WO2012085808A1

WO2012085808A1 - Increased avenasterol production

Info

Publication number: WO2012085808A1
Application number: PCT/IB2011/055759
Authority: WO
Inventors: Bettina Tschiersch; Dietrich Rein; Ralf Flachmann; Rainer Lemke
Original assignee: Basf Plant Science Company Gmbh; Basf (China) Company Limited
Priority date: 2010-12-20
Filing date: 2011-12-19
Publication date: 2012-06-28

Abstract

The invention is directed to methods for manipulating the level of phytosterol-related compounds in a plant organism and especially for increasing the Avenasterol content in the seed.

Description

Increased Avenasterol Production

The invention relates to a method for the production of a transgenic cereal plant with increased A5-Avenasterol and/or A7-Avenasterol content in the kernels characterized in that the wild-type plant is transformed with a vector comprising nucleic acid sequences encoding a SMT1 protein and a HMGR protein both under the control of a constitutive promoter and selecting for transgenic plants in which - in contrast to or comparison with the wild type plant - the A5-Avenasterol and/or Δ7 -Avenasterol content in the kernels is increased.

Furthermore the invention relates to a transgenic wheat plant with increased Δ5- Avenasterol and/or A7-Avenasterol content in the kernels characterized in that said plant contains a gene construct comprising genes encoding a SMT1 and a HMGR protein under the control of a constitutive pZmUbi promoter.

The invention also comprises transgenic cereal kernels with increased A5-Avenasterol and/or Δ7 -Avenasterol content from a transgenic plant produced by the method described above, extracted oil therefrom or flour produced from said transgenic kernels. In addition the invention relates to the use of transgenic cereal kernels having an increased A5-Avenasterol and/or A7-Avenasterol content, flour produced from said kernels and oil extracted from said kernels as ingredient of food, pet food, feed or medical support products. High plasma cholesterol levels in humans, hypercholesterolaemia, have been associated with many diseases, particularly cardiovascular diseases. Dietary plant sterols

(phythosterols) are well known for their ability to lower blood serum cholesterol levels. Phythosterols reduce serum cholesterol and low-density lipoprotein-cholesterol levels thereby lowering the risk of cardiovascular diseases. One of the hypocholesterolemic mechanisms is a competition between the structurally similar cholesterol and phythosterols for micellar solubilization. Phythosterols interfere with cholesterol absorption in the intestine resulting in reduced cholesterol up-take from the gut. In addition, an increased intestinal sterol secretion activity of ATP-binding cassette transporter (ABC) A1 has been proposed as a mechanism underlying the active hypo-cholesterolaemic effect of phythosterols. The scientific evidence for cardioprotective hypo-cholesterolemic effects of dietary sterols was the basis for FDA to approve a health claim for phythosterols and risk of coronary heart disease in the year 2000 (21 CFR 101.83). The claim applies to foods containing at least 0.65 g plant sterol esters equivalent to 0.4 g free plant sterols per reference amount. Plant sterols occur naturally in seeds, vegetables, nuts (peanuts), coniferous trees, oil of corn kernels and corn fibers. Largely unrecognized as a source for dietary plant sterols have been cereals. The chief source of plant sterols for sterol enhanced foods and supplements is deodorizer distillate from soybean oil or palm oil. Sterols and its

hydrogenated form, stanols, are also sourced from tall (pine) oil as phythosterol-rich byproduct from the pulping of pine and other trees (refine - purify - chemical hydrogenation - esterification with food-grade fatty acids).

The most important sources of plant sterols in human diets are currently oils and

margarines. Low fat food ingredients such as cereals and cereal products are also recognized as plant sterol sources. In cereals, plant sterols occur as free sterols, steryl esters (with fatty acids or phenolic acids), steryl glycosides and acylated steryl glycosides. In all cereal grain and milling product samples sitosterol is the most abundant sterol, making up more than 50% of the total sterol content (Table 1 ). Among milling products, whole meal products, bran fractions and germ fractions are the richest sterol sources. The endosperm as the largest compartment of the wheat kernel contributes the largest amount to the total sterol content of the wheat kernel (Table 2).

Table 1 : Plant sterol content of wheat and wheat milling fractions compared with rye (mg/100g)

Data from Piironen et al. Cereal Chemistry. 79(1 ): 148-154, 2002 Table 2: Sterol concentrations in different wheat grain compartments and their contribution to the total sterol content of the whole grain

(1 ) Data from Piironen et al. Cereal Chemistry. 79(1 ):148-154, 2002

Higher dietary plant sterol intake can be achieved through adding sterols to specific items of the food chain or through genetically increasing the production in the crop plant. Even low fat foods can be efficient in reducing serum cholesterol, if phythosterols are appropriately emulsified (Ostlund, Jr. 2004). The cereal sterol content of whole wheat bread (about 50-80 mg/100 g) compared to about 250 mg/100 g refined oil in European food products shows that low fat food items contribute to plant sterol intake (Normen et al. 2001 ). Thus, in addition to oil crops, crops grown for protein or starch such as soy, peas, wheat, rye, triticale, rice, barley, oats, corn, bananas or tomatoes could serve as a source of sterols.

The average sterol/stanol intake with whole wheat bread in a Dutch population was estimated to be about 50 mg with 60 g or a two oz serving (Normen et al. 2001 ), slightly higher than calculated from bread wheat sterol concentrations measured by other groups (Piironen, Toivo, & Lampi 2002;Ruibal-Mendieta et al. 2004).

Unexpectedly, an average ten fold to 15 fold increase in sterols of the whole wheat seed could achieve the 0.4 g sterol per food serving potentially qualifying for a health claim. By increasing the content of plant sterols in whole wheat, bakery products made out of this wheat could become a significant source of dietary sterols or stanols. A food, of which two servings can be consumed per day with 0.4 g free sterols per serving, can carry the claim "may reduce the risk of heart disease" if included into a diet low in saturated fat and cholesterol (21 CFR 101 .83). Bread made from high sterol wheat can be such a food. The high sterol wheat bread has the additional advantage that it is low in fat and easily incorporated into a Western diet.

Therefore increasing phythosterol concentrations in a low-fat vegetable food such as wheat is of special value for human nutrition. In recent years, plant sterols have been isolated from different plant oil sources and added especially to high-fat foods, including margarines and yogurt. High sterol wheat could be used directly to produce phythosterol-enriched bakery products or pasta. Similarly, other low fat crops would be suitable for increasing sterol content by genetic modification to become foods qualifying for a heart health claim.

Currently whole wheat bread contains 40-80 mg sterols plus stanols/serving size

(peer reviewed papers range in sterol concentration of wheat: 500-1500 mg/kg, (Ruibal- Mendieta et al. 2004) (Normen et al. 2001 ; Piironen, Toivo, & Lampi 2002)). Cholesterol lowering efficacy of high sterol wheat may be similar or slightly lower than same amount of plant sterol consumed through other foods including fatty foods (spread, milk products etc.) (Ostlund, Jr., Racette, & Stenson 2003). The idea of high sterols in cereals is new and innovative since experts associated naturally occurring plant sterols in foods to closely correlate with the lipid fractions present in the plant food products. Cereals have higher sterol content than their fat level may suggest.

Current sterol source is for example plant sterol enriched spreads, the most economical source of sterols, contain approximately 80 mg free sterols per gram. A future sterol source could be wheat bread using wheat with 10 fold increased sterols plus stanols from 40-80 mg to 400 or 800 mg/serving (90 g bread) and may result in a possible US health claim that this product "may reduce the risk of heart disease" if included into a diet low in saturated fat and cholesterol (21 CFR 101.83). The present invention aims to increase sterol levels in wheat, especially the wheat kernels. The constitutive expression of one of the key sterol biosynthetic enzymes, 3-hydroxy-3- methylglutaryl-CoA reductase (HMGR) alone or in combination with a Sterol

Methyltransferase 1 (SMT1 ) enhanced the levels of health claim related phythosterols (β- Sitosterol, Campesterol, Stigmasterol) in wheat kernels by up to 2.5 fold. Additionally the sterol profile in transgenic wheat kernel changed completely. Transgenic wheat kernels accumulated high levels of certain sterol pathway intermediates in particular 24-Methylene cycloartenol and Cycloartenol. The overall increase of phythosterol pathway products was up to 10-fold in transgenic kernels. Surprisingly it has been found that A 5-Avenasterol which is a minor sterol in wheat and the direct precursor of β-Sitosterol became the most prominent sterol in wheat kernels. Including Δ 5-Avenasterol, the increase in Δ 5- phythosterols (β-Sitosterol, Campesterol, Stigmasterol, Δ 5-Avenasterol) is up to 4 fold in wheat kernels. Such a dramatic enhancement of Δ 5-Avenasterol (isofucosterol) and changes in composition of Δ 5-sterols were not reported for tobacco or soy seeds over- expressing the HMGR alone or in combination with the SMT1 . Conversion of Δ 5- Avenasterol into β-Sitosterol seems to be a further limiting step in synthesis of phythosterols in wheat. Based on these findings it could be predicted that an over-expression of a 24- Methyl-cholesterol reductase catalyzing the conversion of Δ 5-Avenasterol to β-Sitosterol would further increase the level of health claim related phythosterols and therefore increase the nutritional value. The following constructs were transformed into wheat - see Table 3. All genes of interest (GOI) were expressed using the constitutive p-ZmUbi - promoter from maize.

Table 3: Constructs transformed into wheat

HMGR and SMT1 were codon-optimized for expression in wheat.

Described herein are inventions in the field of genetic engineering of plants, including combinations of polynucleotides encoding phythosterol metabolism proteins (PMP) to improve agronomic, horticultural, and quality traits. This invention also relates to the combination of polynucleotides encoding proteins that are related to the presence of phythosterol compounds in plants. More specifically, the present invention relates to polynucleotides encoding SMT1 and HMGR and the use of these combinations of these sequences, their order and direction in the combination, and the regulatory elements used to control expression and transcript termination in these combinations in transgenic plants. In particular, the invention is directed to methods for manipulating phythosterol-related compounds and altering the phythosterol composition in plants and seeds. The invention further relates to methods of using these novel combinations of polypeptides to increase yield and/or composition of phythosterol compounds. Although the phythosterol related compound content, and/or composition of kernels, can be modified by the traditional methods of plant breeding, the advent of recombinant DNA technology has allowed for easier manipulation of the phythosterol related compound in kernels of a plant. In order to increase or alter the levels of phythosterol related compounds in kernels in plants, nucleic acid sequences and proteins regulating phythosterol metabolism must be identified. Although several compounds are known that generally affect plant and kernel or seed development, there is a clear need to specifically identify factors, and particularly

combinations thereof, that are more specific for the developmental regulation of storage compound accumulation and to identify combination of genes which have the capacity to confer altered or increased oil production to its host plant and to other plant species. One embodiment of this invention discloses combinations of nucleic acid sequences from

Nicotiana tabacum and Hevea brasiliensis. These combinations of nucleic acid sequences can be used to alter or increase the levels of phythosterol compounds in cereal plants, including transgenic cereal plants, such as wheat, barley, triticale, rye, oats, rice and corn. Although several compounds are known that generally affect plant and kernel development, there is a clear need to specifically identify factors that are more specific for the

developmental regulation of storage compound accumulation and to identify genes which have the capacity to confer altered or increased phythosterol production to its host plant and to other plant species.

Thus, this invention, in principle, discloses nucleic acid sequences and combinations thereof which can be used to alter or increase the levels of phythosterol compounds such as avenasterol and/or the composition of phythosterol in plants, including transgenic cereal plants, such as wheat, maize, triticale, oat, rye, barley and rice.

Specifically, the present invention relates to a polynucleotide comprising nucleic acid sequences selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 1 and 3;

a nucleic acid sequence encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 and 4;

a nucleic acid sequence which is at least 80% identical to the nucleic acid sequence of (a) or (b), wherein said nucleic acid sequence encodes a polypeptide having SMT1 protein or HMGR protein activity and wherein said polypeptide comprises at least one of the amino acid sequences shown in SEQ ID NO: 2 and 4; and a nucleic acid sequence being a fragment of any one of (a) to (c), wherein said fragment encodes a polypeptide or biologically active portion thereof having SMT1 protein or HMGR protein activity and wherein said polypeptide comprises at least one of the amino acid sequences shown in SEQ ID NO: 2 and 4. The term "polynucleotide" as used in accordance with the present invention relates to a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide having phytherosterol compound increasing activity, i.e. being capable of specifically increasing phythosterol content in plants and in the kernels of plants. More preferably, the polypeptide encoded by the polynucleotide of the present invention having phythosterol increasing activity shall be capable of increasing the amount of phythosterol compounds, preferably avenasterol, when present in plant kernels. The polypeptides encoded by the

polynucleotide of the present invention are also referred to as phythosterol metabolism proteins (PMP) herein below. Suitable assays for measuring the activities mentioned before are described in the accompanying Examples. Preferably, the polynucleotide of the present invention upon expression in a plant kernel shall be capable of significantly increasing the kernel storage of phythosterol related compounds, e.g. especially avenasterol.

Preferably, the polynucleotide of the present invention upon expression in the kernel of a transgenic cereal plant is capable of significantly increasing the amount by weight of at least one phythosterol related compound. More preferably, such an increase as referred to in accordance with the present invention is an increase of the amount by weight of at least 1 , 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 % as compared to a control. Whether an increase is significant can be determined by statistical tests well known in the art including, e.g., Student^'s t-test. The percent increase rates of a seed storage compound are, preferably, determined compared to an empty vector control. An empty vector control is a transgenic plant, which has been transformed with the same vector or construct as a transgenic plant according to the present invention except for such a vector or construct is lacking the polynucleotide of the present invention. Alternatively, an untreated plant (i.e. a plant which has not been genetically manipulated) may be used as a control.

A polynucleotide encoding a polypeptide having a biological activity as specified above has been obtained in accordance with the present invention, preferably from Nicotiana tabacum and Hevea brasiliensis. The corresponding polynucleotides, preferably, comprises the nucleic acid sequence shown in SEQ ID NO: 1 and 3, respectively, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 and 4, respectively. It is to be

understood that a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 and 4 may be also encoded due to the degenerated genetic code by other polynucleotides as well.

Moreover, the term "polynucleotide" as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. Said variants may represent orthologs, paralogs or other homologs of the polynucleotide of the present invention. The polynucleotide variants, preferably, also comprise a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequences shown in SEQ ID NO: 1 and 3 by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a polypeptide having a biological activity as specified above. Variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1 -6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6 χ sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 χ SSC, 0.1 % SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under "standard hybridization conditions" the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. The hybridization conditions for DNA: DNA hybrids are, preferably, 0.1 χ SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for

DNA:RNA hybrids are, preferably, 0.1 χ SSC and 30°C to 55°C, preferably between 45°C and 55°C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991 , "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants are obtainable by PCR-based techniques such as mixed oligonucleotide primer-based amplification of DNA, i.e. using degenerated primers against conserved domains of the polypeptides of the present invention. Conserved domains of the polypeptide of the present invention may be identified by a sequence comparison of the nucleic acid sequences of the polynucleotides or the amino acid sequences of the polypeptides of the present invention. Oligonucleotides suitable as PCR primers as well as suitable PCR conditions are described in the accompanying Examples. As a template, DNA or cDNA from bacteria, fungi, plants or animals may be used. Further, variants include polynucleotides comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequences shown in SEQ ID NO: 1 or 3 encoding polypeptides retaining a biological activity as specified above. More preferably, said variant polynucleotides encode polypeptides comprising amino acid sequence patterns shown in SEQ ID NOs: 2 and 4. Moreover, also encompassed are polynucleotides which comprise nucleic acid sequences encoding amino acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequences shown in SEQ ID NO: 2 and 4 wherein the

polypeptide comprising the amino acid sequence retains a biological activity as specified above. The percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (J. Mol. Evolution., 25, 351 -360, 1987, Higgins et al., CABIOS, 5 1989: 151 -153) or the programs Gap and BestFit (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981)), which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991 )], are to be used. The sequence identity values recited above in percent (%) are to be determined, preferably, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average

Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences can be also determined using the Vector NTI 7.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814). A gap-opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap-opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two

polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap-opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide sequence is equivalent to an uracil nucleotide.

A polynucleotide comprising a fragment of any of the aforementioned nucleic acid sequences is also encompassed as a polynucleotide of the present invention. The fragment shall encode a polypeptide which still has a biological activity as specified above.

