阿拉伯芥AGL13 基因之功能分析及探討其第一個內含子中的調控因子對AGL13 表現的影響

Abstract

In this study, we characterized the Arabidopsis MADS box gene AGL13. To determine the tissue specific expression of AGL13, the promoter and intron 1 of AGL13 were fused to the GUS reporter gene. This construct was transformed into Arabidopsis, and GUS activity was analyzed. GUS activity was first detected in the anther primordia of early stage 2-3 flowers and was specifically detected from the initiation to maturation of both pollens and ovules. This result suggests that there is a role for AGL13 in regulating early initiation and further development of pollens and ovules. Transgenic Arabidopsis plants ectopically expressing AGL13 have significantly reduced plant size, flower early and lose inflorescence indeterminacy. The ectopic expression of AGL13 also causes the homeotic conversion of the sepals into carpel-like structures with stigmatic papillae and ovules. The ectopic expression of an AGL13 RNAi construct was found to cause sterility by inducing the production of flowers with defective pollens and ovules in transgenic Arabidopsis plants. The defective pollens were flat or collapsed with aberrations in the exine patterning of the outer wall. The defected ovules were aborted during early development. Further analysis indicated that the severity of the mutant phenotype was dependent only on the down-regulation of AGL13. The expression of the closest related gene, AGL6, is not altered in these AGL13 RNAi plants, indicating that AGL13 has a specific function in regulating the initiation and development of the pollens and ovule in Arabidopsis. The first intron of AGL13 is 600 bp in length, implying that the large, first intron could contain regulatory elements necessary for proper spatiotemporal expression. In plants containingAGL13△In1::GUS (the upstream promoter region which lacks the first intron), weak GUS reporter activity was detected in radicles of 7-day-old seedlings . By the AGL13△In1::GUS transgenic plants grown up, reporter activity was no longer detected in all tissues.The GUS reporter activity of AGL13In1::GUS (the upstream promoter region in combination with the first intron)transgenic lines was detected in the shoot apical meristem and root apical meristem of seedlings, meristem of secondary inflorescence, anthers and ovules. This result suggested that the 5’upstream promoter region is responsible for the repression effect of AGL13 expression, whereas the first intron could be an enhancer-like element. We further wanted to know which regions in first intron are important for proper gene expression. There are four MADS-box transcription factor binding sites in first intron by the prediction of AthaMap. To examine the role of the intronic elements in first intron in the expression of the AGL13, three specific deletion derivatives and five progresive DNA deletion derivatives were generated.