Abstract:
Nucleic acid sequence capable of regulating transcription during embryogenesis in plants is provided. This sequence may be used in transgenic plants to promote high levels of expression of foreign and endogenous genes in developing seeds to affect seed lipid metabolism, protein or carbohydrate composition and accumulation, or seed development. In addition, these sequences may be useful for the production of modified seed containing novel recombinant proteins which have pharmaceutical, industrial or nutritional value, or novel products like plastics.

Description:
This application derives priority from U.S. Provisional Patent Application No. 60/206,787, which was filed May 24, 2000. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to a nucleic acid sequence, which regulates transcription during embryogenesis in plants. More specifically, the nucleic acid sequence of the present invention can be used in transgenic plants to promote high levels of expression of foreign and endogenous genes in developing seeds to affect seed lipid metabolism, protein or carbohydrate composition and accumulation, or seed development. In addition, the nucleic acid sequence of the present invention can be useful for the production of modified seed containing novel recombinant proteins which have pharmaceutical, industrial or nutritional value, or novel products like plastics. 
     BACKGROUND 
     Most of the information about seed-specific gene expression comes from studies of genes encoding seed storage proteins like napin, a major protein in the seeds of  Brassica napus , or conglycinin of soybean. Furthermore, upstream DNA sequences directing strong embryo-specific expression of these storage proteins have been used successfully in transgenic plants to manipulate seed lipid composition and accumulation (Voelker et al., 1996). However, expression of storage protein genes begins fairly late in embryogenesis. Thus, promoters of seed storage protein genes may not be ideal for all seed-specific applications. For example, storage oil accumulation commences significantly before the highest level of expression of either napin (Stalberg et al., 1996) or conglycinin (Chen et al., 1988) is achieved. It is, therefore of interest to identify other promoters which control expression of genes in developing embryos with temporal specificity different from that of seed storage proteins. 
     SUMMARY OF THE INVENTION 
     The nucleic acid sequence of the present invention can be used to regulate transcription during embryogenesis in plants. By the present invention it is possible to promote high levels of expression of foreign and endogenous genes in developing seeds to affect seed lipid metabolism, protein or carbohydrate composition and accumulation, or seed development. The present invention can also be useful for the production of modified seed, which contains novel recombinant proteins. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
       The FIGURE shows nucleic acid sequence (SEQ ID NO: 1) of the insert in the plasmid pLfKCS3-GUS. 
     
    
    