Accordingly, the polypeptide may comprise or consist of the domains of the polypeptide of the present invention conferring the said biological activity. A fragment as meant herein, preferably, comprises at least 20, at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of any one of the aforementioned nucleic acid sequences or encodes an amino acid sequence comprising at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids of any one of the aforementioned amino acid sequences. The polynucleotides of the present invention either essentially consist of the aforementioned nucleic acid sequences or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well. Preferably, the polynucleotide of the present invention may comprise in addition to an open reading frame further untranslated sequence at the 3' and at the 5' terminus of the coding gene region: at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5' terminus of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3' terminus of the coding gene region.

Variant polynucleotides as referred to in accordance with the present invention may be obtained by various natural as well as artificial sources. For example, polynucleotides may be obtained by in vitro and in vivo mutagenesis approaches using the above mentioned specific polynucleotides as a basis. Moreover, polynucleotids being homologs or orthologs may be obtained from various animal, plant, bacteria or fungus species. Paralogs may be identified from E. coli.

The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context such as a gene locus) or in genetically modified or exogenously (i.e. artificially) manipulated form. An isolated polynucleotide can, for example, comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. The polynucleotide, preferably, is double or single stranded DNA including cDNA or RNA including antisense-, micro-, and siRNAs. The term encompasses single- as well as double- stranded

polynucleotides. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified ones such as biotinylated polynucleotides. The polynucleotide encoding a polypeptide having a biological activity as specified encompassed by the present invention is also, preferably, a polynucleotide having a nucleic acid sequence which has been adapted to the specific codon-usage of the organism, e.g., the plant species, in which the polynucleotide shall be expressed (i.e. the target organism). This is, in general, achieved by changing the codons of a nucleic acid sequence obtained from a first organism (i.e. the donor organism) encoding a given amino acid sequence into the codons normally used by the target organism whereby the amino acid sequence is retained. It is in principle acknowledged that the genetic code is redundant (i.e.

degenerated). Specifically, 61 codons are used to encode only 20 amino acids. Thus, a majority of the 20 amino acids will be encoded by more than one codon. The codons for the amino acids are well known in the art and are universal to all organisms. However, among the different codons which may be used to encode a given amino acid, each organism may preferably use certain codons. The presence of rarely used codons in a nucleic acid sequence will result in a depletion of the respective tRNA pools and, thereby, lower the translation efficiency. Thus, it may be advantageous to provide a polynucleotide comprising a nucleic acid sequence encoding a polypeptide as referred to above wherein said nucleic acid sequence is optimized for expression in the target organism with respect to the codon usage. In order to optimize the codon usage for a target organism, a plurality of known genes from the said organism may be investigated for the most commonly used codons encoding the amino acids. In a subsequent step, the codons of a nucleic acid sequence from the donor organism will be optimized by replacing the codons in the donor sequence by the codons most commonly used by the target organism for encoding the same amino acids. It is to be understood that if the same codon is used preferably by both organisms, no replacement will be necessary. For various target organisms, tables with the preferred codon usages are already known in the art; see e.g., http://www.kazusa.or.jp/Kodon/E.html. Moreover, computer programs exist for the optimization, e.g., the Leto software, version 1.0 (Entelechon GmbH, Germany) or the GeneOptimizer (Geneart AG, Germany). For the optimization of a nucleic acid sequence, several criteria may be taken into account. For example, for a given amino acid, always the most commonly used codon may be selected for each codon to be exchanged. Alternatively, the codons used by the target organism may replace those in a donor sequence according to their naturally frequency. Accordingly, at some positions even less commonly used codons of the target organism will appear in the optimized nucleic acid sequence. The distribution of the different replacement codons of the target organism to the donor nucleic acid sequence may be randomly. Preferred target organisms in accordance with the present invention are wheat, maize, oat, triticale, rye, barley and rice species. Preferably, the polynucleotide of the present invention has an optimized nucleic acid for codon usage in the envisaged target organism wherein at least 20%, at least 40%, at least 60%, at least 80% or all of the relevant codons are adapted.

Moreover, the polypeptides encoded by the polynucleotides of the present invention are, advantageously, capable of increasing the amount of phythosterol compounds in plants significantly. Thus, the polynucleotides of the present invention are, in principle, useful for the synthesis of phythosterol related compounds such as campesterol, campestanol, stigmasterol, sitosterol, sitostanol, Δ 5-avenasterol, Δ 7-avenasterol or other sterols.

Moreover, they may be used to generate transgenic cereal plants or kernels thereof having a modified, preferably increased, amount of phythosterol related compounds. Such transgenic plants or kernels may be used for the manufacture of kernel oil, kernel flour or other phythosterol containing compositions.

Further, the present invention relates to a vector comprising the polynucleotide of the present invention. Preferably, the vector is an expression vector. The term "vector", preferably, encompasses phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homolgous recombination or heterologous insertion as described in detail below. The vector encompassing the polynucleotides of the present invention, preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. If introduced into a host cell, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous

recombination or heterologous insertion, see below. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. An "expression vector" according to the present invention is characterized in that it comprises an expression control sequence such as promoter and/or enhancer sequence operatively linked to the polynucleotide of the present invention. Preferred vectors, expression vectors and transformation or transfection techniques are specified elsewhere in this specification in detail. Furthermore, the present invention encompasses a host cell comprising the polynucleotide or vector of the present invention.

Host cells are primary cells or cell lines derived from multicellular organisms such as plants or animals. Furthermore, host cells encompass prokaryotic or eukaryotic single cell organisms (also referred to as microorganisms), e.g. bacteria or fungi including yeast or bacteria. Primary cells or cell lines to be used as host cells in accordance with the present invention may be derived from the multicellular organisms, preferably from plants.

Specifically preferred host cells, microorganisms or multicellular organism from which host cells may be obtained are disclosed below.

The polynucleotides or vectors of the present invention may be incorporated into a host cell or a cell of a transgenic non-human organism by heterologous insertion or homologous recombination. "Heterologous" as used in the context of the present invention refers to a polynucleotide which is inserted (e.g., by ligation) or is manipulated to become inserted to a nucleic acid sequence context which does not naturally encompass the said polynucleotide, e.g., an artificial nucleic acid sequence in a genome of an organism. Thus, a heterologous polynucleotide is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous polynucleotides encode proteins that are normally not produced by the cell expressing the said heterologous polynucleotide. An expression control sequence as used in a targeting construct or expression vector is considered to be "heterologous" in relation to another sequence (e.g., encoding a marker sequence or an agronomically relevant trait) if said two sequences are either not combined or operatively linked in a different way in their natural environment. Preferably, said sequences are not operatively linked in their natural environment (i.e. originate from different genes). Most preferably, said regulatory sequence is covalently joined (i.e. ligated) and adjacent to a nucleic acid to which it is not adjacent in its natural environment. "Homologous" as used in accordance with the present invention relates to the insertion of a polynucleotide in the sequence context in which the said polynucleotide naturally occurs. Usually, a heterologous polynucleotide is also incorporated into a cell by homologous recombination. To this end, the heterologous polynucleotide is flanked by nucleic acid sequences being homologous to a target sequence in the genome of a host cell or a non-human organism. Homologous recombination now occurs between the homologous sequences. However, as a result of the homologous recombination of the flanking sequences, the heterologous polynucleotide will be inserted, too. How to prepare suitable target constructs for homologous recombination and how to carry out the said homologous recombination is well known in the art.

Also provided in accordance with the present invention is a method for the manufacture of a polypeptide having lipoprotein activity comprising: (a) expressing the polynucleotide of the present invention in a host cell; and

(b) obtaining the polypeptide encoded by said polynucleotide from the host cell.

The polypeptide may be obtained, for example, by all conventional purification techniques including affinity chromatography, size exclusion chromatography, high pressure liquid chromatography (HPLC) and precipitation techniques including antibody precipitation. It is to be understood that the method may - although preferred - not necessarily yield an essentially pure preparation of the polypeptide. It is to be understood that depending on the host cell which is used for the aforementioned method, the polypeptides produced thereby may become posttranslationally modified or processed otherwise.

The present invention, moreover, pertains to a polypeptide encoded by the polynucleotide of the present invention or which is obtainable by the aforementioned method of the present invention. The term "polypeptide" as used herein encompasses essentially purified polypeptides or polypeptide preparations comprising other proteins in addition. Further, the term also relates to the fusion proteins or polypeptide fragments being at least partially encoded by the polynucleotide of the present invention referred to above. Moreover, it includes chemically modified polypeptides. Such modifications may be artificial modifications or naturally occurring modifications such as phosphorylation, glycosylation, myristylation and the like. The terms "polypeptide", "peptide" or "protein" are used interchangeable throughout this specification. The polypeptide of the present invention shall exhibit the biological activities referred to above, i.e. lipoprotein activity and, more preferably, it shall be capable of increasing the amount of phythosterol related compounds, preferably avenasterol, when present in plant kernels as referred to above.

The present invention also relates to a transgenic non-human organism comprising the polynucleotide, the vector or the host cell of the present invention. Preferably, said non- human transgenic organism is a plant. The term "non-human transgenic organism", preferably, relates to a plant, an animal or a multicellular microorganism. The polynucleotide or vector may be present in the cytoplasm of the organism or may be incorporated into the genome either heterologous or by homologous recombination. Host cells, in particular those obtained from plants or animals, may be introduced into a developing embryo in order to obtain mosaic or chimeric organisms, i.e. non-human transgenic organisms comprising the host cells of the present invention. Preferably, the non-human transgenic organism expresses the polynucleotide of the present invention in order to produce the polypeptide in an amount resulting in a detectable lipoprotein activity. Suitable transgenic organisms are, preferably, all those organisms which are capable of synthesizing phythosterol related compounds. Preferred organisms and methods for transgenesis are disclosed in detail below. A transgenic organism or tissue may comprise one or more transgenic cells. Preferably, the organism or tissue is substantially consisting of transgenic cells (i.e., more than 80%, preferably 90%, more preferably 95%, most preferably 99% of the cells in said organism or tissue are transgenic). The term "transgene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell or which has been manipulated by

experimental manipulations including techniques such as chimerablasty. Preferably, said sequence is resulting in a genome which is significantly different from the overall genome of an organism (e.g., said sequence, if endogenous to said organism, is introduced into a location different from its natural location, or its copy number is increased or decreased). A transgene may comprise an endogenous polynucleotide (i.e. a polynucleotide having a nucleic acid sequence obtained from the same organism or host cell) or may be obtained from a different organism or host cell, wherein said different organism is, preferably an organism of another species and the said different host cell is, preferably, a different microorganism, a host cell of a different origin or derived from a an organism of a different species.

Particularly preferred as a plant to be used in accordance with the present invention are starch producing plant species. Most preferably, the said plant is selected from the group consisting of wheat, maize, oat, triticale, rye, barley and rice. The present invention relates to a method for the manufacture of a phythosterol related compound and/or avenasterol comprising the steps of:

(a) cultivating the host cell or the transgenic non-human organism of the present

invention under conditions allowing synthesis of the said phythosterol related compound and/or avenasterol; and

(b) obtaining the said phythosterol related compound and/or avenasterol from the host cell or the transgenic non-human organism. The term "phythosterol related compound" and "avenasterol" as used herein refer, preferably, to those recited in Table 1 (phythosterol related compounds, e.g. but not limited to campesterol, campestanol, stigmasterol, sitosterol, sitostanol, Δ 5-avenasterol, Δ 7- avenasterol) below. However, the term in principle also encompass other phythosterol related compounds which can be obtained by the phythosterol biosynthetic pathway in a host cell or an organism referred to in accordance with the present invention.

The term "avenasterol" as used herein refers to A5-avenasterol and/or A7-avenasterol.

The term "cereal" as used herein refers to one of the following plants: wheat, oat, barley, triticale, rye, corn or rice.

Moreover, the present invention pertains to a method for the manufacture of a plant having a modified amount of a phythosterol related compounds, preferably A5-avenasterol and/or A7-avenasterol, comprising the steps of:

(a) introducing the polynucleotide or the vector of the present invention into a plant cell; and

(b) generating a transgenic plant from the said plant cell, wherein the polypeptide

encoded by the polynucleotide modifies the amount of the said seed storage compound in the transgenic plant.

The term "phythosterol related compound" as used herein, preferably, refers to compounds being campesterol, campestanol, stigmasterol, sitosterol, sitostanol, Δδ-avenasterol, Δ7- avenasterol or other sterols, more preferably, avenasterol. Preferably, the amount of said phythosterol related compound is significantly increased compared to a control, preferably an empty vector control as specified above. The increase is, more preferably, an increase in the amount by weight of at least 1 , 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 % as compared to a control. It is to be understood that the polynucleotides or the vector referred to in accordance with the above method of the present invention may be introduced into the plant cell by any of the aforementioned insertion or recombination techniques. The nucleic acid variants, preferably, also comprise nucleic acids having a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequences by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a polypeptide having a biological activity as specified above. Variants also encompass nucleic acids comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in

Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1 -6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6 χ sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 ^χ SSC, 0.1 % SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under

"standard hybridization conditions" the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 ^χ SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50%

formamide, the temperature under standard conditions is approximately 42°C. The hybridization conditions for DNA:DNA hybrids are, preferably, 0.1 ^χ SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for DNA:RNA hybrids are, preferably, 0.1 ^χ SSC and 30°C to 55°C, preferably between 45°C and 55°C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991 , "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, nucleic acid variants are obtainable by PCR-based techniques such as mixed

oligonucleotide primer-based amplification of DNA, i.e. using degenerated primers against conserved domains of the polypeptides of the present invention. Conserved domains of the specific polypeptides of the present invention may be identified by a sequence comparison of the nucleic acid sequences or the amino acid sequences of the polypeptides of the present invention. Oligonucleotides suitable as PCR primers as well as suitable PCR conditions are described in the accompanying Examples. As a template, DNA or cDNA from bacteria, fungi, plants or animals may be used. Further, variants include nucleic acids comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical of the specific nucleic acid sequences, wherein the polypeptides encoded by the polynucleotides retain the biological activities of the aforementioned specific polypeptides. Moreover, also encompassed are nucleic acids which comprise nucleic acid sequences encoding amino acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequences of the specific polypeptides encoded by the polynucleotide, wherein the polypeptides encoded by the variant amino acid sequences retain the biological activity of the aforementioned specific polypeptides. The percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (J. Mol.

Evolution., 25, 351 -360, 1987, Higgins et al., CABIOS, 5 1989: 151 -153) or the programs Gap and BestFit (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981 ))), which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)], are to be used. The sequence identity values recited above in percent (%) are to be determined, preferably, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences can be also determined using the Vector NTI 7.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814). A gap-opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap-opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two

polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap-opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide sequence is equivalent to an uracil nucleotide. Variant nucleic acids as referred to in accordance with the present invention may be obtained by various natural as well as artificial sources. For example, nucleic acids may be obtained by in vitro and in vivo mutagenesis approaches using the above mentioned specific nucleic acids as a basis. Moreover, nucleic acids being homologs or orthologs may be obtained from various animal, plant, bacteria or fungus species. Paralogs may be identified from the species from which the specific sequences are derived. This is, in general, achieved by changing the codons of a nucleic acid sequence obtained from a first organism (i.e. the donor organism) encoding a given amino acid sequence into the codons normally used by the target organism whereby the amino acid sequence is retained. It is in principle acknowledged that the genetic code is redundant (i.e.

degenerated). Specifically, 61 codons are used to encode only 20 amino acids. Thus, a majority of the 20 amino acids will be encoded by more than one codon. The codons for the amino acids are well known in the art and are universal to all organisms. However, among the different codons which may be used to encode a given amino acid, each organism may preferably use certain codons. The presence of rarely used codons in a nucleic acid sequence will result a depletion of the respective tRNA pools and, thereby, lower the translation efficiency. Thus, it may be advantageous to provide a fusion polynucleotide comprising a nucleic acid sequence encoding a polypeptide as referred to above wherein said nucleic acid sequence is optimized for expression in the target organism with respect to the codon usage. In order to optimize the codon usage for a target organism, a plurality of known genes from the said organism may be investigated for the most commonly used codons encoding the amino acids. In a subsequent step, the codons of a nuclei acid sequence from the donor organism will be optimized by replacing the codons in the donor sequence by the codons most commonly used by the target organism for encoding the same amino acids. It is to be understood that if the same codon is used preferably by both organisms, no replacement will be necessary. For various target organisms, tables with the preferred codon usages are already known in the art; see e.g.,

http://www.kazusa.or.jp/Kodon/E.html. Moreover, computer programs exist for the optimization, e.g., the Leto software, version 1.0 (Entelechon GmbH, Germany) or the GeneOptimizer (Geneart AG, Germany). For the optimization of a nucleic acid sequence, several criteria may be taken into account. For example, for a given amino acid, always the most commonly used codon may be selected for each codon to be exchanged.

