WO1998010062A1

WO1998010062A1 - Monocot seed gene expression system

Info

Publication number: WO1998010062A1
Application number: PCT/US1997/015340
Authority: WO
Inventors: Gurdip Brar; David R. Russell
Original assignee: Monsanto Company
Priority date: 1996-09-03
Filing date: 1997-09-02
Publication date: 1998-03-12
Also published as: AU4173497A

Abstract

A gene construct comprising the rice glutelin-1 promoter operably connected to a foreign non-plant gene is disclosed. A monocot seed containing a gene construct comprising a rice glutelin-1 promoter operably connected to a foreign non-plant gene is also disclosed. Preferably, the gene product is expressed at a level of at least 1 % of total soluble endosperm protein. Most preferably, the gene product is expressed at a level of at least 2.5 % of the total soluble endosperm protein.

Description

MONOCOT SEED GENE EXPRESSION SYSTEM

Field of the Invention The field of the present invention is plant transformation systems. In particular, the field of the present invention is a system for expressing foreign genes in maize kernels by use of the rice glutelin-1 promoter.

Background Plant Transformation One general object of modern biotechnology is to -genetically engineer crop plants by introducing new genetic traits into the genome of elite plant lines. Plants with new traits, such as insect resistance or herbicide resistance, and artificial manipulations of the agronomic qualities of the crop product are possible once recornbinant genes are introduced into plant lines.

The first widely used plant genetic engineering technique was based on the natural ability of the soil- dwelling microorganism Aqrobacterium tumefaciens to introduce a portion of its DNA into a plant cell as a part of the normal pathogenic process. If a foreign gene is inserted into the bacteria in certain ways, the Aqrobacterium can be used to transfer the foreign gene into a plant. Aqrobacterium transformation techniques have been developed for a number of plants, mostly dicotyledonous, but the usefulness of the technique has -2 - varied from plant species to species. Agrobacterium- based transformation systems are limited because they require cell or tissue culture and plant regeneration techniques. Plants lines vary in their amenability to tissue culture and regenerations methods. Regeneration of maize protoplasts has been accomplished recently, but the efficiency of the process is low. Rhodes, e_£ al.. Bio/technology 6:56-60 (1988). A maize genetic engineering system which obviates tissue culture and regeneration would represent a significant advantage. One new developing technique for creating transformed plants includes bombarding a cell with accelerated particles carrying genetic information. The first indication of the utility of this technique was a demonstration that DNA constructs could be coated onto tungsten particles and accelerated into onion skin where the genes were transiently expressed, as is described in the specification of U.S. Patent No. 4,945,050. A problem in the development of an accelerated particle transformation process to create transgenic plants is the difficulty of obtaining a germline plant transformation. By the term "germline transformation" we mean that the germ cells of the plant are transformed in such a way that the progeny of the plant inherit the inserted foreign genetic construct. I "some species, plant genetic transformation has been achieved by the accelerated particle method. European Patent Application No. 301,749 discloses the germline transformation of soybean plants and plant lines. The method disclosed in that published patent application is based on accelerating DNA-coated particles into the excised embryonic axes of soybean seeds. If the bornbarded soybean embryonic axes are treated with high σytokinin media, shoots are induced from the treated embryonic axes. When the shoots are cultivated into whole soybean plants, a significant percentage of the plants will have transformed ger lines. 62

-3 -

Accelerated particle transformation has also been successful in treatment of maize suspension cultures. Fromm, gt _!• Bio/Technology 8:833-839 (1990), discloses the creation of transformed maize plants from suspension cultures and from calli created from suspension cultures. Klein, e_£. _ . , Proc. Natl. Acad. Sci . USA 85:4305-4309 (1988), discloses accelerated particle transformation of maize suspension culture cells. Spencer, et aJL. , Theor . Appl . Genet . 79:625-631 (1990) , discloses stable transformed maize callus recovered from suspension culture cells bombarded with DNA-coated accelerated particles. Gordon-Kamm, e_t al. , The Plant Cell 2:603-618 (1990), discloses microprojectile bombardment of embryogenic maize suspension cultures. None of these disclosures deal with transformation of tissues not in cell culture. Most elite lines of maize cannot presently be regenerated from suspension cell cultures. Hence, prior techniques may be limited to certain less desirable genotypes of maize plants.

Maize embryos have been bombarded and transient ^"expression achieved by DNA-coated particles. Klein, e_t al. , Bio/Technology 6:559-563 (1988), discloses particle bombardment of surface cells of an excised maize embryo. This reference does not disclose regeneration from the bombarded embryo, nor inheritance in progeny plants. The cells were bombarded with DNA encoding the beta-glucuronidase (GUS) gene. Because it turns a substrate, 5-bromo-4-chloro-3-indolyl glucuronidide, blue in an in situ tissue assay, beta- glucuronidide, can be histochemically detected. After bombardment and treatment with the GUS substrate, blue spots appeared on the embryo, indicating transiently transformed cells. Koziel, e____L., Biol . Technology 11:194-199, 1993, describe the introduction of a synthetic gene encoding a truncated version of the CryΙA(b) protein derived from Bacillus thuringiensis into immature embryos of 62

-4- maize using microprojectile bombardment.

Rice Glutelin-1 Promoter

One recently identified plant promoter has been identified as driving the transcription of the rice glutelin-1 gene. The rice glutelin-1 promoter has been described in Zheng, Z., e_£ ε_L. , The Plant Journal 4:357-366 (1993) and Okita, T.W., etui., The J. Biol . SSasB .264:12573-12581 (1989). Zheng, __t aj,. (Plant Phvsio . 109:777-786, 1995) disclose the attachment of either a 5.1 or 1.8 kb promoter fragment of the rice seed storage protein glutelin GT-1 gene to either the ?-phaseolin geno ic or cDNA sequence. The 3-phaseolin protein was successfully synthesized, processed and accumulated in rice endosperm. What is needed in the art of molecular biology is a maize kernel gene expression system by which gene products can be highly expressed in a maize kernel. Maize kernels containing high levels of specific heterologous gene products will be very useful for modifying seed composition, altering protein

^"nutritional composition and facilitating purification of specific heterologous proteins from maize kernels.

Summary of the Invention In one embodiment, the present invention is a monocot seed, plant, or cell containing a gene construct comprising the rice glutelin-1 promoter operably connected to a foreign non-plant gene, wherein the promoter and the gene are not natively connected. Preferably, the gene is a mammalian gene and, preferably, the seed, plant or cell is a maize seed, plant or cell.

In another embodiment of the present invention, the product encoded by the foreign gene is expressed at a level of at least 1% of the total soluble endosperm protein. More preferably, the product is expressed at a level of at least 2.5% of the total soluble endosperm 62

-5- protein. Most preferably, the product is at least 5% of .'the total soluble endosperm protein.

The present invention is also a gene construct comprising the rice glutelin-l promoter operably connected to a gene encoding a non-plant protein, preferably a protein such as an antibody.

In one embodiment of the invention, the rice glutelin-1 promoter is the 4888 bp fragment described in SEQ ID NO:l. In another embodiment of the present invention, the rice glutelin-l promoter is a 1.8 kb fragment of the 4888 bp promoter. This 1867 bp fragment extends from residue 3021 to residue 4888 of SEQ ID NO:l.

In another embodiment of the present invention, the present invention is method of creating a transformed monocot plant, seed, or cell. This method comprises the steps of creating the gene construct described above, creating a transgenic plant comprising the gene construct, and allowing expression of the non- plant gene.

It is an advantage of the present invention that ^"the rice glutelin-1 promoter operably connected with a foreign non-plant gene provides an expression system in maize kernel that results in the foreign gene expression product comprising a significant amount of the total soluble protein. This abundance of foreign protein will facilitate purification of the product and may also improve the nutritional quality of seed protein. It is a further advantage of the present invention that the foreign gene product may be an antibody protein, thus allowing one to more easily produce or isolate recombinant antibodies.

