Isolation And Identification Of An AP2/ERF Factor That Binds An Allelic Cis-element Of A Rice Yield-related Gene, LRK6.

To practice efficient breeding through genetic engineering techniques, it is important to identify the function and expression regulation of the gene. We previously map-cloned a gene cluster including eight leucine-rich repeat receptor-like kinase (LRK) genes in Dongxiang wild rice(Oryza rufipogon Griff.), which increased grain yield by 16%. Interestingly, we detected allelic expression variation in the LRK gene cluster, in which alleles of some genes were unequally expressed in hybrids of Nipponbare(Oryza sativa L. subsp. Japonica var. Nipponbare)/93-11 (Oryza sativa L. subsp. Indica var.93-11) cross. Both parental alleles of LRK6 were expressed, but they did not contribute equally to the total amount of transcript in the hybrid. In the parental lines, the Nipponbare allele was expressed at a much higher level than the 93-11 allele. In the hybrid, the expression levels of the Nipponbare and 93-11 alleles were similar to that of each parent, respectively. Allelic expression of the rice yield-related gene, LRK6, in the hybrid of 93-11 and Nipponbare is determined by allelic promoter cis-elements. Using deletion analysis of the LRK6 promoter, we identify two distinct regions that might contribute to LRK6 expression. Sequence alignment reveals differences in these regions of LRK6 promoter in 93-11, Dongxiang wild rice and Nipponbare. One of the segments, named DSLP2, contains potential transcription factor binding sites. Using a yeast one-hybrid assay, we isolate an ethylene-responsive factor (ERF) protein that binds to the DSLP2. Sequence analysis and a GCC-box assay showe that the ERF gene, O. sativa ethylene-responsive factor 3 (OsERF3), which belongs to ERF subfamily class?, has a conserved ERF domain and an ERF-associated amphiphilic repression (EAR) repressor motif. We use an in vivo mutation assay to identify a new motif (5'-TAA(A)GT-3') located in DSLP2 that interacts with OsERF3. In ethephon treatment and overexpression of OsERF3 experiments, we suggest that OsERF3 may inhibit the expression of LRK6 in 93-11, but has no effect on the expression of LRK6 in Nipponbare. These results suggest that OsERF3, an AP2/ERF transcription factor, binds the LRK6 promoter at this new motif, which might cause differential expression of LRK6 in the 93-11/Nipponbare hybrid. It has great views for understanding the expression regulation of yield-relative gene and the mechanism of heterosis.