Alternatively, the codons used by the target organism may replace those in a donor sequence according to their naturally frequency. Accordingly, at some positions even less commonly used codons of the target organism will appear in the optimized nucleic acid sequence. The distribution of the different replacment codons of the target organism to the donor nucleic acid sequence may be randomly. Preferred target organisms in accordance with the present invention are soybean or canola (Brassica) species. Preferably, the fusion polynucleotide of the present invention or at least the nucleic acids comprised thereby have an optimized nucleic acid for codon usage in the envisaged target organism wherein at least 20%, at least 40%, at least 60%, at least 80% or all of the relevant codons are adapted.

It has been found in the studies underlying the present invention that the combinations of polypeptides referred to herein above are, advantageously, capable of modulating the amount of phythosterol compounds in plants significantly. Suitable expression control sequences are referred to elsewhere in this specification and include promoters which allow for transcription in plants, preferably, in plant seeds. More preferably, a promoter to be used as an expression control sequence for a nucleic acid sequence comprised by the polynucleotide of the invention is selected from the group consisting of: USP, SBP1000, BnGLP, STPT, LegB4, LuPXR1727, Vicillin, Napin A, LuPXR, Conlinin, pVfSBP, Leb4, pVfVic and Oleosin. It is to be understood that, more preferably, a first nucleic acid is driven by a first expression control sequence while a second nucleic acid comprised by the additional polynucleotide is driven by a second expression control sequence being different from the said first expression control sequence.

The nucleic acid sequences are also, preferably, operatively linked to a terminator sequence, i.e. a sequence which terminates transcription of RNA. Suitable terminator sequences are referred to elsewhere in this specification and include terminator sequences which allow for termination of transcription in plants, preferably, in plant seeds or kernels. More preferably, a terminator sequence for a nucleic acid sequence is selected from the group consisting of: tCaMV35S, OCS, AtGLP, AtSACPD, Leb3, CatpA, t-AtPXR, E9 and t- AtTIP. It is to be understood that, more preferably, the transcription of a first nucleic acid is terminated by a first terminator sequence while the transcription of a second nucleic acid sequence is terminated by a second terminator sequence being different from the said first terminator sequence.

The present invention also contemplates a host cell wherein said first polypeptide is encoded by a nucleic acid selected from the group consisting of:

a) a nucleic acid having a nucleic acid sequence as shown in SEQ ID NO: 1 ;

b) a nucleic acid encoding an amino acid sequence as shown in SEQ ID NO: 2; and c) a nucleic acid being at least 70% identical to the nucleic acid of a) or b), wherein said second polypeptide is encoded by a nucleic acid is selected from the group consisting of:

a) a nucleic acid having a nucleic acid sequence as shown in SEQ ID NO: 3;

b) a nucleic acid encoding an amino acid sequence as shown in SEQ ID NOs: 4; and c) a nucleic acid being at least 70% identical to the nucleic acid of a) or b).

The polypeptides may be encoded by separate polynucleotides comprising the nucleic acids encoding the aforementioned polypeptides. Such separate polynucleotides may be either transiently introduced into the host cell (e.g., by expression vectors) or permanently integrated into its genome (e.g., as an expression cassette). It will be understood that the separate polynucleotides preferably also comprise in addition to the nucleic acid to be expressed (i.e. the nucleic acid encoding the polypeptide of the required combination of polypeptides) suitable expression control and/or terminator sequences. Such expression control and/or terminator sequences shall also be operatively linked to the nucleic acid comprised by the separate polynucleotides as to allow expression of the nucleic acid and/or termination of the transcription.

The present invention also relates to a transgenic non-human organism comprising the fusion polynucleotide, the aforementioned vector or the aforementioned host cell of the present invention. More preferably, said non-human transgenic organism is a plant. The explanations of the terms given elsewhere in this specification, apply accordingly.

The present invention further relates to a method for the manufacture of phythosterol related compounds comprising the steps of:

(a) cultivating the aforementioned host cell or transgenic non-human organism under conditions allowing synthesis of the said phythosterol related compounds; and

(b) obtaining the said phythosterol related compounds from the host cell or the

transgenic non-human organism.

The explanations of the terms given elsewhere in this specification, apply accordingly.

More preferably, the amount of said phythosterol related compound is increased compared to a non-transgenic control plant. Most preferably, said phythosterol related compound is avenasterol. The explanations of the terms given elsewhere in this specification, apply accordingly.

The aforementioned method of the present invention may be also used to manufacture a plant having an altered phythosterol content in its kernels or a plant having an altered avenasterol content in its kernels. Such plants are suitable sources for phythosterol related compounds and especially avenasterol and may be used for the large scale manufacture thereof.

Further methods and uses of the aforementioned polynucleotides, vectors, host cells, organisms, methods and uses of the present invention will be described also below.

Moreover, the terms used above will be explained in more detail.

The present invention further relates to combinations of polynucleotides encoding PMPs and order thereof within the combinations, resulting in coordinated presence of proteins associated with the metabolism of phythosterol compounds in plants.

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included therein.

Before the present compounds, compositions, and methods are disclosed and described, it is to be understood that this invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.

The term "transgenic" or "recombinant" when used in reference to a cell or an organism (e.g., with regard to a wheat, maize, oat, rye, barley and rice plant or plant cell) refers to a cell or organism which contains a transgene, or whose genome has been altered by the introduction of a transgene. A transgenic organism or tissue may comprise one or more transgenic cells. Preferably, the organism or tissue is substantially consisting of transgenic cells (i.e., more than 80%, preferably 90%, more preferably 95%, most preferably 99% of the cells in said organism or tissue are transgenic). The term "transgene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell or which has been manipulated by experimental manipulations by man. Preferably, said sequence is resulting in a genome which is different from a naturally occurring organism (e.g., said sequence, if endogenous to said organism, is introduced into a location different from its natural location, or its copy number is increased or decreased). A transgene may be an "endogenous DNA sequence", "an "exogenous DNA sequence" (e.g., a foreign gene), or a "heterologous DNA sequence". The term "endogenous DNA sequence" refers to a nucleotide sequence, which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence.

The term "wild-type", "natural" or of "natural origin" means with respect to an organism, polypeptide, or nucleic acid sequence, that said organism is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.

The terms "heterologous nucleic acid sequence" or "heterologous DNA" are used interchangeably to refer to a nucleotide sequence, which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. A promoter, transcription regulating sequence or other genetic element is considered to be "heterologous" in relation to another sequence (e.g., encoding a marker sequence or an agronomically relevant trait) if said two sequences are not combined or differently operably linked their natural environment. Preferably, said sequences are not operably linked in their natural environment (i.e. come from different genes). Most preferably, said regulatory sequence is covalently joined and adjacent to a nucleic acid to which it is not adjacent in its natural environment. One aspect of the invention pertains to combinations of isolated nucleic acid molecules that encode PMP polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of a PMP-encoding nucleic acid (e.g., PMP DNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3^' and 5^' ends of the coding region of a gene: at least about 1000 nucleotides of sequence upstream from the 5^' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3^' end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one, which is substantially separated from other nucleic acid molecules, which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is substantially free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5^' and 3^' ends of the nucleic acid) in the genomic DNA of the organism, from which the nucleic acid is derived. For example, in various embodiments, the isolated PMP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences, which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be

substantially free of other cellular material, or culture medium when produced by

recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention (i.e. the polynucleotide or fusion

polynucleotide of the invention), e.g., a nucleic acid molecule consisting of a combination of isolated nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 3 or a portion thereof, can be constructed using standard molecular biology techniques and the sequence information provided herein. For example, an Nicotiana tabacum, Hevea brasiliensis, Arabidopsis thaliana, Helianthus annuus, Escherichia coli, Saccharomyces cerevisiae or Physcomitrella patens, Brassica napus, Glycine max or Linum usitatissimum PMP cDNA can be isolated from an Arabidopsis thaliana, Helianthus annuus, Escherichia coli, Saccharomyces cerevisiae or Physcomitrella patens, Brassica napus, Glycine max or Linum usitatissimum library using all or portion of one of the sequences of SEQ ID NO: 1 and SEQ ID NO: 3 as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor

Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Moreover, a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO: 1 and SEQ ID NO: 3 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO: 1 and SEQ ID NO: 3 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence of SEQ ID NO: 1 and SEQ ID NO: 3). For example, mRNA can be isolated from plant cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. 1979, Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and may contain restriction enzyme sites or sites for ligase independent cloning to construct the combinations described by this invention. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acids so amplified can be cloned into an appropriate vector in the combinations described by the present invention or variations thereof and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a PMP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In still another preferred embodiment, an isolated nucleic acid molecule in the combinations of the invention comprises a nucleotide sequence, which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, or 90- 95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to one or more nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, or a portion thereof. In an additional preferred embodiment, an isolated nucleic acid molecule in the combinations of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one or more of the nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 3, or a portion thereof.

For the purposes of the invention hybridization means preferably hybridization under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2 X SSC, 0. 1 % SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.5 X SSC, 0. 1 % SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 65°C to a nucleic acid comprising 50 to 200 or more consecutive nucleotides. A further preferred, non-limiting example of stringent hybridization conditions includes washing with a solution having a salt concentration of about 0.02 molar at pH 7 at about 60°C.

Moreover, the nucleic acid molecule in the combinations of the invention can comprise only a portion of the coding region of one of the sequences in SEQ ID NO: 1 and SEQ ID NO: 3, for example a fragment, which can be used as a probe or primer or a fragment encoding a biologically active portion of a PMP. The nucleotide sequences determined from the cloning of the PMP from Nicotiana tabacum, Hevea brasiliensis, Arabidopsis thaliana, Brassica napus, Helianthus annuus, Escherichia coli, Saccharomyces cerevisiae or Physcomitrella patens, allows for the generation of probes and primers designed for use in identifying and/or cloning PMP homologues in other cell types and organisms, as well as PMP homologues from other plants or related species. Therefore this invention also provides compounds comprising the combinations of nucleic acids disclosed herein, or fragments thereof. These compounds include the nucleic acid combinations attached to a moiety. These moieties include, but are not limited to, detection moieties, hybridization moieties, purification moieties, delivery moieties, reaction moieties, binding moieties, and the like. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50, or 75 consecutive nucleotides of a sense strand of one of the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3, an anti-sense sequence of one of the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3 can be used in PCR reactions to clone PMP homologues for the combinations described by this inventions or variations thereof. Probes based on the PMP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred

embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic marker test kit for identifying cells which express a PMP, such as by measuring a level of a PMP-encoding nucleic acid in a sample of cells, e.g., detecting PMP mRNA levels, or determining whether a genomic PMP gene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes a combination of proteins or portions thereof, which include amino acid sequences, which are sufficiently homologous to an amino acid encoded by a sequence of SEQ ID NO: 2 or SEQ ID NO: 4, such that the protein or portion thereof maintains the same or a similar function as the wild- type protein. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof, which have amino acid sequences, which include a minimum number of identical or equivalent (e.g., an amino acid residue, which has a similar side chain as an amino acid residue in one of the ORFs of a sequence of SEQ ID NO: 1 or SEQ ID NO: 3) amino acid residues to an amino acid sequence, such that the protein or portion thereof is able to participate in the metabolism of compounds necessary for the production of phythosterol related compounds in plants, construction of cellular membranes in

microorganisms or plants, or in the transport of molecules across these membranes.

Examples of PMP-encoding nucleic acid sequences are set forth in SEQ ID NO: 1 and SEQ ID NO: 3. Portions of proteins encoded by the PMP nucleic acid molecules of the invention are preferably biologically active portions of one of the PMPs. As used herein, the term

"biologically active portion of a PMP" is intended to include a portion, e.g., a domain/motif, of a PMP that participates in the metabolism of compounds necessary for the biosynthesis of phythosterol related compounds, or the construction of cellular membranes in

microorganisms or plants, or in the transport of molecules across these membranes, or has an activity as set forth in Table 3. To determine whether a PMP or a biologically active portion thereof can participate in the metabolism of compounds necessary for the production of phythosterol related compounds and cellular membranes, an assay of enzymatic activity may be performed. Such assay methods are well known to those skilled in the art.

Biologically active portions of a PMP include peptides comprising amino acid sequences derived from the amino acid sequence of a PMP (e.g., an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3 or the amino acid sequence of a protein homologous to a PMP, which include fewer amino acids than a full length PMP or the full length protein which is homologous to a PMP) and exhibit at least one activity of a PMP. Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of a PMP. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of a PMP include one or more selected domains/motifs or portions thereof having biological activity. Additional nucleic acid fragments encoding biologically active portions of a PMP can be prepared by isolating a portion of one of the sequences, expressing the encoded portion of the PMP or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the PMP or peptide. The invention further encompasses combinations of nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 (and portions thereof) due to degeneracy of the genetic code and thus encode the same PMP as that encoded by the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3. In a further embodiment, the combinations of nucleic acid molecule of the invention encode one or more full-length proteins, which are substantially homologous to an amino acid sequence of a polypeptide encoded by an open reading frame shown in SEQ ID NO: 1 and SEQ ID NO: 3. In one embodiment, the full-length nucleic acid or protein, or fragment of the nucleic acid or protein, is from Nicotiana tabacum, Hevea brasiliensis, Arabidopsis thaiiana, Brassica napus, Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens.

In addition to the Nicotiana tabacum and Hevea brasiliensis PMP nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of PMPs may exist within a Nicotiana tabacum and Hevea brasiliensis population. Such genetic polymorphism in the PMP gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a PMP, preferably a Nicotiana tabacum and Hevea brasiliensis, Arabidopsis thaiiana, Brassica napus,

Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens PMP. Such natural variations can typically result in 1 -40% variance in the nucleotide sequence of the PMP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in PMP that are the result of natural variation and that do not alter the functional activity of PMPs are intended to be within the scope of the invention. The invention further encompasses combinations of nucleic acid molecules corresponding to natural variants and non- Nicotiana tabacum and Hevea brasiliensis, Arabidopsis thaiiana, Brassica napus, Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens orthologs of the Arabidopsis thaiiana, Brassica napus, Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens PMP nucleic acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3. Nucleic acid molecules corresponding to natural variants and non- Nicotiana tabacum and Hevea brasiliensis, Arabidopsis thaiiana, Brassica napus, Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens orthologs of the Nicotiana tabacum and Hevea brasiliensis , Arabidopsis thaiiana, Brassica napus, Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens PMP cDNA described in SEQ ID NO: 1 and SEQ ID NO: 3 can be isolated based on their homology to Nicotiana tabacum and Hevea brasiliensis, Arabidopsis thaiiana, Brassica napus, Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens PMP nucleic acid shown in SEQ ID NO: 1 and SEQ ID NO: 3 using the Nicotiana tabacum and Hevea brasiliensis ,

Arabidopsis thaiiana, Brassica napus, Helianthus annuus, Escherichia coii, Saccharomyces cerevisiae or Physcomitreiia patens cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. As used herein, the term "orthologs" refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode proteins having the same or similar functions. Accordingly, in another embodiment, an isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3. In other embodiments, the nucleic acid is at least 30, 50, 100, 250, or more nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing, under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989: 6.3.1 -6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65°C. Preferably, an isolated nucleic acid molecule that hybridizes under stringent conditions to a sequence of SEQ ID NO: 1 or SEQ ID NO: 3 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to a RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

In addition to naturally-occurring variants of the PMP sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3, thereby leading to changes in the amino acid sequence of the encoded PMP, without altering the functional ability of the PMP. For example, nucleotide substitutions leading to amino acid

substitutions at "non-essential" amino acid residues can be made in a sequence of SEQ ID NO: 1 and SEQ ID NO: 3. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the PMPs (SEQ ID NO: 1 and SEQ ID NO: 3) without altering the activity of said PMP, whereas an "essential" amino acid residue is required for PMP activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having PMP activity) may not be essential for activity and thus are likely to be amenable to alteration without altering PMP activity.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding PMPs that contain changes in amino acid residues that are not essential for PMP activity. Such PMPs differ in amino acid sequence from a sequence yet retain at least one of the PMP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3 and is capable of participation in the metabolism of compounds necessary for the production of phythosterol compounds in wheat, maize, oat, rye, barley and rice, or cellular membranes, or has one or more activities set forth in Table 3. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3, more preferably at least about 60-70% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3 even more preferably at least about 70-80%, 80-90%, 90-95% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3, and most preferably at least about 96%, 97%, 98%, or 99% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3.