It is a further advantage of the present invention that a gene product of agricultural importance may be expressed in the maize kernel. Thus, one may have increased expression of a gene product that would add nutritive value or beneficial properties to the maize 2

- 6 - product itself.

Other objects, features and advantages of the present invention will become apparent to one skilled in the art after examination of the specification, drawings and claims .

Description of the Drawings Fig. 1 is an exploded schematic view of a particle acceleration device useful in the present invention.

Fig. 2 is a top plan view of the device of Fig. 1. Fig. 3 is an enlarged drawing of the corn immature embryo stage 1.

Fig. 4 is a schematic diagram of plasmid WRG5084. Fig. 5 is a schematic diagram of plasmid WRG5086. Fig. 6 is a schematic diagram of plasmid WRG456 . Fig. 7 is a schematic diagram of plasmid WRG5236.

Fig. 8 is a schematic diagram of plasmid WRG5243. Fig. 9 is a schematic diagram of plasmid WRG5245.

Description of the Invention 1. In General This invention relates to the economical production of heterologous proteins in monocotyledonous (monocot) plants such as maize. Preferably the heterologous proteins are pharmaceutical proteins such as antibodies." The proteins are preferably expressed in monocot seeds such as maize kernels. More specifically, the proteins are preferably expressed in the maize seed endosperm. High levels of expression of heterologous proteins, especially of monoclonal antibodies, have been difficult to achieve in monocots before, partly because of the lack of suitable seed- specific promoters .

In one embodiment, the present invention is a monocot seed, such as a maize kernel, containing a gene construct comprising the rice glutelin-1 promoter operably connected to a foreign non-plant gene. The gene product encoded by the foreign gene is preferably 62

- 7- expressed at a level of at least 1% of the total s-oluble endosperm protein and most preferably is expressed at a level of at least 5% of the total soluble protein in seed or kernel endosperm. We report below in the Examples the high levels of expression of monoclonal antibody BR96 in maize endosperm when the heavy chain and light chain genes are driven by the rice glutelin-1 promoter.

Additionally, we have described the successful production of a second monoclonal antibody, NeoRx451.

2. Suitable Promoters and Preparation of Nucleic Acid Constructs

The present invention requires a gene construct in which the rice glutelin-1 promoter is operably connected to a foreign gene. The Examples below disclose one particularly useful embodiment of the rice glutelin-1 promoter that was obtained from Dr. N. Murai (Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, Louisiana 70803-1720) as part of clone pGt5.1 (Bglll) . To obtain the promoter, a Hindlll site was inserted into the Bglll site destroying the Bglll site and resulting in pWRG4563. The rice glutelin-1 promoter was removed from pWRG4563 as an approximately 4.9 kb. Kpnl-Hindlll fragment and inserted into the Kpnl-Hindlll site of pWRG2484 to make pWRG4564 (glutelin promoter and leader-amv leader-GUS) . Fig. 6 is a map of pWRG4564. The rice glutelin-1 promoter was then moved as a Xhol-Hindlll fragment into both pWRG2800 and pWRG2801 to make pWRG4565 (glutelin promoter and leader-tobacco extensin leader and signal peptide-BR96 heavy chain) and pWRG4566 (Glutelin promoter and leader-tobacco extensin leader and signal peptide-BR96 light chain respectively) , respectively. The tobacco extensin fragment contains all of the 5' UTR and the first 26 amino acids of extensin coding region (De Loose, e_t ill., Gene 99:95-100, 1991). 35S-hygromycin resistance cassettes were inserted into both of these plasmids 2

-8 - resulting in pWRG5086 and pWRG5084 (Figs. 4 and 5) .

The nucleotide sequence of the Kpnl-Hindlll fragment from pWRG4563 was determined and is listed at SEQ ID NO:l. Sequencing of both strands was performed by Retrogen Inc. using Applied Biosystems automated sequencer. The transcription initiation site as described by Okita, e_t al., is at residue 4861 of SEQ ID₍N0:1. Nucleotides -4861 to +27 of the rice glutelin-1 gene (as numbered by Zheng, et al. The Plant Journal 4:357-366 (1993)) correspond to nucleotides 1- 4888 in SEQ ID N0:1. One may use the nucleotide sequence reported in SEQ ID N0:1 to design primers for direct PCR amplification of the rice glutelin-1 promoter from a rice genomic library. One may also obtain the rice glutelin promoter by following the method described by Okita, e_t a_L . , The J. Biol. Chem. 264:12573-12581 (1989).

Although the full-length (4888 bp) rice glutelin promoter was used in the experiments below, Applicants envision that a smaller fragment would also be suitable, although it is believed that the 4888 bp ^"fragment is preferable. A 1867 bp fragment of the 4888 bp promoter has been demonstrated to promote gene expression, although with less strength than the larger fragment. This 1867 bp fragment extends from the EcoRI site to the Bglll site of pGT5.1 as described in Zheng, gt έi , The Plant Journal 4:357-366 (1993). This fragment corresponds to residues 3021 to 4888 of SEQ ID NO: l. Therefore, a suitable glutelin-1 promoter will contain at least this 1867 bp sequence. Expression levels may be further increased by adding some intron sequences .

One of skill in the art of molecular biology will also realize that minor additions and subtractions to a promoter sequence can result in a promoter with nearly identical activity to the wild type promoter. These manipulated or mutated sequences would be just as suitable and are considered "rice glutelin-1 062

promoters . "

To determine whether a fragment or mutation is suitable for the present invention, one would assay it side-by-side the rice glutelin-1 promoter described above and measure gene expression in a system utilizing a plasmid such as described at Figs . 4 and 5. Any gene expression of at least 75% that displayed by the native rice glutelin-1 promoter indicates that the promoter fragment or modification is suitable. A preferable gene construct will comprise at least a rice glutelin-1 promoter operably connected to a foreign non-plant gene. However, other expression elements are useful in various embodiments of the gene construct of the present invention. In particular, signal peptide sequences are useful for protein products that are expected to be secreted through membranes. For example, the antibodies described below in the Examples were connected to the tobacco extensin signal peptide for proper membrane processing. Additionally, a portion of the rice glutelin untranslated region sequence (UTR) is a preferable addition.

The Examples below describe gene constructs that are preferable for expressing antibodies .in the method of the present invention. Elements in the constructs used below in the Examples and diagramed at Figs . 4 and 5 are -described in the following sources: Adh intron: Callis, J., ej al., (1987) Genes Dev. 1:1183-1200; GUS gene: Jefferson, R.A. , et. al. , (1987) EMBO J. 6:3901-3907; tobacco extensin gene: De Loose, M. , et. al., (1991) Gene 99:95-100; hygromycin: Van den Elzen, P.J.M., et __l., (1985) Plant Mol. Biol . 5:299-302: and vector backbone: Yanisch-Perron, C, e aj,. (1985) Gene 33:103-119. As described above, foreign genetic constructions are often embodied in expression cassette vectors for plant cells, many of which are known in the art. These vectors may be part of a plasmid or a virus. Typically 62

- 10- such a plant expression vector system includes the co_rding sequence for the desired foreign gene and appropriate regulatory sequences . The appropriate regulatory sequences might include a translational terminator and signal or leader sequences . A translation or transcriptibnal enhancer may be incorporated between the promoter and the coding region of the genetic sequence. One with knowledge in the art of molecular biology would know how to use standard techniques to insert the rice glutelin-1 promoter upstream, or 5', of the coding region of any particular gene.

The transforming nucleic acid construct can include a marker gene which can provide selection or screening capability in the treated plant tissues .

Selectable markers are generally preferred for plant transformation events, but are not available for all plant species. A selectable marker encodes for a trait in the transformed plant cells which can be selected for by the exposure of the plant tissues to a selection agent. Suitable selectable markers can be antibiotic or herbicide resistant genes which, when inserted in some cells of a plant in culture, would imbue those particular cells with the ability to withstand exposure to the antibiotic or the herbicide.

