Abstract:

Plant reproductive development involves the coordination of a lot of genes encoding transcription factors.The AP2 domain transcription factors have been proved with critical roles in plant reproductive development.By microarray analysis, we identified a gene which showed higher expression in pod as 500 folds as that in leaf of soybean. The Blast searches indicated this gene encodes a dehydration responsive element binding protein (DREB)-like protein showing highest similarity to Arabidopsis TINY, therefore named as GmTINY1. By searching soybean genome and EST databases, the putative full-length cDNA sequence for GmTINY1 was in silico assembled. The GmTINY1 gene was cloned from soybean seeds at 15 DAF (days after flowering) by RT-PCR. GmTINY1 contained a complete open reading frame (ORF) of 755 bp which encoded a peptide of 244 amino acids. The predicted molecular mass and isoelctric point of GmTINY1 are 26.76 kD and 5.12, respectively. An AP2 domain and a ser-rich domain were identified in GmTINY1 amino acid sequence by Motif Scan server. The GmTINY1 encoding product showed 59% and 62% sequence similarities with Arabidopsis TINY and TINY2, respectively. Multiple sequences alignment revealed that a highly conserved AP2 domain was present in each AP2 domain transcription factor. In this AP2 domain, it was found that a Ser⁶⁶ was specifically present in GmTINY1, DREB1B, TINY and TINY2 proteins but a Cys⁶⁶ in DREB1A and DREB1C proteins, indicating, like Arabidopsis TINY, TINY2 and DREB1B, GmTINY1 might be able to bind both the dehydration responsive element(DRE) and ethylene responsive element (ERE) motifs while DREB1A and DREB1C only bind DRE motif. Besides, it was found that a ser-rich domain probably involving translational modification on GmTINY1 protein was located close to AP2 domain. The neighbor-joining phylogenetic tree showed that the AP2 domain transcription factors were grouped into four subfamilies: DREB, ERF, AP2, and RAV; and GmTINY1, TINY, and TINY2 were grouped into a branch which attributed to the DREB subfamily. With an attempt to understand the biological role for GmTINY1 and to verify the microarray result, we used the Real-time quantitative PCR approach to analyze the expression pattern of GmTINY1 in various soybean organs. We found that expression of GmTINY1 was highest in pod, relatively lower in flower and root but undetectable in leaf, indicating GmTINY1 may play some roles in soybean reproductive organs and root, but not in leaf. The Blast search results against soybean EST database also supported the specific expression of GmTINY1 in soybean pod and root. We analyzed GmTINY1 expression during the course of seed development based on publicly available microarray data and found that GmTINY1 was expressed with a low level at the globular stage and heart stage but highly expressed in hilum at the cotyledon stage embryos. Taken together, it is suggested that GmTINY1 may play some regulatory role in soybean reproductive development, such as the formation of hilum in soybean.

Key words: Soybean, Transcription factory, AP2, TINY, DREB