To determine the percent homology of two amino acid sequences (e.g., one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3 and a mutant form thereof), or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant form of the sequence selected from the polypeptide encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3), then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = numbers of identical positions/total numbers of positions x 100). The sequence identity can be generally based on any one of the full length sequences of SEQ ID NO: 1 and SEQ ID NO: 3 as 100 %.

For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences is determined using the Vector NTI 7.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814). A gap-opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap-opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap- opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide sequence is equivalent to an uracil nucleotide. An isolated nucleic acid molecule encoding a PMP homologous to a protein sequence encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3 such that one or more amino acid

substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the sequences of SEQ ID NO: 1 and SEQ ID NO: 3 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

Preferably, conservative amino acid substitutions are made at one or more predicted non- essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in a PMP is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a PMP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a PMP activity described herein to identify mutants that retain PMP activity. Following mutagenesis of one of the sequences of SEQ ID NO: 1 and SEQ ID NO: 3, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using, for example, assays described in the scientific literature.

Combinations of PMPs are preferably produced by recombinant DNA techniques. For example, one or more nucleic acid molecule is encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described herein), and the PMPs are expressed in the host cell. The PMPs can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, one or more PMP or peptide thereof can be synthesized chemically using standard peptide synthesis techniques.

Moreover, native PMPs can be isolated from cells, for example using an anti-PMP antibody, which can be produced by standard techniques utilizing a PMP or fragment thereof of this invention.

The invention also provides combinations of PMP chimeric or fusion proteins. As used herein, a PMP "chimeric protein" or "fusion protein" comprises a PMP polypeptide operatively linked to a non-PMP polypeptide. A "PMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a PMP, whereas a "non-PMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the PMP, e.g., a protein which is different from the PMP, and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the PMP polypeptide and the non-PMP polypeptide are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used. The non-PMP polypeptide can be fused to the N-terminus or C-terminus of the PMP polypeptide. For example, in one embodiment, the fusion protein is a GST-PMP (glutathione S-transferase) fusion protein in which the PMP sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant PMPs. In another embodiment, the fusion protein is a PMP containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a PMP can be increased through use of a heterologous signal sequence.

Preferably, a combination of PMP chimeric or fusion proteins of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with

conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can

subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A PMP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the PMP.

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a combination of nucleic acids encoding PMPs (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid, to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell, into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes, to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid," and "vector" can be used inter-changeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses,

adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors of the invention comprise a combination of nucleic acids of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence and both sequences are fused to each other so that each fulfills its proposed function (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene

Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and

Biotechnolgy, CRC Press, Boca Raton, Florida, eds.: Glick & Thompson, Chapter 7, 89- 108 including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., PMPs, mutant forms of PMPs, fusion proteins, etc.). The recombinant expression vectors of the invention can be designed for expression of combinations of PMPs in prokaryotic or eukaryotic cells. For example, PMP genes can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos M.A. et al. 1992, Foreign gene expression in yeast: a review, Yeast 8:423-488; van den Hondel, C.A.M.J.J. et al. 1991 , Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, Bennet & Lasure, eds., p. 396-428:Academic Press: an Diego; and van den Hondel & Punt 1991 , Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1 -28, Cambridge University Press: Cambridge), algae (Falciatore et al. 1999, Marine Biotechnology 1 :239-251 ), ciliates of the types:

Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especially of the genus Stylonychia lemnae with vectors following a

transformation method as described in WO 98/01572 and multicellular plant cells (see Schmidt & Willmitzer 1988, High efficiency Agrobacterium tumefaciens-med\a .ed

transformation of Arabidopsis thaliana leaf and cotyledon plants, Plant Cell Rep.:583-586); Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Florida, chapter 6/7,

S.71 -119 (1993); White, Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds.: Kung and Wu, Academic Press 1993, 128-43; Potrykus 1991 , Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225 (and references cited therein) or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA

1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve one or more of the following purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the

recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase.

One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein

(Gottesman S. 1990, Gene Expression Technology: Methods in Enzymology 185:119-128, Academic Press, San Diego, California). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression (Wada et al. 1992, Nucleic Acids Res. 20:21 11 -21 18). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques. In another embodiment, the PMP combination expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSed

(Baldari et al. 1987, Embo J. 6:229-234), pMFa (Kurjan & Herskowitz 1982, Cell 30:933- 943), pJRY88 (Schultz et al. 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel & Punt 1991 , "Gene transfer systems and vector development for filamentous fungi," in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1 -28, Cambridge University Press: Cambridge.

Alternatively, the combinations of PMPs of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow & Summers 1989, Virology 170:31-39).

In yet another embodiment, a combination of nucleic acids of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed 1987, Nature 329:840) and pMT2PC (Kaufman et al. 1987, EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, Fritsh and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In another embodiment, a combination of the PMPs of the invention may be expressed in unicellular plant cells (such as algae, see Falciatore et al. (1999, Marine Biotechnology 1 :239-251 and references therein) and plant cells from higher plants (e.g., the

spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, Kemper, Schell and Masterson (1992, "New plant binary vectors with selectable markers located proximal to the left border," Plant Mol. Biol. 20:1195-1 197) and Bevan (1984, "Binary Agrobacterium vectors for plant transformation," Nucleic Acids Res. 12:871 1 -8721 ; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds.: Kung und R. Wu, Academic Press, 1993, S. 15-38).

A plant expression cassette preferably contains regulatory sequences capable to drive gene expression in plant cells, and which are operably linked so that each sequence can fulfill its function such as termination of transcription, including polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al. 1984, EMBO J. 3:835) or functional equivalents thereof. But also all other terminators functionally active in plants are suitable. As plant gene expression is very often not limited on transcriptional levels a plant expression cassette preferably contains other operably-linked sequences, like translational enhancers such as the overdrive-sequence containing the 5^'-untranslated leader sequence from tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al. 1987, Nucleic Acids Res. 15:8693-871 1 ).

Plant gene expression has to be operably linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner. Preferred are promoters driving constitutive expression (Benfey et al. 1989, EMBO J. 8:2195-2202) like those derived from plant viruses like the 35S CAMV (Franck et al. 1980, Cell 21 :285-294), the 19S CaMV (see also US 5,352,605 and WO 84/02913) or the ptxA promoter (Bown, D.P. PhD thesis (1992) Department of Biological Sciences, University of Durham, Durham, U.K) or plant promoters like those from Rubisco small subunit described in US 4,962,028. Even more preferred are seed-specific promoters driving expression of PMP proteins during all or selected stages of seed development. Seed-specific plant promoters are known to those of ordinary skill in the art and are identified and characterized using seed-specific mRNA libraries and expression profiling techniques. Seed-specific promoters include the napin-gene promoter from rapeseed (US 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al. 1991 , Mol. Gen. Genetics 225:459-67), the oleosin-promoter from Arabidopsis (WO 98/45461 ), the phaseolin- promoter from Phaseolus vulgaris (US 5,504,200), the Bce4-promoter from Brassica (W091 13980) or the legumin B4 promoter (LeB4; Baeumlein et al. 1992, Plant J. 2:233-239), as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice etc. Suitable promoters to note are the Ipt2 or Ipt1 -gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO

99/16890 (promoters from the barley hordein-gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the Sorghum kasirin-gene, and the rye secalin gene).

Plant gene expression can also be facilitated via an inducible promoter (for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are especially suitable if gene expression is desired in a time specific manner. Examples for such promoters are a salicylic acid inducible promoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al. 1992, Plant J. 2:397-404) and an ethanol inducible promoter (WO 93/21334). Promoters responding to biotic or abiotic stress conditions are also suitable promoters such as the pathogen inducible PRP1 -gene promoter (Ward et al., 1993, Plant Mol. Biol. 22:361 - 366), the heat inducible hsp80-promoter from tomato (US 5,187,267), cold inducible alpha- amylase promoter from potato (WO 96/12814) or the wound-inducible pinll-promoter (EP 375091 ). Other preferred sequences for use in plant gene expression cassettes are targeting- sequences necessary to direct the gene-product in its appropriate cell compartment (for review see Kermode 1996, Crit. Rev. Plant Sci. 15:285-423 and references cited therein) such as the vacuole, the nucleus, all types of plastids like amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells. Also especially suited are promoters that confer plastid-specific gene expression, as plastids are the compartment where precursors and some end products of lipid biosynthesis are synthesized. Suitable promoters such as the viral RNA-polymerase promoter are described in WO 95/16783 and WO 97/06250 and the clpP-promoter from Arabidopsis described in WO 99/46394.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is to be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a combination of PMPs can be expressed in bacterial cells, insect cells, fungal cells, mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates, or plant cells. Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection," "conjugation," and "transduction" are intended to refer to a variety of art- recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or

electroporation. Suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and other laboratory manuals such as Methods in Molecular Biology 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, New

Jersey. For stable transfection of mammalian and plant cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, kanamycin, and methotrexate or in plants that confer resistance towards an herbicide, such as glyphosate or glufosinate. A nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a combination of PMPs or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is prepared that contains a combination of at least a portion of a PMP gene, into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the PMP gene. Preferably, this PMP gene is an Arabidopsis thaliana, Brassica napus, Helianthus annuus, Escherichia coli, Saccharomyces cerevisiae or Physcomitrella patens PMP gene, but it can be a homologue from a related plant or even from a mammalian, yeast, or insect source. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous PMP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a knock-out vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous PMP gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous PMP). To create a point mutation via homologous recombination, DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al. 1999, Nucleic Acids Res. 27:1323-1330 and Kmiec 1999, American Scientist 87:240-247). Homologous recombination procedures in Arabidopsis thaliana or other crops are also well known in the art and are contemplated for use herein.

In a homologous recombination vector, within the combination of genes coding for PMPs shown in Table 3 the altered portion of the PMP gene is flanked at its 5' and 3' ends by additional nucleic acid of the PMP gene to allow for homologous recombination to occur between the exogenous PMP gene carried by the vector and an endogenous PMP gene in a microorganism or plant. The additional flanking PMP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundreds of base pairs up to kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas & Capecchi 1987, Cell 51 :503, for a description of homologous recombination vectors). The vector is introduced into a microorganism or plant cell (e.g., via polyethyleneglycol mediated DNA). Cells in which the introduced PMP gene has homologously recombined with the endogenous PMP gene are selected using art- known techniques.

In another embodiment, recombinant microorganisms can be produced which contain selected systems, which allow for regulated expression of the introduced combinations of genes. For example, inclusion of a combination of one two or more PMP genes on a vector placing it under control of the lac operon permits expression of the PMP gene only in the presence of IPTG. Such regulatory systems are well known in the art. A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture can be used to produce (i.e., express) a combination of PMPs. Accordingly, the invention further provides methods for producing PMPs using the host cells of the invention. In one embodiment, the method comprises culturing a host cell of the invention (into which a recombinant expression vector encoding a combination of PMPs has been introduced, or which contains a wild-type or altered PMP gene in it's genome) in a suitable medium until the combination of PMPs is produced.

An isolated PMP or a portion thereof of the invention can participate in the metabolism of compounds necessary for the production of phythosterol compounds in plants such as wheat, maize, oat, rye, barley and rice or of cellular membranes, or has one or more of the activities set forth in Table 3. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3 such that the protein or portion thereof maintains the ability to participate in the metabolism of

compounds necessary for the construction of cellular membranes in plants such as wheat, maize, oat, rye, barley and rice, or in the transport of molecules across these membranes. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, a PMP of the invention has an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3. In yet another preferred embodiment, the PMP has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3. In still another preferred embodiment, the PMP has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70- 80%, 80-90%, 90-95%, and even more preferably at least about 96%, 97%, 98%, 99%, or more homologous to one of the amino acid sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3. The preferred PMPs of the present invention also preferably possess at least one of the PMP activities described herein. For example, a preferred PMP of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3, and which can participate in the metabolism of compounds necessary for the construction of cellular membranes in plants such as wheat, maize, oat, triticale, rye, barley and rice, or in the transport of molecules across these membranes, or which has one or more of the activities set forth in Table 3. In other embodiments, the combination of PMPs is substantially homologous to a combination of amino acid sequences encoded by nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 3 and retain the functional activity of the protein of one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3 yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail above. Accordingly the PMP is a protein which comprises an amino acid sequence which is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more homologous to an entire amino acid sequence and which has at least one of the PMP activities described herein. In another embodiment, the invention pertains to a full Nicotiana tabacum and a Helvea brasiliensis protein which is substantially homologous to an entire amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and SEQ ID NO: 3.

In addition, libraries of fragments of the PMP coding sequences can be used to generate a variegated population of PMP fragments for screening and subsequent selection of homologues of a PMP to be included in combinations. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a PMP coding sequence with a nuclease under conditions, wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA, which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived, which encodes N-terminal, C-terminal and internal fragments of various sizes of the PMP. Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of PMP homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify PMP homologues (Arkin & Yourvan 1992, Proc. Natl. Acad. Sci. USA 89:781 1 -7815;

Delgrave et al. 1993, Protein Engineering 6:327-331 ).

In another embodiment, cell based assays can be exploited to analyze a variegated PMP library, using methods well known in the art.

The nucleic acid molecules, proteins, protein homologues and fusion proteins for the combinations described herein, and vectors, and host cells described herein can be used in one or more of the following methods: identification of Nicotiana tabacum, Helvea brasiliensis, Arabidopsis thaliana, Brassica napus, Helianthus annuus, Escherichia coli, Saccharomyces cerevisiae or Physcomitrella patens and related organisms; mapping of genomes of organisms related to Nicotiana tabacum, Helvea brasiliensis, Arabidopsis thaliana, Brassica napus, Helianthus annuus, Escherichia coli, Saccharomyces cerevisiae or Physcomitrella patens; identification and localization of Nicotiana tabacum, Helvea brasiliensis, Arabidopsis thaliana, Brassica napus, Helianthus annuus, Escherichia coli, Saccharomyces cerevisiae or Physcomitrella patens sequences of interest; evolutionary studies; determination of PMP regions required for function; modulation of a PMP activity; modulation of the metabolism of one or more cell functions; modulation of the

transmembrane transport of one or more compounds; and modulation of phythosterol related compound accumulation.

Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and Examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the claims included herein.

Examples

Materials and General Methods

Unless indicated otherwise, chemicals and reagents in the examples were obtained from Sigma Chemical Company (St. Louis, MO), restriction endonucleases were from New England Biolabs (Beverly, MA) or Roche (Indianapolis, IN), oligonucleotides were synthesized by MWG Biotech Inc. (High Point, NC), and other modifying enzymes or kits regarding biochemicals and molecular biological assays were from Clontech (Palo Alto, CA), Pharmacia Biotech (Piscataway, NJ), Promega Corporation (Madison, Wl), or

Stratagene (La Jolla, CA). Materials for cell culture media were obtained from Gibco/BRL (Gaithersburg, MD) or DIFCO (Detroit, Ml). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, linking DNA fragments, transformation of bacteria, growing bacteria, and sequence analysis of recombinant DNA, are carried out as described by Sambrook (1989). The synthesis of DNA molecules was carried out by Entelechon GmbH (Regensburg, Germany) using state of the art technologies. EXAMPLE 1

Generation of T-DNA constructs for expression of improved DNA sequences encoding for a truncated form of the Hevea brasiliensis HMGR and the Nicotiana tabacum SMT1 in wheat

Improved DNA sequences encoding for the truncated form of the Hevea brasiliensis 3- hydroxy-3-methylglutaryl-CoA reductase (HMGR) and the Nicotiana tabacum Sterol

Methyltransferase 1 (SMT1) were synthesized based on the published sequences (Chye et al., 1991 and Bouvier-Nave et al., 1998). The truncated form of the Hevea brasiliensis HMGR lacking the membrane binding domain comprises the amino 153 to 575 of the HMGR protein (Harker et al., 2003). The improved truncated HMGR and SMT1 sequence were generated by adapting its codon usage to the codon usage of wheat. A list of codon usages for a large range of organisms and organelles may be found in the resources of the Japanese "Kasuza DNA Research Institute" provided in the internet under

http://www.kazusa.or.jp/codon/. In addition, the expression of the HMGR and the SMT1 may be further improved by removing or avoiding signals and/or structures in the sequence negatively interfering with expression efficiency in the respective host organism such as wheat, for example by removing cryptic splice sites, cryptic polyadenylation signals or sequences able to form secondary structures inhibiting expression in particular translation. To optimize both gene sequences the software LETO1.0 from Entelechon GmbH

(Regensburg, Germany) was used. The optimized DNA-sequences of the truncated form of the Hevea brasiliensis HMGR and the Nicotiana tabacum SMT1 are given in SEQ ID NO: 1 and SEQ ID NO: 3. The corresponding protein sequences are given in SEQ ID NO: 2 and SEQ ID NO: 4.

