Isolation of phloem specific gene promoters for use in genetic engineering of insect resistance in rice

Abstract

Towards the aim of producing transgenic rice with enhanced resistance to one of its phloem sap-sucking insect pests, the brown planthopper (BPH), two potential phloem-specific promoters, of the rice sucrose synthase-1 (RSsl) and the cucurbit phloem protein PP2 genes, were isolated and investigated. Using a PGR fragment of the maize sucrose synthase-1 (Shi) gene, genomic clones containing the RSsl and RSs2 (rice sucrose synthase-2) genes were isolated from a genomic library of rice (Oryza sativa L. Japonica) and characterised. A full- length RSsl gene from one of the genomic clones was sequenced, including 1756 bp of 5'-flanking sequence and 710 bp of 3'-flanking sequence. The gene had an identical intron-exon structure (16 exons and 15 introns) to the maize Shi gene. The RSsl 5'- flankmg region contained a number of promoter-like sequences, including putative exacting elements homologous to those found in several endosperm-specific, anaerobiosis-inducible, or phloem-specific promoters. The RSsl promoter region, including 1.9 kb 5'-flanking sequence, the first intron, the first exon, and the translational start codon, was fused with coding sequences for β-glucuronidase (GUS) and snowdrop (Galanthus navalis) lectin (GNA). Tobacco plants were transformed with these chimaeric genes in order to determine the expression pattern directed by the RSsl promoter. Histochemical and immunochemical assays demonstrated that the expression of both GUS and GNA was restricted to phloem cells in various tissues (i.e. stem, root, leaf, petiole) and in different transformants. In addition, GNA was detected by immunological assay in the honeydew excreted by peach potato aphids (Myzus persicae), also a phloem sap- sucking insect, feeding on RSsl-GNA transgenic tobacco plants. This provided direct evidence that GNA was not only expressed in the phloem tissue, but was also present in the phloem sap of transgenic tobacco plants. Since GNA has been shown to have antimetabolic effect against BPH, the RSsl-GNA construct is being used to transform rice plants by collaborating groups elsewhere. In order to isolate the phloem protein PP2 gene promoter, the PP2 polypeptide from 3 months old Cucurbita pepo plants was partially sequenced after in situ cleavage with cyanogen bromide vapour, giving 75 residues of sequence on two cyanogen . bromide fragments. Using an oligonucleotide probe based on this amino acid sequence, cDNA clones encoding PP2 were isolated from a C. pepo cDNA library constructed from mRNA of 3-5 days old seedling hypocotyls. A cDNA clone was used as probe to screen a C. pepo genomic library, and several genomic clones were isolated. Restriction mapping showed that these clones contained different genes, consistent with results from Southern blots of C. pepo genomic DNA probed with PP2 cDNA, in which multiple bands were detected in all restriction endonuclease digests. One of the clones was partially sequenced, and was shown to contain a gene encoding PP2. A 1.2 kb 5'-flanking region of this clone was fused with a GUS reporter gene, and this construct was used to transform tobacco. Initial histochemical analysis showed that this chimaeric gene was not expressed in the putative transgenic plants examined. Possible reasons for this failure are discussed.