Rice Genetics IV - IRRI books - International Rice Research Institute

1998, Kumpatla and Hall 1999). The presence of repeated sequences is also a wellknownsignal for methylation and silencing (Assaad et al 1993, Dorer and Henikoff 1994,Garrick et al 1998, Henikoff 1998, Meyer 1996, Selker 1999, Ye and Signer 1996).Epistatic interactions can silence a single geneEpistatic regulation of transgene expression was first recorded in tobacco by Matzkeet al (1989) and both cis-inactivation (between closely linked genes) and trans-inactivation(between nonlinked transgenes or a transgene and an endogenous gene) occur(Matzke et al 1993). As we were unaware of any previous reports of epistaticinteractions in transgenic monocots, we thought it would be of interest to exploregene silencing in rice lines bearing single and multicopy transgene inserts.A series of transgenic rice lines was obtained using a construct (pJDV, Fig. 1D)that bears three different genes in a single T-DNA element. Silencing of expression ofthe central 35S/int-uidA gene was found to be rare in the R 0 and R 1 generations oflines bearing a single-copy insert, but multicopy insertion often yielded plants thatwere silenced in at least one transgene (Table 2). Silencing could be reversed by treatmentwith AzaC (20 or 50 mg L –1 ) or trichostatin A (TSA) (10 or 20 nM) for all R 1progeny of lines JDV88 (one copy), JDV92 (five copies), JDV83 (eight copies), andJDV 90 (eight copies) but not for line JDV95 (two copies).The silencing seen in JDV95 is most likely transcriptional because it is brieflyreset at meiosis, giving rise to a brief reactivation of GUS expression (48–56 hpostgermination), followed by immediate re-silencing in the R 1 seedling (Fig. 4). Thismay represent a case in which either the transgene is inserted into a locus that ishighly subject to silencing or one at which gene insertion stimulates an as yet unidentifiedand potent silencing mechanism.Line JDV92 has a total of five transgene copies at two separate transgene loci(Fig. 5). The single-copy gene at one locus was silenced in the presence of the locusbearing multiple transgene copies. Segregation from the multiple transgene locus re-Table 2. Analysis of transgene expression in JDV rice lines. Expression of 35S/uidA and mUbi1/bar was assayed using histochemical staining (Jefferson et al 1987) and bialaphos dipping(Kumpatla et al 1997) on representative transformants having a low (JDV85, 86, 88, and 92) ormulticopy (JDV83, 90, and 92) transgene insert. With the exception of line JDV95, all 35S/uidA-silenced lines were reactivated following 7 d of AzaC or TSA treatment in the dark.Transgene R 0R 1AzaC (mg L –1 ) TSA (nM)Line Copy GUS BAR GUS BAR 20 50 10 20JDV85 1 + + + + na a na na naJDV86 1 + + + + na na na naJDV88 1 + + +/– + + + + +JDV95 2 – – – – – – – –JDV83 8 – + – + + + + +JDV90 8 – + – + + + + +JDV92 5 – + – + + + + +ana = not applicable.472 Hall et al