It has been found that at least one antibiotic selectable marker system can be made to work in maize. A gene coding for resistance to the antibiotic hygromycin from E. coli has previously been found to be useful as a selectable marker in tobacco transformation. Waldron, e_t a_L . , Plant Mol. Biol . 5:103-108 (1985). Such selectable markers do not reliably transfer from species to species, particularly when used on callus or differentiated tissue as opposed to protoplasts or suspension culture.

Another type of marker gene is one that can be screened by histochemical or biochemical assay, even though the gene cannot be selected for. A suitable 62

-11 - marker gene found useful in such plant transformation experience is the GUS gene, discussed above. Jefferson, ejt aJL . , EMBO J. 6:3901-3907 (1987) discloses the general protocol for a GUS assay. Thus, the use of a GUS gene provides a convenient colorimetric assay for the expression of introduced DNA in plant tissues by histochemical analysis of the plant tissues. In a typi Ical transformation process, the gene of interest sought to be expressed in the plant can be coupled in tandem With the GUS gene. Then the tandem construct can be transformed into plant tissues and the resulting plant tissues can be analyzed for expression of the GUS enzyme. Tissues and plants expressing the GUS enzyme will also be found to be expressing the gene of interest.

3. Suitable Foreign Genes

In one embodiment, the present invention is a monocot seed, such as a maize kernel, containing a gene construct comprising the rice glutelin-1 promoter operably connected to a foreign non-plant gene. By "foreign non-plant gene" we mean a gene that is not natively connected to the rice glutelin-l promoter and is not isolated from plant sources. Preferably, the gene is of mammalian origins or is synthetically created. A "foreign non-plant gene" may also be a synthetic gene that is created to mimic a non-plant gene. _^ Specifically, a "mammalian gene" may include a synthetic gene designed to mimic a mammalian protein such as an antibody. A "non-plant gene" or a "mammalian gene" may be a chimeric gene.

Preferably, the foreign gene encodes an antibody or a fragment of an antibody. The Examples below demonstrate the successful production of both the heavy and light chains of monoclonal antibody BR96 and antibody NeoRx 51.

4. Preparation of Monocot Embryos

We envision that the present invention will be useful in creating a variety of transgenic 062

-12- monocotyledonous plants. Most preferably, the myention involves the creation of a transgemc maize plant, seed or kernel. Below, we describe a preferred method of transformation of maize embryos. One of skill in the art will realize that these methods may be adapted to work in other monocotyledonous plants such as rice and wheat .

, A gene construct including the rice glutelin-1 promoter will be effective at enhanced gene expression in a variety of transformation systems and methods. Below in the next three sections we list a preferred method of preparing maize embryos, transforming the embryos and regenerating the plants . One of skill in the art of molecular biology would realize that variations of this transformation procedure would also be effective.

Maize kernels are sterilized and isolated from conditioned ears. Preferably, the ears from which these kernels are taken have been conditioned at 4°C from one to eight days with two to four days being preferred. The maize embryo, which is composed of the scutellum ,and the shoot/root axis, is then excised from the kernel. The scutellum is the cotyledon of the maize embryo. Esau, Anatomy of Seed Plants, John Wiley & Sons, New York, p. 477 (1960) .

Preferably, immature embryos are excised. By "immature" we mean that the embryos are approximately between 0.50 mm and 2.50 mm in length are in the developmental stages of early coleoptilar, stage one or stage two, as defined by Abbe and Stein Am, Jour . Bot . 41:285-293 (1954). The embryos just entering stage one are preferred. Mature embryos may be used in the present invention, but it is more difficult to culture these embryos. Very immature embryos may be used, but these smaller embryos are harder to manipulate.

The maize embryos are placed on a medium amenable to immature embryo scutella culturing. The embryonic axis should touch the media, and the scutellum should 62

- 13 - be exposed. Preferably, the medium should contain the salts and carbon source (sucrose or other sugars) needed for growth, an auxin or auxin-like substance, and agar or other gelling agent . In the practice to date, the culturing medium contained chloramben, an aUxin. Other auxins, such as 2,4-D may also be used. The embryos are incubated in the dark for conditioning. Usually, this takes about four days.

Preferably, the callus that develops on the scutellum is a mixture of organogenic and embryogenic callus. Almost every maize line is susceptible to this type of callus culture. For example, McCain and Hodges, Bot . Qaz . 147 [4] :453-460 (1986), disclose a method of obtaining somatic embryos that have developed from scutellum of immature zygotic embryos of maize variety A188.

In transformation efforts using Agrobacterium-based procedures with other crops, was found that wounded tissue often gave a better transformation result. This phenomenon has not been demonstrated to be useful in accelerated particle ^"transformation methods. Nevertheless, in the protocol used here a part of the embryo that is not producing callus is preferably excised to optimize, transformation results. The non-producing tissue is visually identified by the lack of visible callus proliferation. Typically, the callus proliferates mainly at the suspensor end of the immature embryo on abaxial side of scutellum. The portion of the embryo that is removed is from the coleoptile end. This is illustrated in

Fig. 3, illustrating a stage 1 embryo modelled after Abbe and Stein, Am. Jour. Bot. 41:287 (1954) . The suspensor end 2 is where the callus proliferates on the lower one-half to three-quarters of the scutellum. The top one-half to one-quarter of the coleoptile end 3 of the scutellum is the part that is removed. A dashed line 4 indicates the usual locus of the art . The removed scutellum portion is discarded. 2

- 14 -

The dissected embryos are placed on fresh medium and positioned so that the callus tissue is available to be bombarded by the accelerated particles. Preferably, this medium now contains an osmoticum such as 0.4 M mannitol, so that the embryogenic cells will be partly plasmolyzed. This osmotic condition helps to preserve cell integrity through bombardment. Optimally, the ernbryos should remain on this medium one to three hours at room temperature in the dark before they are bombarded.

5. Transformation of Embryos

A. Preparation of Nucleic Acid-Coated Particles Multiple copies of the nucleic acid construct are prepared by known molecular biology techniques . The transformation process requires carrier particles of a durable, dense, biologically-inert material . Gold is a suitable material for use as the carrier particle. The carrier particles are of extremely small size, typically in a range of 0.7 to 3 microns, so that they are small in relation to the size of the maize target cells. Preferably, ^"microcrystalline gold particles are used as carrier particles. A suitable source of microcrystalline gold particles is Degussa/Metz (Lot #7-29020-0, Gold Beads 0.71 μ) . This product consists of gold particles of high surface area and amorphous shape and size. We found_^that microcrystalline carrier particles of irregular size achieve a higher transformation efficiency than that achieved by spherical gold particles.

The genetic material to be inserted into the cells is coated onto the carrier particles. Both heavy chain and light chain plasmids are coated onto the gold beads simultaneously for co- ransformation. The DNA loading ratio for each plasmid was 2 μg/mg of gold particles. This can be readily done by drying solutions of DNA or RNA onto the carrier particles themselves. Suitable stabilizers can be added to the mixture to help with 2

- 15 - the longevity of the genetic material on the carrier particles, such as the preparation based on spermidine described in the Example below.

B. Bombarding Cultured Embryos The apparatus utilized in the present invention must be capable of delivering the nucleic acid-coated particles into plant cells in such a fashion that a suitable number of cells can be transformed. At some frequency, the carrier particles lodge within the maize cells and, through a process we do not understand, the genetic materials leaves the carrier particles and integrates into the DNA of the host maize cells. Many types of mechanical systems can accelerate the carrier particles into plant cells. Possible mechanisms include ballistic explosive acceleration of particles, centrifugal acceleration of particles, electrostatic acceleration of particles, or other analogous systems capable of providing momentum and velocity to small particles. The mechanism we used in the Example is based on the acceleration of particles through an adjustable electric voltage spark discharge device which is capable of accelerating a planar carrier sheet at a target surface. This apparatus will be described further below with reference to Figs. 1 and 2.

The particle acceleration apparatus is generally indicated at 10 of Fig. 1. The apparatus consists of the spark discharge chamber 12 into which are inserted two electrodes 14 spaced apart by a distance of approximately one to two millimeters. The spark discharge chamber 12 is a horizontally extended rectangle having two openings, 16 and 18, extending out its upward end. The opening 16 is covered by an access plate 20. The opening 18, located on the side of the rectangle of the spark discharge chamber opposite from the electrode 14, is ultimately intended to be covered by a carrier sheet 22.

