r family. In addition to transcription factors, the regulated genes were mainly involved in several different metabolic processes relating to hormones, secondary substances, carbohydrates, lipids, and amino acids. Up-regulated genes in possible BnGRF2-regulated pathways Corresponding to the pleiotropic phenotypes of the transgenic lines, there was up-regulation of a number of genes that were thought to be involved in regulatory pathways. In 35S-BnGRF2a and Napin-BnGRF2a transgenic lines, overexpression of BnGRF2a resulted in an enlarged leaf and cotyledon area, respectively, which were induced by increasing cell numbers. According to microarray analyses, some genes related to cell division and the cell cycle were up-regulated in the BnGRF2a transgenic lines. As might be expected from the increased chlorophyll content and photosynthetic 480-44-4 efficiency caused by BnGRF2, some genes relating to light harvesting and chlorophyll biosynthesis were up-regulated. GLK2 , which was up-regulated in the transgenic lines, is known to stimulate expression of genes related to light harvesting and chlorophyll biosynthesis. Partially up-regulated genes such as lhcb2.4, CHI, lhcb3, lhcb6, and LTP were also found to be coincident with the up-regulated genes induced by GLK2. Also, some genes related to light harvesting and chlorophyll biosynthesis such as PUB44, CPN60A, PORA, lhcb2.1, and lhca4 were up-regulated in the transgenic lines. In addition, some up-regulated genes such as KCS16, GPAT, F4I10.40, GDPD6, SAMT, SMO1-3, and oleosin proteins were classified as being involved in lipid and FA biosynthetic and storage processes by GO category. According to the leaf phenotypes of the 35S-BnGRF2a transgenic line, although the expression level of most up-regulated genes in transgenic young leaves was <2-fold compared with the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19811292 WT control, they might be more highly expressed in the adult leaf. To confirm this, six genes, MAPKKK16, CYCA2;1, lhcb2.1, PORA, GPAT, and oleosin, were chosen to detect their expression levels in adult leaf of the 35S-BnGRF2a transgenic lines. The results showed that most genes were expressed more highly in adult leaf than in young leaf. The 
up-regulation of these genes was further verified in seed of the Napin-BnGRF2a-3 line. Discussion The present study began with gene expression analyses of 20 d ovules from the zy036 and 51070 rapeseed lines, which exhibited different levels of seed oil production. The differentially expressed gene BnGRF2 was chosen for further examination to elucidate its function in oil production. BLAST analyses of the Brassica sequence databases indicated that two BnGRF2 homologues are located in the rapeseed A or C genome. Real-time PCR showed that the two homologues were expressed differentially in six rapeseed tissues, and BnGRF2a is expressed dominantly compared with BnGRF2b. In B. rapa and B. oleracea, quite a lot of homologous genes showed differences in expression levels, which is a ubiquitous phenomenon during polyploid genome evolution, and suggested the importance of dominantly expressed genes in plant development. Therefore, BnGRF2a was chosen for further functional research. In a previous study, GRFs were believed to function as transcription activators; therefore, it was not a surprise that BnGRF2 induced pleiotropic phenotypes in Arabidopsis. However, it must be noted that greater phenotypic differences were observed in transgenic overexpression of BnGRF2, compared with previous reports regarding overexp