Rice Genetics IV - IRRI books - International Rice Research Institute

Table 2 summarizes the transformation experiments. The Ac-Ds construct containsthe mobile transposon that can be a Ds or an I transposon and the correspondingimmobile transposase source Ac or En under control of a strong promoter. To observetransposition early after transformation and help make a choice among transformants,the GFP excision marker can be used to monitor excision. Independent excision eventscan be selected at the seed or germinated seedling level so that germinal transposantsare recovered and grown. To be able to select stable transposed elements, we employeda BAR gene on the Ds element conferring resistance to the herbicide Basta(Basta R ) and a negative selection marker SU1 (O’Keefe et al 1994) that converts theproherbicide R7402 into the active herbicide and results in shorter plants (Koprek etal 1999). Using a combination of these greenhouse/field selectable markers, progenyof single T-DNA locus transformants can be used to identify stable transposants(BAR + SU1 – ) (Fig. 5), where the Ds-BAR transposes from the T-DNA and is segregatedaway (to another chromosome or after recombination from a linked site).The enhancer trap construct contains a minimal promoter that can initiate transcriptionupstream of the GUS marker gene. On insertion near enhancers of host genesin the genome, the GUS gene detector can display the expression pattern of the adjacentgene. This will help identify the adjacent plant gene on the basis of its expression.In plants such as Arabidopsis, about 50% of the inserts display expression that issimilarly observed in rice. The activation tag construct has a multiple enhancer of theCaMV 35S promoter inserted near the transposon end and can activate adjacent genes.Enhancer trap and activation tag constructs have been transformed and moleculardata generated in rice (Table 1). They yield about 50% of the active lines that arebeing propagated for developing tagging strategies. Some lines also contain multipleDs elements with active transpositions that are useful for generating multiple transposonlines.Insertional mutagenesis in rice genomicsThe rice genome sequence will probably uncover about 30,000 genes, half of whichwill have no known function. Transposon mutagenesis, using knockout and gene detectioninsertions, is an important tool for discovering these gene functions by reversegenetics strategies. In Arabidopsis, about 100,000 random inserts (Krysan et al 1999)are required for genome saturation and for rice about four times that number. Multipleindependent inserts per plant, averaging four in many of the Ac and Ds lines, willdecrease the required number of plants. The insertional preference of Ac for genes asdescribed here can reduce the required number further by a factor of 3–5. A populationof about 25,000 Ac or Ds plants, with multiple inserts, would therefore be sufficientto recover knockout or gene detection inserts for most genes.Transposon populations for single stable Ds insertions can also be generated froma minimum of 10 active lines, as shown in the strategy outlined in Figure 5, in fourgenerations after transformation and seed multiplication. This could produce a populationof around 150,000 inserts, sufficient for genome saturation assuming preferentialtransposition of Ds in genes. By a concerted international effort, this transposonlibrary is being produced and will be made available to rice researchers worldwide.Transposons and functional genomics in rice 273