CA2355198A1 - Als3 promoter in transformed plants - Google Patents

Abstract

The Brassica ALS3 promoter is operably linked to a foreign structural gene to provide high level, and generally constitutive or tissue general expression of the structural gene in transformed plants. The Brassica ALS3 promoter exhibits a non-tissue-preferred mode of expression at a level comparable to, and in some cases higher than, the widely used CaMV 35S promoter. Accordingly, DNA constructs comprising the Brassica ALS3 promoter operably linked to any number of different gene coding regions can be used for constitutive and tissue-general expression of the gene in transformed plants.
The Brassica ALS3 promoter is used to direct expression of agronomically important genes and selectable marker genes.

Description

This application is a divisional of Canadian Patent application No. 2,216,244 stemming from PCT application PCT/US96/03635 filed on March 22, 1996.
BACKGROUND OF THE INVENTION
I. Field of the Invention The present invention relates to a novel ALS3 promoter which generally drives constitutive and generally non-tissue-preferred expression of operably linked foreign genes in transformed plants. In particular, this invention is directed to, DNA constructs in which a Brassica ALS3 promoter is operably linked to a foreign structural gene, and to using the DNA
construct to produce, in a transformed plant, a protein which is encoded by the structural gene. The Brassica ALS3 promoter is used to direct expression of agronomically important genes and selectable marker genes.
II. Background Acetolactate synthase (ALS), which is also known as acetohydroxy acid synthase (AHAS), catalyses the first step in the biosynthesis of the branched chain amino acids leucine, isoleucine and valine. It has also been shown to be the site of action of sulfonylurea and imidazolinone based herbicides.
See, for example, Chaleff, R.S. and C.J. Mauvais, Science 224:1443 (1984) and Shaver et al., Plant Physiol. 76:545 (1984). A number of different ALS genes from Brassica napus have been cloned and characterized. See, for example Wiersma et al., Mol. Gen. Genetics 219:413 (1989) and Rutledge et al., loc. cit. 229:31 (1991).
Rutledge et a1. (1991) reported that the B. napus rapeseed cultivar Topas contains an ALS multigene family - la -comprised of five genes. DNA sequence analysis of the structural genes revealed that the ALS1 and ALS3 genes shared extensive sequence homology. In contrast, the ALS2 gene has diverged significantly from the ALS1 and ALS3 genes and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding region. The ALS2 gene therefore may encode a ~, _ i6I30530 PCT/US96I036.,_ _ 2 _ polypeptide with a distinct function from that of ALS1 and ALS3. The ALSO and ALSS genes have interrupted coding regions and therefore may be defective.
Experiments conducted with the promoter of the Arabidopsis thaliana ALS gene revealed that the A.
thaliana ALS promoter is significantly less effective in driving gene expression than the CaMV 355 promoter.
Odell et al., Plant Physiol. 94 4 :1647-1654 (1990) replaced the A. thaliana ALS promoter with the CaMV 35S
promoter and observed a 25-fold increase in the level of ALS mRNA accompanied by a 2-fold increase in ALS enzyme level and a 3-fold increase in sulfonylurea tolerance.
These observations indicate that the ALS gene is regulated post-transcriptionally and that the A. thaliana ALS promoter is significantly less effective in driving gene expression than the Ca~'~IV 35S promoter.
The number of isolated and characterized constitutive generally non-tissue-preferred plant promoters available for expression of foreign proteins in transgenic plants is very limited. Well known examples of promoters with constitutive and tissu= generated expression patterns include those associated with the CaMV 3SS, Agrobacterium nopaline synthase, and maize ubiouitin genes. See Odell et al., Plant Mol. Biol. 10 3 :263-272 (1988), Herrera-Estrella et al., Nature 303:209-213 (1983) and Fox et al., Va. J. Sci. 43 2 :287 (1992).
There is a critical need for a broader repertoire of strong constitutive anti generally non-tissue-preferred plant promoters. A broader array of constitutive and generally non-tissue-preferred plant promoters that are expressed at high levels, that is, that drive expression of operably linked genes at a level comparable to the CaMV 35S promoter, would allow the genetic engineer to analyze the relative strengths of the available promoters and select promoters that provide the required level of expression of foreign genes in transformed plants. A

. .. .r 96130530 PCT/U596103~_., selected promoter might provide optimum levels of expression for the first gene but may be either too strong or too weak for use in driving the expression of a second gene. Consequently, additional constitutive and tissue general promoters are needed to optimize foreign gene expression in plants.
. There is also _a need for additional strong constitutive and generally non-tissue preferred promoters for construction of plants transformed with multiple foreign genes. Numerous difficulties have arisen when two or more different genes are introduced into a plant wherein each of the genes are operably linked to the same . or similar promoters. Some of these difficulties include ~(1) gene inactivation; (2) recombination as a result of pairing along homologous regions within the nucleotide sequence of the promoter leading to cross-over events and loss of the intervening region prior, or subsequent to, integration; and (3) competition among different copies of the same promoter region for binding of promoter-specific transcription factors or other regulatory DNA-binding proteins. A need therefore exists for a broader repertoire of strongly constitutive and tissue general promoters to be used for expression of foreign genes in transformed plants.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a strong, constitutive promoter which can effect high level, generally non-tissue-preferred expression of an operably linked, foreign gene in transformed plants.
It is another object of the present invention to add to the limited repertoire of generally non-tissue-preferred promoters available for the transformation of plants with multiple genes.
In achieving these and other objects, there has been provided, in accordance with on=_ aspect of the present ~6I30530 PC'TlUS95/036.._ - d -invention, an isolated DNA molecule comprising a promoter operably linked to a foreign structural gene, wherein said promoter comprises a nucleotide sequence corresponding to the sequence of a polynucleotide from the group consisting of (1) a XbaI/Ncol fragment 5-prime to the Brassica napus ALS3 structural gene or (2) a nucleotide sequence .that has substantial sequence similarity with said XbaI/NcoI fragment. Other objects of the present invention include providing an isolated DNA molecule wherein the promoter is operably linked to an agronomically important gene or a selectable marker gene, an isolated DNA that is part of an expression vector and an expression vector carrying the isolated DNA
molecule that is present in a transformed host.
It is another object of the present invention to provide an isolated DNA molecule comprising a promoter operably linked to a foreign structural gene, wherein said promoter comprises a nucleotide sequence corresponding to the sequence of a polynucleotide from the group consisting of (1) SEQ ID NO: 1 or (2) a nucleotide sequence that has substantial sequence similarity with S~Q ID NO: 1. Other objects of the present invention include providing an isolated~DNA
molecule wherein the proTOter is operably linked to an agronomically important gene or a selectable marker gene, an isolated DNA that is part of an expression vector and an expression vector carrying the isolated DNA molecule that is present in a tra.~.sform~d host.
Another object of the present invention is to provide a method of using an ALS3 promoter to produce a foreign protein in a transformed host plant, comprising the steps of (1) constructing an expression vector comprising a promoter operably linked to a foreign structural gene, wherein the promoter comprises a nucleotide sequence corresponding to the sequence of a polynucleotide from the group consisting of (a) a XhaI/NcoI fragment 5-prime . J 96130530 PCTIUS96IO~~~S