The synthetic DNA fragment SEQ ID NO: 1 corresponding to the optimized truncated Hevea brasiliensis HMGR coding sequence and the synthetic DNA fragment SEQ ID NO: 3 corresponding to the optimized Nicotiana tabacum SMT1 coding sequence were used in subsequent cloning steps to generate the plant transformation vectors SBT490 SEQ ID NO: 5 and SBT493 SEQ ID NO: 6 which were based on the vector VC-SUH401qcz backbone SEQ ID NO: 7. All cloning steps were carried out following standard molecular biology protocols. The T-DNA of SBT490 SEQ ID NO: 5 contains a cassette for regeneration of plants under hygromycin selection pressure comprising the Zea mays ubiquitin promoter, the coding region of the E.coli hygromycin phosphotransferase gene, and the cauliflower mosaic virus (CaMV) 35S-RNA-terminator. A second expression cassette comprises the Zea mays ubiquitin promoter, the Hevea brasiliensis truncated HMGR coding sequence optimized for wheat, and the cauliflower mosaic virus (CaMV) 35S-RNA-terminator. A third expression cassette comprises the Zea mays ubiquitin promoter, the Nicotiana tabacum SMT1 coding sequence optimized for wheat, and the cauliflower mosaic virus (CaMV) 35S-RNA- terminator.

The T-DNA of SBT493 SEQ ID NO: 6 contains a cassette for regeneration of plants under hygromycin selection pressure comprising the Zea mays ubiquitin promoter, the coding region of the E.coli hygromycin phosphotransferase gene, and the cauliflower mosaic virus (CaMV) 35S-RNA-terminator. A second expression cassette comprises the Zea mays ubiquitin promoter, the Hevea brasiliensis truncated HMGR coding sequence optimized for wheat, and the cauliflower mosaic virus (CaMV) 35S-RNA-terminator.

The following sequences were used: SEQ ID NO: 1 : Synthetic DNA fragment corresponding to the optimized truncated Hevea brasiliensis HMGR coding sequence (1275 bp) atggtcgcaccactcgtgagcgaagaggacgagatgatcgtcaactccgtggttgacgggaagatcccaagctactcgctgg agagcaagcttggcgactgcaagagggctgcagccatccgtagagaggcgcttcaacgcatgacgcgtaggagcttggag ggacttccagtcgaaggcttcgactacgagtccatcctcggacaatgctgcgagatgccagtgggctacgtccagatccctgtg ggtatcgcaggaccactcctgctcaacggcagggaatactccgtcccaatggcgactacagagggctgcctcgtcgcctctac caacaggggctgtaaggccatctacctcagcggaggtgctaccagcgtcctgctcaaggacggcatgacgagagctcctgtg gtgcgcttcgcatctgcgacacgtgctgccgagctcaagttcttcctggaagacccagacaacttcgacacgctagctgtggtgt tcaacaagtcgtccagattcgccagactgcaaggcatcaagtgctccatcgctgggaagaacctctacatccgcttctcgtgctc taccggtgacgccatggggatgaacatggtgagcaagggcgtgcagaacgtcctcgagttcctgcaatccgacttctccgaca tggacgtcataggcatctccgggaacttctgctccgacaagaagcctgcggcagtgaactggatcgagggtagagggaaga gtgtcgtgtgtgaggcgatcatcaaggaggaggtggtgaagaaagtcctcaagacgaacgtggccagtttggtggagctgaa catgctgaagaacctcgcaggaagtgctgtggctggagcgctcggtggcttcaacgcccacgctgggaacatcgtgagcgcc atcttcatcgcgactggccaagacccagcgcagaacgtcgagtcatcgcactgcatcaccatgatggaggccgttaacgacg gcaaggacctccacatcagcgtcaccatgccctctatcgaggtgggaactgtgggtggcggtacccaactagcctcccaatct gcgtgcctgaacctgctcggtgtcaagggggcaaacaaggagtcaccagggagcaactctcgcctccttgccgcaatcgtgg caggaagtgtcctagctggggaactgtccctcatgagcgctattgccgctgggcagctcgtgaagagccacatgaagtacaa ccgctcgtccaaggacatgtcgaaggccgctagttga SEQ ID NO: 3: Synthetic DNA fragment corresponding to the optimized Nicotiana tabacum SMT1 coding sequence coding sequence (1038 bp) atgtcgaagcaaggcgcctttgatctcgcatcgggagttggagggaagatcaacaaggaggaggtgttgagtgcggtcgaca agtacgagaagtaccatggctattacggcggtgaagaggaggagaggaagaacaactacacggacatggtgaacaagta ctacgacctctgcaccagcttctacgagtacggatggggtgagagcttccactttgcccctaggtggaagggcgaatctctcca ggaatccatcaagcgtcacgagcatttcctcgctcttcagctaggcctaaagcccggtcagaaggtcttggacgtgggatgtgg tatcggaggtccattgcgtgaaatcgccaggtttagctccactagtgtcactggcctgaacaacaacgagtaccagatctcaag agggcaagtgctgaaccgtaaggtcggacttgaccagacctgcaatttcgtcaagggcgacttcatgaagatgcctttccctga caactcattcgatgcggtttacgcgattgaagccacatgccatgcgccagacccagtggggtgctacaaggagatctacagg gtgctgaagccagggcaatgctttgccgtctacgagtggtgcatgacggacagctacaaccccaacaacgaggagcacaat cggatcaaggccgagatcgagttgggcaatggactaccagaggtcagactgacaacgcaatgtctcgaggccgcaaagca agctggcttcgaagtcgtgtgggataaggatctcgccgatgactctccagtcccatggtatctcccactggatacctcccacttctc tctctccagcttccgtcttacagcggtgggaaggctcttcaccaggaaccttgtgtcagcactcgagtatgtgggcttggctccaa agggcagtcaaagggtgcaagccttcctggagaaggcagctgaaggacttgtgggtggagcgaagaagggcatcttcacc ccaatgtacttcttcgtggttaggaagcccatatccgactcgcaa

SEQ ID NO: 2: Amino acid sequence corresponding to the optimized truncated Hevea brasiliensis HMGR coding sequence (424 aa) mvaplvseedemivnsvvdgkipsyslesklgdckraaairrealqrmtrrsleglpvegfdyesilgqccempvgyvqipvgi agplllngreysvpmattegclvastnrgckaiylsggatsvllkdgmtrapvvrfasatraaelkffledpdnfdtlavvfnkssrfa rlqgikcsiagknlyirfscstgdamgmnmvskgvqnvleflqsdfsdmdvigisgnfcsdkkpaavnwiegrgksvvceaiik eevvkkvlktnvaslvelnmlknlagsavagalggfnahagnivsaifiatgqdpaqnvesshcitmmeavndgkdlhisvtm psievgtvgggtqlasqsaclnllgvkgankespgsnsrllaaivagsvlagelslmsaiaagqlvkshmkynrsskdmskaa s

SEQ ID NO: 4

Amino acid sequence corresponding to the optimized Nicotiana tabacum SMT1 coding sequence (346 aa) mskqgafdlasgvggkinkeevlsavdkyekyhgyyggeeeerknnytdmvnkyydlctsfyeygwgesfhfaprwkges Iqesikrhehflalqlglkpgqkvldvgcgiggplreiarfsstsvtglnnneyqisrgqvlnrkvgldqtcnfvkgdfmkmpfpdns fdavyaieatchapdpvgcykeiyrvlkpgqcfavyewcmtdsynpnneehnrikaeielgnglpevrlttqcleaakqagfev vwdkdladdspvpwylpldtshfslssfrltavgrlftrnlvsaleyvglapkgsqrvqaflekaaeglvggakkgiftpmyffvvrk pisdsq

SEQ ID NO: 5: SBT490 T-DNA (T-DNA region of binary vector)

Position 1 to 214 Left border

Position 226 to 430 CaMV 35S-RNA gene terminator (complementary) Position 442 to 1464 E.coli hygromycin phosphotransferase gene/CDS

(complementary)

Position 1483 to 3463 Zea mays ubiquitin gene promoter (complementary)

Position 3553 to 3757 CaMV 35S-RNA gene terminator (complementary)

Position 3766 to 5040 Hevea brasiliensis truncated HMGR gene/CDS sequence

optimized for expression in wheat (complementary)

Position 5059 to 7039 Zea mays ubiquitin gene promoter (complementary)

Position 7220 to 7424 CaMV 35S-RNA gene terminator (complementary)

Position 7436 to 8473 Nicotiana tabacum SMT1 gene/CDS sequence optimized for

expression in wheat (complementary)

Position 8492 to 10472 Zea mays ubiquitin gene promoter (complementary)

Position 10630 to 10775 Right border gtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttaga caacttaataacacattgcggacgtctttaatgtactgaattaacatccgtttgatacttgtctaaaattggctgatttcgagtgcatct atgcataaaaacaatctaatgacaattattaccaagcaaattcgagctccggtcactggattttggttttaggaattagaaattttatt gatagaagtattttacaaatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataagaaccctaatt cccttatctgggaactactcacacattattctggagaaaaatagagagagatagatttgtagagagagactggtgatttgcggcc gcctattcctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacggc cgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcccaag ctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccggagccgcgg cgatcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctccagaagaag atgttggcgacctcgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgtccgtcagga cattgttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcctcggcccaaagcatcagctcatcgagagc ctgcgcgacggacgcactgacggtgtcgtccatcacagtttgccagtgatacacatggggatcagcaatcgcgcatatgaaat cacgccatgtagtgtattgaccgattccttgcggtccgaatgggccgaacccgctcgtctggctaagatcggccgcagcgatcg catccatggcctccgcgaccggctgcagaacagcgggcagttcggtttcaggcaggtcttgcaacgtgacaccctgtgcacg gcgggagatgcaataggtcaggctctcgctgaattccccaatgtcaagcacttccggaatcgggagcgcggccgatgcaaa gtgccgataaacataacgatctttgtagaaaccatcggcgcagctatttacccgcaggacatatccacgccctcctacatcgaa gctgaaagcacgagattcttcgccctccgagagctgcatcaggtcggagacgctgtcgaacttttcgatcagaaacttctcgac agacgtcgcggtgagttcaggctttttcatggatcccccgggctgcagaagtaacaccaaacaacagggtgagcatcgacaa aagaaacagtaccaagcaaataaatagcgtatgaaggcagggctaaaaaaatccacatatagctgctgcatatgccatcat ccaagtatatcaagatcaaaataattataaaacatacttgtttattataatagataggtactcaaggttagagcatatgaatagatg ctgcatatgccatcatgtatatgcatcagtaaaacccacatcaacatgtatacctatcctagatcgatatttccatccatcttaaact cgtaactatgaagatgtatgacacacacatacagttccaaaattaataaatacaccaggtagtttgaaacagtattctactccga tctagaacgaatgaacgaccgcccaaccacaccacatcatcacaaccaagcgaacaaaaagcatctctgtatatgcatcag taaaacccgcatcaacatgtatacctatcctagatcgatatttccatccatcatcttcaattcgtaactatgaatatgtatggcacac acatacagatccaaaattaataaatccaccaggtagtttgaaacagaattctactccgatctagaacgaccgcccaaccagac cacatcatcacaaccaagacaaaaaaaagcatgaaaagatgacccgacaaacaagtgcacggcatatattgaaataaag gaaaagggcaaaccaaaccctatgcaacgaaacaaaaaaaatcatgaaatcgatcccgtctgcggaacggctagagcca tcccaggattccccaaagagaaacactggcaagttagcaatcagaacgtgtctgacgtacaggtcgcatccgtgtacgaacg ctagcagcacggatctaacacaaacacggatctaacacaaacatgaacagaagtagaactaccgggccctaaccatggac cggaacgccgatctagagaaggtagagagggggggggggggaggacgagcggcgtaccttgaagcggaggtgccgac gggtggatttgggggagatctggttgtgtgtgtgtgcgctccgaacaacacgaggttggggaaagagggtgtggagggggtgt ctatttattacggcgggcgaggaagggaaagcgaaggagcggtgggaaaggaatcccccgtagctgccggtgccgtgaga ggaggaggaggccgcctgccgtgccggctcacgtctgccgctccgccacgcaatttctggatgccgacagcggagcaagtc caacggtggagcggaactctcgagaggggtccagaggcagcgacagagatgccgtgccgtctgcttcgcttggcccgacgc gacgctgctggttcgctggttggtgtccgttagactcgtcgacggcgtttaacaggctggcattatctactcgaaacaagaaaaat gtttcctta gttttttta atttctta aa gg g ta tttgttta attttt^

