# Patent

## Title
Altering the fatty acid composition of rice

## Abstract

The present invention relates to rice oil, rice bran and rice seeds which have altered levels of oleic acid, palmitic acid and/or linoleic acid. The present invention also provides methods for genetically modifying rice plants such that rice oil, rice bran and rice seeds produced therefrom have altered levels of oleic acid, palmitic acid and/or linoleic acid. Specifically this is achieved through modulation of Fad2 and/or FatB expression.

## Background
This application is a §371 national stage of PCT International Application No. PCT/AU2007/000977, filed Jul. 13, 2007, and claims priority of Australian Patent Application No. 2006903810, filed Jul. 14, 2006, the contents of all of which are hereby incorporated by reference into this application.
FIELD OF THE INVENTION
The present invention relates to rice oil, rice bran and rice seeds which have altered levels of oleic acid, palmitic acid and/or linoleic acid. The present invention also provides methods for genetically modifying rice plants such that rice oil, rice bran and rice seeds produced therefrom have altered levels of oleic acid, palmitic acid and/or linoleic acid.
BACKGROUND OF THE INVENTION
Plant oils are an important source of dietary fat for humans, representing about 25% of caloric intake in developed countries (Broun et al., 1999). The current world production of plant oils is about 110 million tones per year, of which 86% is used for human consumption. Almost all of these oils are obtained from oilseed crops such as soybean, canola, sunflower, cottonseed and groundnut, or plantation trees such as palm, olive and coconut (Gunstone, 2001; Oil World Annual, 2004). The growing scientific understanding and community recognition of the impact of the individual fatty acid components of food oils on various aspects of human health is motivating the development of modified vegetable oils that have improved nutritional value while retaining the required functionality for various food applications. These modifications require knowledge about the metabolic pathways for plant fatty acid synthesis and genes encoding the enzymes for these pathways (Liu et al., 2002a; Thelen and Ohlrogge, 2002).
Considerable attention is being given to the nutritional impact of various fats and oils, in particular the influence of the constituents of fats and oils on cardiovascular disease, cancer and various inflammatory conditions. High levels of cholesterol and saturated fatty acids in the diet are thought to increase the risk of heart disease and this has led to nutritional advice to reduce the consumption of cholesterol-rich saturated animal fats in favour of cholesterol-free unsaturated plant oils (Liu et al., 2002a).
While dietary intake of cholesterol present in animal fats can significantly increase the levels of total cholesterol in the blood, it has also been found that the fatty acids that comprise the fats and oils can themselves have significant effects on blood serum cholesterol levels. Of particular interest is the effect of dietary fatty acids on the undesirable low density lipoprotein (LDL) and desirable high density lipoprotein (HDL) forms of cholesterol in the blood. In general, saturated fatty acids, particularly myristic acid (14:0) and palmitic acid (16:0), the principal saturates present in plant oils, have the undesirable property of raising serum LDL-cholesterol levels and consequently increasing the risk of cardiovascular disease (Zock et al., 1994; Hu et al., 1997). However, it has become well established that stearic acid (18:0), the other main saturate present in plant oils, does not raise LDL-cholesterol, and may actually lower total cholesterol (Bonanome and Grundy, 1988; Dougherty et al., 1995). Stearic acid is therefore generally considered to be at least neutral with respect to risk of cardiovascular disease (Tholstrup, et al., 1994). On the other hand, unsaturated fatty acids, such as the monounsaturate oleic acid (18:1) and the polyunsaturates linoleic acid (18:2) and α-linolenic acid (ALA, 18:3), have the beneficial property of lowering LDL-cholesterol (Mensink and Katan, 1989; Roche and Gibney, 2000), thus reducing the risk of cardiovascular disease.
Although nutritionally desirable, highly unsaturated oils are too unstable for use in many food applications, particularly for commercial deep-frying where they are exposed to high temperatures and oxidative conditions for long periods of time. Under such conditions, the oxidative breakdown of the numerous carbon double bonds present in unsaturated oils results in the development of short-chain aldehyde, hydroperoxide and keto derivatives, imparting undesirable flavours and reducing the frying performance of the oil by raising the level of polar compounds (Chang et al., 1978; Williams et al., 1999).
Oil processors and food manufacturers have traditionally relied on hydrogenation to reduce the level of unsaturated fatty acids in oils, thereby increasing their oxidative stability in frying applications and also providing solid fats for use in margarine and shortenings. Hydrogenation is a chemical process that reduces the degree of unsaturation of oils by converting carbon double bonds into carbon single bonds. Complete hydrogenation produces a fully saturated fat. However, the process of partial hydrogenation results in increased levels of both saturated fatty acids and monounsaturated fatty acids. Some of the monounsaturates formed during partial hydrogenation are in the trans isomer form (such as elaidic acid, a trans isomer of oleic acid) rather than the naturally occurring cis isomer (Sebedio et al., 1994; Fernandez San Juan, 1995). In contrast to cis-unsaturated fatty acids, trans-fatty acids are now known to be as potent as palmitic acid in raising serum LDL cholesterol levels (Mensink and Katan, 1990; Noakes and Clifton, 1998) and lowering serum HDL cholesterol (Zock and Katan, 1992), and thus contribute to increased risk of cardiovascular disease (Ascherio and Willett, 1997). As a result of increased awareness of the anti-nutritional effects of trans-fatty acids, there is now a growing trend away from the use of hydrogenated oils in the food industry, in favour of fats and oils that are both nutritionally beneficial and can provide the required functionality without hydrogenation, in particular those that are rich in either oleic acid where liquid oils are required or stearic acid where a solid or semi-solid fat is preferred.
Plant oils are composed almost entirely of triacylglycerols (TAG) molecules, which consist of three fatty acid (acyl) chains esterified to a glycerol backbone and are deposited in specialised oil body structures called oleosomes (Stymne and Stobart, 1987). These storage lipids serve as an energy source for the germinating seedling until it is able to photosynthesise. Edible plant oils in common use are generally comprised of five main fatty acids—the saturated palmitic and stearic acids, the monounsaturated oleic acid, and the polyunsaturated linoleic and α-linolenic acids. In addition to fatty acids, plant oils also contain some important minor components such as tocopherols, phytosterols, terpenes and mixed isoprenoids. These minor constituents are of increasing interest because some have been shown to exert beneficial effects on skin health, aging, eyesight and blood cholesterol or preventing breast cancer or cardiovascular disease (Theriault et al., 1999; Moghadasian and Frohlich, 1999).
Fatty Acid and TAG Synthesis in Seeds
A diagrammatic overview of the metabolic pathways for fatty acids synthesis in developing seeds is shown in FIG. 1. The initial stages of fatty acid synthesis occur in the plastid compartments of the cell, where synthesis of fatty acid carbon chains is initiated with a C2 molecule and extended through a stepwise condensation process whereby additional C2 carbon units are donated from malonyl-ACP to the elongating acyl chains. The first step in this sequence involves acetyl-CoA condensing with malonyl-ACP and is catalysed by the β-ketoacyl synthase III (KASIII) enzyme. The subsequent condensation rounds are catalysed by β-ketoacyl synthase I (KASI) and result in the eventual formation of a saturated C16 acyl chain joined to acyl carrier protein (ACP), palmitoyl-ACP. The final elongation within the plastid is catalysed by β-ketoacyl synthase II (KASII) to form the saturated C18 acyl chain, stearoyl-ACP. When desaturation occurs, the first double bond is introduced into the Δ9 position of the C18 chain by a soluble enzyme in the plastid, stearoyl-ACP Δ9-desaturase, to yield the monounsaturated C18:1 oleoyl-ACP.
Fatty acids thus synthesised are either retained in the plastid for further modification and incorporation into plastidic lipids, or are released from their ACPs by acyl-thioesterases to produce free fatty acids which are exported into the cytosol for further modification and eventual incorporation into TAG molecules. Higher plants have been found to have at least two types of acyl-thioesterase, FatA with substrate specificity towards oleoyl-ACP, an unsaturated acyl-ACP, and FatB with preference for saturated acyl-ACPs (Jones et al., 1995; Voelker et al., 1996).
On exiting the plastids, free fatty acids become esterified to Co-enzyme A (CoA) and are then available for transfer to glycerol 3-phosphate (G-3-P) backbones to form lysophosphatidic acid (LPA), phosphatidic acid (PA) and phosphatidyl-choline (PC). Additionally, in some plants, notably the Brassica species, oleic acid esterified to CoA is able to be elongated to form eicosenoic acid (C20:1) and erucic acid (C22:1). Oleic acid esterified to PC is available for further modification before incorporation into TAG. In edible oils, the principal modifications on PC are the sequential desaturations of oleic acid to form linoleic and α-linolenic acids by the microsomal Δ12-desaturase (Fad2) and Δ15-desaturase (Fad3) enzymes respectively.
Modification of Existing Fatty Acid Biosynthetic Enzymes
Gene inactivation approaches such as post transcriptional gene silencing (PTGS) have been successfully applied to inactivate fatty acid biosynthetic genes and develop nutritionally improved plant oils in oilseed crops. For example, soybean lines with 80% oleic acid in their seed oil were created by cosuppression of the Fad2 encoded microsomal Δ12-desaturase (Kinney, 1996). This reduced the level of Δ12-desaturation and resulted in accumulation of high amounts of oleic acid. Using a similar approach, cosuppression-based silencing of the Fad2 gene was used to raise oleic acid levels in Brassica napus and B. juncea (Stoutjesdijk et al., 2000). Likewise, transgenic expression in cottonseed of a mutant allele of the Fad2 gene obtained from rapeseed was found to be able to suppress the expression of the endogenous cotton Fad2 gene and resulted in elevated oleic acid content in about half of the primary transgenic cotton lines (Chapman et al., 2001). In another variation, transgenic expression in soybean of a Fad2 gene terminated by a self-cleaving ribozyme was able to inactivate the endogenous Fad2 gene resulting in increased oleic acid levels (Buhr et al., 2002). RNAi-mediated gene silencing techniques have also been employed to develop oilseeds with nutritionally-improved fatty acid composition. In cottonseed, transgenic expression of a hairpin RNA (hpRNA) gene silencing construct targeted against ghFad2-1, a seed-specific member of cotton Fad2 gene family, resulted in the increase of oleic acid from normal levels of 15% up to 77% of total fatty acids in the oil (Liu et al., 2002b). This increase was mainly at the expense of linoleic acid which was reduced from normal levels of 60% down to as low as 4%.
Fatty Acids in Cereals
In contrast to the considerable work done on fatty acid biosynthesis and modification in oilseeds, oil modification in cereals is relatively unexplored. This is probably due to the much lower levels of oils (about 1.5-6% by weight) in cereal grains and consequently the perceived lower importance of oils from cereals in the human diet.
Rice (Oryza sativa L.) is the most important cereal crop in the developing world and is grown widely, particularly in Asia which produces about 90% of the world total. The vast majority of rice in the world is eaten as “white rice” which is essentially the endosperm of the rice grain, having been produced by milling of harvested grain to remove the outer bran layer and germ (embryo and scutellum). This is done primarily because “brown rice” does not keep well on storage, particularly under hot tropical conditions.
The oil content of cereal grains such as rice (4%) is quite low relative to oilseeds where oil can make up to 60% of the weight of the grain (Ohlrogge and Jaworski, 1997). However, lipids may still comprise up to 37% of the dry weight of the cereal embryo (Choudhury and Juliano 1980). Most of the lipid content in the rice grain is found in the outer bran layer (Resurreccion et al, 1979) but some is also present in the endosperm, at least some associated with the starch (Tables 1 and 2). The main fatty acids in rice oil are palmitic (16:0) (about 20% of total fatty acids in the TAG), oleic (18:1) (about 40%), and linoleic acids (18:2) (about 34%) (Radcliffe et al., 1997). There is a range of levels naturally occurring in different rice cultivars, for example for oleic acid, from 37.9% to 47.5% and for linoleic (18:2), from 38.2% to 30.4% (Taira et al., 1988).







TABLE 1







Typical fatty acid composition (wt % of total fatty acids)


for selected fatty acids of various plant oils.












Plant
16:0
18:1
18:2
















Barley
18
22
54



Soybean
11
23
51



Peanut
8
50
36



Canola
4
63
20



Olive
15
75
9



Rice bran
22
38
34










While the FatB gene has been shown to have a high affinity to catalysing the production of free palmitic acid which is subsequently converted to palmitoyl-CoA in oilseed plants and dicot plants, no information is reported on the role of FatB in rice or other cereals.







TABLE 2







Fatty acid composition (wt % of total fatty acids) for selected


fatty acids of plant lipids associated with starch.












Plant
16:0
18:1
18:2







Wheat
35-44
6-14
42-52



Barley
55
4
36



Rye
23
41
35



Oat
40
22
35



Maize
37
11
46



Maize- High amylose
36
20
38



Maize- waxy
36
23
36



Millet
36
28
29



Rice
37-48
9-18
29-46







Data adapted from: Morrison (1988).