to the Brassica napes ALS3 structural gene or (b) a nucleotide sequence that has substantial sequence similarity with said XbaI/NcoI fragment; and (2) transforming a host.
Yet another object of the present invention is to provide a method of using an ALS3 promoter to produce a foreign protein in a transformed host plant, comprising the steps of (1) constructing an expression vector comprising a promoter operably linked to a foreign structural gene, wherein said promoter comprises a nucleotide sequence corresponding to the sequence of a polynucleotide from the group consisting of (a) SEQ ID
NO: 1 or (b) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 1; and (2) transforming a host.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way o~ illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 presents the nucleotide sequence (SEQ ID N0:
1] of a 1063 by XbaI/NcoI fragment that comprises the promoter ~-prime to the B. napes ALS3 wild type structural gene. The XbaI and NcoI restriction sites are underlined. The ATG start codon is found within the NcoI
restriction site.
Figure 2 presents a restriction man of a 1063 by XbaI/NcoI fragment which comprises the B. napes ALS3 wild type structural gene.
Figure 3 presents a man of pPHI4960, a binary vector containing the B. napes ALS3 promoter (ALS3-Pro) driving ~. _ 16130530 PCT/US96I036~_ uidA (GUS) gene expression. Plasmid pPHI4960 was used to transform canola and sunflower. LB and RB represent th=_ left and right borders, respectively, of the Agrobacterium Ti plasmid T-DNA region. The region between the left and right borders also includes a CaMV 35S promoter (35S-Pro) driving expression of the NPTII gene for kanamycin selection of transformed- plarits.
Figure 4 presents a map.of pPHI6333 which is a binary vector containing a cassette comprising the B. napus A.LS3 promoter (BNALS3 PROM), NPTII gene and the 3-prime terminator sequence from the potato proteinase inhibitor gene (PINII). Plasmid oPHI6333 was used to transform sunflower to ascertain the suitability of this promote_~
for driving selectable marker expression. LB and R3 represent the left and right borders, respectively, o the Agrobacterium Ti plasmid T-DNA region.
Figure S presents a man of plasmid pPHI3042 whic:~
contains the NPTII and GUS structural genes operably linked to the CaMV 3SS promoter.
Figure 6 presents a map o' plasmid pPHI5765 whic contains the NPTII structural gene operably linked to the CaMV 35S promoter (3SSPR0) and the potato proteinase inhibiter gene terminator sequence (PINII).
Figure 7 is a histogram showing GUS activity in plan=
2S seedlings transformed with expression cassettes in whic:~
the GUS gene is expressed from either the CaMV 35S or ALS3 promoters. GUS activity was measured in cotyledon, hypocotyl, meristem and root tissues.
Figure 8 is a histog=am showing GUS activity in vegetative stage plants transformed with expressio:~
cassettes in which the GUS gene is expressed from either the CaMV 35S or ALS3 promoters. GUS activity was measured in leaf, petiole, meristem, stem and root tissues.
Figure 9 is a histogram showing GUS activity in flowering stage plants transformed with expression.

.. ~ 96/30530 PCT/US96I036~~
cassettes in which the GUS gene is expressed from either the CaMV 35S or ALS3 promoters. GUS activity was measured in flower, leaf stem and root tissues.
Figure 10 is a histogram showing GUS activity in mature plants transformed with expression cassettes in which the GUS gene is expressed from either the CaMV 35S
or ALS3 promoters. GUS activity was measured in leaf, stem, pod and seed tissues..
DETAILED DESCRIPTION
I. Definitions In the description that follows, a number of terms are used extensively. The following definitions a=a provided to facilitate understanding of the invention.
A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a seauence of amino acids characteristic of a specific polypeptide.
A promoter is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the S' region of a gene, proximal to the transcriptional start site of a structural gene.
If a promoter is an inducible promoter, then the rate of transcription incr eases in response to an inducing agent .
For example, a promoter may be regulated in a tissue-2S specific manner such that it is only active in transcribing the associated coding region in a specific tissue types) such as leaves, roots or meristem.
In contrast, the rate of transcription is not generally regulated by an inducing agent it the promoter is a constitutive promoter. The promoter may be tissue general, also known as non-tissue-preferred, such that i t is active in transcribing th=_ associated coding region i n a variety of different tissue twes.
A core promoter contains essential nucleotide 3S sequences for promoter function, including the TATA box ~ . .. 96130530 PCTlUS96/036_ and start of transcription. By this definition, a cor=_ promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity.
S An isolated DNA molecule is a~~fragment of DNA that is not integrated in the genomic DNA of an organism. For example, the promoter of the ALS3 gene is a DNA fragment that has been separated from the genomic DNA of Brassica napes.
Complementary DNA (cDNA) is a single-stranded DNA
molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
To operably link one nucleotide sequence to another refers to joining two heterologous DNA fragments to produce a chimeric DNA construct that has biological activity. For exampl=, an isolated DNA fragme::t comprising a promoter from a first gene, such as the ALS3 gene, is operably linked to an isolated DNA fragment comprising the structural gene from a second heterologous gene. The resulting chimeric DNA construct is functional when the AL3 promoter is shown to initiate transcription of the heterologous structural gene.
The term expression =e~ers to the biosynthesis of a gene product. For example, in the case of a structural gene, expressior.involv~s transcription of the structural gene into mRNA and the translation of mRNA into one o_~
more polypeptides.
A cloniny vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of reel icati ng autonomously in a host cell .
Cloning vectors typically contain one or a small number ~ 96!30530 PCTlUS96103~__ of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable S for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes. that provide tetracycline resistance or ampicillin resistance.
An expression vector is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible . promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked to" the regulatory elements.
A foreign gene refers in the present description to a DNA sequence that is operably linked to at least one heterologous regulatory element. For example, any gene other than the ALS3 structural gene is considered to be a foreign gene ii the expression of that gene is controlled by the ALS3 promoter.
A recombinant host may be any prokaryotic or eukaryotic cell that contains either a cloning vector o.
expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned genes) 'n the chromosome or genome of the host cell.
A transQenic plant is a plant having one or mora plant cells that contai-~ an expression vector.
In eukaryotes, RNA pclymerase IT catalyzes t:~:e transcription of a structural gene to produce mRNA. A
DNA molecule can b= designed to contain an RNA polymerase II template in wi:ich the RNA transcript has a sequence that is complementary tc that of a specific mRNA. The RNA transcript is termed an antisense RNA and a DNA
sequence that encodes the antisense RNA is termed an 96130530 PCTlUS96I0~.