aatagagttttagttttcttaatttagaggctaaaatagaataaaatagatgtactaaaaaaattagtctataaaaaccattaaccct aaaccctaaatggatgtactaataaaatggatgaagtattatataggtgaagctatttgcaaaaaaaaaggagaacacatgca cactaaaaagataaaactgtagagtcctgttgtcaaaatactcaattgtcctttagaccatgtctaactgttcatttatatgattctcta aaacactgatattattgtagtactatagattatattattcgtagagtaaagtttaaatatatgtataaagatagataaactgcacttca aacaagtgtgacaaaaaaaatatgtggtaattttttataacttagacatgcaatgctcattatctctagagaggggcacgaccgg gtcacgctgcactgcaggaattcgatctcggtacctcgagcccgggcgatatcgattacgccaagctatcaactttgtatagaaa agttgggtacccctccggtcactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatacta agggtttcttatatgctcaacacatgagcgaaaccctataagaaccctaattcccttatctgggaactactcacacattattctgga gaaaaatagagagagatagatttgtagagagagactggtgatttgcggccgctcaactagcggccttcgacatgtccttggac gagcggttgtacttcatgtggctcttcacgagctgcccagcggcaatagcgctcatgagggacagttccccagctaggacactt cctgccacgattgcggcaaggaggcgagagttgctccctggtgactccttgtttgcccccttgacaccgagcaggttcaggcac gcagattgggaggctagttgggtaccgccacccacagttcccacctcgatagagggcatggtgacgctgatgtggaggtcctt gccgtcgttaacggcctccatcatggtgatgcagtgcgatgactcgacgttctgcgctgggtcttggccagtcgcgatgaagatg gcgctcacgatgttcccagcgtgggcgttgaagccaccgagcgctccagccacagcacttcctgcgaggttcttcagcatgttc agctccaccaaactggccacgttcgtcttgaggactttcttcaccacctcctccttgatgatcgcctcacacacgacactcttccct ctaccctcgatccagttcactgccgcaggcttcttgtcggagcagaagttcccggagatgcctatgacgtccatgtcggagaagt cggattgcaggaactcgaggacgttctgcacgcccttgctcaccatgttcatccccatggcgtcaccggtagagcacgagaag cggatgtagaggttcttcccagcgatggagcacttgatgccttgcagtctggcgaatctggacgacttgttgaacaccacagcta gcgtgtcgaagttgtctgggtcttccaggaagaacttgagctcggcagcacgtgtcgcagatgcgaagcgcaccacaggagc tctcgtcatgccgtccttgagcaggacgctggtagcacctccgctgaggtagatggccttacagcccctgttggtagaggcgac gaggcagccctctgtagtcgccattgggacggagtattccctgccgttgagcaggagtggtcctgcgatacccacagggatctg gacgtagcccactggcatctcgcagcattgtccgaggatggactcgtagtcgaagccttcgactggaagtccctccaagctcct acgcgtcatgcgttgaagcgcctctctacggatggctgcagccctcttgcagtcgccaagcttgctctccagcgagtagcttggg atcttcccgtcaaccacggagttgacgatcatctcgtcctcttcgctcacgagtggtgcgaccatggatcccccgggctgcagaa gtaacaccaaacaacagggtgagcatcgacaaaagaaacagtaccaagcaaataaatagcgtatgaaggcagggctaa aaaaatccacatatagctgctgcatatgccatcatccaagtatatcaagatcaaaataattataaaacatacttgtttattataata gataggtactcaaggttagagcatatgaatagatgctgcatatgccatcatgtatatgcatcagtaaaacccacatcaacatgta tacctatcctagatcgatatttccatccatcttaaactcgtaactatgaagatgtatgacacacacatacagttccaaaattaataa atacaccaggtagtttgaaacagtattctactccgatctagaacgaatgaacgaccgcccaaccacaccacatcatcacaac caagcgaacaaaaagcatctctgtatatgcatcagtaaaacccgcatcaacatgtatacctatcctagatcgatatttccatccat catcttcaattcgtaactatgaatatgtatggcacacacatacagatccaaaattaataaatccaccaggtagtttgaaacagaa ttctactccgatctagaacgaccgcccaaccagaccacatcatcacaaccaagacaaaaaaaagcatgaaaagatgaccc gacaaacaagtgcacggcatatattgaaataaaggaaaagggcaaaccaaaccctatgcaacgaaacaaaaaaaatcat gaaatcgatcccgtctgcggaacggctagagccatcccaggattccccaaagagaaacactggcaagttagcaatcagaac gtgtctgacgtacaggtcgcatccgtgtacgaacgctagcagcacggatctaacacaaacacggatctaacacaaacatga acagaagtagaactaccgggccctaaccatggaccggaacgccgatctagagaaggtagagagggggggggggggag gacgagcggcgtaccttgaagcggaggtgccgacgggtggatttgggggagatctggttgtgtgtgtgtgcgctccgaacaac acgaggttggggaaagagggtgtggagggggtgtctatttattacggcgggcgaggaagggaaagcgaaggagcggtgg gaaaggaatcccccgtagctgccggtgccgtgagaggaggaggaggccgcctgccgtgccggctcacgtctgccgctccgc cacgcaatttctggatgccgacagcggagcaagtccaacggtggagcggaactctcgagaggggtccagaggcagcgac agagatgccgtgccgtctgcttcgcttggcccgacgcgacgctgctggttcgctggttggtgtccgttagactcgtcgacggcgttt aacaggctggcattatctactcgaaacaagaaaaatgtttccttagtttttttaatttcttaaagggtatttgtttaatttttagtcactttat tttattctattttatatctaaattattaaataaaaaaactaaaatagagttttagttttcttaatttagaggctaaaatagaataaaatag atgtactaaaaaaattagtctataaaaaccattaaccctaaaccctaaatggatgtactaataaaatggatgaagtattatatag gtgaagctatttgcaaaaaaaaaggagaacacatgcacactaaaaagataaaactgtagagtcctgttgtcaaaatactcaat tgtcctttagaccatgtctaactgttcatttatatgattctctaaaacactgatattattgtagtactatagattatattattcgtagagtaa agtttaaatatatgtataaagatagataaactgcacttcaaacaagtgtgacaaaaaaaatatgtggtaattttttataacttagac atgcaatgctcattatctctagagaggggcacgaccgggtcacgctgcactgcaggaattcgatatcaagcttggcgtaatcat ggcaagtttgtacaaaaaagcaggctggtacccggggatcctctagcatatgctcgaggcggccgcagatatcagatctggtc gacggcatgcaagcttggctaatcatggacccagctttcttgtacaaagtggggtacccctccggtcactggattttggttttagga attagaaattttattgatagaagtattttacaaatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctat aagaaccctaattcccttatctgggaactactcacacattattctggagaaaaatagagagagatagatttgtagagagagact ggtgatttgcggccgctcattgcgagtcggatatgggcttcctaaccacgaagaagtacattggggtgaagatgcccttcttcgct ccacccacaagtccttcagctgccttctccaggaaggcttgcaccctttgactgccctttggagccaagcccacatactcgagtg ctgacacaaggttcctggtgaagagccttcccaccgctgtaagacggaagctggagagagagaagtgggaggtatccagtg ggagataccatgggactggagagtcatcggcgagatccttatcccacacgacttcgaagccagcttgctttgcggcctcgaga cattgcgttgtcagtctgacctctggtagtccattgcccaactcgatctcggccttgatccgattgtgctcctcgttgttggggttgtag ctgtccgtcatgcaccactcgtagacggcaaagcattgccctggcttcagcaccctgtagatctccttgtagcaccccactgggt ctggcgcatggcatgtggcttcaatcgcgtaaaccgcatcgaatgagttgtcagggaaaggcatcttcatgaagtcgcccttga cgaaattgcaggtctggtcaagtccgaccttacggttcagcacttgccctcttgagatctggtactcgttgttgttcaggccagtgac actagtggagctaaacctggcgatttcacgcaatggacctccgataccacatcccacgtccaagaccttctgaccgggctttag gcctagctgaagagcgaggaaatgctcgtgacgcttgatggattcctggagagattcgcccttccacctaggggcaaagtgga agctctcaccccatccgtactcgtagaagctggtgcagaggtcgtagtacttgttcaccatgtccgtgtagttgttcttcctctcctcct cttcaccgccgtaatagccatggtacttctcgtacttgtcgaccgcactcaacacctcctccttgttgatcttccctccaactcccgat gcgagatcaaaggcgccttgcttcgacatggatcccccgggctgcagaagtaacaccaaacaacagggtgagcatcgaca aaagaaacagtaccaagcaaataaatagcgtatgaaggcagggctaaaaaaatccacatatagctgctgcatatgccatca tccaagtatatcaagatcaaaataattataaaacatacttgtttattataatagataggtactcaaggttagagcatatgaatagat gctgcatatgccatcatgtatatgcatcagtaaaacccacatcaacatgtatacctatcctagatcgatatttccatccatcttaaac tcgtaactatgaagatgtatgacacacacatacagttccaaaattaataaatacaccaggtagtttgaaacagtattctactccg atctagaacgaatgaacgaccgcccaacca caeca catcatcacaaccaagcgaacaaaaagcatctctgtatatgcatca gtaaaacccgcatcaacatgtatacctatcctagatcgatatttccatccatcatcttcaattcgtaactatgaatatgtatggcaca cacatacagatccaaaattaataaatccaccaggtagtttgaaacagaattctactccgatctagaacgaccgcccaaccaga ccacatcatcacaaccaagacaaaaaaaagcatgaaaagatgacccgacaaacaagtgcacggcatatattgaaataaa ggaaaagggcaaaccaaaccctatgcaacgaaacaaaaaaaatcatgaaatcgatcccgtctgcggaacggctagagcc atcccaggattccccaaagagaaacactggcaagttagcaatcagaacgtgtctgacgtacaggtcgcatccgtgtacgaac gctagcagcacggatctaacacaaacacggatctaacacaaacatgaacagaagtagaactaccgggccctaaccatgg accggaacgccgatctagagaaggtagagagggggggggggggaggacgagcggcgtaccttgaagcggaggtgccg acgggtggatttgggggagatctggttgtgtgtgtgtgcgctccgaacaacacgaggttggggaaagagggtgtggagggggt gtctatttattacggcgggcgaggaagggaaagcgaaggagcggtgggaaaggaatcccccgtagctgccggtgccgtga gaggaggaggaggccgcctgccgtgccggctcacgtctgccgctccgccacgcaatttctggatgccgacagcggagcaa gtccaacggtggagcggaactctcgagaggggtccagaggcagcgacagagatgccgtgccgtctgcttcgcttggcccga cgcgacgctgctggttcgctggttggtgtccgttagactcgtcgacggcgtttaacaggctggcattatctactcgaaacaagaa aaatgtttccttagtttttttaatttcttaaagggtatttgtttaatttttagtcactttattttattctattttatatctaaattattaaataaaa actaaaatagagttttagttttcttaatttagaggctaaaatagaataaaatagatgtactaaaaaaattagtctataaaaaccatt aaccctaaaccctaaatggatgtactaataaaatggatgaagtattatataggtgaagctatttgcaaaaaaaaaggagaaca catgcacactaaaaagataaaactgtagagtcctgttgtcaaaatactcaattgtcctttagaccatgtctaactgttcatttatatg attctctaaaacactgatattattgtagtactatagattatattattcgtagagtaaagtttaaatatatgtataaagatagataaactg cacttcaaacaagtgtgacaaaaaaaatatgtggtaattttttataacttagacatgcaatgctcattatctctagagaggggcac gaccgggtcacgctgcactgcaggaattcgatatcaagcttggcgtaatcatggcaactttattatacatagttgataattcactgg ccggatatcggatccactagtctagagtcgacctgcaggcatgcaagcttggcgtaatcatggtcatagctgtttcctactagatc tgattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttatta gaataatcggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtc

SEQ ID NO: 6: SBT493 T-DNA (T-DNA region of binary vector)

Position 1 to 214 Left border

Position 226 to 430 CaMV 35S-RNA gene terminator (complementary)

Position 442 to 1464 E.coli hygromycin phosphotransferase gene/CDS

(complementary)

Position 1483 to 3463 Zea mays ubiquitin gene promoter (complementary)

Position 3553 to 3757 CaMV 35S-RNA gene terminator (complementary)

Position 3766 to 5040 Hevea brasiliensis truncated HMGR gene/CDS sequence

optimized for expression in wheat (complementary)

Position 5059 to 7039 Zea mays ubiquitin gene promoter (complementary)

Position 7416 to 7561 Right border gtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttaga caacttaataacacattgcggacgtctttaatgtactgaattaacatccgtttgatacttgtctaaaattggctgatttcgagtgcatct atgcataaaaacaatctaatgacaattattaccaagcaaattcgagctccggtcactggattttggttttaggaattagaaattttatt gatagaagtattttacaaatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataagaaccctaatt cccttatctgggaactactcacacattattctggagaaaaatagagagagatagatttgtagagagagactggtgatttgcggcc gcctattcctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacggc cgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcccaag ctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccggagccgcgg cgatcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctccagaagaag atgttggcgacctcgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgtccgtcagga cattgttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcctcggcccaaagcatcagctcatcgagagc ctgcgcgacggacgcactgacggtgtcgtccatcacagtttgccagtgatacacatggggatcagcaatcgcgcatatgaaat cacgccatgtagtgtattgaccgattccttgcggtccgaatgggccgaacccgctcgtctggctaagatcggccgcagcgatcg catccatggcctccgcgaccggctgcagaacagcgggcagttcggtttcaggcaggtcttgcaacgtgacaccctgtgcacg gcgggagatgcaataggtcaggctctcgctgaattccccaatgtcaagcacttccggaatcgggagcgcggccgatgcaaa gtgccgataaacataacgatctttgtagaaaccatcggcgcagctatttacccgcaggacatatccacgccctcctacatcgaa gctgaaagcacgagattcttcgccctccgagagctgcatcaggtcggagacgctgtcgaacttttcgatcagaaacttctcgac agacgtcgcggtgagttcaggctttttcatggatcccccgggctgcagaagtaacaccaaacaacagggtgagcatcgacaa aagaaacagtaccaagcaaataaatagcgtatgaaggcagggctaaaaaaatccacatatagctgctgcatatgccatcat ccaagtatatcaagatcaaaataattataaaacatacttgtttattataatagataggtactcaaggttagagcatatgaatagatg ctgcatatgccatcatgtatatgcatcagtaaaacccacatcaacatgtatacctatcctagatcgatatttccatccatcttaaact cgtaactatgaagatgtatgacacacacatacagttccaaaattaataaatacaccaggtagtttgaaacagtattctactccga tctagaacgaatgaacgaccgcccaaccacaccacatcatcacaaccaagcgaacaaaaagcatctctgtatatgcatcag taaaacccgcatcaacatgtatacctatcctagatcgatatttccatccatcatcttcaattcgtaactatgaatatgtatggcacac acatacagatccaaaattaataaatccaccaggtagtttgaaacagaattctactccgatctagaacgaccgcccaaccagac cacatcatcacaaccaagacaaaaaaaagcatgaaaagatgacccgacaaacaagtgcacggcatatattgaaataaag gaaaagggcaaaccaaaccctatgcaacgaaacaaaaaaaatcatgaaatcgatcccgtctgcggaacggctagagcca tcccaggattccccaaagagaaacactggcaagttagcaatcagaacgtgtctgacgtacaggtcgcatccgtgtacgaacg ctagcagcacggatctaacacaaacacggatctaacacaaacatgaacagaagtagaactaccgggccctaaccatggac cggaacgccgatctagagaaggtagagagggggggggggggaggacgagcggcgtaccttgaagcggaggtgccgac gggtggatttgggggagatctggttgtgtgtgtgtgcgctccgaacaacacgaggttggggaaagagggtgtggagggggtgt ctatttattacggcgggcgaggaagggaaagcgaaggagcggtgggaaaggaatcccccgtagctgccggtgccgtgaga ggaggaggaggccgcctgccgtgccggctcacgtctgccgctccgccacgcaatttctggatgccgacagcggagcaagtc caacggtggagcggaactctcgagaggggtccagaggcagcgacagagatgccgtgccgtctgcttcgcttggcccgacgc gacgctgctggttcgctggttggtgtccgttagactcgtcgacggcgtttaacaggctggcattatctactcgaaacaagaaaaat gtttccttagtttttttaatttcttaaagggtatttgtttaatttttagtcactttattttattctattttatatctaaattattaaataaaaaaac aatagagttttagttttcttaatttagaggctaaaatagaataaaatagatgtactaaaaaaattagtctataaaaaccattaaccct aaaccctaaatggatgtactaataaaatggatgaagtattatataggtgaagctatttgcaaaaaaaaaggagaacacatgca cactaaaaagataaaactgtagagtcctgttgtcaaaatactcaattgtcctttagaccatgtctaactgttcatttatatgattctcta aaacactgatattattgtagtactatagattatattattcgtagagtaaagtttaaatatatgtataaagatagataaactgcacttca aacaagtgtgacaaaaaaaatatgtggtaattttttataacttagacatgcaatgctcattatctctagagaggggcacgaccgg gtcacgctgcactgcaggaattcgatctcggtacctcgagcccgggcgatatcgattacgccaagctatcaactttgtatagaaa agttgggtacccctccggtcactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatacta agggtttcttatatgctcaacacatgagcgaaaccctataagaaccctaattcccttatctgggaactactcacacattattctgga gaaaaatagagagagatagatttgtagagagagactggtgatttgcggccgctcaactagcggccttcgacatgtccttggac gagcggttgtacttcatgtggctcttcacgagctgcccagcggcaatagcgctcatgagggacagttccccagctaggacactt cctgccacgattgcggcaaggaggcgagagttgctccctggtgactccttgtttgcccccttgacaccgagcaggttcaggcac gcagattgggaggctagttgggtaccgccacccacagttcccacctcgatagagggcatggtgacgctgatgtggaggtcctt gccgtcgttaacggcctccatcatggtgatgcagtgcgatgactcgacgttctgcgctgggtcttggccagtcgcgatgaagatg gcgctcacgatgttcccagcgtgggcgttgaagccaccgagcgctccagccacagcacttcctgcgaggttcttcagcatgttc agctccaccaaactggccacgttcgtcttgaggactttcttcaccacctcctccttgatgatcgcctcacacacgacactcttccct ctaccctcgatccagttcactgccgcaggcttcttgtcggagcagaagttcccggagatgcctatgacgtccatgtcggagaagt cggattgcaggaactcgaggacgttctgcacgcccttgctcaccatgttcatccccatggcgtcaccggtagagcacgagaag cggatgtagaggttcttcccagcgatggagcacttgatgccttgcagtctggcgaatctggacgacttgttgaacaccacagcta gcgtgtcgaagttgtctgggtcttccaggaagaacttgagctcggcagcacgtgtcgcagatgcgaagcgcaccacaggagc tctcgtcatgccgtccttgagcaggacgctggtagcacctccgctgaggtagatggccttacagcccctgttggtagaggcgac gaggcagccctctgtagtcgccattgggacggagtattccctgccgttgagcaggagtggtcctgcgatacccacagggatctg gacgtagcccactggcatctcgcagcattgtccgaggatggactcgtagtcgaagccttcgactggaagtccctccaagctcct acgcgtcatgcgttgaagcgcctctctacggatggctgcagccctcttgcagtcgccaagcttgctctccagcgagtagcttggg atcttcccgtcaaccacggagttgacgatcatctcgtcctcttcgctcacgagtggtgcgaccatggatcccccgggctgcagaa gtaacaccaaacaacagggtgagcatcgacaaaagaaacagtaccaagcaaataaatagcgtatgaaggcagggctaa aaaaatccacatatagctgctgcatatgccatcatccaagtatatcaagatcaaaataattataaaacatacttgtttattataata gataggtactcaaggttagagcatatgaatagatgctgcatatgccatcatgtatatgcatcagtaaaacccacatcaacatgta tacctatcctagatcgatatttccatccatcttaaactcgtaactatgaagatgtatgacacacacatacagttccaaaattaataa atacaccaggtagtttgaaacagtattctactccgatctagaacgaatgaacgaccgcccaaccacaccacatcatcacaac caagcgaacaaaaagcatctctgtatatgcatcagtaaaacccgcatcaacatgtatacctatcctagatcgatatttccatccat catcttcaattcgtaactatgaatatgtatggcacacacatacagatccaaaattaataaatccaccaggtagtttgaaacagaa ttctactccgatctagaacgaccgcccaaccagaccacatcatcacaaccaagacaaaaaaaagcatgaaaagatgaccc gacaaacaagtgcacggcatatattgaaataaaggaaaagggcaaaccaaaccctatgcaacgaaacaaaaaaaatcat gaaatcgatcccgtctgcggaacggctagagccatcccaggattccccaaagagaaacactggcaagttagcaatcagaac gtgtctgacgtacaggtcgcatccgtgtacgaacgctagcagcacggatctaacacaaacacggatctaacacaaacatga acagaagtagaactaccgggccctaaccatggaccggaacgccgatctagagaaggtagagagggggggggggggag gacgagcggcgtaccttgaagcggaggtgccgacgggtggatttgggggagatctggttgtgtgtgtgtgcgctccgaacaac acgaggttggggaaagagggtgtggagggggtgtctatttattacggcgggcgaggaagggaaagcgaaggagcggtgg gaaaggaatcccccgtagctgccggtgccgtgagaggaggaggaggccgcctgccgtgccggctcacgtctgccgctccgc cacgcaatttctggatgccgacagcggagcaagtccaacggtggagcggaactctcgagaggggtccagaggcagcgac agagatgccgtgccgtctgcttcgcttggcccgacgcgacgctgctggttcgctggttggtgtccgttagactcgtcgacggcgttt aacaggctggcattatctactcgaaacaagaaaaatgtttccttagtttttttaatttcttaaagggtatttgtttaatttttagtcactttat tttattctattttatatctaaattattaaataaaaaaactaaaatagagttttagttttcttaatttagaggctaaaatagaataaaatag atgtactaaaaaaattagtctataaaaaccattaaccctaaaccctaaatggatgtactaataaaatggatgaagtattatatag gtgaagctatttgcaaaaaaaaaggagaacacatgcacactaaaaagataaaactgtagagtcctgttgtcaaaatactcaat tgtcctttagaccatgtctaactgttcatttatatgattctctaaaacactgatattattgtagtactatagattatattattcgtagagtaa agtttaaatatatgtataaagatagataaactgcacttcaaacaagtgtgacaaaaaaaatatgtggtaattttttataacttagac atgcaatgctcattatctctagagaggggcacgaccgggtcacgctgcactgcaggaattcgatatcaagcttggcgtaatcat ggcaagtttgtacaaaaaagcaggctggtacccggggatcctctagcatatgctcgaggcggccgcagatatcagatctggtc gacggcatgcaagcttggctaatcatggacccagctttcttgtacaaagtggggtacccggggatcctctagcatatgctcgag gcggccgcagatatcagatctggtcgacggcatgcaagcttggcgtaatcatggcaactttattatacatagttgataattcactg gccggatatcggatccactagtctagagtcgacctgcaggcatgcaagcttggcgtaatcatggtcatagctgtttcctactagat ctgattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttatt agaataatcggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtc SEQ ID NO: 7: SUH401 qcz (binary vector backbone)