Some of the fatty acid desaturases have been characterized in rice but not Fad2. Akagai et al, (1995) published the nucleotide sequence of a gene on rice chromosome 4 encoding stearoyl-acyl carrier protein desaturase from developing seeds. The gene product participates in the production of oleoyl ACP from stearoyl ACP. Kodama et al. (1997) reported the structure, chromosomal location and expression of Fad3 in rice.
The proportion of linolenic acid (18:3) in rice seed oil has been increased ten-fold by using soybean Fad3 expression (Anai et al., 2003). More recently, there have been reports of the production of conjugated linoleic acid in rice by introduction of a linoleate isomerase gene from bacteria (Kohno-Murase et al., 2006); conjugated linoleic acid is reported to have anti-carcinogenic activity. In a similar vein, in vitro modification of rice bran oil to incorporate capric acid, which may improve dietary lipid utilisation in some diseases, using immobilized microbial enzymes has also been reported (Jennings and Akoh, 2000). In maize, Fad2 and FA-6 desaturase genes have been sequenced and mapped to chromosomes (Mikkilinen and Rocheford, 2003). The Fad2 and Fad6 clones could not be mapped to any QTLs for oleic/linoliec acid ratios in the maize grain. There are no published reports of other Fad2 or FatB genes characterized from rice, maize or wheat.
Storage of Rice
Storage of rice for prolonged times at high temperatures impairs grain quality due to hydrolytic and oxidative deterioration of bran oil. Dehulling the outer husk during harvest to produce brown rice disturbs the outer bran layers, which allows the oil to diffuse to the outer layers. Endogenous and microbial lipases then catalyse the hydrolysis of triglycerides to free fatty acids (FFA) which are then oxidised to produce an off-flavour (Yasumatsu et al., 1966, Tsuzuki et al., 2004, Zhou et al., 2002 Champagne and Grimm, 1995).
Hexanal is the major component increased in the headspace of raw and cooked brown rice stored at high temperatures (Boggs et al., 1964, Shibuya et al., 1974, Tsugita et al., 1983). However, hexanal itself is not the main cause of the ‘off’ smell; the unattractive smell and flavour of deteriorated rice is probably due to a mixture of the volatiles that are increased after storage. These include alkanals, alkenals, aromatic aldehydes, ketones, 2-pentylfuran, 4-vinylphenol and others (Tsugita et al., 1983). Nevertheless, hexanal levels during storage have been shown to be associated with the oxidation of linoleic acid (18:2) in brown rice and therefore are a good indicator of oxidative deterioration (Shin et al., 1986).
The production of hexanal from linoleic acid can be catalysed by the enzyme lipoxygenase (LOX) (St Angelo et al., 1980). Suzuki et al. (1999) identified rice varieties lacking Lox3 and found that on storage of the mutant grain at 35° C. for 8 weeks, less hexanal was formed in the headspace vapour, both for raw and cooked brown rice grain. They also found that mutant rice formed less pentanal and pentanol.
The nutrient-rich outer rice bran layer obtained through polishing the outer layers of the rice grain is an excellent food source, containing antioxidant compounds such as tocotrienols and gamma-oryzanol which is also a phytoestrogen (Rukmini and Raghuram, 1991). The bioactive compounds present in rice bran oil have been found to lower cholesterol in humans (Most et al., 2005). These bioactive components have also been shown to improve lipid profiles in rats fed a high cholesterol diet (Ha et al., 2005). Another important component found primarily in the bran is vitamin A precursors. However, these nutritional and health benefits are lost through the polishing of rice and the consumption of white rice.
There is still a need for cereal, such as rice, varieties that produce grain with an improved oil composition for health benefits, which at the same time is more stable on storage, allowing greater use of, for example, brown rice in the human diet.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides rice oil having a fatty acid composition comprising greater than about 48% oleic acid, less than about 17% palmitic acid, less than about 30% linoleic acid and/or any combination thereof.
In an embodiment, the ratio of oleic acid to linoleic acid is greater than 1.5:1, preferably greater than 2:1, more preferably greater than 3:1, and even more preferably greater than 4:1.
In another embodiment, the rice oil has a fatty acid composition comprising greater than about 48% oleic acid, less than about 17% palmitic acid, and less than about 30% linoleic acid.
In a further embodiment, the rice oil has a fatty acid composition comprising greater than about 40% oleic acid, preferably greater than about 50% oleic acid, and even more preferably greater than about 60% oleic acid. In an embodiment, the fatty acid composition of the rice oil is in the range 48-80% oleic acid, 6-16% palmitic acid and 10-25% linoleic acid.
In yet another embodiment, the rice oil has a fatty acid composition comprising less than about 16% palmitic acid, preferably less than about 15% palmitic acid, more preferably less than about 14% palmitic acid, more preferably less than about 13% palmitic acid, and even more preferably less than about 12% palmitic acid.
In another embodiment, the rice oil has a fatty acid composition comprising less than about 25% linoleic acid, preferably less than about 20% linoleic acid, even more preferably less than about 15% linoleic acid.
In another aspect the present invention provides rice bran having a fatty acid composition comprising greater than about 48% oleic acid, less than about 17% palmitic acid, less than about 30% linoleic acid and/or any combination thereof.
In an embodiment, the ratio of oleic acid to linoleic acid is greater than 1.5:1, preferably greater than 2:1, more preferably greater than 3:1, and even more preferably greater than 4:1.
In another embodiment, the rice bran has a fatty acid composition comprising greater than about 48% oleic acid, less than about 17% palmitic acid, and less than about 30% linoleic acid.
In a further embodiment, the rice bran has a fatty acid composition comprising greater than about 40% oleic acid, preferably greater than about 50% oleic acid, and even more preferably greater than about 60% oleic acid. In an embodiment, the fatty acid composition of the rice bran is in the range 48-80% oleic acid, 6-16% palmitic acid and 10-25% linoleic acid.
In yet another embodiment, the rice bran has a fatty acid composition comprising less than about 16% palmitic acid, preferably less than about 15% palmitic acid, more preferably less than about 14% palmitic acid, more preferably less than about 13% palmitic acid, and even more preferably less than about 12% palmitic acid.
In another embodiment, the rice bran has a fatty acid composition comprising less than about 25% linoleic acid, preferably less than about 20% linoleic acid, even more preferably less than about 15% linoleic acid.
In a further aspect, the present invention provides a rice seed having a fatty acid composition comprising greater than about 48% oleic acid, less than about 17% palmitic acid, less than about 30% linoleic acid and/or any combination thereof.
In an embodiment, the ratio of oleic acid to linoleic acid is greater than 1.5:1, preferably greater than 2:1, more preferably greater than 3:1, and even more preferably greater than 4:1.
In another embodiment, the rice seed has a fatty acid composition comprising greater than about 48% oleic acid, less than about 17% palmitic acid, and less than about 30% linoleic acid.
In a further embodiment, the rice seed has a fatty acid composition (i.e. of the oil inside the seed) comprising greater than about 40% oleic acid, preferably greater than about 50% oleic acid, and even more preferably greater than about 60% oleic acid. In an embodiment, the fatty acid composition of the rice seed is in the range 48-80% oleic acid, 6-16% palmitic acid and 10-25% linoleic acid.
In yet another embodiment, the rice seed has a fatty acid composition comprising less than about 16% palnitic acid, preferably less than about 15% palmitic acid, more preferably less than about 14% palmitic acid, more preferably less than about 13% palmitic acid, and even more preferably less than about 12% palmitic acid.
In another embodiment, the rice seed has a fatty acid composition comprising less than about 25% linoleic acid, preferably less than about 20% linoleic acid, even more preferably less than about 15% linoleic acid.
In any of the embodiments described above for rice bran or rice seed, the fatty acid composition is typically determined by extraction of the oil and analysis by FAME/GC as described in Example 1. Compositions for individual fatty acids are expressed as percent (w/w) of the total fatty acids in the oil.
In a further aspect, provided is a rice plant which produces rice oil of the invention, rice bran of the invention, and/or a rice seed of the invention.
In another aspect, the present invention provides an isolated polynucleotide which, when present in a cell of a rice plant, down-regulates the level of activity of a Fad2 and/or FatB polypeptide in the cell when compared to a cell that lacks said polynucleotide.
Preferably, the polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in a cell of a rice plant.
In an embodiment, the polynucleotide down-regulates mRNA levels expressed from at least one Fad2 and/or FatB gene.
Examples of suitable polynucleotides include, but are not limited to, a polynucleotide selected from: an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, a polynucleotide which encodes a polypeptide which binds a Fad2 or FatB polypeptide and a double stranded RNA.
In an embodiment, the polynucleotide is an antisense polynucleotide which hybridises under physiological conditions to a polynucleotide comprising any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25.
In a further embodiment, the polynucleotide is a catalytic polynucleotide capable of cleaving a polynucleotide comprising any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25.
In another embodiment, the polynucleotide is a double stranded RNA (dsRNA) molecule comprising an oligonucleotide which comprises at least 19 contiguous nucleotides of any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25, wherein the portion of the molecule that is double stranded is at least 19 basepairs in length and comprises said oligonucleotide.
Preferably, the dsRNA is expressed from a single promoter, wherein the strands of the double stranded portion are linked by a single stranded portion. Examples of the construction of vectors to produce such dsRNA molecules is provided in Example 5.
In a preferred embodiment, the polynucleotide, or a strand thereof, is capable of hybridising to a polynucleotide comprising any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25 under stringent conditions.
In a further aspect, the present invention provides a method of identifying a polynucleotide which, when present in a cell of a rice plant, down-regulates the level of activity of a Fad2 and/or FatB polypeptide in the cell when compared to a cell that lacks said polynucleotide, the method comprising
i) determining the ability of a candidate polynucleotide to down-regulate the level of activity of a Fad2 and/or FatB polypeptide in a cell, and
ii) selecting a polynucleotide which down-regulated the level of activity of a Fad2 and/or FatB polypeptide in the cell.
Step i) can rely on, for example, analysing the amount or enzymatic activity of a Fad2 and/or FatB polypeptide, or the amount of mRNA encoding a Fad2 and/or FatB polypeptide in the cell. Alternatively, step i) may comprise analysing the fatty acid content of the cell, or a seed or plant comprising said cell. Preferably, step i) comprises the introduction of the candidate polynucleotide or a chimeric DNA including a promoter operably linked to the candidate polynucleotide into a plant cell, more preferably into a rice cell, and even more preferably comprises the step of regenerating a transgenic plant from the plant cell and the production of seed from the transgenic plant. The candidate gene may be one of a collection of candidate genes, at least 2 or 3 in number. The invention therefore provides for the use of the polynucleotides of the invention in a screening method.
The polynucleotide can be, but not limited to, an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, a polynucleotide which encodes a polypeptide which binds a Fad2 or FatB polypeptide and a double stranded RNA.
In an embodiment, the antisense polynucleotide hybridises under physiological conditions to a polynucleotide comprising any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25.
In another embodiment, the catalytic polynucleotide is capable of cleaving a polynucleotide comprising any one or more of the sequence of nucleotides provided as SEQ D NOs 5 to 8, 11 to 14 or 19 to 25.
In a further embodiment, the double stranded RNA (dsRNA) molecule comprises an oligonucleotide which comprises at least 19 contiguous nucleotides of any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25, wherein the portion of the molecule that is double stranded is at least 19 basepairs in length and comprises said oligonucleotide.
Also provided is an isolated polynucleotide identified using a method of the invention.
In a further aspect the present invention provides a vector comprising or encoding a polynucleotide of the invention.
Preferably, the polynucleotide, or sequence encoding the polynucleotide, is operably linked to a promoter. Preferably, the promoter confers expression of the polynucleotide preferentially in the embryo, endosperm, bran layer and/or seed of a rice plant relative to at least one other tissue or organ of said plant.
Also provided is a cell comprising the vector of the invention, and/or the polynucleotide of the invention.
In an embodiment, the polynucleotide or vector was introduced into the cell or a progenitor of the cell.
In a further embodiment, the cell is a rice cell or an Agrobacterium cell.
Preferably, the polynucleotide is integrated into the genome of the cell.
In another aspect, the present invention provides a rice plant comprising a cell of the invention.
In yet another aspect, the present invention provides a method of producing the cell of the invention, the method comprising the step of introducing the polynucleotide of the invention, or a vector of the invention, into a cell.
Preferably the method further comprises the step of regenerating a transgenic plant from the cell.
Also provided is the use of the polynucleotide of the invention or a vector of the invention to produce a recombinant cell.
In yet another aspect, the present invention provides a genetically modified rice plant, wherein the plant has decreased expression of a polypeptide having Fad2 and/or FatB activity relative to a corresponding non-modified plant.
Preferably, the plant has been transformed such that it comprises a polynucleotide of the invention, or a progeny plant thereof which comprises said polynucleotide.
In a further aspect the present invention provides a method of producing rice oil of the invention, rice bran of the invention and/or rice seed of the invention, the method comprising exposing a rice plant to an antagonist of a Fad2 or FatB polypeptide.
In another aspect, the present invention provides a method of obtaining a genetically modified rice plant which can be used to produce brown rice seed with an increased storage life when compared to unmodified brown rice seed, the method comprising genetically manipulating the plant such that the activity and/or level of production of a Fad2 and/or FatB polypeptide is reduced in the rice seed when compared to a corresponding plant which produces unmodified brown rice seed.
Preferably, the activity and/or level of production of a Fad2 and/or FatB polypeptide is only reduced in the seed of the plant.
In an embodiment, the activity and/or level of production of a Fad2 polypeptide is reduced.
In a further embodiment, the transgenic plant comprises a polynucleotide of the invention or a vector of the invention.
In a further aspect, the present invention provides a genetically modified rice plant produced using a method of the invention, or progeny thereof.
In a further aspect, the present invention provides a method of selecting a rice plant which can be used to produce rice oil of the invention, rice bran according to the invention and/or rice seed of the invention, the method comprising;
i) screening a mutagenized population of rice seeds or rice plants, and
ii) selecting a seed or plant which is capable of producing rice oil of the invention, rice bran of the invention and/or rice seed of the invention.
In one embodiment, step i) comprises analysing a mutagenized seed and/or plant for a polypeptide which has Fad2 or FatB activity.
In another embodiment, step i) comprises analysing the sequence and/or expression levels of a Fad2 or FatB gene of the mutagenized seed and/or plant.
In a further embodiment, step i) comprises analysing the fatty acid composition of the oil, bran and/or seed of the mutagenized seed and/or plant.
In a further aspect, the present invention provides a method of selecting a rice plant which can be used to produce rice oil of the invention, rice bran of the invention and/or rice seed of the invention, the method comprising
i) analysing the fatty acid content of said rice oil, rice bran and/or seed obtained from a candidate rice plant, and
ii) selecting a rice plant which can be used to produce rice oil of the invention, rice bran of the invention and/or rice seed of the invention.
In yet a further aspect, the present invention provides a method of selecting a rice plant which can be used to produce rice oil of the invention, rice bran of the invention and/or rice seed of the invention, the method comprising
i) analysing a sample from a candidate plant for a polypeptide which has Fad2 or FatB activity, and
ii) selecting a rice plant which can be used to produce rice oil of the invention, rice bran of the invention and/or rice seed of the invention based on the sequence, level of production and/or activity of said polypeptide.
In another aspect, the present invention provides a method of selecting a rice plant which can be used to produce rice oil of the invention, rice bran of the invention and/or rice seed of the invention, the method comprising
i) analysing the sequence and/or expression levels of a Fad2 or FatB gene of a candidate plant, and
ii) selecting a rice plant which can be used to produce rice oil of the invention rice bran of the invention and/or rice seed of the invention based on the sequence and/or expression levels of said gene.
In yet another aspect the present invention provides a method of identifying a rice plant which can be used to produce rice oil of the invention, rice bran of the invention and/or rice seed of the invention, the method comprising detecting a nucleic acid molecule of the plant, wherein the nucleic acid molecule is linked to, and/or comprises at least a part of, a Fad2 gene and/or FatB gene in the plant
In a further aspect, the present invention provides a method of identifying a rice plant which can be used to produce brown rice seed with an increased storage life, the method comprising detecting a nucleic acid molecule of the plant, wherein the nucleic acid molecule is linked to, and/or comprises at least a part of, a Fad2 gene and/or FatB gene in the plant.
In an embodiment, the above two methods comprise:
i) hybridising a second nucleic acid molecule to said nucleic acid molecule which is obtained from said plant,
ii) optionally hybridising at least one other nucleic acid molecule to said nucleic acid molecule which is obtained from said plant; and
iii) detecting a product of said hybridising step(s) or the absence of a product from said hybridising step(s).
In an embodiment, the second nucleic acid molecule is used as a primer to reverse transcribe or replicate at least a portion of the nucleic acid molecule.
The nucleic acid can be detected using any technique such as, but not limited to, restriction fragment length polymorphism analysis, amplification fragment length polymorphism analysis, microsatellite amplification and/or nucleic acid sequencing.
In an embodiment, the method comprises nucleic acid amplification. In another embodiment, the method analyses expression levels of the gene.
Also provided is a method of obtaining a rice plant or seed, the method comprising;
i) crossing a first parental rice plant which comprises a Fad2 allele which confers an increased proportion of oleic acid in oil of the grain of the plant with a second parental rice plant which comprises a FatB allele which confers a decreased proportion of palmitic acid in oil of the grain of the plant;
ii) screening progeny plants or grain from the cross for the presence of both alleles; and
iv) selecting a progeny plant or grain comprising both alleles and having an increased proportion of oleic acid and a decreased proportion of palmitic acid in oil of the grain of the plant.
In another aspect, the present invention provides a method of introducing a Fad2 allele into a rice plant, the method comprising
i) crossing a first parental rice plant with a second parental rice plant, wherein the second plant comprises said allele, and
ii) backcrossing the progeny of the cross of step i) with plants of the same genotype as the first parent plant for a sufficient number of times to produce a plant with a majority of the genotype of the first parent but comprising said allele
iii) selecting a plant with the majority of the genotype of the first plant and comprising said allele;
wherein said allele confers an increased proportion of oleic acid in oil, bran and/or seed of the plant.
In yet another aspect, the present invention provides a method of introducing a FatB allele into a rice plant, the method comprising
i) crossing a first parental rice plant with a second parental rice plant, wherein the second plant comprises said allele, and
ii) backcrossing the progeny of the cross of step i) with plants of the same genotype as the first parent plant for a sufficient number of times to produce a plant with a majority of the genotype of the first parent but comprising said allele
iii) selecting a plant with the majority of the genotype of the first plant and comprising said allele;
wherein said allele confers a decreased proportion of palmitic acid in oil, bran and/or seed of the plant.
Further, provided is a method of increasing the proportion of oleic acid in oil, bran and/or seed of a rice plant, the method comprising genetically manipulating said plant such that the production of a Fad2 polypeptide is decreased when compared to a wild-type plant, wherein the polypeptide has Δ12 desaturase activity.
In yet another aspect, the present invention provides a method of decreasing the proportion of palmitic acid in oil, bran and/or seed of a rice plant, the method comprising genetically manipulating said plant such that the production of a FatB polypeptide is decreased when compared to a wild-type plant, wherein the polypeptide has (FatB) activity.
Also provided is a rice plant obtained using a method of the invention, or progeny plant thereof.
In a further aspect, the present invention provides rice oil obtained from a plant of the invention.
In a further aspect, the present invention provides rice bran obtained from a plant of the invention.
In a further aspect, the present invention provides rice seed obtained from a plant of the invention.
Further, provided is a method of producing seed, the method comprising;
a) growing a plant of the invention, and
b) harvesting the seed.
In yet another aspect, the present invention provides a food product comprising rice oil of the invention, rice bran of the invention and/or rice seed of the invention.
In another aspect, the present invention provides a method of preparing food, the method comprising cooking an edible substance in rice oil of the invention.
As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
FIG. 1—Principal fatty acid biosynthetic pathways in plastid and cytosol of higher plants.
FIG. 2—Alignment of rice FatB proteins. ProteinFATB2=SEQ ID NO:1, proteinFATB3=SEQ ID NO:2, proteinFATB1=SEQ ID NO:3 and proteinFATB4=SEQ ID NO:4.
FIG. 3—Rice FatB gene structures. LOC_Os02g4=SEQ ID NO:5, LOC_Os11g4=SEQ ID NO:6, LOC_Os06g0=SEQ ID NO:7 and LOC_Os06g3=SEQ ID NO:8.
FIG. 4—Alignment of FatB gene sequences by ClustalW. Default parameters were used and note that the ‘genes’ are of different lengths.
FIG. 5—The exon-intron structures of the FatB gene corresponding to LOC_Os6g05130. The top line corresponds to the start of the coding sequence in mRNA (SEQ ID NO:9) and the second line of the pair corresponds to the gene (SEQ ID NO:10).
FIG. 6—Alignment of coding sequence of four FatB isoforms showing consecutive exons in alternating lower case and upper case respectively and location of primers (underlined) used to distinguish the isoforms by RT-PCR. The initiating codon (start position 1) is in bold. AC108870=SEQ ID NO:11, AP005291=SEQ ID NO:12, AP000399=SEQ ID NO:13 and AP004236=SEQ ID NO:14.
FIG. 7—Clustal W alignment of deduced polypeptide sequence of Fad2 isoforms. Note that line F—1 corresponds to Os02g48560, F—2 corresponds to Os07g23410, F—3 to Os07g23430 and O—4 to Os07g23390. The program ClustalW (Fast) with default parameters was used. ProteinF—1=SEQ ID NO:15, ProteinF—3=SEQ ID NO:16, ProteinF—2=SEQ ID NO:17 and ProteinF—4=SEQ ID NO:18.
FIG. 8—Alignment of Fad2 sequences showing location of 5′ UTR in isoform AP004047 (lowercase) and location of primers used for amplification by RT-PCR (underlined). The location of the stop codon is indicated by a box and untranslated regions downstream of the stop codon are in lower case. AP005168=SEQ ID NO:19, AP004047=SEQ ID NO:20 and Contig2654=SEQ ID NO:21.
FIG. 9—Rice Fad2 gene structures.
FIG. 10—Alignment of the nucleotide sequences of the protein coding regions for rice Fad2 genes. Line 0—2 corresponds to Os07g23410, O—4 to Os07g23390, O—1 to Os02g48560 and 0—3 to Os07g23410. The program ClustalW with default parameters was used. CdsFAD2O—2=SEQ ID NO:22, CdsFAD2O—4=SEQ ID NO:23, CdsFAD2O—1=SEQ ID NO:24 and CdsFAD2O—3=SEQ ID NO:25.
FIG. 11—Graphical representation of the relative percentages of palmitic, oleic and linoleic acid as determined by GC analysis of total oil fraction from grains of indicated genotypes.
FIG. 12—Graphical representation of the relative percentages of palmitic and oleic acid in a GC total lipid analysis from grains of the genotypes indicated.
FIG. 13—Graphical representation of the relative percentages of linoleic and oleic acid in a GC total lipid analysis from grains of the genotypes indicated.
FIG. 14—Scatterplot showing the percentage of linoleic versus oleic acid in the grain of rice plants of the indicated genotypes. Note that the relationship (reflected in the slope of the line) between the amounts of these two fatty acids is essentially the same in all of the lines analysed but the pool size capacity appears to be different (as reflected in the displacements of the different lines along the space analysed.
FIG. 15—Scatterplot of the percentage of linoleic versus palmitic acid among different genotypes. Note the different slopes of the lines affected in FatB compared to those affected in Fad2. This suggests the relationship between these components is different in the different genotypes, probably reflecting the differences in the steps affected.
FIG. 16—Scatterplot of the percentage of oleic versus palmitic acid for various genotypes. Note the differences in slopes.
FIG. 17—Graphical representation of Principal Component Analysis of variation in oil composition for Fad2 RNAi plants and FatB RNAi plants. The results show that Principal Component 2 is Linoleic versus Oleic and Principal Component 2 is Palmitic versus Linoleic plus Oleic.
FIG. 18—Western blot showing the reaction of antisera raised against peptide. FatB-99 against total leaf protein extracts analysed by SDS-PAGE. A peptide of approx 20 kDa is missing in the Tos-17 line. The reaction with preimmune sera is indicated. R refers to FatB RNAi lines and T is the Tos-17 line. W1 and W2 are wildtype.