antisense ctene. Antisense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
A first nucleotide sequence has substantial sevuence S similarity to the nucleotide sequence of Figure 1 [SEQ.
ID NO: 1] if the former sequence share a similarity of at . least 65% with the Figure 1 sequence and is a constitutive promoter active in directing the transcription of an operably linked foreign structural gene in plants. Sequence similarity determinations can be performed, for example, using the FASTA program (Genetics Computer Group; Madison, WI). Alternatively, sequence similarity determinations can be performed using BLASTP (Basic Local Alignment Search Tool) of the 1S Experimental GENIFO~ BLAST Network Service. See Altschul et al., J. Mol. Biol. 215:403 (1990). Also,. see Pasternak et al., "Sequence Similarity Searches, Multiple Sequence Alignments, and Molecular Tree Building," in METHODS IN PLP.NT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et aI. (eds.), pages 251-20'7 (CRC Press 1993).
Promoter activity o~ the isolated nucleotide sea~uence can be assayed by means of fusing the nucleotide sequence to a coding region of a foreign reporter gene. Promoter activity is measured by assaying reporter expression.
See, for example, An et al., "Techniques for Isolating and Characterizing Transcription Promoters, Enhancers, and Terminators," in METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY, Glick et al. (eds.), pages 15S-155 (CRC Press, 1993).
II. Cloning of ALS3 Promoters A 1063 by Xbal/NcoT_ Fragment comprising the promoter region of the ALS3 gene ~=om wi ld type Brassica napes was cloned into pGEMS (Promega Corporation, Madison, WI) and characterized. The nucleotid=_ sequence of the 1063 b~
Xbal/NcoI fragment was determined by the dideoxy sequencing protocol. Sanger et ai., Proc. Nat'1 Acad.