The electrodes 14 are connected to a suitable adjustable source of electric discharge voltage (not shown) . A suitable source of electric discharge voltage includes a capacitor in the size range of one to two microfarad. The voltage of the charge introduced into the capacitor should be adjustable. An adjustable voltage can be introduced readily into such a capacitor through the use of an autotransformer which can be adjustable between a range of one and fifty thousand volts. Preferably, a high voltage electric switch is provided so that the capacitor can be discharged safely through the electrodes 14 without harm to the operator.

A carrier sheet 22 is placed upon the opening 18 of the spark discharge chamber 12. The carrier sheet 22 is a planar sheet of relatively stiff material which is capable of carrying small, inert carrier particles thereon toward the target surface. Preferably, the carrier sheet 22 is a small sheet of aluminized, saran-coated mylar. We envision that other relatively stiff, planer materials may be used for the carrier sheet 22. The function of the carrier sheet 22 is to crbnvert an outwardly outstanding force produced by the electrodes to a broadly distributed horizontal force capable of accelerating a large number of carrier particles in parallel with an even force. Other kinds of force othe than electric discharge can be used to propel the carrier sheet 22 upward. The force should be adjustable so that the force of travel of the carrier sheet 22 can be adjusted. Again referring to the apparatus of Figs. 1 and 2, a retaining screen 24 is approximately 15 millimeters above the opening 18 and the discharge chamber 12. A target surface 26 is placed above the retaining screen 24 at a distance of between 5 and 25 millimeters. The target surface 26 is any suitable culture surface onto which the material to be transformed can readily be placed. An overturned petri dish can conveniently be used for the transformation of plant tissues. Using a 62

- 17- semisolid or solid agar-based medium in the bottom of a petri dish, it is possible to place tissues on the agar where they will be retained. The petri dish itself can serve as the target surface while retaining the plant tissues on the agar.

The DNA-coated particles are layered onto the top of the carrier sheet 22. The layering is done so as to distribute a relatively even pattern of carrier particles across the entire top surface of the carrier sheet 22. Preferably, the coated carrier particles are applied to the carrier sheet at a loading rate of .025 to .050 milligrams of coated carrier particles per square centimeter of carrier sheet . The carrier sheet 22 is placed upon opening 18. An oil or water droplet is used to adhere the carrier sheet 22 in place. The target surface 26, with the living plant material thereon, is placed in position above the retaining screen 24. A small droplet of water, preferably 10 microliters, is placed in the chamber bridging between the ends of the two electrodes 14. The access cover 20 is placed in position on top of the spark discharge chamber 12.

At this point, the entire apparatus is enclosed in a vacuum chamber and a vacuum is drawn down into the range of approximately 400-500 millimeters of mercury. As the vacuum is drawn, a supply of helium is bled into the vacuum chamber. Thus, the vacuum chamber contains a relative vacuum compared to the atmosphere and the atmosphere within the vacuum contains helium. The lower density of helium, combined with the reduced pressure, lowers the drag on both the carrier sheet 22 and the carrier particles.

The accelerated particle transformation process is initiated at this point. The voltage from the capacitors is electrically discharged to the electrodes 14. The voltages used in the present process have been in the range of 9-25 kV. The range of 9-10 kV is preferred. The voltage is discharged through the use 62

- 18 - of appropriate electric switching described above . The fqrce of the electric discharge initiates a spark which leaps the gap between the electrodes 14 and vaporizes the small droplet of water which was placed between the electrodes. The vaporization force creates a violent atmospheric shock wave within spark discharge chamber 12. The shock wave radiates outward from the electrodes in all directions. Because of the immovable

I sides of the chamber, the impact of the radiating shock wave upon the interior of the discharge chamber 12 is focused towards the carrier sheet 22, which is then propelled upward with great velocity. The upwardly traveling carrier sheet 22 accelerates upward at great force until it contacts the retaining screen 24. The displacement of the remaining atmosphere in the chamber with helium assists in the travel of the carrier sheet 22, since helium provides less drag on the flight of the carrier sheet as well as on the carrier particles themselves. At the retaining screen 24, the carrier sheet 22 impacts the retaining screen 24 and is retained. The nucleic acid-coated particles, in contrast, fly off of the carrier sheet and travel freely toward the target tissues. The small carrier particles then hit the living tissue on the target surface and proceed into the tissue cells. C. Regeneration of Maize Plants Plants must be created from the bombarded conditioned IES tissue. At either the cellular or plant level, the plants must be screened or selected to segregate the transformed plants from the nontransformed plants. In most particle-mediated plant transformation procedures done without selection, the nontransformed plants will be the large majority of the recovered plants. If a selection agent like hygromycin is used, the number of plants recovered is smaller but the relative proportions of transformant plants recovered is much higher. The selection regimen may not kill all of the non-transformed embryogenic cells, 62

- 19 - but it has been found to be useful in enriching the pool of recovered plants so that germ line transformant plants are recovered with greater frequency.

After bombardment, the embryo tissue is fragile. It is considered advantageous to let the bombarded tissues remain on the culture medium for approximately four hours. If a gene encoding a selection marker has been incorporated into the cells, the regenerating plant must be placed on medium containing the selection agent at some point in the growth process. Many independent transformants have been obtained.

First, shoots must be induced from the transformed cells. Shoot induction methods are well-known in the art. Green and Phillips, Crop. Sci . 15:417-420 (1975) disclose one practical method for regenerating maize shoots from callus culture. Second, mature plants must be grown from the shoots. Kernels from mature transformed plants may then be analyzed for gene expression. 6. Gene Expression Assay Protocols

One will wish to determine the level of gene "expression with the gene constructs of the present invention in many independent transformants . Preferably, the expression level is at least 1% of total soluble protein (TSP) in endosperm. More preferably, the level is at least 2.5% of total soluble endosperm protein. Even more preferably, the level is at least 5% of total soluble endosperm protein, and, most preferably, the level is at least 10% of this total soluble protein in endosperm.

Although we have expressed the protein connected to the rice glutelin-1 promoter in the maize kernel as a percent of endosperm protein, the product can also be expressed as a percentage of total soluble seed protein. It is more convenient to express the protein product as a percentage of total soluble endosperm protein because rice glutelin promoter is endosperm- specific. Thus, we expect to have maximal/optimal P 62

-20 - expression in that tissue. Additionally, it is possible and convenient to separate the maize endosperms (for ELISA assays) from the scutella (to obtain next generation plant and seeds) . 64% of the total soluble protein and 97% of the BR96 (in a recombinant plant) is in seed endosperm. In corn seeds hull (pericarp and testa) contain ~4% of TSP and 2% of BR96 and germ has 32% of TSP and 0.4% of BR96. Most, if hot all, of the BR96 antibody is concentrated in the endosperm. Therefore, BR 6 as percent of endosperm TSP is approximately twice what it would be in the whole seeds.

To determine these seed and endosperm TSP levels, one needs to first extract protein from dry maize seeds or endosperm and then determine the level of gene product within that protein preparation. We detail below two preferable methods for extracting protein and performing an ELISA assay, but one of skill in the art would realize that there are many substitutions that can be made in these procedures. All that is really necessary is to determine the level of gene expression within a soluble protein extraction.

A. Protein Extraction from dry seed and endosperm Surface sterilized seeds are placed. on wet sterile filter paper overnight at room temperature to imbibe water. Holding seed firmly in one hand and using a scalpel, one then cuts around the perimeter of scutellum to remove the pericarp. The scutellum are removed by cutting deeper around it in the endosperm. The part of kernel remaining after removing scutellum is treated as endosperm because the remaining pericarp contains only 4% of total soluble protein and 2% of the total antibody.

Total soluble protein from the endosperm is essentially extracted the same way as the whole seeds except that the endosperms (because they are wet) are ground in pestle and mortar.