KEY TO SEQUENCE LISTING
SEQ ID NO:1—Rice FatB2 protein.
SEQ ID NO:2—Rice FatB3 protein.
SEQ ID NO:3—Rice FatB1 protein.
SEQ ID NO:4—Rice FatB4 protein.
SEQ ID NO:5—Rice FatB3 gene.
SEQ ID NO:6—Rice FatB2 gene.
SEQ ID NO:7—Rice FatB1 gene.
SEQ ID NO:8—Rice FatB4 gene.
SEQ ID NO:9—cDNA encoding rice FatB1 protein.
SEQ ID NO:10—Gene encoding rice FatB1 protein partial sequence only, see FIG. 5—includes all exon sequences and some flanking and intron sequence).
SEQ ID NO:11—Open reading frame encoding rice FatB2 protein.
SEQ ID NO:12—Open reading frame encoding rice FatB3 protein.
SEQ ID NO:13—Open reading frame encoding rice FatB1 protein.
SEQ ID NO:14—Open reading frame encoding rice FatB4 protein.
SEQ ID NO:15—Rice Fad2 isoform 1.
SEQ ID NO:16—Rice Fad2 isoform 3.
SEQ ID NO:17—Rice Fad2 isoform 2.
SEQ ID NO:18—Rice Fad2 isoform 4.
SEQ ID NO:19—Rice Fad2-3 cDNA.
SEQ ID NO:20—Rice Fad2-1 cDNA.
SEQ ID NO:21—Rice Fad2-2 cDNA.
SEQ ID NO:22—Open reading frame encoding rice Fad2-2.
SEQ ID NO:23—Open reading frame encoding rice Fad2-4.
SEQ ID NO:24—Open reading frame encoding rice Fad2-1.
SEQ ID NO:25—Open reading frame encoding rice Fad2-3.
SEQ ID NO:26—FatB consensus sequence.
SEQ ID NOs 27 to 33—Fad2 consensus sequences.
SEQ ID NOs 34 to 55 and 60 to 63—Oligonucleotide primers.
SEQ ID NOs 56 to 59—Antigenic rice FatB peptides.
SEQ ID NOs 64 to 83—Sequence of one strand of molecules that can be used for RNAi.
DETAILED DESCRIPTION OF THE INVENTION
General Techniques
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, plant molecular biology, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
SELECTED DEFINITIONS
As used herein, the term “Fad2 polypeptide” refers to a protein which performs a desaturase reaction converting oleic acid to linoleic acid. Thus, the term “Fad2 activity” refers to the conversion of oleic acid to linoleic acid. These fatty acids may be in an esterified form, such as, for example, as part of a phospholipid. Examples of rice Fad2 polypeptides include proteins comprising an amino acid sequence provided in FIG. 7 and SEQ ID NOs 15 to 18, as well as variants and/or mutants thereof. Such variants and/or mutants may be at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to any one of the polypeptides provided in FIG. 7 and SEQ ID NOs 15 to 18.
A “Fad2 polynucleotide” or “Fad2 gene” encodes a Fad2 polypeptide. Examples of Fad2 polynucleotides include nucleic acids comprising a nucleotide sequence provided in FIG. 8 or 10 and SEQ ID NOs 19 to 25, as well as allelic variants and/or mutants thereof. Examples of Fad2 genes include nucleic acids comprising a nucleotide sequence provided in FIG. 8 and SEQ ID NOs 19 to 21, as well as allelic variants and/or mutants thereof. Such allelic variants and/or mutants may be at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to any one of the polynucleotides provided in FIGS. 8 and/or 10, and/or SEQ ID NOs 19 to 25.
As used herein, the term “FatB polypeptide” refers to a protein which hydrolyses palmitoyl-ACP to produce free palmitic acid. Thus, the term “FatB activity” refers to the hydrolysis of palmitoyl-ACP to produce free palmitic acid. Examples of rice FatB polypeptides include proteins comprising an amino acid sequence provided FIG. 2 SEQ ID NOs 1 to 4, as well as variants and/or mutants thereof. Such variants and/or mutants may be at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to any one of the polypeptides provided in FIG. 2 and SEQ ID NOs 1 to 4.
A “FatB polynucleotide” or “FatB gene” encodes a FatB polypeptide. Examples of FatB polynucleotides include nucleic acids comprising a nucleotide sequence provided in FIGS. 4 and 6 and SEQ ID NOs 5 to 8 and 11 to 14, as well as allelic variants and/or mutants thereof. Examples of FatB genes include nucleic acids comprising a nucleotide sequence provided in FIG. 4 and SEQ ID NOs 5 to 8, as well as allelic variants and/or mutants thereof. Such allelic variants and/or mutants may be at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to any one of the polynucleotides provided in FIGS. 4 and/or 6, and/or SEQ ID NOs 5 to 8 and 11 to 14.
As used herein, the term “rice” refers to any species of the Genus Oryza, including progenitors thereof, as well as progeny thereof produced by crosses with other species. It is preferred that the plant is of a Oryza species which is commercially cultivated such as, for example, a strain or cultivar or variety of Oryza sativa or suitable for commercial production of grain.
As used herein, the term “rice oil” refers to a composition obtained from the seed/grain, or a portion thereof such as the bran layer, of a rice plant which comprises at least 60% (w/w) lipid. Rice oil is typically a liquid at room temperature. Preferably, the lipid comprises fatty acids that are at least 6 carbons in length. The fatty acids are typically in an esterified form, such as for example as triacylglycerols, phospholipid. Rice oil of the invention comprises oleic acid. Rice oil of the invention may also comprise at least some other fatty acids such as palmitic acid, linoleic acid, myristic acid, stearic acid and/or linolenic acid. The fatty acids may be free fatty acids and/or be found as triacylglycerols (TAGs). In an embodiment, at least 50%, more preferably at least 70%, more preferably at least 80% of the fatty acids in rice oil of the invention be found as TAGs. Rice oil of the invention can form part of the rice grain/seed or portion thereof such as the aleurone layer or embryo/scutellnum, which together are referred to as “rice bran”. Alternatively, rice oil of the invention has been extracted from rice grain/seed or rice bran. An example of such an extraction procedure is provided in Example 1. Thus, in an embodiment, “rice oil” of the invention is “substantially purified” or “purified” rice oil that has been separated from one or more other lipids, nucleic acids, polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified rice oil is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. In a preferred embodiment, upon extraction the ratio of oleic acid to linoleic acid, palmitic acid to oleic acid and/or palmitic acid to linoleic acid has not been significantly altered (for example, no greater than a 5% alteration) when compared to the ratio in the intact seed/grain or bran. In a further embodiment, the rice oil has not been exposed to a procedure, such as hydrogenation, which may alter the ratio of oleic acid to linoleic acid, palmitic acid to oleic acid and/or palmitic acid to linoleic acid when compared to the ratio in the intact seed/grain or bran. Rice oil of the invention may further comprise non-fatty acid molecules such as, but not limited to, γ-oryzanols and sterols.
Rice oil may be extracted from rice seed or bran by any method known in the art. This typically involves extraction with nonpolar solvents such as diethyl ether, petroleum ether, chloroform/methanol or butanol mixtures. Lipids associated with the starch in the grain may be extracted with water-saturated butanol. The rice oil may be “de-gummed” by methods known in the art to remove polysaccharides or treated in other ways to remove contaminants or improve purity, stability or colour. The triacylglycerols and other esters in the oil may be hydrolysed to release free fatty acids, or the oil hydrogenated or treated chemically or enzymatically as known in the art.
Rice oil after extraction from rice seed or bran typically comprises the group of lipids called γ-oryzanols. As used herein, “comprises γ-oryzanol” refers to the presence of at least 0.1% (w/w) γ-oryzanol compounds in the oil. The levels of γ-oryzanol in rice oil after extraction and before removal from the TAG is typically 1.5-3.5% (w/w). The compounds are typically a mixture of steryl and other triterpenyl esters of ferulic acid (4-hydroxy-3-methoxy cinnamic acid). Cycloartenyl ferulate, 24-methylene cycloartanyl ferulate and campesteryl ferulate are the predominant ferulates in oryzanol, with lower levels of β-sitosteryl ferulate and stigmasteryl ferulate. The presence of γ-oryzanols is thought to help protect consumers of rice oil against chronic diseases such as heart disease and cancer and therefore the presence of γ-oryzanol is advantageous.
As used herein, the term “rice bran” refers to the layer (aleurone layer) between the inner white rice grain and the outer hull of a rice seed/grain as well as the embryo/scutellum of the grain. The rice bran is the primary by product of the polishing of brown rice to produce white rice.
As used herein, the term “increased storage life” refers to a method of the invention producing a seed/grain, which upon harvesting, can be stored as brown rice for an enhanced period of time when compared to, for example, brown rice harvested from a wild type (non-genetically modified) rice plant As described herein, one measure for “increased storage life” of brown rice is hexanal production following storage for at least 8 weeks at 40° C. (see Example 8).
The term “plant” includes whole plants, vegetative structures (for example, leaves, stems), roots, floral organs/structures, seed (including embryo, endosperm, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), cells and progeny of the same.
A “transgenic plant”, “genetically modified plant” or variations thereof refers to a plant that contains a gene construct (“transgene”) not found in a wild-type plant of the same species, variety or cultivar. A “transgene” as referred to herein has the normal meaning in the art of biotechnology and includes a genetic sequence which has been produced or altered by recombinant DNA or RNA technology and which has been introduced into the plant cell. The transgene may include genetic sequences derived from a plant cell. Typically, the transgene has been introduced into the plant by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes.
The terms “seed” and “grain” are used interchangeably herein. “Grain” generally refers to mature, harvested grain but can also refer to grain after imbibition or germination, according to the context. Mature grain commonly has a moisture content of less than about 18-20%.
As used herein, the term “corresponding non-modified plant” refers to a wild-type plant. “Wild type”, as used herein, refers to a cell, tissue or plant that has not been modified according to the invention. Wild-type cells, tissue or plants may be used as controls to compare levels of expression of an exogenous nucleic acid or the extent and nature of trait modification with cells, tissue or plants modified as described herein. Wild-type rice varieties that are suitable as a reference standard include Nipponbare.
“Nucleic acid molecule” refers to a oligonucleotide, polynucleotide or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity defined herein. The terms “nucleic acid molecule” and “polynucleotide” are used herein interchangeably.
The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
“Oligonucleotides” can be RNA, DNA, or derivatives of either. Although the terms polynucleotide and oligonucleotide have overlapping meaning, oligonucleotide are typically relatively short single stranded molecules. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length.
As used herein, the term “nucleic acid amplification” refers to any in vitro method for increasing the number of copies of a nucleic acid molecule with the use of a DNA polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a DNA molecule or primer thereby forming a new DNA molecule complementary to a DNA template. The newly formed DNA molecule can be used a template to synthesize additional DNA molecules.
“Operably linked” as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
As used herein, the term “gene” is to be taken in its broadest context and includes the deoxyribonucleotide sequences comprising the protein coding region of a structural gene and including sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of at least about 2 kb on either end and which are involved in expression of the gene. The sequences which are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region which may be interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term “gene” includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.
As used herein, the term “genetically linked” or similar refers to a marker locus and a second locus being sufficiently close on a chromosome that they will be inherited together in more than 50% of meioses, e.g., not randomly. This definition includes the situation where the marker locus and second locus form part of the same gene. Furthermore, this definition includes the embodiment where the marker locus comprises a polymorphism that is responsible for the trait of interest (in other words the marker locus is directly “linked” or “perfectly linked” to the phenotype). In another embodiment, the marker locus and a second locus are different, yet sufficiently close on a chromosome that they will be inherited together in more than 50% of meioses. The percent of recombination observed between genetically linked loci per generation (centimorgans (cM)), will be less than 50. In particular embodiments of the invention, genetically linked loci may be 45, 35, 25, 15, 10, 5, 4, 3, 2, or 1 or less cM apart on a chromosome. Preferably, the markers are less than 5 cM apart and most preferably about 0 cM apart.
An “allele” refers to one specific form of a genetic sequence (such as a gene) within a cell, an individual plant or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene. The sequences at these variant sites that differ between different alleles are termed “variances”, “polymorphisms”, or “mutations”.
A “polymorphism” as used herein denotes a variation in the nucleotide sequence between alleles of the loci of the invention, of different species, cultivars, strains or individuals of a plant. A “polymorphic position” is a preselected nucleotide position within the sequence of the gene. In some cases, genetic polymorphisms are reflected by an amino acid sequence variation, and thus a polymorphic position can result in location of a polymorphism in the amino acid sequence at a predetermined position in the sequence of a polypeptide. In other instances, the polymorphic region may be in a non-polypeptide encoding region of the gene, for example in the promoter region such may influence expression levels of the gene. Typical polymorphisms are deletions, insertions or substitutions. These can involve a single nucleotide (single nucleotide polymorphism or SNP) or two or more nucleotides.
The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms “proteins” and “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the invention as described herein.
The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
Antisense Polynucleotides
The term “antisense polynucleotide” shall be taken to mean a DNA or RNA, or combination thereof, molecule that is complementary to at least a portion of a specific mRNA molecule encoding a FatB or Fad2 polypeptide and capable of interfering with a post-transcriptional event such as mRNA translation. The use of antisense methods is well known in the art (see for example, G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)). The use of antisense techniques in plants has been reviewed by Bourque, 1995 and Senior, 1998. Bourque, 1995 lists a large number of examples of how antisense sequences have been utilized in plant systems as a method of gene inactivation. She also states that attaining 100% inhibition of any enzyme activity may not be necessary as partial inhibition will more than likely result in measurable change in the system. Senior (1998) states that antisense methods are now a very well established technique for manipulating gene expression.
An antisense polynucleotide of the invention will hybridize to a target polynucleotide under physiological conditions. As used herein, the term “an antisense polynucleotide which hybridises under physiological conditions” means that the polynucleotide (which is fully or partially single stranded) is at least capable of forming a double stranded polynucleotide with mRNA encoding a protein, such as those provided in FIG. 2 or 7 or SEQ ID NOs 1 to 4 or 15 to 18 under normal conditions in a cell, preferably a rice cell.
Antisense molecules may include sequences that correspond to the structural genes or for sequences that effect control over the gene expression or splicing event. For example, the antisense sequence may correspond to the targeted coding region of the genes of the invention, or the 5′-untranslated region (UTR) or the 3′-UTR or combination of these. It may be complementary in part to intron sequences, which may be spliced out during or after transcription, preferably only to exon sequences of the target gene. In view of the generally greater divergence of the UTRs, targeting these regions provides greater specificity of gene inhibition.
The length of the antisense sequence should be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides. The full-length sequence complementary to the entire gene transcript may be used. The length is most preferably 100-2000 nucleotides. The degree of identity of the antisense sequence to the targeted transcript should be at least 90% and more preferably 95-100%. The antisense RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule.
Catalytic Polynucleotides
The term catalytic polynucleotide/nucleic acid refers to a DNA molecule or DNA-containing molecule (also known in the art as a “deoxyribozyme”) or an RNA or RNA-containing molecule (also known as a “ribozyme”) which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate. The nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T (and U for RNA).
Typically, the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity (also referred to herein as the “catalytic domain”). The types of ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach, 1988; Perriman et al., 1992) and the hairpin ribozyme (Shippy et al., 1999).
The ribozymes of this invention and DNA encoding the ribozymes can be chemically synthesized using methods well known in the art. The ribozymes can also be prepared from a DNA molecule (that upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase. Accordingly, also provided by this invention is a nucleic acid molecule, i.e., DNA or cDNA, coding for a catalytic polynucleotide of the invention. When the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, the ribozyme can be produced in vitro upon incubation with RNA polymerase and nucleotides. In a separate embodiment, the DNA can be inserted into an expression cassette or transcription cassette. After synthesis, the RNA molecule can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase.