.96/30530 PCT/US96103.
- II -Sci. USA, 74:5463 (1977). The nucleotide sequence of the 1063 by XbaI/NcoI fragment is shown in Figure 1 [SEQ ID
NO: 1J and a detailed restriction map of this same fragment is shown in Figure 2.
Other ALS3 promoters having substantial sequence similarity with the nucleotide sequence shown in Figure 1 can be cloned by conventional methods.
Oligonucleotides of defined sequence are chemically synthesized. Itakura et al., Annu. Rev. Biochem. 53:323 (1984). Numerous automated and commercially available DNA synthesizers are currently available. The probe can be a single and relatively short oligonucleotide of defined sequence, pools of short olgonucleotides whose sequences are highly degenerate or pools of long oligonucleotides of lesser degeneracy. Sambrook et al., Molecular Cloning: A Laooratory Manual, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989).
The oligonucleotid~ hybridization probes based on S3Q
ID NO: 1 are labeled, fo. example radio labeled, by conventional methods and used to detect related nucleotides sequences in Brassica genomic libraries by means of DNA hybridiza~ion. See, for example, Sambroo'.<
supra.
A plant genomic DNA library can be prepared by mews well-known in the art. See, for example, Slightom et a1.
"Construction of ~ Clone Banks," in METHODS IN PLA_~i MOLECULAR BIOLOGY AND 3IOTECHNOLOGY, Glick et a1 . (eds. ) , pages 121-146 (CRC Press, 1993). Genomic DNA can be isolated from Hrassica tissue, for example, by lysi::g plant tissue with the d~t~rgent Sarkosyl, digesting t~~
lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isoDropanol, and purifying resuspended DNA on a cesium chloride density gradient.
3S Ausubel at a1. (eds.). CURRENT PROTOCOLS IN MOLECUI~.3~
BIOLOGY, pages 2.3.1-2.3.3. (1990), .. ~ 96/30530 PCTIUS961036_ DNA fragments that a=a suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases. See, for example, Ausubel et al.. supra, at pages 5.3.2-5.4.4, and Slightom et al., supra.
Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or~cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining o. DNA molecules, and ligation . with appropriate ligases. Techniques for such manipulation are disclosed by Slightom et al., supra, and are well-known in the art. Also see Ausubel et al., supra, at pages 3Ø5-3.17.5.
A library contain_ng genomic clones is screened with DNA hybridization probes based on the nucleotides sequence of the ALS3 promoter shown in Figure 1 (SEQ ID
NO: 1] using standard 'echniques. See, for example, Ausubel et al., supra, at pages 6Ø3-6.6.1; Slightom et al., supra.
III. Characterization of ALS3 Promoters Genomic clones can b~ analyzed using a variety of techniques such as rest_iction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis. Primer extension analysis or S1 nuclease protection analysi s, nor ~xa«,pl ~, can be used to localize the putative start site oz transcription of the cloned gene. Ausubel et al., supra, at pages 4.8.1-4.8.5;
Walmsley et al., "Quantitative and Qualitative Analysis of Exogenous Gene expression by the S1 Nuclease Protection Assay," in MST:-iODS IN MOLECULAR BIOLOGY, VOL.
7: GENE TRANSFER A:VD EXPRESSION PROTOCOLS, Murray (ed.), pages 271-281 (Humana ~=ess Inc. 1991). Structural J 96130530 PCT/U596/0~,,_~
analysis can be combined with functional analysis for a complete characterization of the promoter region.
The general approach of such functional analysis involves subcloning fragments of the genomic clone into an expression vector which contains a reporter gene, introducing the expression vector into various plant tissues, and assaying_the tissue to detect the transient expression of the reporter gene. The presence of a constitutive, tissue-general promoter is verified by the observation of reporter gene expression in diverse plant tissues including roots, stems or leaves.
Methods for generating fragments of a genomic clone . are well-known. Preferably, enzymatic digestion is used to form nested deletions of genomic DNA fragments. See, for example, Ausubel et al., supra, at pages 7.2.1-7.2.20; An et al., supra.
Alternatively, DNA tha~ resides "upstream," or S-ward, of the transcriptional start site can be tested by subcloning a DNA fragment that contains the upstream region, digesting the DNA i=agmer.t in either the 5' to 3' direction or in t:-~e 3' to 5' direction to produce nested deletions, and subcloning the small fragments into expression vectors for c=ansient expression.
The selection of an appropriate expression vector will depend upon the method of introducing the expression vector into host ells. Typically, an expression vector contains: (1) prokaryotic DNA elements coding for a bacterial replication crigin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in the bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; (3> DNA elements that control the processing of transcrip t, suc:~. as a transcription termination/polyadenylation sequence; and (4) a reporter gene that is operably linked to the DNA elements that control transcription. in_=iation. Useful reporter genes '. .. 96/30530 PCTIUS96/036__ include a-glucuronidase,~3-galactosidase,chloramphenicol acetyl transferase, luciferase, and the like.
Preferably, the reporter gene is either the ~i-glucuronidase (GUS) gene or the luciferase gene. See, for example, Jefferson et al., Plant Molecular Biology Reporter 5 4 :387 (1987). General descriptions of plant expression vectors and reporter genes can be found in Gruber et al., "Vectors for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et a1. (eds.), pages 89-119 (CRC Press, 1993).
Moreover, GUS expression vectors and GUS gene cassettes are available from Clontech Laboratories, Inc. (Palo Alto, CA), whip luciferase expression vectors and luciferase gene cassettes are available from Promega Corporation (Madison, P1I) .
Expression vectors containing test genomic fragments can be introduced into protoplasts, or into intact tissues or isolated cells. Preferably, expression vectors are introduced into intact tissues. General methods of culturing plant tissues are provided, for example, by Miki et al., "Procedures for Introducing Foreign DNA into ?lants," in METHODS IN PLANT MOLECUI:A.R
BIOLOGY AND BIOT~CHNOLOG'!, Glick et a1 . (eds. ) , pages 57-88 (CRC Press, 1993). Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation o~ plan= tissue with Agrobacterium tumefaciens. Horsch et al., Science 227:1229 (1985).
Descriptions of Agrobact~=ium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, and Miki et al., supra. Methods of introducing expression vectors into plant tissue also include direct gene Transfer methods such as microprojectile-mediated delivery, DNA injection., electroporation, and the like. See, for example, Gruber et al., supra; Mik= et al., supra.