     DETAILED DESCRIPTION 
     The inventors have determined that a more suitable gene regulatory region for directing gene expression aimed at seed oil modification would originate from a seed lipid metabolic gene expressed in a seed-specific manner. One such gene is LfKCS3, which encodes a condensing enzyme of very long chain fatty acid biosynthesis in  Lesquerella fendleri . LfKCS3 condensing enzyme is thought to be localized in the endoplasmic reticulum where it catalyzes the sequential elongation of C18 fatty acyl chains to C20 in length. RNA blot analyses showed that the LfKCS3 gene transcript was present only in developing embryos. The inventors isolated the 5′ regulatory region of the LfKCS3 gene and in the present application demonstrate that it is useful in promoting early seed-specific transcription of heterologous genes in  Arabidopsis . Regulatory 5′ DNA sequences promoting early seed-specific transcription found upstream of other plant KCS genes have also been isolated and disclosed previously (U.S. Provisional Patent Application filed Aug. 4, 1999, Inventors Kunst and Clemens). 
     Plasmid pLfKCS3-GUS was deposited in GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) on Dec. 27, 2001 and can be found as  Lesquerella fendleri  3-ketoacyl-CoA synthase (KCS3) gene under the Accession No. AF367052. 
     Isolated transcription regulatory region from the LpfKCS3 gene is capable of directing expression of desired genes at an early stage of development in a seed-specific manner. Because this regulatory sequence can also promote transcription in developing seeds of a different plant species, it can be used in a variety of dicotyledonous plants for modification of the seed phenotype. 
     Examples of applications wherein the nucleic acid sequence of the present invention can be useful include, for example: 
     (1) altered seed fatty acid compositionor seed oil composition and accumulation, 
     (2) altered seed protein or carbohydrate composition or accumulation, 
     (3) enhanced production of desirable seed products, 
     (4) suppression of production of undesirable seed products using antisense, co suppression or ribozyme technologies, 
     (5) production of novel recombinant proteins for pharmaceutical, industrial or nutritional purposes, 
     (6) production of novel compounds/products in the seed, ie. secondary metabolites, plastics, etc. 
     The methods employed in the isolation of the nucleic acid of the present invention and the uses thereof are discussed in the following non-limiting examples: 
     EXAMPLES 
     Isolation of a Seed-Specific Promoter Region form  Lesquerella fendleri A  Lesquerella fendleri  genomic DNA library was obtained from Dr. Chris Somerville, Carnegie Institution of Washington, Stanford, Calif. The genomic library was plated on  E. coli  LE392 (Promega) and about 150,000 clones were screened using  Arabidopsis  FAE1 gene (James et al., 1995) as a probe. The probe was prepared by PCR using pGEM7-7Zf(+)-FAE1 (Millar and Kunst, 1997) as a template with FAE1 upstream primer (SEQ ID NO: 2); 5′-CCGAGCTCAAAGAGGATACATAC-3′(SEQ ID NO: 2) and FAE1 downstream primer (SEQ ID NO: 3). 5′-GATACTCGAGAACGTTGGCACTCAGATAC-3′(SEQ ID NO: 3). PCR was performed in a 10 μl reaction containing 10 ng of the template, 2 mM MgCl 2 , 1.1 μM of each primer, 100 μM of (dCTP+dGTP+dTTP) mix, 50 μCi of [α-32P]dATP, 1×PCR buffer and 2.5 units of Taq DNA polymerase (Life Technologies). Amplification conditions were: 2 min of initial denaturation at 94° C., 30 cycles of 94° C. for 15 sec, 55° C. for 30 sec, 72° C. for 1 min and 40 sec, followed by a final extension at 72° C. for 7 min. The amplified radiolabeled probe was purified by QIAquick PCR Purification Kit (Qiagen) and denatured by boiling before adding to the hybridization solution. Hybridization took place overnight at 65° C. in a solution containing 6×SSC, 20 mM NaH 2 PO 4 . 0.4% SDS, 5×Denhardt&#39;s solution, and 50 μg/ml sonicated, denatured salmon sperm DNA (Sigma) and washing was performed three times for 20 min each in 2×SSC, 0.5% (w/v) SDS at 65° C. 
     Nine clones with sequences corresponding to the  Arabidopsis  FAE1 gene were isolated from the  Lesquerella fendleri  genomic library. The phage DNA from those nine clones was extracted and purified using QIAGEN Lambda Mini Kit (Qiagen) according to the manufacturer&#39;s protocol. One of them was digested with EcoRI and a 4.3 kb fragment was subcloned into the pGEM-7Zf(+) vector (Promega) cut with EcoRI, resulting in the vector pMHS15. The whole insert was sequenced with ABI automatic 373 DNA sequencer using fluorescent dye terminators. Approximately 573 bp of the 5′ upstream region of the 4.3 kb genomic DNA was amplified using the high fidelity Pfu polymerase (Stratagene) with a forward primer (SEQ ID NO: 4)5′-CGCAAGCTTGAATTCGGAAATGGGCCAAG-3′(SEQ ID NO: 4) and a reverse primer (SEQ ID NO: 5)5′-CGCGTCGACTGTTTTGAGTTTGTGTCGGG-3′(SEQ ID NO: 5). The amplified fragment was inserted upstream of the GUS gene in pBI101 (Clontech) cut with HindIII and SalI, resulting in the vector pLfKCS3-GUS. The sequence of the insert in the plasmid pLfKCS3-GUS is shown in  FIG. 1  (SEQ ID NO: 1) (SEQ ID NO: 1). 
     Functional Analysis of the LfKCS3 5′ Upstream Region 
     To evaluate the ability of the 5′ upstream fragment of the LfKCS3 gene to confer seed-specific and temporal regulation of gene expression in plants, the pLfKCS3-GUS construct was introduced into  Agrobacterium tumefaciens  strain GV3101 (pMP90) (Koncz and Schell, 1986) by heat-shock and selected for resistance to kanamycin (50 μg/mL).  A. thaliana  ecotype Columbia was transformed with  A. tumefaciens  harbouring the pLfKCS3-GUS construct using floral dip method (Clough and Bent, 1998). Screening for transformed seed was done on 50 μg/mL kanamycin as described previously (Katavic et al., 1994). Approximately 100 transgenic lines were generated for each construct. 
     Histochemical localization of GUS activity in transgenic plants was done on tissue sections as follows. Sections were incubated in 50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, 0.05%(w/v) triton X-100, and 0.35 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) for 4 to 7 hours at 37° C. (Jefferson, 1987). Following staining the blue-stained samples were fixed in 70°/ethanol. 
     Using this assay, over 30 independent transgenic  Arabidopsis lines were examined for the embryo-specific expression of the GUS gene. In addition, leaves, stems, inflorescences, roots, and siliques at different stages of development were histochemically stained for β-glucuronidase activity. The GUS reporter gene fused to the LfKCS3 promoter was not expressed in any of the vegetative tissues, whereas it was highly expressed in developing embryos. We also compared the LfKCS3 promoter with the LFAH12 promoter that was reported to be an early and seed-specific promoter active already at the torpedo stage of  Arabidopsis (Broun et al., 1998). Our results suggest that the LfKCS3 promoter is active even earlier. Thus, the onset of the LfKCS3 promoter activity coincides with or precedes that of storage oil accumulation. GUS activity in all the examined transgenic lines persisted throughout subsequent embryo development. Thus the LfKCS3 promoter is useful for seed-specific expression of foreign genes in transgenic plants. 
     In conclusion, we have demonstrated that the elements which confer both tissue specific and developmental regulation of a reporter gene linked to the LfKCS3 promoter reside within the 573 bp upstream of the AUG translation initiation codon. Our experiments also show that the  Lesquerell afenaleri  LfKCS3 promoter directs seed-specific expression at least as early as the torpedo stage embryo and that the it is capable of promoting transcription in plants other than  Lesquerell afendleri.    
     It should also be mentioned that the seed-specific expression conferred by the LfKCS3 promoter is independent of the native terminator at the LfKCS3 gene 3′ end. In all our constructs, a terminator derived from the  Agrobacterium nopaline synthase gene was used. Thus, the sequence in the 573 bp promoter construct is sufficient for the desired expression profile independent of ancillary sequences. 
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