RESUMO
Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
Assuntos
Inativação Gênica , Vetores Genéticos , Técnicas de Sonda Molecular , Plantas Geneticamente Modificadas/genética , RNA Antissenso , Arabidopsis/genética , Íntrons , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oryza , Sondas RNA , Splicing de RNA , Projetos de Pesquisa , Nicotiana/genética , Transformação GenéticaAssuntos
Inativação Gênica , Íntrons , Splicing de RNA , RNA/genética , Arabidopsis , Endopeptidases , Ácidos Graxos Dessaturases , Regulação da Expressão Gênica de Plantas , Regulação Viral da Expressão Gênica , Engenharia Genética , Conformação de Ácido Nucleico , Plantas Tóxicas , Potyvirus/genética , RNA/química , RNA de Plantas/química , RNA de Plantas/genética , Nicotiana/genética , Proteínas Virais/genéticaRESUMO
Genetic engineering methods have been used successfully to modify the fatty acid profile of elite Australian germplasm of Brassica napus and B. juncea. Co-suppression plasmids carrying oleate desaturase genes from each species have been constructed and transferred into Australian elite breeding lines of B. napus and B. juncea using Agrobacterium tumifaciens plant-transformation techniques. Modifications to existing Brassica transformation protocols and the use of an intron-interrupted hygromycin-resistance gene as the selectable marker have resulted in improved transformation efficiencies. Silencing of the endogenous oleate desaturase genes has resulted in substantial increases in oleic acid levels, up to 89% in B. napus and 73% in B. juncea.