Position 1 to 146 Right border

Position 320 to 11 1 1 Adenyltransferase [aadA] gene coding region

Position 1560 to 2241 ColE1 E. coli origin of replication

Position 2615 to 2809 pVS1 origin (complementary)

Position 2413 to 5682 pVS1 replicon (complementary)

Position 5691 to 5904 Left border

Position 5605 to 5916 Placeholder (to be replaced by respective T-DNA) gattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattag aataatcggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtccatggaacgcagtggcggttttcatggctt gttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatg gagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatcatgggggaagcggtgatcgccgaag tatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgc agtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgag ctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacg acgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatctt cgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcgg cggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccg actgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaag gatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttgga caagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtag tcggcaaataatgtctagctagaaattcgttcaagccgacgccgcttcgcggcgcggcttaactcaagcgttagatgcactaag cacataattgctcacagccaaactatcaggtcaagtctgcttttattatttttaagcgtgcataataagccctacacaaattgggag atatatcatgcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttct tgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagag ctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggcca ccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgt gtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaaggga gaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcc tggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaa aaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtgg ataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagg aagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatct gctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaac acccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtg tcagaggttttcaccgtcatcaccgaaacgcgcgaggcagggtgccttgatgtgggcgccggcggtcgagtggcgacggcgc ggcttgtccgcgccctggtagattgcctggccgtaggccagccatttttgagcggccagcggccgcgataggccgacgcgaa gcggcggggcgtagggagcgcagcgaccgaagggtaggcgctttttgcagctcttcggctgtgcgctggccagacagttatg cacaggccaggcgggttttaagagttttaataagttttaaagagttttaggcggaaaaatcgccttttttctcttttatatcagtcactta catgtgtgaccggttcccaatgtacggctttgggttcccaatgtacgggttccggttcccaatgtacggctttgggttcccaatgtac gtgctatccacaggaaagagaccttttcgacctttttcccctgctagggcaatttgccctagcatctgctccgtacattaggaaccg gcggatgcttcgccctcgatcaggttgcggtagcgcatgactaggatcgggccagcctgccccgcctcctccttcaaatcgtact ccggcaggtcatttgacccgatcagcttgcgcacggtgaaacagaacttcttgaactctccggcgctgccactgcgttcgtagat cgtcttgaacaaccatctggcttctgccttgcctgcggcgcggcgtgccaggcggtagagaaaacggccgatgccgggatcg atcaaaaagtaatcggggtgaaccgtcagcacgtccgggttcttgccttctgtgatctcgcggtacatccaatcagctagctcgat ctcgatgtactccggccgcccggtttcgctctttacgatcttgtagcggctaatcaaggcttcaccctcggataccgtcaccaggc ggccgttcttggccttcttcgtacgctgcatggcaacgtgcgtggtgtttaaccgaatgcaggtttctaccaggtcgtctttctgctttc cgccatcggctcgccggcagaacttgagtacgtccgcaacgtgtggacggaacacgcggccgggcttgtctcccttcccttcc cggtatcggttcatggattcggttagatgggaaaccgccatcagtaccaggtcgtaatcccacacactggccatgccggccgg ccctgcggaaacctctacgtgcccgtctggaagctcgtagcggatcacctcgccagctcgtcggtcacgcttcgacagacgga aaacggccacgtccatgatgctgcgactatcgcgggtgcccacgtcatagagcatcggaacgaaaaaatctggttgctcgtcg cccttgggcggcttcctaatcgacggcgcaccggctgccggcggttgccgggattctttgcggattcgatcagcggccgcttgcc acgattcaccggggcgtgcttctgcctcgatgcgttgccgctgggcggcctgcgcggccttcaacttctccaccaggtcatcacc cagcgccgcgccgatttgtaccgggccggatggtttgcgaccgctcacgccgattcctcgggcttgggggttccagtgccattgc agggccggcagacaacccagccgcttacgcctggccaaccgcccgttcctccacacatggggcattccacggcgtcggtgc ctggttgttcttgattttccatgccgcctcctttagccgctaaaattcatctactcatttattcatttgctcatttactctggtagctgcgcga tgtattcagatagcagctcggtaatggtcttgccttggcgtaccgcgtacatcttcagcttggtgtgatcctccgccggcaactgaa agttgacccgcttcatggctggcgtgtctgccaggctggccaacgttgcagccttgctgctgcgtgcgctcggacggccggcact tagcgtgtttgtgcttttgctcattttctctttacctcattaactcaaatgagttttgatttaatttcagcggccagcgcctggacctcgcg ggcagcgtcgccctcgggttctgattcaagaacggttgtgccggcggcggcagtgcctgggtagctcacgcgctgcgtgatac gggactcaagaatgggcagctcgtacccggccagcgcctcggcaacctcaccgccgatgcgcgtgcctttgatcgcccgcg acacgacaaaggccgcttgtagccttccatccgtgacctcaatgcgctgcttaaccagctccaccaggtcggcggtggcccat atgtcgtaagggcttggctgcaccggaatcagcacgaagtcggctgccttgatcgcggacacagccaagtccgccgcctggg gcgctccgtcgatcactacgaagtcgcgccggccgatggccttcacgtcgcggtcaatcgtcgggcggtcgatgccgacaac ggttagcggttgatcttcccgcacggccgcccaatcgcgggcactgccctggggatcggaatcgactaacagaacatcggcc ccggcgagttgcagggcgcgggctagatgggttgcgatggtcgtcttgcctgacccgcctttctggttaagtacagcgataacct tcatgcgttccccttgcgtatttgtttatttactcatcgcatcatatacgcagcgaccgcatgacgcaagctgttttactcaaatacac atcacctttttagacggcggcgctcggtttcttcagcggccaagctggccggccaggccgccagcttggcatcagacaaaccg gccaggatttcatgcagccgcacggttgagacgtgcgcgggcggctcgaacacgtacccggccgcgatcatctccgcctcga tctcttcggtaatgaaaaacggttcgtcctggccgtcctggtgcggtttcatgcttgttcctcttggcgttcattctcggcggccgcca gggcgtcggcctcggtcaatgcgtcctcacggaaggcaccgcgccgcctggcctcggtgggcgtcacttcctcgctgcgctca agtgcgcggtacagggtcgagcgatgcacgccaagcagtgcagccgcctctttcacggtgcggccttcctggtcgatcagctc gcgggcgtgcgcgatctgtgccggggtgagggtagggcgggggccaaacttcacgcctcgggccttggcggcctcgcgccc gctccgggtgcggtcgatgattagggaacgctcgaactcggcaatgccggcgaacacggtcaacaccatgcggccggccg gcgtggtggtaacgcgtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgta aacaaattgacgcttagacaacttaataacacattgcggacgtctttaatgtactgaattaacatccgtttgatacttgtctaaaatt ggctgatttcgagtgcatctatgcataaaaacaatctaatgacaattattaccaagcagctgatcatgag

EXAMPLE 2 Transformation of wheat (Triticum aestivum cultivar Bobwhite)

An Agrobacterium tumefaciens-mediated transformation system for wheat was used to transform isolated immature embryos. The immature wheat embryos were inoculated with a hypervirulent derivative of the disarmed Agrobacterium tumefaciens strain LBA4404 harboring the binary vectors SBT490 or SBT493 containing a cassette for regeneration of transgenic plants under hygromycin selection pressure comprising the Zea mays ubiquitin promoter, the coding region of the E.coli hygromycin phosphotransferase gene, and the cauliflower mosaic virus (CaMV) 35S-RNA-terminator. The conditions for growing the donor plants, transformation of embryos, selection and regeneration of transgenic plants are described in detail in Hensel et al., 2009. Putative transgenic plants were analyzed by PCR for the presence of the corresponding transgenes. The PCR reactions were carried out under standard conditions using the following specific primers to detect the Hevea brasiliensis HMGR SEQ ID NO: 8 and SEQ ID NO: 9 or the Nicotiana tabacum SMT1 SEQ ID NO: 10 and SEQ ID NO: 1 1 coding regions in wheat plants.

SEQ ID NO: 8: HMGfor: TG CTCAACG G C AG G G AATAC

SEQ ID NO: 9: HMGrev: CATGTCGGAGAAGTCGGATTG SEQ ID NO: 10: SMTfor: TTTGATCTCGCATCGGGAGT

SEQ ID NO: 1 1 : SMTrev: GCGTAAACCGCATCGAATGA

EXAMPLE 3

Sample preparation for sterol analysis

For sterol analysis single wheat kernels were weighted and flatten using a hammer. The flatten kernel was transferred into a 2ml Eppendorf-tube and grinded to a fine powder using a grinding mill. After this the homogenous material was extracted in 1 ml hexane/ ethyl acetate 7:3 (v/v) containing 5 g cholesterol as an internal standard and incubated for 45 min at 50°C at constant shaking. The sample was centrifuged for 1 min at 13.000 rpm. The pellet was re-extracted twice with 0.5 ml hexane/ethyl acetate 7:3 (v/v). The hexane extracts were combined and evaporated at 45°C to dryness using a speedvac concentrator. For saponification, 0.4 ml of 2M KOH (in methanol) was added. The sample was incubated for 1 hour under constant shaking (400 rpm) at 60°C. After adding 0.3 ml water to the sample the mixture was extracted two times with heptane. The heptane and water layers were separated by centrifugation for 1 min at 13.000 rpm. The heptane fractions were combined and evaporated at 50°C to dryness using a speedvac. Sterols were dissolved in 50 μΙ acetonitrile and silylated by addition of 50 μΙ N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) followed by an incubation for 20 min at 70°C at constant shaking.

EXAMPLE 4 Sterol analysis

All standards including cholesterol, campesterol, β-sitosterol, stigmasterol and stigmastanol were obtained from Sigma-Aldrich. The analysis of sterol samples prepared according to the procedure described above was carried out in an Agilent GC/MSD system

(6890N/5975B) equipped with an Agilent HP-5MS column (5% Phenyl Methyl Siloxane, max temperature: 325 °C, nominal length: 30.0 m, nominal diameter: 250.00 μιη, nominal film thickness: 0.25 μιη, mode: constant flow). The TMS-sterols were analyzed under the following conditions: 1 μΙ injection volume, helium carrier gas at constant flow of 1.0 ml/ min, split ration 1 :20, injector temperature 250°C, oven temperature 280°C for 25 min, post run 5 min at 320°C, transfer line temperature 280°C. TMS-sterols were identified by a scan technique (m/z 100-600) according to their retention time and MS spectra. Characteristic mass spectral data for different plant sterols/stanols are available from mass spectral databases (NIST/EPA/NIH Mass Spectral Library) and have been reported in literature (Li et al., 2007, Yang et al., 2003). Quantification of sterols was performed using cholesterol as an internal standard.

EXAMPLE 5

Molecular analysis of T1 wheat kernel

Since sterol analysis was performed on single wheat kernels from segregating T1 seed population of primary transgenic lines it had to be tested if the kernels analyzed for sterol content carried the respective transgenes or not . Therefore the disrupted wheat kernel presenting the pellet after the first sterol extraction with hexane/ ethyl acetate was kept (see Example 3), dried and dissolved in bufferl from a DNeasy Plant Mini-Kit (Qiagen) which was used to isolate the genomic DNA from the pellet.

The qualitative qPCR analysis of DNA samples prepared according to the procedure described above was done under standard PCR condition using the primers described in Example 2 to detect the Hevea brasiliensis HMGR (SEQ ID NO: 8 and SEQ ID NO: 9) the Nicotiana tabacum SMT1 or (SEQ ID NO: 10 and SEQ ID NO: 1 1 ) coding regions. A significant increase in phythosterol concentration had been correlated to presence of transgene sequences in all analyzed seed samples. EXAMPLE 6

Expression of the a truncated form of the Hevea brasiliensis HMGR in wheat (SBT493)

Single mature seeds from wheat plants expressing the truncated form of the Hevea brasiliensis HMGR under the control of the constitutive Zea mays ubiquitin promoter were analyzed for their sterol content using a GC/MS method as described above. Sterol extraction and analysis was performed as described in Example 3 and Example 4. All analyzed kernels were simultaneously analyzed for the presence of the transgene by PCR according to a method described in Example 5. Single seeds from independent transgenic lines were analyzed together with seeds from non-transformed Bobwhite lines originated from the transformation procedure and seeds from untreated Bobwhite lines (Table 4). Sitosterol was the most abundant sterol in wild type wheat kernels comprising about 50 to 60% of the total sterol content, campesterol, campestanol and sitostanol were the other major sterols in wheat. The total sterol content in non-transformed Bobwhite kernel was on average between 40 to 50 mg per 100g fresh weight. There was no significant difference in sterol profile or content between kernels from non-transformed Bobwhite plants passed through the transformation process and seeds from untreated Bobwhite controls. Best transgenic lines harboring the T-DNA of SBT493 showed up to 10 fold increase in total sterols and in terms of sterol composition a strongly altered sterol profile. The key proportion of the total sterol increase was attributed to accumulation of pathway

intermediates like cycloartenol, 24-methylene cycloartenol, citrostadienol, Δ 7-avenasterol, Δ 5-avenasterol, lanosterol and ergosta-7,22-dien-3 -ol (Figure 1 B) which have not been detected in non-transformed controls using the current detection method (Figure 1A).