~ 96130530 pCT/US96/0~
_ 15 _ The above-described methods have been used to identify and characterize the Brassica ALS3 promoter that is constitutively expressed in transformed plants. In particular, the ALS3 constitutive and generally non-tissue-preferred promoter was found to reside within a 1063 by DNA fragment shown in Figure 1 (SEQ ID NO: 1).
Thus, the present invention encompasses a DNA molecule having a nucleotide sequence of SEQ ID NO: 1 and having the function of a constitutive plant promoter.
Variants of the 1063 by generally constitutive and non-tissue-preferred promoter can be produced by deleting, adding and/or substituting nucleotides for the nucleotides recited in SEQ ID~NO: 1. Such variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerise chain reaction, and the like.
Ausubel et al., supra, at pages 8Ø3-8.5.9. Also see generally, McPherson (ed.), DIpECTED MUTAGENESIS: A
PRACTICAL APPROAC:~, IRL PreSS (1991). Thus, the present invention also encompasses DNA molecules comprising nucleotide sequences that have substantial sequence similarity with SEQ ID NO: i and function as a tissue-general promoter.
Moreover, additional deletion analyses are performed to further localize the core promoter region within the 1063 by promoter. Thus, the present invention also encompasses fragments oT the DNA molecule having nucleotide sequence of SEQ ID NO: ?, as long as the DNA
fragments function as a constitutive promoter.
IV. Vector Construction The 1063 by Xbal/NcoI fragment shown in Figure 1 [S?Q
ID NO: 1] was fused to a GUS r~po~ter gene cassette which includes the 3-prim. terminator sequence from the potato proteinase inhibitor gene (PINIi). The ALS3 promoter was cloned as an XbaI/EcoRI fragment into the corresponding sites of the binary vector pP:T.I1741 to create plasmid 95/30530 PCTIL1S961036_ _ _ 1' _ pPHI4960 (Figure 3). Plasmid pPHI1741 differs from plasmid pBI101.1, taught by Jefferson et al., supra, by having the CaMV 35S rather than the nopaline synthase 5 prime and 3-prime regulatory sequences driving the NPTII
selectable marker gene.
The vector pPHI6333 was constructed by replacing the region between the.PmeI and EcoRI sites of pPHI1741 with a cassette containing the ALS3 promoter driving an NPTII
selectable marker gene and the PINII 3' terminator sequence (Figure 4).
v. Agronomic Genes and Selectable Marker Genes for Brassica Transformation By means of the present invention, agronomic genes ' 'and selectable marker genes can be operably linked to the ALS3 promoter and constitutively expressed in transformed plants. More particularly, Brassica can be genetically engineered to express various Dhenotypes of agronomic interest. The genes implicated in this regard include, but are not limit'd to, those categorized below.
1. Genes That Confer Resistance To Pests or Disease And That Encode:
(A) Plant dis=_ase resistance genes. Plant defenses are oLt~n activated by specific interaction between the o=oduct of a disease resistance gene (R) in th~ plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, fog example Jones et al., Science 265:789 (1994) (cloning of the tomato Cf-9 gene fo= resistance to Cladosporium fulvum) ; Mar ti n et a1 . , Science 262 :1432 (1993) (tomato Pto aen~ ~o= resistance to Pseudomonas syringa= pv. tomato encodes a protein kinase);
Mindrinos et a?., Cel.I 78:1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringa2) .
(B) A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48:109 (1986), who disclose the cloning and nucleotide sequence of a Bt 8-endotoxin gene.
Moreover, DNA molecules encoding b-endotoxin genes can be purchased from American Type Culture Collection (Rockville, MD), under ATCC accession Nos. 40098, 67136, 31995 and 31998.
(C) A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24:825 (1994), who di,~close the nucleotide sequences of several Clivia miniata mannose-binding lectin genes.
(D) A vitamin-binding protein such as avidin. See PCT patent publication serial No. W09400992. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.
An enzyme inhibitor, for example, a protease inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al., Plant Molec.
Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci.
Biotech. Biochsm. 57:1243 (1993) (nucleotide sequence of Strsptomyces nitrosporsus cc-amylasa inhibitor).
(F) An insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof.
See, for 96!30530 PCTIUS96I03.
_ i8 _ example, the disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
S (G) An insect-specific peptide or neuropeptide . which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Res. Comm.
163:1243 (1989) (an allostatin is identified in Diploptera puntata). See also U.S. patent No.
' 5,266,317 to Tomalski et al., who disclose genes encoding insect-specific, paralytic neurotoxins.
(H) An insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 116:i6S (1992), for disclosure of heterologous e:<pression in plants of a gene coding for a scorpion insectotoxic peptide.
(I) An enzyme responsible for an hyperaccumulation of a monterpen~, a sesquicerpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.
(J) An enzyme involved in the modification, includin3 the pose-~~anslational modification, of a biologically ac~ive molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphacase, a kinase, a phosphorylase, a polymerase, an elascase, a chitinase and a glucanase, whech~r natural or synthetic. See PCT applicatio:: ~rlC 73/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al., Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence of tie parsley vbi4-2 polyubiquitin gene.
(K) A molecule that stimulates signal transduction.
For example, see the disclosure by Botella et al., Plant Mole.
Biol. 24:757 (1994), of nucleotide sequences for mung bean c~:lmodulin cDNR clones, and Griess et al., Plant Physiol.
104:1467 (1994), who provide the nucleotide sequence of a maze calmodulin cDNA clone.
(L) A hydrophobic moment peptide. See U.S. patent serial No. 5,580,852 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and serial No. 5,607,914 (teaches synthetic antimicrobial peptides that confer disease resistance).
(M) A membrane permease, a channel former or a channel blocker. For example, see the disclosure by Jaynes et al., Plant Sci. 89:43 (1993), of heterologous expression c.
a cecropin-~ lytic peptide analog to render transgenic tobac~~
plants resistant to Pseudomonas solanacearvm.
(N) A viral-invasive protein or a complex toxin derived therefrom. For example, the .~ 96/30530 PCTIUS96/0~.._..

accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. Rev.

Phytopathol._ 28:451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

(O) An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cr. Taylor et al., Abstract r497, S~V~NTH INT'L SYMPOSIUM ON

MOLECULA.? PLAiVT-f'ICROB~ INTERP,CTIONS (1994) (enzymatic inactivation in transgenic tobacco via production o' single-chain antibody fragments) .

(P) A virus-specific a:~tibody. See, for example, Tavlado=aki et al., Natur= 366:469 (1993), who show that transg=nit plants expressing recombinant antibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo cr-1,4-D-polygalacturonases facilitate funga'_ colonization and plant nutrient release by solubilizing plant cell wall homo-a-1,4-D-galacturonase. See Lamb et al., 9io/Technoloay 10:1436 (1992). The cloning and characterization of a gene which 96!30530 PCTlUS96103.
-, .
_ c. _ encodes a bean endopolygalac-turonase-inhibiting protein is described by Toubart et al., Plant J. 2:367 (1992).
(R) A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10:305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.