As with antisense polynucleotides described herein, catalytic polynucleotides of the invention should also be capable of hybridizing a target nucleic acid molecule (for example an mRNA encoding any polypeptide provided in FIG. 2 or 7 or SEQ ID NOs 1 to 4 or 15 to 18) under “physiological conditions”, namely those conditions within a cell (especially conditions in a plant cell such as a rice cell).
RNA Interference
RNA interference (RNAi) is particularly useful for specifically inhibiting the production of a particular protein. Although not wishing to be limited by theory, Waterhouse et al. (1998) have provided a model for the mechanism by which dsRNA (duplex RNA) can be used to reduce protein production. This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest or part thereof, in this case an mRNA encoding a polypeptide according to the invention. Conveniently, the dsRNA can be produced from a single promoter in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules for the present invention is well within the capacity of a person skilled in the art, particularly considering Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.
In one example, a DNA is introduced that directs the synthesis of an at least partly double stranded RNA product(s) with homology to the target gene to be inactivated. The DNA therefore comprises both sense and antisense sequences that, when transcribed into RNA, can hybridize to form the double-stranded RNA region. In a preferred embodiment, the sense and antisense sequences are separated by a spacer region that comprises an intron which, when transcribed into RNA, is spliced out. This arrangement has been shown to result in a higher efficiency of gene silencing. The double-stranded region may comprise one or two RNA molecules, transcribed from either one DNA region or two. The presence of the double stranded molecule is thought to trigger a response from an endogenous plant system that destroys both the double stranded RNA and also the homologous RNA transcript from the target plant gene, efficiently reducing or eliminating the activity of the target gene.
The length of the sense and antisense sequences that hybridise should each be at least 19 contiguous nucleotides, preferably at least 30 or 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides. The full-length sequence corresponding to the entire gene transcript may be used. The lengths are most preferably 100-2000 nucleotides. The degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, preferably at least 90% and more preferably 95-100%. The RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule. The RNA molecule may be expressed under the control of a RNA polymerase II or RNA polymerase III promoter. Examples of the latter include tRNA or snRNA promoters.
Preferred small interfering RNA (‘siRNA’) molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA. Preferably, the target mRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the plant (preferably rice) in which it is to be introduced, e.g., as determined by standard BLAST search.
Examples of dsRNA molecules of the invention are provided in Example 5. Further examples include those which comprise a sequence as provided in one or more of SEQ ID NOs 64 to 73 (for Fad2) and SEQ ID NOs 74 to 83 (for FatB).
MicroRNA
MicroRNA regulation is a clearly specialized branch of the RNA silencing pathway that evolved towards gene regulation, diverging from conventional RNAi/PTGS. MicroRNAs are a specific class of small RNAs that are encoded in gene-like elements organized in a characteristic inverted repeat. When transcribed, microRNA genes give rise to stem-looped precursor RNAs from which the microRNAs are subsequently processed. MicroRNAs are typically about 21 nucleotides in length. The released miRNAs are incorporated into RISC-like complexes containing a particular subset of Argonaute proteins that exert sequence-specific gene repression (see, for example, Millar and Waterhouse, 2005; Pasquinelli et al., 2005; Almeida and Allshire, 2005).
Cosuppression
Another molecular biological approach that may be used is co-suppression. The mechanism of co-suppression is not well understood but is thought to involve post-transcriptional gene silencing (PTGS) and in that regard may be very similar to many examples of antisense suppression. It involves introducing an extra copy of a gene or a fragment thereof into a plant in the sense orientation with respect to a promoter for its expression. The size of the sense fragment, its correspondence to target gene regions, and its degree of sequence identity to the target gene are as for the antisense sequences described above. In some instances the additional copy of the gene sequence interferes with the expression of the target plant gene. Reference is made to WO 97/20936 and EP 0465572 for methods of implementing co-suppression approaches.
Nucleic Acid Hybridization
In an embodiment, polynucleotides of the invention, or a strand thereof, hybridize under physiological conditions to a polynucleotide comprising any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25. In a further embodiment, polynucleotides of the invention, or a strand thereof, also hybridize to a polynucleotide comprising any one or more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25 under stringent conditions.
As used herein, the phrase “stringent conditions” refers to conditions under which a polynucleotide, probe, primer and/or oligonucleotide will hybridize to its target sequence(s), but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel et al. (supra), Current Protocols In Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, as well as the Examples described herein. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C., followed by one or more washes in 0.2.×SSC, 0.01% BSA at 50° C. In another embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well-known within the art, see, e.g., Ausubel et al. (supra), and Kriegler, 1990; Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY. In yet another embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art, see, e.g., Ausubel et al. (supra) and Kriegler, 1990, Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY, as well as the Examples provided herein.
Nucleic Acid Constructs, Vectors and Host Cells
The present invention includes the production of genetically modified rice plants, wherein the plant has decreased expression of a polypeptide having Fad2 and/or FatB activity relative to a corresponding non-modified plant.
Nucleic acid constructs useful for producing the above-mentioned transgenic plants can readily be produced using standard techniques.
When inserting a region encoding an mRNA the construct may comprise intron sequences. These intron sequences may aid expression of the transgene in the plant. The term “intron” is used in its normal sense as meaning a genetic segment that is transcribed but does not encode protein and which is spliced out of an RNA before translation. Introns may be incorporated in a 5′-UTR or a coding region if the transgene encodes a translated product, or anywhere in the transcribed region if it does not. However, in a preferred embodiment, any polypeptide encoding region is provided as a single open reading frame. As the skilled addressee would be aware, such open reading frames can be obtained by reverse transcribing mRNA encoding the polypeptide.
To ensure appropriate expression of the gene encoding an mRNA of interest, the nucleic acid construct typically comprises one or more regulatory elements such as promoters, enhancers, as well as transcription termination or polyadenylation sequences. Such elements are well known in the art.
The transcriptional initiation region comprising the regulatory element(s) may provide for regulated or constitutive expression in the plant Preferably, expression at least occurs in cells of the embryo, endosperm, bran layer developing seed and/or mature seed (grain). In an alternate embodiment, the regulatory elements may be promoters not specific for seed cells (such as ubiquitin promoter or CaMV35S or enhanced 35S promoters).
Examples of seed specific promoters useful for the present invention include, but are not limited to, the wheat low molecular weight glutenin promoter (Colot et al., 1987), the promoter expressing α-amylase in wheat seeds (Stefanov et al., 1991), and the hordein promoter (Brandt et al., 1985).
A number of constitutive promoters that are active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliform virus promoter, the commelina yellow mottle virus promoter, the light-inducible promoter from the small subunit of the ribulose-1,5-bis-phosphate carboxylase, the rice cytosolic triosephosphate isomerase promoter, the adenine phosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 gene promoter, the mannopine synthase and octopine synthase promoters, the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll α/β binding protein gene promoter. These promoters have been used to create DNA vectors that have been expressed in plants; see, e.g., PCT publication WO 8402913. All of these promoters have been used to create various types of plant-expressible recombinant DNA vectors.
The promoter may be modulated by factors such as temperature, light or stress. Ordinarily, the regulatory elements will be provided 5′ of the genetic sequence to be expressed. The construct may also contain other elements that enhance transcription such as the nos 3′ or the ocs 3′ polyadenylation regions or transcription terminators.
The 5′ non-translated leader sequence can be derived from the promoter selected to express the heterologous gene sequence of the polynucleotide of the present invention, and can be specifically modified if desired so as to increase translation of mRNA. For a review of optimizing expression of transgenes, see Koziel et al. (1996). The 5′ non-translated regions can also be obtained from plant viral RNAs (Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic virus, among others) from suitable eukaryotic genes, plant genes (wheat and maize chlorophyll a/b binding protein gene leader), or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5′ non-translated sequence that accompanies the promoter sequence. The leader sequence could also be derived from an unrelated promoter or coding sequence. Leader sequences useful in context of the present invention comprise the maize Hsp70 leader U.S. Pat. No. 5,362,865 and U.S. Pat. No. 5,859,347), and the TMV omega element.
The termination of transcription is accomplished by a 3′ non-translated DNA sequence operably linked in the chimeric vector to the polynucleotide of interest. The 3′ non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3′ end of the RNA. The 3′ non-translated region can be obtained from various genes that are expressed in plant cells. The nopaline synthase 3′ intranslated region, the 3′ untranslated region from pea small subunit Rubisco gene, the 3′ untranslated region from soybean 7S seed storage protein gene are commonly used in this capacity. The 3′ transcribed, non-translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.
Typically, the nucleic acid construct comprises a selectable marker. Selectable markers aid in the identification and screening of plants or cells that have been transformed with the exogenous nucleic acid molecule. The selectable marker gene may provide antibiotic or herbicide resistance to the rice cells, or allow the utilization of substrates such as mannose. The selectable marker preferably confers hygromycin resistance to the rice cells.
Preferably, the nucleic acid construct is stably incorporated into the genome of the plant. Accordingly, the nucleic acid comprises appropriate elements which allow the molecule to be incorporated into the genome, or the construct is placed in an appropriate vector which can be incorporated into a chromosome of a plant cell.
One embodiment of the present invention includes a recombinant vector, which includes at least one polynucleotide molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al. Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred host cells are plant cells, more preferably cells of a cereal plant, and even more preferably a rice cell.
Transgenic Plants
Transgenic rice (also referred to herein as genetically modified rice) can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology—The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).
In a preferred embodiment, the transgenic plants are homozygous for each and every polynucleotide that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype. The transgenic plants may also be heterozygous for the introduced transgene(s), such as, for example, in F1 progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.
Four general methods for direct delivery of a gene into cells have been described: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87/06614, U.S. Pat. No. 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).
Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required. An illustrative embodiment of a method for delivering DNA into Zea mays cells by acceleration is a biolistics α-particle delivery system, that can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension. A particle delivery system suitable for use with the present invention is the helium acceleration PDS-1000/He gun is available from Bio-Rad Laboratories.
For the bombardment, cells in suspension may be concentrated on filters. Filters containing the cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded.
Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus that express the exogenous gene product 48 hours post-bombardment often range from one to ten and average one to three.
In bombardment transformation, one may optimize the pre-bombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.
In another alternative embodiment, plastids can be stably transformed. Method disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S. Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265.
Accordingly, it is contemplated that one may wish to adjust various aspects of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors by modifying conditions that influence the physiological state of the recipient cells and that may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.
Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310, U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome.
Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203 (1985). Moreover, technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant varieties where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
A transgenic plant formed using Agrobacterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.
It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both exogenous genes. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. Application of these systems to different plant varieties depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).
Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.
The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.
Methods for transformation of cereal plants such as rice for introducing genetic variation into the plant by introduction of an exogenous nucleic acid and for regeneration of plants from protoplasts or immature plant embryos are well known in the art, see for example, Canadian Patent Application No. 2,092,588, Australian Patent Application No 61781/94, Australian Patent No 667939, U.S. Pat. No. 6,100,447, International Patent Application PCT/US97/10621, U.S. Pat. No. 5,589,617, U.S. Pat. No. 6,541,257, and other methods are set out in Patent specification WO99/14314. Preferably, transgenic rice plants are produced by Agrobacterium tumefaciens mediated transformation procedures. An example of Agrobacterium mediated transformation of rice is provided herein in Example 5. Vectors carrying the desired nucleic acid construct may be introduced into regenerable rice cells of tissue cultured plants or explants, or suitable plant systems such as protoplasts.
The regenerable rice cells are preferably from the scutellum of immature embryos, mature embryos, callus derived from these, or the meristematic tissue.
To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.
Marker Assisted Selection
Marker assisted selection is a well recognised method of selecting for heterozygous plants required when backcrossing with a recurrent parent in a classical breeding program. The population of plants in each backcross generation will be heterozygous for the gene of interest normally present in a 1:1 ratio in a backcross population, and the molecular marker can be used to distinguish the two alleles of the gene. By extracting DNA from, for example, young shoots and testing with a specific marker for the introgressed desirable trait, early selection of plants for further backcrossing is made whilst energy and resources are concentrated on fewer plants. To further speed up the backcrossing program, the embryo from immature seeds (25 days post anthesis) may be excised and grown up on nutrient media under sterile conditions, rather than allowing full seed maturity. This process, termed “embryo rescue”, used in combination with DNA extraction at the three leaf stage and analysis for the desired genotype allows rapid selection of plants carrying the desired trait, which may be nurtured to maturity in the greenhouse or field for subsequent further backcrossing to the recurrent parent.
Any molecular biological technique known in the art which is capable of detecting a Fad2 or FatB gene can be used in the methods of the present invention. Such methods include, but are not limited to, the use of nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization with suitably labeled probes, single-strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage or a combination thereof (see, for example, Lemieux, 2000; Langridge et al., 2001). The invention also includes the use of molecular marker techniques to detect polymorphisms linked to alleles of (for example) a Fad2 or FatB gene which confer the desired phenotype. Such methods include the detection or analysis of restriction fragment length polymorphisms (RFLP), RAPD, amplified fragment length polymorphisms (AFLP) and microsatellite (simple sequence repeat, SSR) polymorphisms. The closely linked markers can be obtained readily by methods well known in the art, such as Bulked Segregant Analysis, as reviewed by Langridge et al. (2001).
