create new variation in planta, therefore directly modifying starch composition in wheat becomes a realistic target.
Starch is the major form of carbon reserve in plants, constituting 50% or more of the dry weight of many storage organs, e.g. tubers, seeds of cereals. Starch is used in numerous food and industrial applications. In many cases, however, it is necessary to modify the native starches, via chemical or physical means, in order to produce distinct properties to suit particular applications. It would be highly desirable to be able to produce starches with the required properties directly in the plant, thereby removing the need for additional modification. To achieve this via genetic engineering requires knowledge of the metabolic pathway of starch biosynthesis. This includes characterisation of genes and encoded gene products which catalyse the synthesis of starch. Knowledge about the regulation of starch biosynthesis raises the possibility of “re-programming” biosynthetic pathways to create starches with novel properties that could have new commercial applications.
The most significant property of starch derives from the ability of the native granular form to lose its order and to swell and absorb water upon suitable treatment, thereby conferring viscosity and texture, in a process known as gelatinisation. Gelatinisation has been defined (W A Atwell et al, 1988) as “. . . the collapse (disruption) of molecular orders within the starch granule manifested in irreversible changes in properties such as granular swelling, native crystallite melting, loss of birefringence, and starch solubilisation. The point of initial gelatinisation and the range over which it occurs is governed by starch concentration, method of observation, granule type, and heterogeneities within the granule population under observation”.
14 molecules of water per molecule of anhydrous glucose, i.e. a minimum of 75 % water, are necessary for full starch gelatinisation (Donovan, 1979). Starch gelatinisation is usually caused by heat, but can be caused by physical damage and some chaotropic agents, mainly dimethylsulphoxide (DMSO), urea, calcium chloride, strong base and acid.
The various events taking place during gelatinisation can be followed by various methods, including birefringence, X-ray diffraction, differential scanning calorimetry (DSC),
13
C NMR. Swelling can be monitored by various methods, particularly rheology.
Differential scanning calorimetry (DSC) is a destructive method which records an endothermic event on heating of granules, generally thought to measure the temperature and the endothermic energy (delta H) required for the melting of the native crystallites. Starch gelatinisation temperature is independent of water content above 75% water (described as excess water), but increases when water is limited (Donovan, 1979).
The rate and extent of starch granule swelling upon heating dictate the type of viscosity development of aqueous starch suspensions on heating. Swelling behaviour is therefore of utmost technological importance. Viscosity increase on heating can be conveniently measured by a Brabender amylograph (Brabender is a Trade Mark) (Kennedy and Cabalda, 1991) or using a Rapid Visco analyser (Rapid Visco is a Trade Mark from Newport Scientific, Australia).
FIG. 1
is a typical viscoamylgraph profile for wheat starch, produced in this way, showing changes in starch during and after cooking. As starch granules swell on uptake of water, in a process known as pasting, their phase volume increases, causing an increase in viscosity. The onset of pasting is indicated at A in FIG.
1
. Peak viscosity, indicated at B in
FIG. 1
, is achieved when maximum phase volume is reached. Shear will then disrupt/cause fragmentation of the swollen granules, causing the viscosity to decrease. Complete dispersion is indicated at C in FIG.
1
. This has been confirmed by an oscillatory rheology study of starch pastes at various stages of the viscosity profile (Svegmark and Hermansson, 1990). The viscosity onset temperature and peak viscosity are indicative of the initiation and extent of swelling, respectively. On cooling, leached amylose forms a network in a process involving reassociation of molecules, or retrogradation, causing an increase in viscosity as indicated at D in FIG.
1
. Retrogradation (or set-back) viscosity is therefore indicative of the amount of amylose leached out of the granules.
The properties of wheat starch are useful in a large number of applications and also non-food (paper, textiles, adhesives etc.) applications. However, for many applications, properties are not optimum and various chemical and physical modifications well known in the art are undertaken in order to improve useful properties. Two types of property manipulation which would be of use are: the controlled alteration of gelatinisation and pasting temperatures; and starches which suffer less granular fragmentation during pasting than conventional starches.
Currently the only ways of manipulating the gelatinisation and pasting temperatures of starch are by the inclusion of additives such as sugars, polyhydroxy compounds of salts or by extensive physical or chemical pre-treatments. The reduction of granule fragmentation during pasting can be achieved either by extensive physical pre-treatments or by chemical cross-linking. Such processes are inconvenient and inefficient. It is therefore desirable to obtain plants which produce starch which intrinsically possesses such advantageous properties.
Starch consists of two main glucose polysaccharides: amylose and amylopectin. Amylose is a generally linear polymer comprising &agr;-1,4 linked glucose units, while amylopectin is a highly branched polymer consisting of an &agr;-1,4 linked glucan backbone with &agr;-1,6 linked glucan branches. In wheat endosperm amylopectin constitutes approximately 70% of the total starch content, with the balance being amylose. Amylopectin is synthesised through the concerted action of several enzymes, including soluble starch synthase(s) (SSS), starch branching enzyme(s) (SBE), starch de-branching enzyme(s) (DBE). The physical properties of starch are strongly affected by the relative abundance of amylose and amylopectin, therefore SSSs, SBEs and DBEs play a key role in determining both starch quantity and quality. As such, one approach to manipulating starch structure would be to modify the expression of the enzymes involved in starch biosynthesis in the endosperm using a transgenic approach.
SBE catalyses the formation of the &agr;-1,6 linkages, creating branch points in the growing starch molecule, via hydrolysis of an &agr;-1,4 linkage followed by reattachment of the released &agr;-1,4-glucan chain to the same or another glucosyl chain. This reaction also provides a new non-reducing end for further elongation of t
Colliver Steve
Goldsbrough Andrew
Fox David T.
Howrey Simon Arnold & White , LLP
Monsanto UK Ltd.
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