Usually these minor sterols account for 5-12% of the total sterol content in wheat. The level of the sterol pathway end product such as campesterol and β-sitosterol was up to 2 fold higher in kernels transformed with the truncated form of the Hevea brasiliensis HMGR than in untransformed control kernels. Surprisingly it has been found that Δ 5-Avenasterol which is a minor sterol in wheat and the direct precursor of β-Sitosterol became the most prominent sterol in transgenic wheat kernels. Conversion of Δ 5-Avenasterol into β- Sitosterol seems to be a limiting step in synthesis of phythosterol end products in wheat. EXAMPLE 7

Expression of a truncated form of the Hevea brasiliensis HMGR in combination with the Nicotiana tabacum SMT1 in wheat (SBT490)

In order to examine whether the total sterol content in wheat kernels could be further increased by over-expression of other enzymes of the sterol pathway the catalytic domain of the Hevea brasiliensis HMGR was over-expressed in combination with the

Nicotiana tabacum SMT1. Wheat plants transformed with the construct SBT490 expressed both enzymes under the control of the constitutive Zea mays ubiquitin promoter. Mature single seeds from 53 independed transgenic plants and non-transgenic control plants were analyzed for their sterol content. Sterol extraction and analysis of four individual kernels per plant was carried out as described in Examples 3 and 4. At the same time all kernels were tested for the presence of the transgene using a qualitative PCR approach as described in Example 5. Seeds from untransformed Bobwhite lines originated from the transformation procedure and seeds from untreated Bobwhite lines were used as non-transgenic controls (Figure2A). Fifteen lines out of 53 lines show an at least 1.7-fold increase in sterol pathway end products like β-sitosterol, campestanol and stigmasterol. Eight lines among those 15 lines show more than 2-fold increase in sterol pathway end products. Best lines were analyzed for the presence of sterol pathway intermediates. Sterol composition of best transgenic wheat kernels harboring the Hevea brasiliensis HMGR and the Nicotiana tabacum SMT1 is presented in Figure 2 B and Table 5.

Co-expression of the truncated Hevea brasiliensis HMGR and the Nicotiana tabacum SMT1 via the constitutive maize ubiquitin promoter enhanced total sterols in kernels to a about equal level than that achieved by expression of HMGR alone. Transgenic kernel of best performing transgenic lines showed an dramatic increase in phythosterol pathway intermediates like 24-methylene cycloartenol, citrostadienol,A 7-avenasterol, Δ 5- avenasterol, lanosterol and ergosta-7,22-dien-3 -ol. In transgenic lines expressing in addition the SMT1 catalyzing conversion of cycloartenol into 24-methylene cycloartenol the levels of 24-methylene cycloartenol are particularly elevated whilst levels of cycloartenol are comparable to wild type levels. As in transgenic kernels harboring the truncated

HMGR alone Δ 5-Avenasterol which is a minor sterol in wheat and the direct precursor of β-Sitosterol became again the most abundant sterol in the profile.

Brief description of the drawings and tables

Figure 1 : Representative GC chromatogram of single wheat kernels (A) wild type kernel (B) single kernel expressing the truncated H. brasilisiensis HMG-CoA Reductase (SBT 493). Peak identification: A, Cholesterol-TMS; B, Campesterol-TMS; C, Campestanol-TMS; D, β- Sitosterol-TMS; E, β-Sitostanol-TMS; F, Δ 5-Avenasterol-TMS; G, Ergosta-7,22-dien-3 -ol- TMS; H, Cycloartenol-TMS; I, Δ 7-Avenasterol-TMS; J, 24-Methylene cycloartenol-TMS; K, Citrostadienol-TMS.

Figure 2: Representative GC chromatogram of single wheat kernels (A) wild type kernel (B) single kernel expressing the truncated H. brasilisiensis HMG-CoA Reductase and N.

tabacum Sterol Methyltransferase 1 (SBT490). Peak identification: A, Cholesterol-TMS; B, Campesterol-TMS; C, Campestanol-TMS; D, β-Sitosterol-TMS; E, β-Sitostanol-TMS; F, Δ 5-Avenasterol-TMS; G, Ergosta-7,22-dien-3 -ol-TMS; H, Cycloartenol-TMS; I, Δ 7- Avenasterol-TMS; J, 24-Methylene cycloartenol-TMS; K, Citrostadienol-TMS. Table 4: Sterol composition of transgenic T1 wheat kernels expressing the truncated H. brasilisiensis HMG-CoA Reductase (SBT493) compared to non-transformed control kernels (WT, wild type kernel; TC, kernel of a non-transformed plant originated from the

transformation procedure. Values refer to fresh weight.

SBT493-22 1 27,05 4,93 0,82 50,73 32,48 34,12 37,45 53,44 47,00 26,60 314,62 6,3 +

2 11,29 3,98 0,39 31,60 9,00 5,86 0,46 1,59 4,05 0,00 68,22 1,4 -

3 10,92 3,91 0,56 29,80 0,00 0,00 0,00 0,00 1,28 0,00 46,47 0,9 -

4 9,59 3,58 0,43 27,13 0,00 0,00 0,00 0,00 0,91 0,00 41,64 0,8 -

SBT493-23 1 16,39 3,15 0,41 24,73 21,10 23,39 26,26 48,10 50,01 9,31 222,84 4,5 +

2 10,32 4,53 0,39 28,09 7,60 5,52 0,69 2,02 4,66 0,00 63,82 1,3 -

3 15,90 3,76 0,39 23,84 22,96 24,04 28,35 54,22 48,63 19,49 241,58 4,9 +

4 11,82 4,29 0,44 30,44 10,20 6,63 0,74 2,24 6,35 0,00 73,14 1,5 -

SBT493-28 1 15,60 3,05 0,26 26,84 16,00 20,31 24,73 45,23 58,35 6,48 216,84 4,4 +

2 11,62 3,51 0,48 33,95 6,77 4,62 0,64 1,97 5,20 0,00 68,76 1,4 -

3 9,21 3,65 0,21 29,34 4,38 3,54 0,00 0,00 4,12 0,00 54,44 1,1 - n

4 20,19 4,06 0,52 31,53 33,83 33,33 37,46 70,17 70,18 14,96 316,22 6,4 +

SBT493-38 1 9,70 4,89 0,44 29,69 0,00 0,00 0,00 0,00 1,20 0,00 45,93 0,9 -

2 30,78 5,22 1,17 60,63 34,26 33,99 30,91 68,97 71,35 15,29 352,58 7,1 +

3 9,49 4,16 0,51 28,07 0,00 0,00 0,00 0,00 1,31 0,00 43,54 0,9 -

4 8,70 4,10 0,48 28,19 0,00 0,00 0,00 0,00 1,33 0,00 42,80 0,9 -

SBT493-27 1 20,03 4,41 0,53 42,23 24,70 26,91 27,80 53,88 60,20 5,82 266,49 5,4 +

2 17,44 4,69 0,47 36,92 19,65 25,69 21,31 40,14 58,95 3,77 229,04 4,6 +

3 12,96 5,17 0,34 36,16 13,92 8,91 0,89 3,15 6,12 0,00 87,62 1,8 -

4 28,83 4,29 0,78 46,72 46,61 45,76 46,42 74,13 67,62 29,74 390,91 7,9 +

WT 1360 1 12,26 5,32 0,71 32,89 0,00 0,00 0,00 0,00 1,03 0,00 52,20 1,0 -

2 9,43 3,07 0,39 23,68 0,00 0,00 0,00 0,00 1,08 0,00 37,65 0,8 -

WT 1359 1 8,77 2,97 0,40 24,51 0,00 0,00 0,00 0,00 1,06 0,00 37,71 0,8 -

2 8,93 2,97 0,47 26,10 0,00 0,00 0,00 0,00 0,89 0,00 39,36 0,8 -

TC 061 1 7,93 2,19 0,74 17,19 0,00 0,00 0,00 0,00 0,92 0,00 28,97 0,6 -

2 11,23 7,15 1,43 39,65 0,00 0,00 0,00 0,00 1,37 0,00 60,83 1,2 -

TC 060 1 12,21 0,64 0,64 36,48 0,00 0,00 0,00 0,00 0,94 0,00 50,91 1,0 -

2 12,86 0,69 0,69 39,52 0,00 0,00 0,00 0,00 0,97 0,00 54,73 1,1 -

TC 063 1 12,48 7,31 0,70 39,39 0,00 0,00 0,00 0,00 1,02 0,00 60,90 1,2 -

2 8,49 3,02 1,06 33,80 0,00 0,00 0,00 0,00 3,81 0,00 50,16 1,0 -

Table 5: Sterol composition of transgenic T1 wheat kernels expressing the truncated H. brasilisiensis HMG-CoA Reductase and N. tabacum Sterol Methyltransferase 1 (SBT490) compared to non -transformed control kernels (WT, wild type kernel; TC, kernel of a non- transformed plant originated from the transformation procedure. Values refer to fresh weight.

SBT490-13 1 27,54 0,00 61,23 0,00 0,00 146,01 n.d n.d 89,49 36,59 360,87 7,1 +

2 32,34 14,68 50,21 0,00 0,00 148,72 32,55 43,62 79,57 48,51 450,21 8,9 +

3 31,42 15,27 57,96 0,00 0,00 162,39 34,07 39,38 78,32 45,13 463,94 9,2 +

4 28,52 18,79 77,52 0,00 0,00 130,87 n.d 38,26 81,88 0,00 375,84 7,4 +

SBT490-28 1 21,35 10,96 59,13 0,00 23,46 57,44 20,08 20,93 47,33 40,73 301,40 5,9 +

i

2 16,28 12,35 57,99 0,00 0,00 54,36 0,00 n.d 40,26 0,00 181,25 3,6 +

3 21,34 12,31 53,27 0,00 21,18 56,07 0,00 22,27 47,98 19,78 254,21 5,0 +

4 15,99 12,39 51,87 0,00 n.d 46,40 0,00 n.d 39,19 0,00 165,85 3,3 +

SBT490-14 1 11,33 3,50 30- ,83 0,00 5,40 36,55 1,91 7,20 38,56 0,00 135,28 2,7 +

2 19,39 3,50 49,0 mg7 0,00 9,35 82,71 4,44 14,02 50,93 0,00 233,41 4,6 +

n

3 9,04 3,19 28,19 0,00 2,13 22,47 0,00 1,46 21,01 0,00 87,50 1,7 +

4 19,93 1,69 37,16 0,00 10,47 73,31 6,08 15,88 50,34 17,23 232,09 4,6 +

SBT490-51 1 9,03 2,98 26,34 0os-,00 4,32 81,17 10,70 17,49 53,09 13,27 218,40 4,3 +

2 14,17 0,63 21,22 0,00 1,89 85,71 3,57 7,56 50,00 2,73 187,49 3,7 +

3 19,86 3,54 23,98 0,00 2,45 53,41 8,04 15,80 42,64 6,95 176,68 3,5 +

4 19,28 3,77 34,08 0,00 7,71 111,99 15,24 20,89 53,60 26,71 293,25 5,8 +

SBT490-52 1 14,42 1,95 24,71 0,00 3,78 66,02 10,53 14,87 46,45 11,56 194,28 3,8 +

2 10,13 0,39 45,06 0,00 0,00 100,65 15,58 28,44 10,91 45,06 256,23 5,1 +

3 9,36 0,37 38,95 0,00 0,00 162,17 14,61 28,28 19,29 38,95 311,99 6,2 +

4 5,28 0,28 15,83 0,00 4,17 9- 3,06 5,56 14,44 53,89 2,50 195,00 3,8 +

cycoar

WT 1359 1 9,21 7,60 26,12 0,00 0,00 0,00 0,00 0,00 0,00 0,00 42,93 0,8 -

2 12,89 10,80 34,67 0,00 0,00 0,00 0,00 0,00 0,00 0,00 58,36 1,2 -

3 11,00 9,09 29,33 0,00 0,00 0,00 0,00 0,00 0,00 0,00 49,41 1,0 -

4 9,57 8,11 25,80 0,00 0,00 0,00 Cⁱd^tt 0rosa,00 0,00 0,00 0,00 43,48 0,9 -

WT 1360 1 10,49 8,52 28,15 0,00 0,00 0,00 0,0 mg0 0,00 0,00 0,00 47,16 0,9 -

2 10,48 8,75 27,45 0,00 0,00 0,00 0,00 0,00 0,00 0,00 46,68 0,9 -

WT 1361 1 11,22 9,04 33,53 0,00 0,00 0,00 0,00 0,00 0,00 0,00 53,79 1,1 -

Δ-

2 9,77 7,61 28,75 0,00 0,00 0,00 0,00 0,00 0,00 0,00 46,14 0,9 -

TC 002 1 8,94 9,38 24,49 0,00 0,00 0,00 0,00 0,00 mg 0,00 0,00 42,82 0,8 -

2 9,95 8,62 25,99 0,00 0,00 0,00 0,00 0,00 0,00 0,00 44,56 0,9 -

TC 008 1 13,57 12,27 36,62 0,00 0,00 0,00 0,00 0,00 Δ- 0,00 0,00 62,45 1,2 -

2 15,35 13,49 41,49 0,00 0,00 0,00 0,00 0,00 0,00 0,00 70,33 1,4 -

a o . References

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Harker, M., Holmberg, N., Clayton, J.C., Gibbard, C.L., Wallace, A.D., Rawlins, S., Hellyer, S.A., Lanot, A., Safford, R. (2003) Enhancement of seed phythosterol levels by expression of an N-terminal truncated Hevea brasiliensis (rubber tree) 3-hydroxy-3-methylglutaryl-CoA reductase. Plant Biotechnology J 1 :1 13-121

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Claims

A method for the production of a transgenic cereal plant with increased Avenasterol content in the kernels characterized in that the wild-type plant is transformed with a vector comprising nucleic acid sequences encoding a SMT1 protein and a HMGR protein both under the control of a constitutive promoter and selecting for transgenic plants in which - in contrast to or comparison with the wild type plant - the Avenasterol content in the kernels is increased. 2) A method for the production of a transgenic cereal plant with increased Avenasterol content in the kernels according to Claim 1 characterized in that

a) the SMT1 nucleic acid sequence is SEQ ID NO: 1 and the HMGR nucleic acid sequence is SEQ ID NO: 3 or

b) the nucleic acid sequences are at least 80% identical to the nucleic acid sequences of (a), wherein said nucleic acid sequences encode polypeptides being a SMT1 protein and a HMGR protein.

3) A method for the production of a transgenic cereal plant with increased Avenasterol content in the kernels according to Claim 1 characterized in that

a) the SMT1 nucleic acid sequence encodes the SMT1 amino acid sequence

SEQ ID NO: 2 and the HMGR nucleic acid sequence encodes the HMGR amino acid sequence SEQ ID NO: 4 or

b) the nucleic acids sequences being functional equivalents of a) with an identity of at least 80% of the amino acid sequence as set forth in SEQ ID NO: 2 and SEQ ID NO: 4.

A method for the production of a transgenic cereal plant with increased Avenasterol content in the kernels according to any of Claims 1 to 3 characterized in that the constitutive promoter is the pZmUbi promoter.

A method for the production of a transgenic cereal plant with increased Avenasterol content in the kernels according to any of Claims 1 to 4 characterized in that the Avenasterol increased is A5-Avenasterol.

A method for the production of a transgenic cereal plant with increased Avenasterol content in the kernels according to any of Claims 1 to 4 characterized in that the Avenasterol increased is Δ7 -Avenasterol.

A method for the production of a transgenic cereal plant with increased Avenasterol

content in the kernels according to any of Claims 1 to 6 characterized in that the cereal plant is a wheat plant. A transgenic cereal plant with increased Avenasterol content in the kernels characterized in that said plant contains a gene construct comprising nucleic acid sequences encoding a SMT1 protein and a HMGR protein both under the control of a constitutive promoter.

A transgenic cereal plant with increased Avenasterol content in the kernels according to Claim 8 characterized in that

a) the SMT1 nucleic acid sequence encodes the SMT1 amino acid sequence SEQ ID NO: 2 and the HMGR nucleic acid sequence encodes the HMGR amino acid sequence SEQ ID NO: 4 or

b) the nucleic acids being functional equivalents of a) with an identity of at least 80% of the amino acid sequence as set forth in SEQ ID NO: 2 and SEQ ID NO: 4.

A transgenic cereal plant with increased Avenasterol content in the kernels according to any of Claims 8 to 10 characterized in that said genes are both under the control of the constitutive pZmUbi promoter.

A transgenic cereal plant with increased Avenasterol content in the kernels according to any of Claims 8 to 1 1 characterized in that the Avenasterol increased is A5-Avenasterol.

A transgenic cereal plant with increased Avenasterol content in the kernels according to any of Claims 8 to 1 1 characterized in that the Avenasterol increased is A7-Avenasterol.

A transgenic cereal plant with increased Avenasterol content in the kernels according to any of Claims 8 to 13 characterized in that the cereal plant is a wheat plant. 15) Kernels from a transgenic cereal plant according to any of Claims 8 to 14. 16) Oil extracted from kernels according to Claim 15.

17) Flour produced from kernels according to Claim 15. 18) Use of kernels, oil extracted from kernels and flour produced from kernels according to Claims 15, 16 and 17 as ingredient of food, pet food, feed or medical support products.