The “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using a “pair of primers” or “set of primers” consisting of “upstream” and a “downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are known in the art, and are taught, for example, in “PCR” (Ed. M. J. McPherson and S. G Moller (2000) BIOS Scientific Publishers Ltd, Oxford). PCR can be performed on cDNA obtained from reverse transcribing mRNA isolated from plant cells. However, it will generally be easier if PCR is performed on genomic DNA isolated from a plant.
A primer is an oligonucleotide sequence that is capable of hybridising in a sequence specific fashion to the target sequence and being extended during the PCR. Amplicons or PCR products or PCR fragments or amplification products are extension products that comprise the primer and the newly synthesized copies of the target sequences. Multiplex PCR systems contain multiple sets of primers that result in simultaneous production of more than one amplicon. Primers may be perfectly matched to the target sequence or they may contain internal mismatched bases that can result in the introduction of restriction enzyme or catalytic nucleic acid recognition/cleavage sites in specific target sequences. Primers may also contain additional sequences and/or contain modified or labelled nucleotides to facilitate capture or detection of amplicons. Repeated cycles of heat denaturation of the DNA, annealing of primers to their complementary sequences and extension of the annealed primers with polymerase result in exponential amplification of the target sequence. The terms target or target sequence or template refer to nucleic acid sequences which are amplified.
Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art and can be found for example in Ausubel et al. (supra) and Sambrook et al. (supra). Sequencing can be carried out by any suitable method, for example, dideoxy sequencing, chemical sequencing or variations thereof. Direct sequencing has the advantage of determining variation in any base pair of a particular sequence.
Hybridization based detection systems include, but are not limited to, the TaqMan assay and molecular beacons. The TaqMan assay (U.S. Pat. No. 5,962,233) uses allele specific (ASO) probes with a donor dye on one end and an acceptor dye on the other end such that the dye pair interact via fluorescence resonance energy transfer (FRET). A target sequence is amplified by PCR modified to include the addition of the labeled ASO probe. The PCR conditions are adjusted so that a single nucleotide difference will effect binding of the probe. Due to the 5′ nuclease activity of the Taq polymerase enzyme, a perfectly complementary probe is cleaved during PCR while a probe with a single mismatched base is not cleaved. Cleavage of the probe dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.
An alternative to the TaqMan assay is the molecular beacon assay (U.S. Pat. No. 5,925,517). In the molecular beacon assay, the ASO probes contain complementary sequences flanking the target specific species so that a hairpin structure is formed. The loop of the hairpin is complimentary to the target sequence while each arm of the hairpin contains either donor or acceptor dyes. When not hybridized to a donor sequence, the hairpin structure brings the donor and acceptor dye close together thereby extinguishing the donor fluorescence. When hybridized to the specific target sequence, however, the donor and acceptor dyes are separated with an increase in fluorescence of up to 900 fold. Molecular beacons can be used in conjunction with amplification of the target sequence by PCR and provide a method for real time detection of the presence of target sequences or can be used after amplification.
Tilling
Plants of the invention can be produced using the process known as TILLING (Targeting Induced Local Lesions IN Genomes). In a first step, introduced mutations such as novel single base pair changes are induced in a population of plants by treating seeds (or pollen) with a chemical mutagen, and then advancing plants to a generation where mutations will be stably inherited. DNA is extracted, and seeds are stored from all members of the population to create a resource that can be accessed repeatedly over time.
For a TILLING assay, PCR primers are designed to specifically amplify a single gene target of interest. Specificity is especially important if a target is a member of a gene family or part of a polyploid genome. Next, dye-labeled primers can be used to amplify PCR products from pooled DNA of multiple individuals. These PCR products are denatured and reannealed to allow the formation of mismatched base pairs. Mismatches, or heteroduplexes, represent both naturally occurring single nucleotide polymorphisms (SNPs) (i.e., several plants from the population are likely to carry the same polymorphism) and induced SNPs (i.e., only rare individual plants are likely to display the mutation). After heteroduplex formation, the use of an endonuclease, such as Cel I, that recognizes and cleaves mismatched DNA is the key to discovering novel SNPs within a TILLING population.
Using this approach, many thousands of plants can be screened to identify any individual with a single base change as well as small insertions or deletions (1-30 bp) in any gene or specific region of the genome. Genomic fragments being assayed can range in size anywhere from 0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the ends of fragments where SNP detection is problematic due to noise) and 96 lanes per assay, this combination allows up to a million base pairs of genomic DNA to be screened per single assay, making TILLING a high-throughput technique.
TILLING is further described in Slade and Knauf (2005) and Henikoff et al. (2004).
In addition to allowing efficient detection of mutations, high-throughput TILLING technology is ideal for the detection of natural polymorphisms. Therefore, interrogating an unknown homologous DNA by heteroduplexing to a known sequence reveals the number and position of polymorphic sites. Both nucleotide changes and small insertions and deletions are identified, including at least some repeat number polymorphisms. This has been called Ecotilling (Comai et al., 2004).
Each SNP is recorded by its approximate position within a few nucleotides. Thus, each haplotype can be archived based on its mobility. Sequence data can be obtained with a relatively small incremental effort using aliquots of the same amplified DNA that is used for the mismatch-cleavage assay. The left or right sequencing primer for a single reaction is chosen by its proximity to the polymorphism. Sequencher software performs a multiple alignment and discovers the base change, which in each case confirmed the gel band.
Ecotilling can be performed more cheaply than full sequencing, the method currently used for most SNP discovery. Plates containing arrayed ecotypic DNA can be screened rather than pools of DNA from mutagenized plants. Because detection is on gels with nearly base pair resolution and background patterns are uniform across lanes, bands that are of identical size can be matched, thus discovering and genotyping SNPs in a single step. In this way, ultimate sequencing of the SNP is simple and efficient, made more so by the fact that the aliquots of the same PCR products used for screening can be subjected to DNA sequencing.
Mutagenesis Procedures
Techniques for generating mutant rice plant lines are known in the art. Examples of mutagens that can be used for generating mutant rice plants include irradiation and chemical mutagenesis. Mutants may also be produced by techniques such as T-DNA insertion and transposon-induced mutagenesis. The mutagenesis procedure may be performed on any parental cell of a rice plant, for example a seed or a parental cell in tissue culture.
Chemical mutagens are classifiable by chemical properties, e.g., alkylating agents, cross-linking agents, etc. Useful chemical mutagens include, but are not limited to, N-ethyl-N-nitrosourea (ENU); N-methyl-N-nitrosourea (MNU); procarbazine hydrochloride; chlorambucil; cyclophosphamide; methyl methanesulfonate (MMS); ethyl methanesulfonate (EMS); diethyl sulfate; acrylamide monomer; triethylene melamine (TEM); melphalan; nitrogen mustard; vincristine; dimethylnitrosamine; N-methyl-N′-nitro-Nitrosoguani-dine (MNNG); 7,12 dimethylbenzanthracene (DMBA); ethylene oxide; hexamethylphosphoramide; and bisulfan.
An example of suitable irradiation to induce mutations is by gamma radiation, such as that supplied by a Cesium 137 source. The gamma radiation preferably is supplied to the plant cells in a dosage of approximately 60 to 200 Krad., and most preferably in a dosage of approximately 60 to 90 Krad.
Plants are typically exposed to a mutagen for a sufficient duration to accomplish the desired genetic modification but insufficient to completely destroy the viability of the cells and their ability to be regenerated into a plant.
Antibodies
Monoclonal or polyclonal antibodies which bind specifically to FatB or Fad2 polypeptides can be useful for some of the methods of the invention.
The term “binds specifically” refers to the ability of the antibody to bind to a FatB or Fad2 polypeptide but not other proteins of rice, especially proteins of rice seeds.
As used herein, the term “epitope” refers to a region of a FatB or Fad2 polypeptide which is bound by the antibody. An epitope can be administered to an animal to generate antibodies against the epitope, however, antibodies useful for the methods described herein preferably specifically bind the epitope region in the context of the entire polypeptide.
If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide such as those provided in FIG. 2 or 7 or SEQ ID NOs 1 to 4 or 15 to 18. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides peptides of the invention or fragments thereof haptenised to another peptide for use as immunogens in animals.
Monoclonal antibodies directed against polypeptides of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced can be screened for various properties; i.e., for isotype and epitope affinity.
An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.
For the purposes of this invention, the term “antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab′) and F(ab′)2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
Antibodies may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
Preferably, the antibodies are detectably labeled. Exemplary detectable labels that allow for direct measurement of antibody binding include radiolabels, fluorophores, dyes, magnetic beads, chemiluminescers, colloidal particles, and the like. Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a coloured or fluorescent product. Additional exemplary detectable labels include covalently bound enzymes capable of providing a detectable product signal after addition of suitable substrate. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art. Further exemplary detectable labels include biotin, which binds with high affinity to avidin or streptavidin; fluorochromes (e.g., phycobiliproteins, phycoerythrin and allophycocyanins; fluorescein and Texas red), which can be used with a fluorescence activated cell sorter; haptens; and the like. Preferably, the detectable label allows for direct measurement in a plate luminometer, e.g., biotin. Such labeled antibodies can be used in techniques known in the art to detect polypeptides of the invention.
EXAMPLES
Example 1
Materials and Methods
Extraction of Oil with Sodium Methoxide
For fatty acid and other analyses, total lipid was extracted from rice grains as follows unless stated otherwise. In some cases, samples each consisting of a half grain were used for the extraction, with the second half-grain containing the embryo being used for embryo rescue. The technique can also be used with other cereals.
A single developing seed or half-seed was squashed between filter papers and placed in a tube. 2 ml 0.5M sodium methoxide was added, the tube sealed tightly and then incubated at 80° C. for 10 min. After the tube was cooled, 0.1 ml glacial acetic acid was added followed by 2 ml of distilled water and 2 ml of petroleum spirit. The mixtures were vortexed for 10 sec and, after the phases had separated, the upper petroleum layer was transferred to a small test tube. Approximately 1 g of potassium bicarbonate/sodium sulphate mixture was added to the test tubes and the mixture vortexed. The sample solutions were transferred to autosampler vials and stored in a freezer at −20° C. until GC analysis was performed as described by Soutjesdic et al. (2002).
Extraction of Lipids from Tissues with High Water Content (Bligh-Dyer Method)
This method was adapted from Bligh and Dyer (1959). 1.5 ml of CHCl3/MeOH (1:2) was added to a tissue sample in 0.4 ml of buffer and the sample vortexed vigorously. A further 0.5 ml of CHCl3 was added and the sample vortexed again. 0.5 ml of H2O was added and the sample vortexed again. The tube was centrifuged briefly at 3000 rpm to separate phases; a white precipitate appeared at the interface. The organic (lower) phase was transferred to a new tube and concentrated under vacuum. If acidic lipids were to be extracted, 0.5 ml of 1% HClO4 was added instead of 0.5 ml of H2O. The volumes in the above procedure could be modified as long as the ratios of CHCl3/MeOH/H2O were maintained.
Preparation of Fatty Acid Methyl Esters (FAME) for Quantitative Determination of Fatty Acid Content
To prepare FAME directly from grain, 10-15 seed were weighed accurately and transferred to a glass tube. An internal standard of 10 μl of 1 mg/ml 17:0-methyl ester was added to each seed sample. 0.75 ml 1 N methanolic-HCl (Supelco) was added to each tube which was capped tightly and refluxed at 80° C. for at least 2-3 hrs or overnight. Samples were cooled, 0.5 ml NaCl (0.9% w/v) added followed by 300 μl hexane. The tubes were capped again and vortexed vigorously. The upper hexane phase (200-250 μl) was carefully transferred to an eppendorf tube. The sample was dried down completely under nitrogen. The dried FAME samples were dissolved in 20 μl of hexane and transferred to conical glass inserts in vials for GC analysis.
FA Analysis by Gas Chromatography
Fatty acid methyl esters were prepared by alkaline transmethylation as follows. Single seed samples were squashed between filter paper disks. The fatty acids in the lipid transferred to the filter paper disks were then methylated in 2 mL of 0.02 M sodium methoxide for 10 minutes at 80° C., followed by cooling for 30 minutes. 0.1 mL of glacial acetic acid was then added, followed in order by 2 mL of distilled water and 2 mL of hexane. After vortexing and phase separation, the upper hexane layer containing the fatty acid methyl esters was transferred to a microvial. Fatty acid methyl esters were analysed by gas-liquid chromatography as previously described (Stoutjesdijk et al., 2002).
Transformation of Rice
Rice (cv. Nipponbare) was transformed as follows.
i) Callus Induction and Culture
Husks were removed from mature grain, which were then soaked in 70% EtOH for 30 sec to remove any outer layer of wax. The cleaned grains were washed 3 times with sterile H2O and soaked in a solution of 25% bleach (with 2 drops Tween-20 detergent added) for 20 minutes with shaking to surface sterilize them. Under asceptic conditions, the grains were rinsed briefly with 70% EtOH, washed thoroughly with sterile H2O 8-10 times and plated onto N6D medium. Plates were sealed with Micropore-tape and incubated under full light at 28° C. Calli were produced after 6-8 weeks and then transferred to NB media They were sealed with paraffin paper and left in the at 28° C. with sub-culturing every 4 weeks onto fresh NB plates. Calli were not used for transformation after more than 5 subcultures.
ii) Transformation
Healthy looking calli were picked from subculture plates and transferred onto fresh NB plates at a density of 25-30 calli/plate. Two days later, a fresh culture was established of the Agrobacterium strain containing the construct to be used, and incubated at 28° C. The culture medium was NB medium supplemented with 100 μM acetosyringone. Calli were immersed in a suspension of the cells for 10 minutes. After draining off excess suspension, calli were placed on NB medium supplemented with 100 μM acetosyringone and incubated in the dark at 25° C. for 3 days (co-cultivation). After the co-cultivation step, calli were washed gently three times in a tube with sterile H2O containing 150 mg/ml Timetin. Calli were blotted dry on filter paper and plated well spaced onto NBCT plates (containing 100 μg/ml kanamycin if a kanamycin selectable marker gene was used or other selection agent as appropriate, 150 μg/ml Timentin and 200 μg/ml Claroforan). The plates were incubated in the dark for 3-4 weeks at 26-28° C. Resistant calli were observed after about 10 days and transferred to NBCT+ selection plates and incubated in the dark for a further 14-21 days. Healthy calli were transferred onto PRCT+selection plates and incubated in the dark for 8-12 days, then transferred onto RCT+selection plates and incubated in fill light at 28° C. for 30 days. After this time, plantlets that had developed were transferred to 1⁄2MS medium in tissue culture pots and incubated under light for 10-14 days for further growth before transfer to soil.
iii) Media Composition and Components for Rice Tissue Culture
N6 macro-elements (20×) (g/l): (NH4)2SO4, 9.3; KNO3, 56.6; KH2PO4, 8; MgSO4.7H2O, 3.7; CaCl2.2H2O, 3.3.
N6 micro (100×) (mg/100 ml): MnSO4.4H2O, 440; ZnSO4.7H2O, 150; H3 BO3, 160; KI, 80.
N6 Vitamins (100×) (mg/100 ml): Glycine, 20; Thiamine-HCl, 10; Pyridoxine-HCl, 5; Nicotinic acid, 5.
B5 micro-elements (100×) (mg/1000 ml): MnSO4.4H2O, 1000; Na2MoO4.2H2O, 25; H3BO3, 300; ZnSO4.7H2O, 200; CuSO4.5H2O, 3.87; CoCl2.6H2O, 2.5; KI, 75.
B5 vitamins (100×) and Gamborg's vitamin solution (1000×) from Sigma cell culture.