The following extraction buffer is useful for the 062

-21- extraction of protein from dry seeds and endosperm :

Extraction Buffer (for 500 mis ) 100 mis 0.5M NaP04 (pH 7 .0) final cone . - 100 mM NaP04 20 mis 0 . 5M EDTA (pH 8 . 0) 20 mM EDTA 10 mlβ 10* Triton X-100 0 .2% Triton X-100

370 mis dH₂0

(The buffer should be filter sterilized or made from sterile stocks.)

'j Individual maize seeds are crushed between layers of glassine weighing papers on an Arbor seed press.

Ground material is poured into a 1.5 ml microcentrifuge tube. Approximately 1.5 ml extraction buffer is added. The mixture is vortexed on high for 5-10 seconds and then soaked at 4°C for at least 2 hours. The extractions are sonicated for 1.5-2 minutes. The tubes are centrifuged in a microcentrifuge set at highest speed for 20 minutes at 4°C to remove cell debris. All of the supernatant is transferred to fresh tube. A set of protein standards is prepared using l ug/ul human IgG and unknowns using Coomassie Plus Protein Assay kit from Pierce. Absorbance is read at 595 nM. Unknowns are compared to IgG standard curve to determine total protein in seed extract .

Whole maize seed should typically have 8-12 ug total soluble protein (TSP)/ul extract. Endosperm only should have abor.ut 5-6 ug TSP/ul extract.

B. ELISA Assay

5- ul of capture antibody (goat anti-human, gamma-specific) are combined with 5.5 mis of Capture Buffer (50 mM Na₂ C0₃ pH 9.6) . 50 ul are loaded per well in a microtiter plate to coat plate and stored O/N at 4°C.

Wells are washed 3X with PBS + 0.25% Tween20 (PBST) , using plate washer. Blocking Solution (0.5 g Carnation Milk Powder in 25 mis PBST) is prepared and 200 ul is loaded per well. The plate is incubated at RT for 1 hour. Wells are washed 3X with PBST using plate washer. 62

-22-

The dilution plate is prepared as follows: load samples into head wells (vertical row) , make 3 or more serial 1:2 dilutions (in PBS) across plate using the 8-channel pipettor. Start with 200 ul (total volume) in head well and 100 ul in each dilution well. Pipet 100 ul from head well into first dilution well and mix. Remove 100 ul from first dilution well and mix with the next well, etc.

I

For a standard curve, use 2 ng of protein standard/50 ul in head well (Pipet 4 ul of 2 ng/ul BR96 into 196 ul PBS in headwell of dilution plate to give 2 ng/50 ul on ELISA plate) . (For example, in the examples below, we used two ng of BR96 standard per 50 ul in the headwells.) 50 ul of each dilution is loaded onto ELISA plate.

Incubate at 37°C for 1 hour. Wells are washed 3X with

PBST using plate washer.

5 ul of detection antibody (goat anti-human, kappa-specific--HRP conjugate) is prepared in 5.5 mis PBST. 50 ul is loaded per well and incubated at 37°C for 1 hour. Wells are washed 3X with PBST using plate

^"washer.

Substrate solution is prepared according to BioRad kit directions (9 mis TMB peroxidase solution A + 1 ml solution B) , loaded 100 ul per well and incubated at RT for exactly 10 "minutes. The reaction is stopped by loading 100 ul of 1M H₂S0₄ per well. Absorbance is read at 450 nM.

Unknown samples are compared to protein standard curve to get ng in the protein extract. Divide ng protein extract by ug total soluble protein (TSP) /ul extract to get % protein/TSP.

Examples

1. Comparison of Various Promoters We wanted to investigate various promoters and their ability to drive gene expression in corn kernels.

To that end, we selected the promoters described below at Table 1 and Table 2 and created gene constructs 062

-23- where the promoters were operably connected to the BR96 heavy chain and light chain genes.

For two promoters (rice glutelin-1 and 27-kD- zein) , the heavy chain and light chain were on two different plasmids. For all other plasmids, the heavy chain and light chain genes were on the same plasmid.

If two plasmids were necessary, the plasmids were transformed together. For a positive ELISA reaction, i both the light chain and heavy chain have to be expressed. If light and heavy chains are expressed at different levels, the ELISA assay would only indicate expression indicative of the lower expression level .

We chose this monoclonal antibody BR96 as an example of an antibody that would be usefully commercially produced. Monoclonal antibody BR96 binds selectively to carcinomas of the colon, breast, ovary and lung; This monoclonal antibody is described in Hellstrom, e_£ al. , Cancer Research 50:2183-2190, 1990, and Trail, e_fc. _ιl. , Science 261:212-215, 1993. The gene constructs containing both the light and heavy chain of BR96 and the rice glutelin-1 promoter ^"are described above and at Figs . 4 and 5.

These gene constructs were transformed, as described above and below at Example 2, into maize embryos and transgenic plants were ultimately derived from the bombarded embryos . The plants were transformed by using WRG5084 and WRG5086 plasmid DNA. For preparing the gold bead preparation, for bombarding the conditioned immature embryo scutella, the DNA were mixed in equal proportions (for co-transformations) . As described above, in both plasmids, the heavy chain (HC) or light chain (LC) of BR96 is driven by the rice glutelin promoter. "ITE" designates independent transformation events. This designation is based on which callus the plants originated from. Table 1 tabulates the ELISA values (as percentage of total soluble endosperm protein) in the R_x progeny of these transgenic plants. 062

-24- TABLE 1

Effect of Promoter on Monoclonal Antibody BR96 Expression (heavy chain and light chain) in Endosperm of RI Corn Seeds

# of

# of RI Seed Endosperm

Independent Endosperm and/or

Transgenic ELISA' * Seeds

Plasmid # Promoter Plants MΞ/UsT RANGE Assayed

1 WRG2841 35S 4 0.29 0.03 to 0.71 67

WRQ2926 35S-ADH 5 0 0 68**

WRG2981 U i-1 9 0.61 0.10 to 0.98 8 _ 38**

WRG5003/5005 maize 27 D-ADH 26 0.07 0.01 to 0.39 227 RG5084/5086 Rice glutelin-1 9 3.67 0.15 to 10.1 122 RG51I7 Soy-7S 11 0.01 0.01 to 0.02 64**

WRG5193 35S/ppdK 4 0.02 0.02 60**

WRG5216 maize glob- 3 10 0.08 0.01 to 0.17 17 _ 157**

WRG5217 35S-glob- 3 5 0.07 0.0 to 0.26 4 & 53**

'ELISA values were expressed as percent of total soluble protein. ELISA values of RI endosperms only. Scutella were used to obtain R2 plants. "ELISA assays performed on whole seed protein. ELISA values for whole seed protein were similar to endosperm values in transformants with plasmids WRG5117, WRG5216 and WRG5217.

As Table 1 indicates, gene constructs containing rice glutelin-1 promoter express the foreign mammalian gene, in this case antibody BR96 heavy chain and light chain, to a level of between .15 to 10.1% of the total soluble protein in endosperms . This level stands in sharp contrast to the other promoters evaluated, which never expressed BR96 gene at a level of more than approximately 1% of the TSP.

Additionally, another rice glutelin promoter, GT3, was examined in transgenic rice. 850 bp of the GT3 promoter (Leisy, v_J_ al., Plant Mol. Biol . 14:41-50, 1989) was fused to the GUS gene. This construct was used to transform Gulfmont rice. Transformants were negative for GUS expression in rice seed (Cooley, e_ a_L., Theoretical and Applied Genetics. 90:97-104, 1995) .

