Isolation of five rice nonendosperm tissue‐expressed promoters and evaluation of their activities in transgenic rice

Using promoters expressed in nonendosperm tissues to activate target genes in specific plant tissues or organs with very limited expression in the endosperm is an attractive approach in crop transgenic engineering. In this article, five putative nonendosperm tissue‐expressed promoters were cloned from the rice genome and designated P(O) (s) (NETE) (1), P(O) (s) (NETE) (2), P(O) (s) (NETE) (3), P(O) (s) (NETE) (4) and P(O) (s) (NETE) (5). By qualitatively and quantitatively examining GUSplus reporter gene expression in transgenic rice plants, P(O) (s) (NETE) (1)‐P(O) (s) (NETE) (5) were all found to be active in the roots, leaves, stems, sheaths and panicles but not in the endosperm of plants at different developmental stages. In addition, P(O) (s) (NETE) (2), P(O) (s) (NETE) (4) and P(O) (s) (NETE) (5) were also inactive in rice embryos. Among these promoters, P(O) (s) (NETE) (4) and P(O) (s) (NETE) (5) exhibited higher activities in all of the tested tissues, and their activities in stems, leaves, roots and sheaths were higher than or comparable to those of the rice Actin1 promoter. We also progressively monitored the activities of P(O) (s) (NETE) (1)‐P(O) (s) (NETE) (5) in two generations of single‐copy lines and found that these promoters were stably expressed between generations. Transgenic rice was produced using P(O) (s) (NETE) (4) and P(O) (s) (NETE) (5) to drive a modified Bt gene, mC ry1Ab. Bt protein expressed in the tested plants ranged from 1769.4 to 4428.8 ng/g fresh leaves, whereas Bt protein was barely detected in the endosperm. Overall, our study identified five novel nonendosperm tissue‐expressed promoters that might be suitable for rice genetic engineering and might reduce potential social concern regarding the safety of GMO crops.