.1. Separation Structure Of The T-dna (gus) Gene Capture System Rice Genome 2.osrrm, A Rice Endosperm-specific Expression Of Genes Using Gene Capture System

Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterlessβ-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 T0 plants had various patterns of the reporter gene expression, and the copy number of the T-DNA inserts was determined in the GUS-positive transgenic plants. In addition, the flanking sequences of the T-DNA were isolated by inverse-PCR in 14 GUS-positive plants, and the insertion sites of T-DNAs of GUS-postive transgenic plants transformed with plasmid p13GUS were analyzed by BLAST. To further reveal the GUS expression pattern of other GUS-negative transgenic lines, these calli were induced from the seeds of the T1 generation, and subjected to stress and hormone treatments. Histochemical GUS assays revealed that calli from 21 lines displayed differential GUS expression after treatment. All these data demonstrated that the gene trap system constructed in this study is suitable for identifying rice genes, including those that are sensitive to induction.In the current study, we utilized the promoter trap method to identify a rice plant, named 107#, in which theβ-glucuronidase (GUS) reporter gene was expressed specifically in endosperm. A single copy of T-DNA was inserted into the plant and a candidate gene OsRRM was obtained by separating the flanking sequence and Basic Local Alignment Search Tool (BLAST) comparison. OsRRM promoter drove GUS expression specifically in rice endosperm which was basically identical to GUS expression pattern in 107#. OsRRM, a single copy gene in rice, encodes a nuclear protein containing 1005 amino acid residues with two RNA recognition motifs (RRMs) and one Spen paralog and ortholog C-terminal (SPOC) domain and OsRRM is a member of Spen gene family in rice. Western blot analysis revealed that OsRRM protein specifically expressed in rice endosperm. Ectopic expression of OsRRM resulted in transgenic plants that presented abnormalities such as short stature and retarded growth or even an inability to survive. Considering of the specialized cells in endosperm and the reported function of Spen genes, it suggested that OsRRM might regulate the cell development in endosperm.