Table 2 below describes the various promoters used in the Table 1 analysis. TABLE 2

Promoter Size ( b) Plasmid(s) Reference

Rice Glutelin 1 4.9 WRG5084 BR96 HC Zheng, _t _i. (1993)

-4861 to +27 WRG5086 BR96 LC The Plant Journal

WRG4564 GUS 4, 357-366;

WRG51 4 MC451 SEQ ID NO: 1

CaMV 35S 0.44 WRG2119 GUS Gardner, et al. (1981)

-440 TO +1 WRG2841 BR96 HC+LC Nucleic Acids Res 9:2871-2888

CaMV 35S ADH 0.96 WRG5154 GUS Gardner, el al. (1981) intron WRG2121 GUS Nucleic Acids Res

-440 to +l of 35S 9:2871-2888 fused to +120 to

+672 of ADH intron WRG2926 BR96 HC+LC Callis, et al. (1988)

Genes and Development

1:1183-1200

27kD Zein^* 1.14 WRG2952 GUS Das, et al. (1990)

-1140 to +60 Genomics 11:849-856

27kD Zein ADH 1.69 WRG5003 BR96 HC Das, _t aj. (1990) intron" Genomics 11:849-856

-1140 to +60 of

27 D fused to +120 to +672 of ADH intron WRG5005 BR96 LC Callis, et aj. (1988)

WRG2973 GUS Genes and Development 1:1183-1200

Maize Globulin 1.32 WRG5216 BR96 HC+LC Belanger & Kriz (1991)

-1314 to +4 Genetics 129:863-872

WRG5149 GUS

Maize Ubiquitin 1"^* 1.99 WRG2943 GUS Christensen, et al. (1992)

-900 to +1090 Plant Mol Biol

WRG2980 BR96 HC+LC 18:675-689

Soy cpnglycinin (7S) 0.9 WRG5117 BR96 HC+LC Chen, et aj. (1986)

-900 to +7 Proc. Natl Acad Sci USA

WRG2728 GUS 83:8560-8564

CaMV 35S -430 to 0.44 WRG5193 BR96 HC+LC Jen Sheen (1991)

• 40 fused to Maize The Plant Cell 3:225-245

PPDK -51 to +139

'-60 to - 70 was "a 40 bp linker/leader '"promoter and 1st replaced with pUC between intron and exon w/ intron fused to polylinker and Nc l first AUG is from Ncol linker CMC1208nco 62

- 26 -

2. BR96 Expression in R_λ and R, Maize Kernels of Independent Transformation Events. Table 3 below tabulates the individual rice glutelin-1 promoter transformants that were summarized in Table 1 above. In the Table 3 experiments, we assayed the expression levels of BR96 as percentage of total soluble protein in endosperms of transgenic R maize kernels. Endosperms were separated from scutella to run the ELISA assays, and scutella were used to grow R_x plants. The resulting R₂ seeds from R_x plants were assayed to determine the transgene transmission and stability of level of expression. The results are presented in Table 3. High BR96 expression levels were obtained for many different transformants in both RI and R2 generations suggesting the stability of transgene expression over generations. In general, for rice glutelin-1 promoter, the endosperm ELISA values for BR96 were about twice that of whole kernel ELISA values .

TABLE 3

Rice Glutelin-1 Promoter Provides High Expression Levels of BR96 in Endosperms and Whole Corn Seeds

R_x Seed Endosperm ELISA R₂ Whole Seed ELISA ELISA* positive R ELISA** positive R₂

Expt ft R_ plant # mean range see-ls/total mean range seeds/total

C982 12A1 2.9 1/15 0 0 0/8

C982 12A2 5.1 3.5-7.6 2/13 0 0 0/12

C983 A634xC983 6B1 3.3 1/10 4. 1.8 to 11/18

10 C983 8A1 2.7 1.2-4.1 10/20 2. 0.01 to 105/130

C983 A634XC983 8A1 5.5 4.0-6.9 2/5 1. 1. to 6/15

C984 16A1 4.5 1.1-10.1 34/50 2. 0. to 172/214

C984 16A2 2.5 1.4-3.5 6/10

C984 16A3 3.5 1.9-5.3 10/15 1. 0.5 to 3.7 28/38

15 C984 16A4 3.0 1.8-4.7 9/10 3. 2.8 to 5.8 4/5

C984 16A5 3.0 1.8-4.9 7/10 1. 0.8 to 1. .88 11/15

C984 16A6 3.8 2.0-5.6 13/20 2. 0.5 to 8..11 67/70

C984 A634x984 16A1 4.2 3.8-4.7 4/11 1. 0.9 to 3..44 32/53

C984 A634X984 8A3 0.4 0.2-0.6 4/10 0. 0.05 to 0.1 12/35

20 C985 9A1 5.0 1.0-8.9 24/37 3. 1.2 to 6 .8 36/48

'ELISA values of R_x endosperms only. Scutella were used to grow plants to obtain the next generation. "ELISA values for whole R₂ seeds. Only ELISA positive seeds were used to calculate the means and range of values .

62

-28 -

As Table 3 indicates, the mean percentage of BR96 ill, TSP for different transformants was 0.4 to 5.5% and ranges from 0.4 to 10.1%. 3. pfeoRx451 Expression in Maize Kernels We have assayed another antibody gene to demonstrate that the increased expression due to the rice glutelin-1 promoter would work with other mammalian genes . Table 4 (below) describes an

I experiment in which the rice glutelin-1 promoter was placed upstream of the NeoRx451 heavy chain and light chain genes. The transforming plasmids containing the NeoRx451 light and heavy chains respectively, WRG5243 and WRG5236, are illustrated in Figs. 8 and 7. The heavy chain of this antibody is naturally glycosylated. A version of the heavy chain has been modified to remove the glycon addition site. Both the glycosylated (WRG5245 Fig. 9) and the nonglycosylated (WRG5236 Fig. 7) versions of the heavy chain have been used in these experiments . Seven different transformation events produced transformants containing the nonglycosylated antibody from WRG5243 and WRG5236. The RI seed of these transformants was analyzed with the following results: The NeoRx451 monoclonal antibody was expressed as 0.4 to 3.5% of endosperm total soluble protein. When whole R₂ seeds were measured, mean NeoRx451 ELISA values were 5.0% and ranged from 1.6 to 12.8% of total soluble seed protein.

For producing the glycosylated version of NeoRx 451, the transforming plasmids containing the NeoRx- light and heavy chains (glycosylated) respectively, WRG5243 and WRG5245, are illustrated in Figs. 7 and 9. Ten independent transformants were produced. The _x seed of these transformants was analyzed with the following results: The NeoRx451 monoclonal antibody was expressed as 11.0 to 25.7% of endosperm total soluble protein and 0.3 to 15.0% of whole seed TSP (Table 4) . Mean whole seed ELISA values, for different „,_««„_, 8/10062

-29- transformants, varied from 0.3 to 9.8%, whereas mean ELISA values for the endosperm was 16.7% (C2015-7A1 transformant) . These are quite high expression levels for transgenic protein.

TABLE 4

Rice Glutelin-1 Promoter Provides High Expression Levels of Glycosylated and Non-Glycosylated Forms Of Monoclonal Λntibody NeoRx451 in Endosperms and Whole Corn Seeds

R_x Seed ft of ELISA ELISA ELISA ELISA Values Positive R_x R, Seeds Positive lasmid ft Promoter Genes Exp. ft R~ Plant ft mean ranσe Seeds/Total mean ranσe R- Seeds/Total RG5236/5243 Rice NeoRx451

Glutelin-1 HC&LC 1076 5A1 1.6 0.4-3.5 29/35 5.4 1.6-12.8 15/16 RG5236/5243 Rice NeoRx451

Glutelin- 1 HC&LC 1076 5A2 1.2 0.6-2.6 16/25 4.7 2.5-6.9 5/9 RG5236/5243 Rice NeoRx451

Glutelin-1 HC&LC 1079 5A1 0.4 0.1-0.8* 8/10 RG5236/5243 Rice NeoRx451

Glutelin-1 HC&LC 1094 5AlxA634 2.3 1.8-2.8 3/5 RG5236/5243 Rice NeoRx451

Glutelin-1 HC&LC 1094 A634X5A1 1.1 0.8-1.4 2/5 RG5245/5243 Rice NeoRx451

Glutelin-1 HC&LC 2019 A634x2019-4A1 2.5 2.3-2.6 2/5 RG5245-5243 Rice NeoRx451

Glutelin-1 HC&LC 2019 A634x2019-8A3 0.7 0.5-0.8 2/5 RG5245-5243 Rice NeoRx451

Glutelin-1 HC&LC 2010 A634x2010-1A2 0.3 0.3 1/5 RG5245/5243 Rice NeoRx451 7A1 9.8 7.3-15.0 8/10

Glutelin-1 HC&LC 2015 7A1 16.7* 11.0-25.7* 7/10 LISA values of R, Endosperms only. Scutella were used to grow plants to obtain the next generation. Only ELISA- ositive kernels were used to calculate the means and range of values.

