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:
[0001]    This application derives priority from U.S. Provisional Patent Application No. 60/206,787, which was filed May 24, 2000. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    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  
         [0003]    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 TE INVENTION  
         [0004]    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  
       [0005]    The FIGURE shows nucleic acid sequence of the insert in the plasmid pLfKCS3-GUS. 
     
    
     DETAILED DESCRIPTION  
       [0006]    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).  
         [0007]    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.  
         [0008]    Examples of applications wherein the nucleic acid sequence of the present invention can be useful include, for example:  
         [0009]    (1) altered seed fatty acid compositionor seed oil composition and accumulation,  
         [0010]    (2) altered seed protein or carbohydrate composition or accumulation,  
         [0011]    (3) enhanced production of desirable seed products,  
         [0012]    (4) suppression of production of undesirable seed products using antisense, co suppression or ribozyme technologies,  
         [0013]    (5) production of novel recombinant proteins for pharmaceutical, industrial or nutritional purposes,  
         [0014]    (6) production of novel compounds/products in the seed, ie. secondary metabolites, plastics, etc.  
         [0015]    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  
       [0016]    Isolation of a Seed-Specific Promoter Region form  Lesquerella fendleri    
         [0017]    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 pGEM-7Zf(+)-FAE1 (Millar and Kunst, 1997) as a template with FAEE upstream primer, 5′-CCGAGCTCAAAGAGGATACATAC-3′ and FAE1 downstream primer, 5′-GATACTCGAGAACGTTGGCACTCAGATAC-3′. PCR was performed in a 100 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.  
         [0018]    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 5′-CGCAAGCTTGAATTCGGAAATGGGCCAAG-3′ and a reverse primer 5′-CGCGTCGACTG=TGAGTTTGTGTCGGG-3′. 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.  
         [0019]    Functional Analysis of the LfKCS3 5′ Upstream Region  
         [0020]    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 (MP90) (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 501 g/mL kanamycin as described previously (Katavic et al., 1994). Approximately 100 transgenic lines were generated for each construct.  
         [0021]    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-bromo4-chloro-3-indolyl-p-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.  
         [0022]    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 p-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.  
         [0023]    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.    
         [0024]    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.  
       REFERENCES  
       [0025]    Broun, P., Boddupalli, S., and Somerville, C. (1998) A bifunctional oleate 12-hydroxylase: desaturase from  Lesquerell afendleri.  Plant J. 13, 201-210  
         [0026]    Chen, Z. L., Pan, N. S., and Beachy, R. N. (1988) A DNA sequence element that confers seed-specific enhancement to a constitutive promoter.  EMBO J.  6: 3559-3564.  
         [0027]    Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of  Arabdiopsis thaliana. Plant J.  16: 735-743.  
         [0028]    James, D. W., Jr., Lim, E., Keller, J., Plooy, I., Ralston, E., and Dooner, H. K. (1995) Directed tagging of the Arabidopsis  FATTY ACID ELONGATION  ( FAE 1) gene with the maize transposon  Activator. Plant Cell  7: 309-319.  
         [0029]    Jefferson, R. A., Kavanaugh, T. and Bevan, M. W. (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker system in higher plants.  EMBO J.  6: 3901-3907.  
         [0030]    Katavic, V., Haughn, G. W., Reed, D., Martin, M., and Kunst, L. (1994)  In planta transformation of Arabidopsis thaliana Mol. Gen. Genet.  245: 363-370.  
         [0031]    Koncz, C. and Schell, J. (1986) The promoter of T L -DNA gene S controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector.  Mol. Gen. Genet.  204: 383-396.  
         [0032]    Stalberg, K., Ellerstoem, M., Ezcurra, I., Ablov, S., and Rask, L. (1996) Disruption of an overlapping e-box-ABRB motif abolished high transcription of the napA storage-protein promoter in transgenic  Brassica napus  seeds.  Planta  199: 515-519.  
         [0033]    Voelker, T. A., Hayes, T. R., Cranmer, A. M., Turner, J. C., and Davies H. M. (1996) Genetic engineering of a quantitative trait: Metabolic and genetic parameters influencing the accumulation of laurate in rapeseed.  Plant J.  9: 229-241.  
     
       
       
         1 
         
           
             5  
           
           
             1  
             588  
             DNA  
             Lesquerella fendleri  
           
            1 

gaattcggaa atgggccaag tgaaatggaa atagagcttc aatccattta gtcccactca     60 

aaatggtgct cgaattatat ttagttacgt tcgaatcaga caaccaagta tttggttaat    120 

aaaaaccact cgcaacaaag gaaaaacacc aagcgcgtgc gtccaacatc cgacggaagg    180 

ggggtaatgt ggtccgaaaa ccttacaaaa atctgacgtc atctaccccc gaaaacgttg    240 

aatcgtcaac gggggtagtt ttcgaattat ctttttttta ggggcagttt tattaatttg    300 

ctctagaaat tttatgattt taattaaaaa aagaaaaaga atatttgtat atttattttt    360 

tatactcttt ttttgtccaa ctatttctct tattttggca actttaacta gactagtaac    420 

ttatgtcaat gtgtatggat gcatgagagt gagtatacac atgtctaaat gcatgcctta    480 

tgaaagcaac gcaccacaaa acgaagaccc ctttacaaat acatctcatc ccttagtacc    540 

ctcttactac tgtcccgaca caaactcaaa acaatgacat ctctaaac                 588 

 
           
             2  
             23  
             DNA  
             Artificial Sequence  
             
               FAE1 PCR Primer  
             
           
            2 

ccgagctcaa agaggataca tac                                             23 

 
           
             3  
             29  
             DNA  
             Artificial Sequence  
             
               FAE1 PCR Primer  
             
           
            3 

gatactcgag aacgttggca ctcagatac                                       29 

 
           
             4  
             29  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            4 

cgcaagcttg aattcggaaa tgggccaag                                       29 

 
           
             5  
             29  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

cgcgtcgact gttttgagtt tgtgtcggg                                       29