    FeEDTA(200×) (g/200 ml): Ferric-sodium salt, 1.47.
    2,4-D (1 mg/ml): Dissolve 100 mg of 2,4-dichloro-phenoxyacetic acid in 1 ml absolute ethanol, add 3 ml of 1N KOH, adjust to pH 6 with 1N HCl.


BAP (1 mg/ml): 6-benzyl amino purine from Sigma cell culture.
NAA (1 mg/ml): Naphthalene acetic acid from Sigma cell culture.
ABA (2.5 mg/ml): Dissolve 250 mg of Abscisic acid in 2 ml of 1M NaOH, make up to 100 ml with sterile distilled water. Final background concentration to be 20 mM NaOH.
Hygromycin (50 mg/ml): Hygromycin solution from Roche.
Timetin (150 mg/ml): Dissolve 3100 mg of Timetin in 20.66 ml of sterile water. Final concentration to be 150 mg/ml.
Claforan (200 mg/ml): Dissolve 4 gm of Claforan in 20 ml of sterile water.
MS salts: Murashige-Skoog minimal organic medium.
N6D medium (amount per liter):

















N6 macro (10X)
100
ml



N6 micro (1000X)
1
ml



N6 vitamins (1000X)
1
ml



MS iron/EDTA
5
ml



Myoinositol
100
mg



Casamino acid
300
mg



Proline
2.9
g



2,4-D (1 mg/ml)
2
ml










    Sucrose 30 g


pH adjusted to 5.8 with 1M KOH, add 3 g phytogel/liter, autoclave.
NB medium (amount per liter):

















N6 macro-elements (20X)
50
ml



B5 micro-elements (100X)
10
ml



B5 vitamins (100X)
10
ml



FeEDTA (200X)
5
ml



2,4-D (1 mg/ml)
2
ml



Sucrose
30
g



Proline
500
mg



Glutamine
500
mg



Casein enzymatic hydrolysate (CEH)
300
mg










NBO: NB media, plus 30 g/l mannitol and 30 g/l sorbitol, added before pH adjustment.
NBCT+selection (Hygromycin-H30): NB media plus 30 mg/l hygromycin, Timentin 150 mg/ml, Claforan 200 mg/l.

    NBCT+selection (Hygromycin-H50); NB media plus selection 50 mg/l hygromycin, 200 mg/l Claforan and Timentin 150 mg/ml.


