Isolation of Anther-specific Gene Promoters Suitable for Transgene Expression in Rice

Abstract

To isolate rice anther-specific promoters that allow strong and constitutive expression of transgenes under chilling stress conditions, a genome-wide screen using a microarray was conducted. Six candidate genes for anther-specific expression were selected after comparison of expression levels in leaf and anther tissues from booting stage plants. These genes encoded a mandelonitrile lyase (OsACE1), a sterol desaturase (OsCER1), a MADS-box protein (OsMADS58), a polygalacturonase (OsPGT1), an ERF-like AP2 domain-containing protein (OsERFL1), and a ribosome-inactivating protein-like gene (OsRIP1). Reverse transcriptase-polymerase chain reaction analyses confirmed anther-specific expression of these genes under controlled and in-field chilling conditions. Promoter regions of the selected genes were isolated and fused to the β-glucuronidase (GUS) reporter gene, and the constructs were introduced into rice plants. GUS expression in all transgenic plants except for those carrying OsERFL1and OsRIP1 constructs was only detectable in anthers within the panicles, and no GUS activity was detected in leaves. However, OsERFL1 and OsRIP1 promoters were shown to drive GUS expression in paleae and glumes as well as anthers. GUS expression levels of four transgenic plants were also tested with 2-week-old seedlings and grains. Promoters for OsACE1, OsCER1, OsMADS58, and OsPGT1 were shown to be silent in roots and aerial parts of the seedlings and mature grains. Therefore, these four promoters were deemed suitable for anther-specific transgene expression in rice.