Rice Genetics IV - IRRI books - International Rice Research Institute

homology to known proteins. The transposon inserts are therefore biased about threetimes higher than expected on a random basis to insert in sequences predicted to codefor proteins. To calculate the genome sequence represented by ESTs, with 20,000different ESTs based on the gene index calculation (Quackenbush et al 2000) and anaverage size of 400-bp sequence, 8 Mb of unique EST sequence or 1.86% (8/430) ofthe genome sequence is present in the public databases. We observe 9.6% of the totalITSs with identity to ESTs, meaning that the ITSs are biased for insertions in transcribedgenes five times more than randomly expected.These two calculations reveal a bias of three to five times more insertions in genesand strongly suggest that Ac inserts preferentially into genes in rice. This confirms theearlier results of Ac insertional preference in rice, in which 4% of the inserts in ESTswere observed (Enoki et al 1999), and suggests that the preferential transposon insertionsin genes could be a valuable asset for generating mutants in rice.Gene detection strategies in riceSeveral constructs for gene detection were developed in rice. The general structure ofthe enhancer trap (ET) and activation tag (AT) constructs is outlined in Figure 4 andTransposition markerTPaseMobileBARtransposonAmp ROri35S enhancer sequences35S 4enh HygloxRGUS MPGene detectionmarkerRBTPase lox GFP SU1Hyg RLBImmobilizedtransposase Excision marker Negativeselection markerTransformationmarkerFig. 4. Model gene detection rice construct. The construct has two major parts: the mobiletransposon (Ds or I) drawn above and inserted in the resident T-DNA drawn below. The mobiletransposon, with transposon borders denoted by outward pointing thick solid arrows, containsa transposition marker (BAR gene) to select transposants as Basta R (resistant). Plasmid originand bacterial selection markers help in the recovery of large fragments of genomic-flankingDNA in E. coli. The gene detection marker for an enhancer trap consists of a GUS gene with aCaMV 35S minimal promoter (MP) located very near the transposon border. This is replaced bya multiple 35S enhancer (4enh) sequence to create an activation tag construct. To createchromosomal deletions, lox sites (Cre-lox system) are located in the mobile transposon as wellas in the resident T-DNA. The T-DNA contains the immobilized transposase (Ac or En) undercontrol of a strong promoter. The mobile transposon is inserted in the excision marker greenfluorescent protein (GFP) gene to select transpositions. The transformation marker Hyg R isused for selection of transformants using hygromycin and a negative selection marker, the SU1gene, used to select for stable transposants. LB = left border, RB = right border, Ori = origin,Amp R = ampicillin resistance.272 Greco et al