PRCT+Selection: NB media with no 2,4-D, plus following added after autoclave: BAP, 2 mg/l; NAA, 1 mg/l; ABA, 5 mg/l; Claforan, 100 g/l; Timetin; 150 mg/ml+selection.
RCT+Selection: NB media with no 2,4-D, plus following added after autoclave: BAP; 3 mg/l; NAA, 0.5 mg/l; Timetin, 150 mg/ml; Claforan, 100 mg/l+selection.
1⁄2 MS media: MS salts and vitamin mixture, 2.21 g; Sucrose, 10 g per liter, add 2.5 g phytogel/l, after autoclaving add 0.05 mg/l NAA and Timentin 150 mg/ml.
Example 2
Identification and Isolation of FatB Genes from Rice
FatB genes encode the enzyme palmitoyl-ACP thioesterase which have the activity of preferentially transferring fatty acids that have a length of 16 carbons or less from acyl carrier protein to acyl-CoA and thus prevent further elongation of the fatty acid carbon chain. Putative rice FatB sequences were identified using homology based searches using the sequence for the Genbank accession Arabidopsis locus AtACPTE32 and Iris locus AF213480. The program used was Megablast available at NCBI (www.ncbi.nlm.nih.gov/). Default parameters used by NCBI were used and the databases used were both non-redundant (nr) and high throughput gene sequences (htgs) for rice, Oryza sativa. The most similar sequences from rice selected by the Megablast program were then translated and examined for the presence of the conserved sequence NQHVNN(SEQ ID NO:26) found in all FatB sequences. A further amino acid residue believed to be essential in Arabidopsis is cysteine 264 and along with asparagine 227 and histidine 229 (which are both present in the conserved sequence NQHVNN) comprise the proposed catalytic triad. The Chinese Rice Database (current Website address rise.genomics.org.cn/rice/index2.jsp) was also used to a limited extent and default parameters for BLAST searching were used with the Arabidopsis sequence and Iris sequences. The translated sequences are aligned in FIG. 2.
An overview of the structures of all the FatB genes is shown as in FIG. 3. The genes described correspond as follows to the protein sequences discussed—AC108870 corresponds to Os11g43820, AP005291 corresponds to Os02g43090, AP000399 corresponds to Os06g5130 and AP004236 corresponds to Os06g39520. Note the possibility of multiple transcripts from one gene as indicated on the diagram. FIG. 4 shows a CLUSTAL W (fast) alignment of the ‘gene’ sequences using default parameters—note that the ‘genes’ are of different lengths.
The genes comprise 6 exons. The structure of the gene described by Os06g5130 is shown in detail FIG. 5.
FIG. 6 shows an alignment of the coding sequences of the FatB cDNAs indicating the different primers used for selective PCR amplification.
The sequence identity between the translated peptide sequence of Os06g5130 and Os06g39520 (corresponding to sequence ap000399 and ap004236 in FIG. 2) was 74% overall, and the identity at the nucleotide level over the entire coding sequence was 69%. In both cases the program BESTFIT with default parameters was used. The polypeptides deduced from Os02g43090 (one transcript), Os06g05130, Os06g39520 and Os011g43820 correspond to 298, 427, 423 and 425 amino acids respectively.
Activity of the encoded proteins was surmised from the high degree of sequence identity with known polypeptides shown to have this activity. Furthermore, the observed effect on palmitic acid levels of gene inactivating constructs based on these sequences was also consistent with this conclusion (see below).
Expression of the gene family was complex, with at least seven transcripts predicted from these four genes. On the basis of RT-PCR experiments that were performed and relative numbers of clones recovered from EST libraries, it appeared that RNA from Os06g5130 was relatively abundant in the grain, while Os11g43820 was expressed at a moderate levels in that tissue and the other genes were only expressed at a low level.
Example 3
Identification and Isolation of Fad2 Genes from Rice
Proteins encoded by Fad2 genes (Fatty acid desaturase 2) are responsible for the introduction of a double bond into 18:1 fatty acids—they are Δ12 desaturases. The Genbank sequence from the Arabiodopsis locus athd12aaa was used to search the nr and htgs databases for Oryza sativa using Megablast at default settings. The most similar sequences retrieved from rice were translated and examined for the presence of conserved hydrophobic motifs FSYVVHDLVIVAALLFALVMI (SEQ ID NO:27), AWPLYIAQGCVLTGVWVIA (SEQ ID NO:28), ISDVGVSAGLALFKLSSAFGF (SEQ ID NO:29), VVRVYGVPLLIVNAWLVLITYLQ (SEQ ID NO:30) and the histidine rice sequences HECGHH (SEQ ID NO:31), HRRHHA (SEQ ID NO:32) and HVAHH (SEQ ID NO:33). The translated amino acid sequences of the isoforms obtained are shown in FIG. 7.
FIG. 8 provides an alignment of Fad2 sequences showing location of 5′ UTR in isoform AP004047 (lowercase) and location of primers used for amplification by RT-PCR (underlined). The location of the stop codon is indicated by a box and untranslated regions downstream of the stop codon are in lower case.
Four gene sequences are recovered as being highly similar from the rice genome when the nucleotide sequence encoding the protein with the amino acid sequence of AP004047 was used to search the rice genome using the program BLAST with the default parameters. The overall structure of these genes are shown in FIG. 9. The genes correspond to the protein sequences as follows. The protein sequence AP004047 (also called FAD2-1 herein) corresponded to the gene Os02g48560, the sequence AP005168 (also called FAD2-2 herein) corresponded to Os07g23410, and the sequence contig2654 corresponded to Os07g23430. In addition there was a sequence that shared an intriguing extent of sequence identity but was clearly different to these sequences and may be a pseudo-gene. This sequence was Os07g23390.
Unlike the FatB genes, the Fad2 genes did not contain any introns. An alignment of all the protein coding sequences is shown in FIG. 10. The sequence identity over the entire coding region for Os02g48560 to Os07g23410 was 79%.
The molecular weight of the polypeptide encoded by Os02g48560 (ie AP004047) was 44.35 kDa before processing and contained 388 amino acids. The molecular weight of the polypeptide encoded by Os07g23410 was 44.9 kDa and contained 390 amino acids. These deduced polypeptides had 77% sequence identity as determined by the program BESTFIT using default parameters. The deduced polypeptide from Os07g23430 had a molecular weight of 41 kDa (363 amino acids) and from Os07g23390 was 24 kDa (223 amino acids). An alignment of all the deduced polypeptide sequences is shown in FIG. 7.
The sequence corresponding to Os02g48560 was expressed in the grain and this observation was consistent with data from the relative frequencies of clones for this gene in EST libraries where cognate sequences were recovered from grain cDNA libraries. Sequences corresponding to two of the isoforms encoded on chromosome 7 (Os07g23430,23410) have been recovered from a leaf EST library but the sequence corresponding to the other gene (Os07g23390) has not yet been reported; we concluded it may be expressed at low levels if at all.
In conclusion, the rice Fad2 gene family was also a complex gene family. The two transcripts deduced from Os02g48560 were indistinguishable by sequence. The sequence deduced from Os02g48560 was clearly expressed in the grain.
Example 4
Expression of FatB and Fad2 Genes in Rice
To determine which, if any, of the 4 putative FatB and 3 Fad2 genes identified in rice as described in Examples 2 and 3 might be expressed in developing rice grain, reverse transcription polymerase chain reaction (RT-PCR) assays were carried out. Since the genes were closely related in sequence, primers had to be designed that were specific for each of the genes, to assay transcripts for each gene specifically. Regions of sequence divergence were identified from the alignments (FIGS. 6 and 8) and several primer pairs were designed and tested. Primer sequences for detecting expression of the putative FatB genes are given in Tables 3 and 4. As an internal standard against which to compare expression levels, RT-PCR was also carried out on the RNA for expression of the rice gene encoding alpha-tubulin, OsTubA1. This gene was known to be expressed in all actively dividing tissues and was not affected by the hormone ABA and so was suitable as a constitutively expressed control for leaf and grain analysis.
RNA was prepared from rice grains approx 15 days after flowering using the Qiagen RNeasy kit following protocols supplied by the manufacturer. DNAse treatment (DNA-free kit, Ambion) was then used to remove contaminating DNA from the RNA preparation. The RT-PCR mix contained 5 μl of 5× Qiagen OneStep RT-PCR buffer, 1 μl of dNTP mix (containing 10 mM of each dNTP), 15 pmol of each primer and approximately 20 pg of RNA to a final volume of 25 μl. The following RT-PCR cycling program was used for the RT-PCR amplifications: 30 min at 50° C. (reverse transcription), 15 min at 95° C. (initial PCR activation), 30 cycles of (1 min at 94° C., 1 min at 57° C., 1 min at 72° C.) (PCR amplification), then a final extension of 10 min at 72° C.








TABLE 3







Primers designed to amplify and discriminate



relative expression of the putative FatB


transcripts using one-step RT-PCR












Genbank






ID/Chi-




Gene
nese


Ampli-
Contig
Primer


fied
no. ID
name
Primer Sequence





FatB-1
AP000399
p0399F2
CGCTGCTACCAAACAATTCA






(SEQ ID NO: 34)




p0399R2
TTCTGTGTTGCCATCATCG





(SEQ ID NO: 35)


 


FatB-2
AC108870
p5291_F2
CAGGAAATAAAGTTGGTGATGATG





(SEQ ID NO: 36)




p8870R
CTTCACAATATCAGCTCCTGACTC





(SEQ ID NO: 37)


 


FatB-3
AP005291
p5291_F2
CAGGAAATAAAGTTGGTGATGATG





(SEQ ID NO: 38)




p5291R
CTTCACAATGTCAGCCTTCAC





(SEQ ID NO: 39)


 


FatB-4
AP004236
p4236F2
ACAGGCCTGACTCCACGAT





(SEQ ID NO: 40)




p4236R2
GTCCAGAGTGCTTGTTGCAG





(SEQ ID NO: 41)


 


OsTubA1
AF182523
OSTUBA1_F
TACCCACTCCCTCCTTGAGC





(SEQ ID NO: 42)




OSTUBA1_R
AGGCACTGTTGGTGATCTCG





(SEQ ID NO: 43)

















TABLE 4







Primers designed to amplify and discriminate



relative expression of the putative Fad2


transcripts using one-step RT-PCR.












Genbank ID/





Gene
Chinese


Ampli-
Contig
Primer


fied
no. ID
name
Primer Sequence





Fad2-1
AP004047
pFad2-1F
CACAAAGAGGGAGGGAACAA






(SEQ ID NO: 44)




pFad2-1R
GAAGGACTTGATCACCGAGC





(SEQ ID NO: 45)


 


Fad2-2
Contig2654
UTR_2654
CACAACATCACGGACACACA




_F
(SEQ ID NO: 46)




UTR_2654
GCAAGACCGACATGGCTAAT




_R
(SEQ ID NO: 47)


 


Fad2-3
AP005168
UTR_5168
ACGTCCTCCACCACCTCTT




_F
(SEQ ID NO: 48)




UTR_5168
CAGAAGCAGTGACATACCCAAG




_R
(SEQ ID NO: 49)


 


OsTubA1
AF182523
OSTUBA1
TACCCACTCCCTCCTTGAGC




_F
(SEQ ID NO: 50)




OSTUBA1
AGGCACTGTTGGTGATCTCG




_R
(SEQ ID NO: 51)









Results from the RT-PCR experiments demonstrated that the sequence Fad2-1 gene (TIGR rice database identifier LOC_Os02g48560) was highly expressed in both grain and leaf relative to the genes encoding the other isoforms. The other two Fad2 genes (LOC_Os07g23410 and LOC_Os07g23430) appeared to be expressed at low levels and primarily in the leaf. The analysis of genes encoding the FatB isoforms showed that FatB-1 (Genbank identifier AP000399, Tigr LOC_Os06g05130) and FatB-2 (Genbank identifier AC108870, TIGR identifier Os11g43820) were more highly expressed in the grain than the other two genes. The Tos-17 transpositional insertion mutant had an insertion in the gene encoding AP000399.
In leaf tissue, FatB-1 (Genbank identifier AP000399, TIGR LOC_Os06g05130) and FatB-4 (AP004236, TIGR LOC_Os06g39520) were more highly expressed, referring to the relative abundance of mRNA transcripts from the genes. When compared to the abundance of the tubulin gene transcripts as a standard, which was a moderately abundant mRNA in wheat grain and was therefore expected to similarly abundant in rice grain, all the FatB and Fad2 mRNAs accumulated to low levels as determined by RT-PCR. However, this conclusion was based on the assumption that the primers, for each of the genes tested, hybridized to the target transcripts with similar efficiency to the tubulin primers/transcript.
This conclusion is corroborated by EST database searching. When the Fad2-1 (TIGR Os02g48560) sequence was used to search rice EST sequences, transcript sequences were present in panicle, root and whole plant cDNA libraries. In contrast, Fad2-2 (TIGR Os07g23410) and Fad2-3 (TIGR Os07g23430) sequences were present only in leaf, shoot or whole plant libraries. The sequence for Os07g23390, the Fad2-like gene considered to be a pseudogene, was not present in any of the EST libraries. Similarly, FatB-1 (TIGR Os06g05130) sequences were present in both leaf and panicle EST libraries, whereas FatB-2 sequences (TIGR Os11g43820) were present only in panicle or whole plant EST libraries and FatB-4 (TIGR Os06g39520) only in a leaf library. FatB-3 (AP005291, Os02g430900) sequences although expressed at a relatively low level to the others in both leaf and grain as judged by RT-PCR, were present in panicle and whole plant EST libraries. These searches used the BLAST program on the NCBI website (ncbi.nlm.nih.gov/BLAST/Blast.cgi) using the EST database and limiting the search to sequences from rice.
Based on these data, Fad2-1 and FatB-2 were considered to be the genes that should be down-regulated for alteration of grain lipid. FatB-3 was also thought be important in this regard.
Example 5
Construction of Gene Silencing Constructs and Transformation of Rice
Creation of DuplexRNA Construct for Inhibition of Fad2 Expression
A construct was designed to express a duplex RNA (hairpin RNA) in developing rice grain in order to reduce expression of Fad2-1. By targeting common regions of the three Fad2 gene sequences, the construct was designed so that it would be effective for all three of the identified Fad2 genes in rice grain in order to potentially maximize the effect on fatty acid composition. To improve silencing efficiency, the construct contained an intron between the sense and antisense portions of the inverted repeat sequences as described by Smith et al. (2000).
A 505 basepair fragment was amplified by PCR from the 5′ end of the Fad2-1 gene using the oligonucleotides pFad2-F 5′-AAAGGATCCTCTAGAGGGAGGAGCAGCAGAAGC-3′ (SEQ ID NO:52) and pFad2-R 5′-AAAACTAGTGAATTCTACACGTACGGGGTGTACCA-3′ (SEQ ID NO:53). The PCR product was ligated into pGEM-Teasy, transformed into E. coli and colonies containing the insert identified. The XbaI/EcoRI fragment from the plasmid designated pGEM-T-Fad2, containing the 505 bp Fad2 fragment, was then ligated in the sense direction into the vector ZLRint9_BC3895 (obtained from Zhongyi Li, CSIRO Plant Industry) containing the intron Rint9. A BamHI/SpeI fragment from pGEM-T-Fad2 was then ligated (in the antisense orientation) into the resultant plasmid so that an intron containing hairpin construct was formed. The BamHI/KpnI fragment containing the Fad2-1 intron containing hairpin was then inserted into the same restriction sites of the pBx17casNOT vector (Zhongyi Li, personal communication), containing the Bx17 seed specific promoter containing a Nos terminator/polyadenylation sequence so that the silencing gene would be expressed in developing seed in the order (promoter) sense-intron-antisense (terminator). The HindIII/NotI fragment containing the Bx17 promoter and Fad2-1 inverted repeat region was then inserted into the same restriction sites of the binary vector pWBvec8 (Wang et al., 1998) that contained a selectable maker gene conferring hygromycin resistance. This vector was then introduced into Agrobacterium and used for rice transformation as described in Example 1. The duplex RNA construct was designated dsRNA-Fad2-1.
Creation of DuplexRNA Construct for Inhibition of FatB Expression
A 665-basepair fragment of the rice palmitoyl-ACP thioesterase FatB-1 gene (Tigr LOC_Os06g05130) was amplified by PCR using primers with the following sequences: Rte-s1, 5′-AGTCATGGCTGGTTCTCTTGCGGC-3′ (SEQ ID NO:54) and Rte-a1, 5′-ACCATCACCTAAGAGACCAGCAGT-3′ (SEQ ID NO:55). This PCR fragment was used to make an inverted repeat construct with one copy of the fragment in the sense orientation and a second copy in the antisense orientation, separated by the intron sequence from the 5′ UTR of the cotton microsomsal ω6-desaturase GhFad2-1 gene (Liu et al., 1999). The inverted repeat construct was subsequently inserted into the SacI site between the Ubil promoter and Nos terminator of pUbilcasNOT (from Zhongyi Li based on sequence described in Li et al., 1997). The inverted repeat portion of rice FatB with the pUbil promoter was then inserted in the NotI site of the binary vector pWBVec8 and introduced into Agrobacterium as for the Fad2 construct described above. The duplex RNA construct was designated dsRNA-FatB-1.
Analysis of Fatty Acid Composition in Transformed Rice
Ten independent fertile transgenic plants obtained with dsRNA-Fad2-1 were tested for the presence of the dRNA gene by PCR using one primer corresponding to a site within the promoter region and a second primer for the end of the Fad2 sequence. Nine of the ten plants were found to be positive in the PCR reaction and therefore contained the Fad2 RNAi construct. Similarly, 23 fertile transgenic lines were tested for the FatB RNAi construct and 20 lines were found to be PCR positive.
To analyse the effect of the transgenes on fatty acid composition, total lipid was isolated from grain and leaf samples of the transformed rice plants. This was also done for a rice mutant line containing a Tos17 insertional sequence in the gene for FatB-1 (TIGR locus Os06g5130 corresponding to the protein identified by the Accession No. AP000399) to compare the effect of specifically inactivating this gene. Fatty acid composition was determined for each lipid extract by GC-FAME as described in Example 1. The data are presented in Tables 5 to 7 and some of that data is presented graphically in FIGS. 11 to 13. The proportion of each fatty acid was expressed as a percentage of the total fatty acid in the seed oil of the grain as determined by HPLC as described in Example 1.
The most striking and surprising aspect of the results was the extent of the change in grain oleic acid and linoleic acid composition in the Fad2 dsRNA plants. The proportion of oleic acid in some lines increased from 36% to at least 65% (w/w) and that of linoleic acid decreased from 37% to about 13% in the rice line most affected (Line 22A). Surprisingly, the proportion of palmitic acid in the Fad2 lines was also decreased, for example in Line 22A to less than 14% as compared to the control (18%).
In the FatB dsRNA lines, the reduction in the proportion of palmitic acid in the grain was correlated with an increase in the linoleic acid content while the proportions of oleic acid and linolenic acids were essentially unchanged. It was noted that the extent of the decrease in the proportion of palmitic acid in both Fad2 and FatB transgenic lines was similar, but the extent of increase in the linoleic acid level in the FatB lines was much less than the extent of the decrease observed in the Fad2 lines. That is, the extent of the change in levels of linoleic was greater with the Fad2 construct.
An interesting insight into the FatB catalysed step of the pathway is provided by analysis of the Tos-17 insertional knockout of one isoform of FatB, FatB-1. In the Tos-17 mutant, the extent of the change in the proportions of palmitic acid and oleic acid was reduced compared to the FatB dsRNA lines. These results indicated that genes encoding the FatB isoforms differed in their function.
When the results of the proportions of linoleic acid versus oleic acid were plotted as a scatter plot (FIG. 14), it was clear that the relationship between these two fatty acids in the Fad2 knockouts was the same as in wildtype rice, although the proportion of linoleic acid was vastly reduced. In contrast, with the FatB knockout and