2. Genes That Confer Resistance To A Herbicide For Example:
(A) A herbicide that inhibits the growing point or ~ . meristem, such as an imidazalinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for examqle, by~Lee et al., EMBO J.
7 : 1241 ( 1988 ) , ar.~d ~Iiki et a1 . , Theor. Appl .
Gene . 80:449 (1990?, respectively.
(B) Glyphosate (resistance imparted by mutant EPSP
synthase and aro.4 genes, respectively) and other phosphono co.a,pounds such as glufosinate (PAT and bay genes;, and pyridinoxy or- phenoxy proprionic acids an:: cycloshexones (ACCase inhibitor-encodinc genes). See, for example, U.S. patent No. 4,340,835 to Shah et al., which discloses the nucleotide sequence of a form o.
EPSP which can core= glyphosate resistance.
A DNA molecule encoding a mutant aroA gene can be obtained under ATC'C accession No. 39256, and the nucleotide secu_nce of the mutant gene is disclosed in U.S. patent No. 4,769,061 to Comai. European gatent application No. 0 333 033 to Kumada ~~ a~. and U.S. patent No.
4, 975, 37-~.~. to Goodman et a1. disclose nucleotide sequences oL alLtam=ne synthetase genes which 96130530 PCTIUS96I03w - 2~ -confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in European application No. 0 242 246 to Leemans et a1. ~De Greef et al., Bio/Tecnnology 7:61 (1989), describe the production of transgenic plants that express chimeric bar~genes coding for phosphinothricin acetyl transferase activity. Exemplary of genes conferring resistance to phenoxy proprionic acids and cycloshexones, such as sethoxydim and haloxy=op, are the Acc1-S1, Acct-S2 and Accl-~3 genes described by Marshall ' et al., Theor. ADD1. Genet. 83:435 (1992).
(C) A herbicide that i::hibits photosynthesis, such as a triazine (psbA and gs+ genes) and a benzonitrile (nit:ilase gene). Przibilla et al., Planc Cell 3:10'9 (1991), describe the transformation o~ !'::lamydomonas with plasmids encodirc mutant psb.4 genes. Nucleotide sequences for ~=t_~?ase genes are disclosed in U.S. patent No. 4,310,548 to Stalker, and DNA
molecules containinc these genes are available under ATCC acc~ssio:~ Nos. 53435, 67441 and 67442. Cloning and expression of DNA coding for a giu.tathione ~-transferase is described by Hayes et al., 9io'nem. J. 285:173 (1992).

3. Genes That Confer Or Contribute To A Value-Added Trait, Such As:
(A) Modified Latty ac_;~etabolism, for example, by transforming maiz=_ 3rassica with an antisense gene of stearoyl-~C? desaturase to increase stearic aci~ co.~.t~:.t o. the plant . See Knultzoz ec a1. , P=cue. lVat' 1 Acad Sci. USA
89:2624 (19921.

~ 96130530 PCT/US96/0~.

(B) Decreased phytate content (1) Introduction of a phytase-encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For examDle,~see Van Hartingsveldt et al., Gene 127:87 (1993), for a disclosure of the nucleotide sequence of an Aspergillus niger phytase gene.

(2) A gene could be introduced that reduces phytate content. In maize, this, for example, could be accomplished, by cloning and then ~_-introducing DNA associated with the singly allele which is responsible for maize mutants characterized by low levels of phytic acid. See ~aboy et al., Maydica 35:383 (1990) .

(C) Modif'_ed carbohydrate composition effected, for example, by transforming plants with a gene coding fo= a~ e::zym~ that alters the branching patte_n of start~. See Shiroza et al., J.

Bact~riol. 170:810 (1988) (nucleotide sequence of Streptococcus mutans fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet.

200:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Pen et al., Bio/Tecnnology 10:292 (1992) (production of transgenic plants shat express Bacillus lichenirormis or-amylase), Elliot et al., Plar.t Molec. Biol. 21:S1S (1993) (nucleotide sequences o= tomato invertase genes), SOgaard et a:.., J. Biol. Chum.. 268:22480 (1993) (site-directed mutagenesis of barley amylase gene), and :ishe=- et a'., Plant Physiol. 102:1045 (1993) (ma_ze endosperm starch branching enzyme II) .

.. ~! 96130530 PCT/US961030.,~
- 2~-_ -4. Selectable Marker Genes:
(A) Numerous selectable marker genes are available for use in plant transformation including, but not limited to, neomycin~.phophotransferase I, S hygromycin phophotransferase, EPSP synthase and dihydropteroate. See Miki et al., "Procedures for Introducing Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR BIOLOGY AND
BIOTECHNOLOGY, Glick et aI. (eds.), pages 67-88 (CRC P2-ess, 1993) .
Synthesis of g=nes suitably employed in the present invention can be e_~ect~d by mans or mutually priming, long oligonucleotides. Se" Lor example, Ausubel et a1.
(eds.), CURRENT PROTOCOLS IN htOLECUI~AR BIOLOGY, pages 8.2.8 to 8.2.13 (Wiley Inte=science 1990) , and Wosnick et al., Gene 60:115 (1987). Mo_eover, current techniques which employ the polvmerase chain reaction permit the synthesis or genus as large as 1.8 kilobases in length.
See Adang et al., Plant hlol°c. 9iol. 21:1131 (1993), and Bambot et a1. , PCR :~hthods and ~.pplications 2:266 (1993) .
The present invention, thus generally described, will be understood more =eadily by =eference to the following examples, which are provided by way of i'_lustration and are not intended to be limiting of the present invention.
Example I
Plant Trans~ormation and expression Analysis The vector p?::I4960, shown in Figure 3, was introduced into the Agrobacterium strain GV3101 by transformation. S. rapes cultivar Westar was used throughout this experiment. Transgenic B. napes plants were generated by Agro.bacterium co-cultivation of cotyledonary petioles and microspore derived embryos.
See Arnoldo et al., G~ne:n~ 35:58 (1992). Parallel transformations w~r~ carr~~d out using pPHI3042 in order to compare the Stre:lgt:1 G. ~h~ ALS3 prompter with that of ~ 96/30530 PCT1US96/0~_ _ 25 _ the CaMV 35S promoter. Plasmid pPHI3042 is shown in Figure S and contains the CaMV 35S promoter operably linked to the GUS gene. Tissues from plants containing independent transformation events were quantitatively analyzed for GUS expression using a histochemical assay method based on-that of Jefferson R.A., Plant Mol. Biol.
Rep. 5:387 (1987).
Table 1 summarizes quantitative GUS expression data from B. napes plants independently transformed with _ plasmid pPHI4960 (HWSC63E, HWSC63F, HWSC63G, HWSC63H, PWSC174C, PWSC174D, PWSC174G, PWSC174J, PWSC174M, PWSC174N, and PWSC176A). Tissue from the primary transgenic plant (TO) was analyzed. Plasmid pPHI4960 is shown in Figure 3 and contains the 9. napes ALS3 promoter operably linked to the GUS acne. The controls consisted of the same nontransformed cultivar 'Westar' and 4 independent B. napes plants __ansformed with the plasmid pPHI3042 (P10SB, P105=, P1253 and P145).