-31- SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Brar, Gurdip

Russell, David

(ii) TITLE OF INVENTION: MONOCOT SEED GENE EXPRESSION SYSTEM (iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Quarles & Brady

(B) STREET: 411 East Wisconsin Avenue

(C) CITY: Milwaukee (D) STATE: Wisconsin

(E) COUNTRY: U.S.A.

(F) ZIP: 53202-4497

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(viii)^"ATTORNEY/AGENT INFORMATION:

(A) NAME: Baker, Jean C.

(B) REGISTRATION NUMBER: 35,433 (C) REFERENCE/DOCKET NUMBER: 110229.91195

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (414) 277-5709

(B) TELEFAX: (414) 271-3552

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4891 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -32 -

(Xi) _%SEQUENCE DESCRIPTION: SEQ ID NO:l:

GGTACCCCTC GCTTCACTCC AAGCTCCACG GCGGCGGCTT CTTGCCCCTG CAATCACTGG 60

CCAGCCTGCC CAAGAGGATA AAAGTGAGAG AAAGAGAGGA GGAAGGGAGA TGAGGGGAAA 120

GAGGGAGGTG ATGACATGGA TTACTGATAT GTAGGGTTCA CGTGGGTTCC ACGCTGACTC 180 AGCCGCCACG TCGGATAAAA CCGGGATCAA AGCTACCGAA TGACCTAAAG TGAACAGTTT 240

TGTAAATTGA tGGGATGTCAT GTATCCGGTT TTGTGGTTGA TGGACGATTT TGTAACTCGA 300

I

TGACAAATTG AGCGACCTGC GGTGTACTTT TTCCTTCCGC CCTGTGTGGA GGCCCAAACA 360 TTCAGCCCAT TCCCAACCTG GCACTGACAT GCGGGCCATT CCAAAGCCTT GCACAGTTTC 420 ACCTCTCACC CGCGCCTCCG CTTCCTCCCG CCTCCCCAAA CGATGCCGCC TCCGCCTCCG 480 TCTTCCCGTC TCCTCGCCCT CCTCTCCGCG CGCCGCCCGC CGCCCCCGCT CCGCCGCCTC 540 CTCCAGATCC ACGCCCACCT CCTCGCCGCC GGCCTCCTTC AAGACTTCTC CTCCCTCCTC 600 GCCGCCGCCT ACGCGCTCTC CACCACCGCC ACCGCCACGG ACGCCCGCAC CTCGCCGCCC 660 TCCCCGCTCC GCCACGCGCT CGCGCTCCTC TCCTCGCTCC CGGCCTCCGC CTACAACGCC 720 GCCATCCGAG CACTCTCCCT CTCCGACGAC GGCGACCGCC ATGGCCACGG CGTCGTCCGC 780 CGCTGCCTCC CGCTCTACCG CGCCCTCCTC CGCTCCGGGA CCGCGCGCCC CGACCACCTC 840 ACGTTCCCGT TCCTGCTCAA GGCCTGCGCG CGCCTGCGGG AGTGGGGATA CGGCGACGCG 900 GCCCTCGCGC ACGTCCTCCG CCTCGGCCTC GACTCCGACG TCTTCGTGGT GAACGCGGCC 960 ACGCACTTCC TATCGATCCG CGGGCCCATG GAGGACGCAC GCAGGCTGTT CGACCGAAGT 1020 CCTGTGAGGG ACTTGGTGTC GTGGAACACG CTGATCGGAG GGTACGTGCG GCGGGGGAAC 1080 CCAGCGGAGG CGCTGGAGCT GTTCTGGAGG ATGGTGGCAG AGGATGCAGT GGTGAGGCCT 1140 GATGAGGTCA CGATGATCGC GGCTGTGTCG GGGTGTGGGC AGATGCGTGA CCTGGAGCTT 1200 GGGAGGCGGC TTCATGGGTT CGTGGATAGT GACGGAGTGA GTTGCACTGT GAGGCTGATG 1260 AATGCGCTGA TGGATATGTA CATCAAGTGT GGCAGTTTAG AGATGGCAAA GTCTGTGTTC 1320 GAGAGGATCG AGCACAGGAC AGTTGTCTCT TGGACGACGA TGATCGTGGG GTTTGCCAAG 1380 TTCGGATTGA TGGACGATGC ACGTAAAGTG TTTGATGAGA TGCCTGAAAG GGATGTGTTC 1440 CCATGGAATG CACTCATGAC CGGTTATGTG CAGTGTAAGC AGTGCAAGGA GGCCCTTTCC 1500 TTGTTTCATG AGATGCAGGA AGCAAGTGTG GTGCCTGATG AGATCACAAT GGTCAATCTT 1560 CTAACTGCTT GTTCGCAGCT CGGAGCATTA GAAATGGGGA TGTGGGTTCA CCGGTACATT 1620 -33- GAGAAACATC GCCTTGTATT TAGTGTTGCG CTTGGCACAT CTCTCATTGA CATGTACGCT 1680 AAGTGTGGAft' ACATTGAGAA AGCTATCCAC ATTTTCAAAG AAATTCCCGA GAAAAATGCA 1740 CTCACATGGA CAGCAATGAT ATGTGGTCTA GCAAATCATG GACATGCCAA TGAGGCCATA 1800 GAGCACTTCC GGACAATGAT AGAGCTTGGA CAGAAGCCAG ATGAGATTAC GTTTATAGGT 1860 GTTCTTTCAG CATGCTGTCA TGCTGGTTTG GTGAAAGAAG GTCGGGAATT TTTCTCTCTG 1920

