TaBV transcriptional control elements, chimeric constructs and uses therefor

#This application is a national stage of PCT/AU2003/000919 filed Jul. 17, 2003, which claims priority to U.S. Ser. No. 60/396,912 filed Jul. 17, 2002, the entireties of which are hereby incorporated by reference.

#This invention relates generally to transcriptional control elements for use in plant genetic engineering. More particularly, the present invention relates to a constitutive promoter for expression of foreign or endogenous coding sequences in plants, including dicotyledonous and monocotyledonous plants. The invention is also concerned with a chimeric nucleic acid construct comprising the promoter of the invention operably linked to a foreign or endogenous polynucleotide that codes for a protein of interest or a transcript capable of modulating expression of a target gene. The invention is further concerned with transformed plant cells, as well as differentiated plants and plant parts, containing the construct.

#A primary goal of genetic engineering is to obtain plants having improved characteristics or traits. Many different types of characteristics or traits are considered advantageous, but those of particular importance include resistance to plant diseases, resistance to viruses or insects and resistance to herbicides. Other advantageous characteristics or traits include tolerance to cold or soil salinity, enhanced stability or shelf life of the ultimate consumer product obtained from a plant, or improvement in the nutritional value of edible portions of a plant.

#FIG. 1 is a diagrammatic representation of the genome organisation of TaBV. The positions of ORF 1, ORF 2, and ORF 3 are shown. The location of the putative met tRNA binding site, putative movement protein domains, RNA binding domain (RB), cysteine-rich region, conserved aspartic protease domain, conserved reverse transcriptase domains and conserved RNAse H domains are indicated Positions for designing primers Badna FP, Badna RP, TRBR, 5F (starts at nucleotide 7,344), 1F (starts at nucleotide 5,534) and G2R (starts at nucleotide 5,556) are indicated by FP, RP, R, a, b, and c, respectively.

#FIG. 2 is a diagrammatic representation of the TaBV genomic region, from which the TaBV promoter fragments were derived. Positions and sequences of a region resembling the CaMV 35S promoter as 1 element and of putative TATA box and tRNAmet binding site are indicated by dash lines. The predicted position of open reading frame 1 (ORF 1) and ORF 3 are also indicated. All promoter fragments within bracket A were created with restriction sites as indicated by labeled boxes, except for the XbaI site which exists within the native TaBV sequence. Promoter fragments within bracket B were generated by directional cloning of fragments in bracket A, as indicated by grey arrows using indicated restriction sites. Promoter fragment T600L was generated by removal of the Pst I/Xba I fragment from T1200L and, in the process, destroying the Xba I site (SEQ ID NOS 31-33 disclosed respectively in order of appearance).

#FIG. 3 is a schematic representation of plasmids used for either comparison of commonly used promoters with TaBV derived promoters and/or as selectable markers. Ubi-1=promoter region from maize polyubiquitin-1 gene, Ubi-1 intron=first exon and intron from maize polyubiquitin-1 gene, GFP=gene coding for green fluorescent protein, nos 3′=3′ untranslated region from gene coding for Agrobacterium tumefaciens nopaline synthase, CaMV 35S=promoter region from gene coding for cauliflower mosaic virus (CaMV) 35S RNA, uidA=gene coding for β-glucuronidase (GUS), RbcS 3′=3′ untranslated region from gene coding for Nicotiana tabacum rubisco, BT6.3=promoter region from DNA-6 from banana bunchy top virus (Dugdale et al 1998), nptII=gene coding for neomycin phosphotransferase, 35S 3′=3′ untranslated region from gene coding for CaMV virus 35S RNA, D35S=double CaMV 35S promoter, RB=right border of binary vector T-DNA region, LB=left border of binary vector T-DNA region.

#TABLE A Sequence ID Number Sequence Length SEQ ID NO: 1 Genomic sequence of Papua New 6520 nts Guinea isolate of Taro bacilliform virus encoding ORFs 1, 2 and 3 SEQ ID NO: 2 Entire genomic sequence of 7458 nts Papua New Guinea isolate of Taro bacilliform virus. SEQ ID NO: 3 Putative Polypeptide product 146 aa ORF 1, encoded by nucleotides 353-793 of SEQ ID NO: 1 SEQ ID NO: 4 Putative Polypeptide product 144 aa ORF 2, encoded by nucleotides 792-1227 of SEQ ID NO: 1 SEQ ID NO: 5 Putative Polypeptide product 1881 aa ORF 3, encoded by nucleotides 1230-6872 of SEQ ID NO: 1 SEQ ID NO: 6 Polynucleotide representing 1190 nts promoter region 6281-12, designated T1200 SEQ ID NO: 7 Polynucleotide representing 598 nts promoter region 6873-12, designated T600 SEQ ID NO: 8 Polynucleotide representing 529 nts promoter region 6942-12, designated T500 SEQ ID NO: 9 Polynucleotide representing 261 nts promoter region 7210-12, designated T250 SEQ ID NO: 10 Badna FP primer 23 nts SEQ ID NO: 11 Badna RP primer 24 nts SEQ ID NO: 12 1F primer 23 nts SEQ ID NO: 13 TRBR primer 26 nts SEQ ID NO: 14 5F primer 22 nts SEQ ID NO: 15 G2R primer 25 nts SEQ ID NO: 16 cytoplasmic initiator 12 nts methionine tRNA (tRNAmet) binding site SEQ ID NO: 17 F-GTN primer 25 nts SEQ ID NO: 18 R-GTN primer 24 nts SEQ ID NO: 19 P527-F primer 20 nts SEQ ID NO: 20 P257-F primer 23 nts SEQ ID NO: 21 P114-F primer 23 nts SEQ ID NO: 22 FP-as-1 primer 24 nts SEQ ID NO: 23 TRBR-Bam primer 24 nts SEQ ID NO: 24 FP-6765-pro primer 21 nts SEQ ID NO: 25 P600Not-F primer 28 nts SEQ ID NO: 26 P600Bgl-R primer 30 nts SEQ ID NO: 27 RP-leader primer 27 nts SEQ ID NO: 28 GUS1 primer 15 nts SEQ ID NO: 29 GUS2 primer 12 nts nts = nucleotides; aa = amino acids

#Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

#The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.