~6I30530 PCT/US96/03a.
_ 2~ _ Table 1 SUMMARY OF BRASSICA NAPUS TRANSFORMED
WITH pPHI4960 AND pPH13042 Transgenic GUS Expression.(TO) Southern Fluorogenic PI1VII Probe (pmoles/MU/h/~cg j protein) ' leaf root HWSC63E~ 0.2 6.2 5 HWSC63F~ 131 . 167 1 HWSC63G~ 416. ( 246 1 HWSC63H~ 21 29 1 PWSC174C~ 7.S 63.6 1 PWSC174D~ 11 I 17 1 PWSC174G~ 309 nc root 0/1 PWSC174J~ 1 PWSC174M~ 0/1 PWSC174N~ 1 PWSC176A~ 0.2 37 4 P105B' 315 I 143 P10SEZ 24. f 279 P126B2 0 . -~. ~ 1 . 2 Pl4Sz 0.2 I 0.6 WESTAR3 0.1 I 11.2 B. napes trans~orm~d wig:z pPH_T4960 B. napes trans:.ormed wi~:z oPH:3042 Non-transgenic co::crc 96130530 PCTIUS9510_ The data reveal that there is variability in the level of expression in the transformants. Variability in the level of expression of transformed genes has been noted in numerous transformation experiments described in the literature and has been attributed to such factors as differences in copy number, position effects and co-suppression. However, plants expressing high levels, or a desired level, of the foreign protein can' be identified and selected using the routine screening methods of the present invention.
The data also reveal that there are essentially two classes of expression levels in plants transformed with plasmid pPHI4960. The first class consists of transformed plants expressing high levels of assayable product (131-416 pmal of methylumbelliferone (MU)/h/~eg protein). The second class consists of transformed plants expressing 1 ow levels of assayable product (0 .2-37 pmol MU/h/~g protzin). The low expression level is generally comparable to the non-transformed control plants examined. These values do not imply absolute expected ranges but represent the maximum and minimum values for each class in the present study.
Southern blot analysis of total genomic DNA isolated from transformed ?ants was undertaken to determine the copy number of the GUS gene cassette in each transformant. The PINI_ terminator was used as the radiolabeled hybridization probe. Total genomic DNA was isolated from eacr plant transformant using a variation of a CTAB protocol. See, .or example, Dellaporta et al., 1983, Plant MoI. Biol. Reo. 1(4):19-21, or Saghai-Maroot et al., 1984, PNAS 8:8014-8018. The total genomic DNA
was digested with a restriction enzyme(s), separated on agarose gels by means of electrophoresis and hybridized with the radiolab_led PINIT terminator sequence as a ~ probe. Plasmid pP:iI4960 was digested with either HindIII
alone to assess number o= irt_grations, or double digested with Hindi=T plus EcoRI to determine copy number. Similarly, pP:-iI3042 was digested with HindIII

X6130530 PCTIUS96/03.

alone to assess number of integrations, or with EcoRI
alone to evaluate copy number (see enzyme cut sites on maps in Figures 3 and 5, respectively). Although an exact copy number for each integration event was not determined, the relative intensities of hybridizing bands were used to compare Southern data between transgenics with different vectors.
Plants transformed with multiple copies of the plasmid pPHI4960 (HWSC63E~and PWSC176A) produced low levels of MU. On the other hand, 50% of the single integration events produced high levels of MU. Plants with single transformation events produced high levels of MU irrespective of whether the GUS gene was expressed from the CaMV 35S or the ALS3 promoter.
15~ ' Among the transformed plants producing high levels of MU, plants transformed with the ALS3 GUS cassette produced just as much MU as plants transformed with the CaMV 35S GUS cassette, irrespective of whether enzyme activity was measured in the roots or shoots. Since the CaMV 35S promoter is regarded as a strong constitutive, generally non-tissue-preferred promoter, with wide application in expressing genes in transgenic plants, the Brassica ALS3 promoter offers a suitable alternative to the CaMV 35S promoter.
The level of GUS expression measured in leaf tissue of HwSC63G was higher than the GUS level produced in leaf or root tissue of plants transformed with the CaMV 35S
GUS cassette. Accordingly, the ALS3 promoter may be mor=_ effective than the CaMV 35S promoter for expression of certain genes at high levels. For example, the ALS3 promoter may be preferable to the CaMV 35S promoter in those instances where high level expression of the transformed gene is required. It is understood by those skilled in the art that the range of expression in transgenic plants can vary among transformation events.
The present data indicates that it is possible to recover at least some events with activity comparable to that . .. .~ 96130530 PCT/US96/0~_ _ obtained with the 35S promoter by means of routine screening using the methods disclosed herein.
To test the usefulness of the ALS3 promoter for expression of operably linked genes in other species, transformations were also conducted in sunflower.
Sunflower leaf discs were ~~co-cultivated with Agrobacterium carrying either pPHI4960 or pPHI6333. See Malone-Schoneberg, et al., 1994, Plant Science 103: 199-207. Stably transformed calli were selected using 100 mg/L of kanamycin. GUS or NPTII expression levels were determined for a number of independent transformation events, using histochemical staining for GUS expression analysis, and semi-quantitative ELISAs for NPTII
. expression analysis. While GUS expression varied among individual plants, the ranges of expression from the two promoters in leaf tissues overlapped substantially (73 3119 fluorescence units for the ALS3 promoter and 40-2788 fluorescence units for the 35S promoter) . Limited ELISAs also yielded comparable results for NPTII expression from the two promoters (data not shown), although extensive whole plant analysis was not carried out.
Example 2 Activity of the ALS3 Promoter in Transgenic 9. napus ir.
Selected Tissues from Seedling, vegetative, Flowering and Mature Stage Plants B. napus cv. Westar was transformed with DPHI4960 as described in Example 1. Four independent transformants were analyzed (HWSC63F, HWSC63G, HWSC63E and PWSC176A).
The primary transformants are designated T0. The seed produced by TO plants are T1 seeds and the plants produced by T1 seeds are referred to as T1 plants.
GUS expression was quantitatively determined, by the method described in Example 1, in T1 plants at t seedling stage (less than 2 true leaves), the vegetative stage (4-5 true leaves) , the early flowering stage (about 20-80% full bloom), and in mature plants. In addition, GUS expression was quantitatively determined in specific plant tissues, including meristem, cotyledon, hypocotyl, 96130530 PCT/US96J0~.