TABLE 5







GC analysis of fatty acid composition in rice grain of


FatB and Fad2 mutants













Grain-
Myristic
Palmitic
Stearic
Oleic
Linoleic
Linolenic


Mutant
(C14:0)
(C16:0)
(C18:0)
(C18:1)
(C18:2)
(C18:3)





Wild type
0.70
18.83
1.64
38.41
36.42
1.29


(WH12)


FatB Tos17
0.72
15.13
2.33
37.15
38.61
1.87


insertional


mutant


FatB dsRNA
0.58
11.43
1.81
34.92
46.60
1.91


transformed


line


Fad2 dsRNA
0.58
14.26
2.11
64.94
12.62
1.23


transformed


line


Control for
0.85
17.91
2.60
33.23
39.84
1.76


Tos17


Control for
1.03
18.86
1.84
34.08
39.63
1.75


FatB


dsRNA


Control for
1.02
17.47
2.38
36.03
37.42
1.50


Fad2


dsRNA
















TABLE 6







GC analysis of fatty acid composition in rice leaves of FatB and Fad2 mutants















Lauric
Myristic
Palmitic
Stearic
Oleic
Linoleic
Linolenic


Leaf-mutant
(C12:0)
(C14:0)
(C16:0)
(C18:0)
(C18:1)
(C18:2)
(C18:3)

















Wild-type (WF2)

0.80
14.43
1.90
2.32
8.99
68.96


FatB Tos17

0.34
11.55
1.84
2.24
15.04
67.70


insertional


mutant


FatB dsRNA
0.56
1.01
14.39
2.09
2.07
13.21
64.56


transformed


line


Fad2 dsRNA


transformed


line


Control for

0.39
10.52
1.80
2.30
15.61
67.65


Tos17


Control for
0.72
1.15
13.30
2.03
4.04
24.62
52.28


FatB dsRNA
















TABLE 7







Relative amounts of grain fatty acids in mutant lines compared to


corresponding control lines (% in mutant/% in corresponding control)














Myristic
Palmitic
Stearic
Oleic
Linoleic
Linolenic



(C14:0)
(C16:0)
(C18:0)
(C18:1)
(C18:2)
(C18:3)

















Grain fatty








acids


FatB Tos17
0.85
0.8451*
0.899
1.118*
0.9691
1.06


insertional


mutant


FatB dsRNA
0.57*
0.6060*
0.980
1.025
1.176*
1.09


transformed


line


Fad2 dsRNA
0.570*
0.8167*
0.887*
1.802*
0.3373*
0.822*


transformed


line


Leaf fatty


acids


FatB Tos17
nd
0.911*
0.977
1.03
1.037
0.9992


insertional


mutant


FatB dsRNA
nd
0.9247
0.969
1.95*
1.864*
0.8098*


transformed


line





*Statistically significant change






to a lesser extent in the FatB Tos-17 line, although the relationship between linoleic acid and oleic was similar, it is shifted by a constant. This meant that there was more linoleic acid in the FatB knockout plants for a given amount of oleic acid than in wild-type or Fad2 knockout plants. This was consistent with the idea that knockout of FatB influenced the pathways controlling the amounts of oleic and linoleic acid but not the step directly linking oleic acid and linoleic acid which was controlled by Fad2.

The results of the proportion of linoleic acid versus palmitic were also plotted for all of the rice lines analysed. A positive linear relationship was observed for the Fad2 knockout lines, inverse of what was observed for linoleic versus oleic acid. For the FatB lines, however, a different relationship is observed (FIG. 15). A difference in the relationship between oleic and palmitic acid was also observed when the Fad2 dsRNA plants and FatB dsRNA plants were plotted as a scatter plot (FIG. 16). These results confirmed that the relationship between palmitic and linoleic acid (and oleic acid) was different for the two perturbations of the pathway.
Principal component analysis of the proportions of the various fatty acids under different perturbations of the pathway confirm that principal component 1 (which indicates the axes that contribute the greatest variation) was composed of the proportions of linoleic versus oleic whereas the principal component 2 (the second most important set of axes) was composed of proportions of linoleic and oleic acid versus palmitic acid). This is illustrated in FIG. 17.
We also concluded from the results presented in this Example that crossing the dsRNA Fad2 plants with the dsRNA FatB plants or Tos-17 FatB plants to combine the mutations would further increase the relative proportion of oleic acid and further decrease palmitic and linoleic acid levels.
Example 6
Production and Use of Antibodies
Antibodies were raised by synthesizing 15 or 16-mer peptides that were present in the deduced sequence of FatB. The peptides used to raise antibodies against FatB were:

    FatB-U1 Ac-CGMNKNTRRLSKMPDE (SEQ ID NO:56). This corresponds to the translated sequence of FatB (Accession No. AP000399) from amino acid position number 259 and was also found in sequences translated from AP005291, AC108870 and AC004236. However, the sequence was only identical between the four isoforms in the sequence TRRLSKMPDE (SEQ ID NO:57).
    FatB-U2. Ac-CGEKQWTLLDWKPKKPD (SEQ ID NO:58). This sequence was found in the translated sequence of all four FatB isoforms.
    FatB-99. Ac-CGAQGEGNMGFFPAES (SEQ ID NO:59). This sequence was found only in AP00399 and was at the very C terminus of the deduced polypeptide.


After synthesis the peptides were coupled to either Ovalbumin or Keyhole Limpet Haemocyanin protein (KLH) using the cross-linker MBS (maleimidobenzoic acid N-hydroxyl succinamide ester) using standard techniques. The cross-linked peptide was dialysed and lyophilized and injected into rabbits at two weekly intervals for two months with Friends incomplete adjuvant at a concentration of approx 1 mg/ml. Antisera raised against FatB-99 detected a clear difference between FatB isoforms in that a polypeptide of 20 kDa present in wild-type rice was missing in the Tos-17 mutant line having an insertion in the gene corresponding to TIGR identifier Os06g5130, the product corresponds to FatB-1, the sequence is represented by accession No. AP000399 (FIG. 18). Although this was different to the expected size of approx 40 kDa, such a discrepancy had been noticed before with FatB isoforms Different size of FatB products have been observed in developing and mature Cuphea wrightii seeds showing five different FatB isoforms, longer size in mature seeds and shorter product in mature seeds (Leonard et al., 1997).
The antisera can be used to detect FatB protein in the transgenic or mutant plant samples and confirm the extent of gene inactivation.
Example 7
Identification of Mutants in Rice Fad2
PCR products spanning the active sites (essentially positions 330 to 1020 if the initiating ATG is taken as the start of the numbering in TIGR Loc_Os2g48560) of Fad2-1 (coding sequence corresponding to AP00 4047 in NCBI database, TGR locus identifier LOC_Os02g48560) will be produced from rice DNA extracted from a large number of different rice accessions. The size of the products will be up to 800 bp depending on the primers used. Two sets of overlapping primer pairs may need to be used. One possible set of primers is:









Set A




GTGCCGGCGGCAGGATGACGG
(SEQ ID NO: 60)








(positions 5-20 in the alignment shown in FIG. 10)



 









GCCGACGATGTCGTCGAGCAC
(SEQ ID NO: 61)









(reverse complement of positions 379-398 in the



alignment shown in FIG. 10)


 


Set B









TGCCTTCTCCGACTACTCGG
(SEQ ID NO: 62)









(positions 360-379 in FIG. 10)



 









CCTCGCGCCATGTCGCCTTGG
(SEQ ID NO: 63)









(reverse complement of positions 1099-1118 in FIG.



10).






The annealed products will then be subjected to melting in a Rotorgene 6000 instrument (Corbett Life Science) or a comparable instrument where differences in melting of heteroduplexes can be sensitively detected by means of alteration of fluorescence of a dye LC Green. Hundreds and possibly thousands of rice lines can be screened daily by this technique.
The PCR products from samples showing an altered thermal profile will be sequenced and the mutations in Fad2 identified. Selected mutations which inactivate Fad2 can then be crossed into elite rice lines to produce rice lines with reduced Fad2 activity and therefore high oleic acid. If two or all of the Fad2 isoforms need to be eliminated then this can be achieved by identifying lines with the required mutations in different isoforms and combining the mutations in an elite rice line through marker assisted breeding. At least the Fad2-1 gene needs to be inactive for substantial increases in oleic acid content or decreased linoleic acid content. Inactivation of one or more of the additional Fad2 genes will further increase oleic acid content and decrease linoleic acid content. Mutants having mutations in additional Fad2 genes may be identified in the same way as for Fad2-1, using specific primers for the additional Fad genes.
Example 8
Stability Analysis—Detection of Hexanal Production in Storage
Experiments are underway with FSA, Werribee to detect the production of hexanal on storage in wildtype rice. This involves GC using a sampler to detect the volatiles in the headspace of grain stored at high temperature (40° C.). Once the system is optimized (and also we have sufficient quantity of grain) we will undertake a comparison of the production of volatiles, particularly hexanal, upon storage of wildtype and Fad2 RNAi and FatB RNAi rice lines and suitable combinations of genotypes. This is an important quality issue in the rice industry for storage of grain and also of storage of bran. The production and detection of other volatiles, which could have a role in affecting grain quality, is also being investigated, both with FSA, Werribee and CSIRO Entomology.
Around 10 g of raw brown rice is required for headspace analysis. The brown rice is stored at 4° C. (control) and 35° C. for 8 weeks. The gas sample released from brown rice can be obtained in the headspace of a vial by either heating at 80° C. or by natural diffusion. The volatile components in the headspace can then be analysed by direct injection into a GC or GC-MS machine and analysis of the gas chromatographic profiles (Suzuki et al., 1999). Another method for analysis of volatiles in the headspace is through the trapping of volatiles onto a suitable matrix (eg 250 mg Tenax GR) as described by Nielsen et al. (2004) using nitrogen as a purge gas. The desorption of the aroma compounds is then done thermally and the trapped molecules are analysed by GC and identified using standards.
The expectation that storage of rice would be improved in rice lines with low linoleic acid is based on a number of observations. Suzuki et al. (1999). have presented data that the amount of free linoleic acid increases during storage and that the amount of volatile aldehydes such as pentanal, hexanal and pentanol increase three fold at 35 C. The correlation of hexanal and volatile aldehydes with odour has been noted by other authors as well. On the other hand, Zhou et al. (2002) found a reduction of total linoleic acid upon storage and related this to the decomposition of linoleic acid to other products, including volatiles responsible for off-odours. The differences in the results may be due to differences in the extraction and analytical methods.
The production of hexanal for linoleic acid in vitro has been demonstrated by Nielsen et al. (2004).
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
All publications discussed and/or referenced herein are incorporated herein in their entirety.
This application claims priority from AU 2006903810, the entire contents of which are incorporated herein by reference.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
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## Claims

1. Rice oil obtained from rice grain, the rice oil having a fatty acid composition comprising greater than 60% w/w oleic acid, less than 15% w/w palmitic acid and less than 25% w/w linoleic acid, wherein the ratio of oleic to linoleic acid in the rice oil differs by no more than 5% when compared to the ratio of oleic acid to linoleic acid in the rice grain.

2. The rice oil of claim 1, wherein the weight ratio of oleic acid to linoleic acid is greater than 3.0:1.

3. The rice oil of claim 1, having a fatty acid composition comprising greater than 60% w/w oleic acid, less than 12% w/w palmitic acid, and/or less than 15% w/w linoleic acid.

4. The rice oil of claim 1, further comprising γ-oryzanol.

5. A food product comprising the rice oil of claim 1.

6. The rice oil of claim 1, which is substantially purified.

7. The substantially purified rice oil of claim 6, which is at least 75% free from other components with which it is naturally associated.

8. The substantially purified rice oil of claim 7, which is at least 90% free from other components with which it is naturally associated.