ATGGAGACAA AATATCATCT TGAGAGGAAA ATGAAACATT ATTCATGTAT GATAGACTTA 1980 t

CTAGGCAGGG CAGGCCATTT AGACGAAGCA GAGCAGCTAG TAAACACTAT GCCTATGGAA 2040 CCTGATGCAG TAGTTTGGGG TGCTATCTTC TTTGCTTGTA GGATGCAAGG TAATATCTCT 2100 CTTGGAGAAA AGGCAGCAAT GAAATTGGTA GAAATTGATC CTAGTGATAG TGGAATCTAT 2160 GTGCTACTGG CTAATATGTA TGCAGAAGCG AACATGAGGA AGAAGGCTGA CAAAGTCAGG 2220 GCTATGATGA GACATTTGGG AGTGGAGAAA GTTCCTGGGT GTAGCTGCAT TGAGTTGAAT 2280 GGTGTGGTTC ATGAATTTAT CGTGAAGGAC AAGTCACATA TGGATAGTCA TGCTATTTAT 2340 GACTGCTTGC ATGAGATCAC CCTACAAATA AAGCATACTG CAGATTTGCT TAGCATTTCT 2400 GCGGCTGGTG CGGTGTAGTG TTCTGTTGGC TGGAACAGCT GGCTGAGCTG TGCAAGATGA 2460 TATGTGCAGT TGTGATGCAC AATTCACAGA TGCAGGAACT CGATCATGCT GATTTGTGCT 2520 GGTTTGCCAG GCCATGTTCT GAGAAGGGTA TACTTCATGT TGATTACTAT CTGAGGCATT 2580 CCCCGAGAAT TTTCTGGTCG TTCTTTTGCA GCTTGATGTC AATGGAAACA ATATGTTCCA 2640 CTACATATTG CAAAGTTCTT GTATGCTCTT TACTCAACCC TCACGTGCGG AGCACTTCCT 2700 GGGTAAGTGT GGTTCTCATG CTCTGTTTTG CCTCCTCCAT TTCTCCTCCG TTGCATTTAA 2760 AGTCACATCC CCCTCCTCAG GTTTTCTCCA TTAGCTCTCT GTAGTCCTTG CTGTACTCTC 2820 CTTGGTATTC CATGCTGTCC TACTACTTGC TTCATCCCCT TCTACATTTT GTTCTGGTTT 2880 TTGGCCTGCA TTTCGGATCA TGATGTATGT GATTTCCAAT CTGCTGCAAT ATGAATGGAG 2940 ACTCTGTGCT AACCATCAAC AACATGAAAT GCTTATGAGG CCTTTGCTGA GCAGCCAATC 3000 TTGCCTGTGT TTATGTCTTC ACAGGCCGAA TTCCTCTGTT TTGTTTTTCA CCCTCAATAT 3060 TTGGAAACAT TTATCTAGGT TGTTTGTGTC CAGGCCTATA AATCATACAT GATGTTGTCG 3120 TATTGGATGT GAATGTGGTG GCGTGTTCAG TGCCTTGGAT TTGAGTTTGA TGAGAGTTGC 3180 TTCTGGGTCA CCACTCACCA TTATCGATGC TCCTCTTCAG CATAAGGTAA AAGTCTTCCC 3240 TGTTTACGTT ATTTTACCCA CTATGGTTGC TTGGGTTGGT TTTTTCCTGA TTGCTTATGC 3300 -34- CATGGAAAGT CATTTGATAT GTTGAACTTG AATTAACTGT AGAATTGTAT ACATGTTCCA 3360 TTTGTGTTGT ACTTCCTTCT TTTCTATTAG TAGCCTCAGA TGAGTGTGAA AAAAACAGAT 3420 TATATAACTT GCCCTATAAA TCATTTGAAA AAAATATTGT ACAGTGAGAA ATTGATATAT 3480 AGTGAATTTT TAAHGAGCAT GTTTTCCTAA AGAAGTATAT ATTTTCTATG TACAAAGGCC 3540 ATTGAAGTAA TTGTAGATAC AGGATAATGT AGACTTTTTG GACTTACACT GCTACCTTTA 3600 AGTAACAATC ATGAGCAATA GTGTTGCAAT GATATTTAGG CTGCATTCGT TTACTCTCTT 3660 GATTTCCATG AGCACGCTTC CCAAACTGTT AAACTCTGTG TTTTTTGCCA AAAAAAAATG 3720 TATAGGAAAG TTGCTTTTAA AAAATCATAT CAATCCATTT TTTAAGTTAT AGCTAATACT 3780 TAATTAATCA TGCGCTAATA AGTCACTCTG TTTTTCGTAC TAGAGAGATT GTTTTGAACC 3840 AGCACTCAAG AACACAGCCT TAACCCAGCC AAATAATGCT ACAACCTACC AGTCCACACC 3900 TCTTGTAAAG CATTTGTTGC ATGGAAAAGC TAAGATGACA GCAACCTGTT CAGGAAAACA 3960 ACTGACAAGG TCATAGGGAG AGGGAGCTTT TGGAAAGGTG CCGTGCAGTT CAAACAATTA 4020 GTTAGCAGTA GGGTGTTGGT TTTTGCTCAC AGCAATAAGA AGTTAATCAT GGTGTAGGCA 4080 ACCCAAATAA AACACCAAAA TATGCACAAG GCAGTTTGTT GTATTCTGTA GTACAGACAA 4140 AACTAAAAGT AATGAAAGAA GATGTGGTGT TAGAAAAGGA AACAATATCA TGAGTAATGT 4200 GTGAGCATTA TGGGACCACG AAATAAAAAG AACATTTTGA TGAGTCGTGT ATCCTCGATG 4260 AGCCTCAAAA GTTCTCTCAC CCCGGATAAG AAACCCTTAA GCAATGTGCA AAGTTTGCAT 4320 TCTCCACTGA CATAATGCAA AATAAGATAT CATCGATGAC ATAGCAACTC ATGCATCATA 4380 TCATGCCTCT CTCAACCTAT TCATTCCTAC TCATCTACAT AAGTATCTTC AGCTAAATGT 44 0 TAGAACATAA ACCCATAAGT CACGTTTGAT GAGTATTAGG CGTGACACAT GACAAATCAC 4500 AGACTCAAGC AAGATAAAGC AAAATGATGT GTACATAAAA CTCCAGAGCT ATATGTCATA 4560 TTGCAAAAAG AGGAGAGCTT ATAAGACAAG GCATGACTCA CAAAAATTCA TTTGCCTTTC 4620 GTGTCAAAAA GAGGAGGGCT TTACATTATC CATGTCATAT TGCAAAAGAA AGAGAGAAAG 4680 AACAACACAA TGCTGCGTCA ATTATACATA TCTGTATGTC CATCATTATT CATCCACCTT 4740 TCGTGTACCA CACTTCATAT ATCATGAGTC ACTTCATGTC TGGACATTAA CAAACTCTAT 4800 CTTAACATTT AGATGCAAGA GCCTTTATCT CACTATAAAT GCACGATGAT TTCTCATTGT 4860 TTCTCACAAA AAGCATTCAG TTCATTAG 4888

Claims

0062-35- ClaimsWe claim:

1. A gene construct comprising: a rice glutelin-1 promoter operably connected to a foreign non-plant gene.

2. The gene construct of claim 1 wherein the promoter is SEQ ID NO:l.

3. The gene construct of claim 1 wherein the promoter comprises nucleotides 3021 to 4888 of SEQ ID N0:1.

. The gene construct of claim 1 wherein the gene construct is part of a plasmid or viral vector.

5. The gene construct of claim 1 wherein the foreign gene is mammalian.

6. The gene construct of claim 1 wherein the foreign gene is an antibody or antibody fragment.

7. A monocot plant containing the gene construct of claim 1.

8. The plant of claim 7 wherein the plant is a maize plant.

9. A monocot plant cell containing the gene construct of claim 1.

10. The cell of claim 9 wherein the cell is a maize cell. 62

-sell . A monocot seed containing a gene construct comprising a rice glutelin-1 promoter operably connected to a foreign non-plant gene, wherein the promoter and gene are not natively connected.

12. The seed of claim 11 wherein the seed is a maize kernel.

13. The seed of claim 11, wherein a product encoded by the foreign gene is expressed at a level of at least 1% of total soluble endosperm protein.

14. The seed of claim 13 wherein the level is at least 2.5% of total soluble endosperm protein.

15. The seed of claim 14 wherein the level is at least 5% of total soluble endosperm protein.

16. The seed of claim 15 wherein the level is at least 10% of total soluble endosperm protein.

17. The seed of claim 11 wherein the non-plant gene is an antibody.

18. The seed of claim 12 wherein the non-plant gene is an antibody.

19. A method of expressing a non-plant gene in a monocot cell, comprising the steps of a. creating a gene construct comprising a rice glutelin-1 promoter operably connected to a foreign non-plant gene, b. creating a transgenic monocot cell comprising the gene construct, and c. allowing expression of the non-plant gene in the monocot cell. 062

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20. The method of claim 19 wherein the monocot is maize .

21. A method of expressing a non-plant gene in a monocot plant, comprising the steps of a. creating a gene construct comprising a rice glutelin-1 promoter operably connected to a foreign non-plant gene, b. creating a transgenic monocot plant comprising the gene construct, and c. allowing expression of the non-plant gene in the monocot plant .

22. The method of claim 21 wherein the monocot is maize.

23. A method of expressing a non-plant gene in a monocot seed, comprising the steps of a. creating a gene construct comprising a rice glutelin-1 promoter operably connected to a foreign non-plant gene, b. creating a transgenic monocot seed comprising the gene construct, and c. allowing expression of the non-plant gene in the monocot seed.

3, . The method of claim 23 wherein the monocot is maize.