leaf, petiole, stem, root, pod, seed and flowers.
"Meristem" refers to the meristematic apex and includes much of the tissue surrounding the true meristem.
"Flowers" refers to all organs associated with the flowers, including ovaries, anthers and petals. "Pods"
refers to the developing green pods containing green and brown seed. "Seed" refers to the seed still on the plant, hence the seeds were not desiccated. The results of quantitative enzyme assays were confirmed by means of histochemical assays as in example 1.
GUS expression in B. napes cv. Westar transformed with pHI4960 was compared to plants of the same cultivar transformed with pPHI3042. A non-transgenic Westar line was used as a control. Although T1 stage plants are segregating with respect to the transformed genes, only T1 plants that had a transgene, as evidenced by expression of neomycin phosphotransferase or survival on kanamycin, were analyzed. Seeds were sown on an agar plate containing 100 ~g/ml of kanamycin. Those seeds which germinated and resulted in green seedlings were selected as transformed segregants. Those which bleached white were non-transformed segregants and were discarded or used as negative controls.
Table 2 summarizes Southern, expression and segregation data for several transgenics. GUS expression was measured in leaf tissue from plants at the 3-7 leaf stage. In those primary transgenics with a simile integration pattern, which either showed no segregation (and were therefore fixed) o. whose subsequent progeny segregated in approximately a 3:1 ratio with respect to the transformed genes, the levels of GUS expression from both the 35S and ALS3 promoters was relatively high. In contrast, those with complex integration patterns and/or with complex segregation had low GUS expression.

X6130530 PCTIUS96/03.

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Figures 7, 8, 9 and 10 present GUS expression data in specific tissues taken from plants at the seedling, vegetative, flowering and mature stage, respectively. As evidenced by the results shown in Figure 7, the ALS3 S promoter functions in seedlings, specifically in cotyledons, hypocotyls and "meristeinatic' apex. " The ALS3 promoter drives low level expression of GUS in the roots at the seedling stage. At the 4-5 true leaf stage,~the ALS promoter functions in leaves, petioles, stem, "meristematic apex" and roots, as shown in Figure 8. At the flowering stage, the ALS3 promoter functions in flowers, leaves, stem and roots (Figure 9).
Histochemical analysis of flowers revealed expression of GUS in petals, stigma, anthers and pollen. GUS activity 1S was detected in all tissues (leaf, stem pod and seed)' taken from mature B. napes plants transformed with pPHI4960. Accordingly, the ALS3 promoter is constitutively expressed in diverse plant tissues throughout the development of the plant.
Exam~Ie 3 Selectable Marker Genes Expressed from the ALS3 Promoter The ALS3 promoter can be used to dr ive expression of selectable marker genes for plant transformation. B.
2S napes was transformed with plasmids pPHI1741, pPHI3042, pPHI576S and pPHI6333 (Figure 4) using the methods described in Example 1. Transformants were selected on medium containing 100 ~.g/ml kanamycin. Plasmid pPHI1741 contains the CaMV 3SS promoter operably linked to the NPTII structural gene and the terminator sequence of the 19S gene. Plasmid pPHI3042 contains two expression cassettes with the CaMV 3SS promoter operably linked to the NPTII structural gene and the CaMV 3SS promoter operably linked to the GUS gene. Plasmid pPHI576S
3S contains the CaMV 3SS promoter operably linked to the NPT'II structural gene. Finally, plasmid pHI6333, shown in Figure 4, contains the ALS3 promoter operably linked .. ~ 96/30530 PCTlUS96f03~_., to the NPTII structural gene and the terminator sequence of the potato proteinase inhibitor gene (PINII).
Table 3 Selectable Marker Genes Expressed From the ALS3 Promoter TOTAL NUMBER TOTAL PERCENT
VECTOR OF EXPLANTS NUMBER OF TRANS-COCULTIVATED POSITIVES FORMED

pPHI1741 1350 64 4.7%

' pPHI3042 350 2 0.9%

pPHI5765 1935 60 3.1%

pPHI6333 1935 12 0.6%

As shown in Table 3, the ALS3 promoter is effective in driving expression of the NPT'II selective marker gene in canola. Transformation of plasmids pPHI1741, pPH3042 and pPHI5765 resulted in the recovery of 4.7%, 0.9% and 3.1% transformants among the total B. napus explants cocultivated with these plasmids, respectively. A total of 12 transformants were obtained from experiments in which pPHI6333 was cocultivated with 1,935 B. napus explants, or 0.6% of the explants were transformed. Ten of the 12 transformants expressed low levels of NPTII.
In sunflower, pPHI6333 was as effective as pPHI4960 for selection of transformed plants; that is, both the 35S
and the ALS3 promoter allowed selection of transformed tissues at 100 mg/L of kanamycin. Accordingly, the ALS3 promoter is effective in driving expression of a selective marker gene for plant transformation.
Although the foregoing refers to particular preferred embodiments, it will be understood that the '9-137 present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention, which is defined by the following claims.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those in the art to which the invention pertains.

Claims (3)

1. A transformed plant comprising an expression vector comprising an isolated DNA molecule, wherein the isolated DNA
molecule comprises a promoter operably linked to a foreign structural gene, wherein the promoter comprises a nucleotide sequence which is a XbaI/NcoI fragment 5-prime to the Brassica napus ALS3 structural gene.

2. A transformed plant comprising an expression vector comprising an isolated DNA molecule, wherein the isolated DNA
molecule comprises a promoter operably linked to a foreign structural gene, wherein said promoter comprises a nucleotide sequence which is SEQ ID NO: 1.

3. The transformed plant according to claim 1 or 2, wherein the foreign structural gene is an agronomically important gene or a selectable marker gene.