STUDY OF GENE SILENCING IN RICE: A ROOT PREFERENTIAL GENE RCG2

The RCg2 promoter was identified in a search for root-specific genes to combat the rice water weevil (RWW) but expressed at low frequency (~10%). Spatial expression of RCg2 was investigated using two reporter constructs YXA (RCg2-gus-ocs) and YXB (RCg2-gus-RCg2) that included 1.6 kb of the RCg2 5' sequence fused to the ?-glucuronidase (gus) coding region. YXB plants were generated via Agrobacterium-mediated transformation but only 8 of 158 plants analyzed showed strong GUS activity despite the presence of an intact construct. Reactivation of RCg2 gene in rice was investigated by treatment of R0 and R1 of YXB transgenic plants with 5-azacytidine. Reactivation of RCg2-gus was observed in some transgenic plants indicating different mechanisms involved in the gene silencing of the YXB lines. DNA methylation analysis, northern blotting, RT-PCR and small RNA analysis supported the conclusion that PTGS and TGS are present in the silenced plants. Promoter analysis in silico and using promoter deletion assays predicted that the RCg2 promoter contains a complex region that includes miRNA homologs, MITEs and repetitive sequences. The high frequency of promoter-related silencing suggests functional interactions of these elements of the transgene and the homologous endogenous gene. To identify key elements contributing to the root-preferential expression of RCg2 and the high frequency of silencing observed in transgenic (YXB) lines, several RCg2 promoter deletion constructs were designed. These include 5' deletions MC1, MC2, MC4, MC7 and MC8 and internal deletions MC5, MC11, MC12 and MC13. The frequency with which silencing was encountered in populations of the deletion mutants was used to characterize the effects of various promoter elements. Deletion of the region from -406 to -208 (compared MC11 to YXB, and MC13 to MC1) revealed that region contains a negative element. Among 36 independent transformants, 33% with MC11 expressed GUS and 85% with MC13 showed GUS expression. Comparing MC7 transgenic plants to MC1 revealed that the region ?888 to ?729 is another negative regulatory element, and comparing MC11 to MC12, the proportion of expression of transgenic plants indicated the region ?729 to ?406 is a positive regulatory element.