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標題: 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
Functional analysis of AGL13 gene from Arabidopsis thaliana and molecular dissection of intronic regulatory elements in first intron
作者: 葉琮任
Yeh, Tsung-Jen
關鍵字: MADS box gene
MADS box 基因
defective pollens
出版社: 生物科技學研究所
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摘要: 在此研究中,我們對阿拉伯芥MADS box 基因--AGL13,進行功能性分析。為了瞭解AGL13 於哪些組織中專一性表現,我們將AGL13 的promoter 包含它的第一個intron 與GUS 報導基因融合,然後將此構築體轉殖到阿拉伯芥中並分析GUS活性。GUS 活性最初在花早期階段(Stage2-3)的花藥原基(anther primordia)偵測到,在花粉、胚珠發育的起始階段到成熟階段也有GUS 活性的表現,這結果顯示AGL13 具有調控早期的起始作用跟後續的花粉、胚珠的發育。大量表現AGL13 的轉基因阿拉伯芥其植株明顯變小、早開花與花序無法繼續生長等性狀。AGL13的異位表現也觀察到花萼產生同源異型(homeotic)的轉化,具有柱頭狀的papillae 與胚珠的似心皮(carpel-like)結構出現。AGL13 RNAi 構築體之轉基因植物中,缺陷的花粉與胚珠導致植株出現不孕(sterility)。缺陷的花粉呈現扁平狀或外壁的exine 結構毀壞,而缺陷的胚珠則在發育早期就退化了。進一步的分析顯示,這些AGL13 RNAi 構築體之轉基因植物不孕性狀的嚴重程度只與AGL13 表現量下降有關,而與其親源性最相近的基因AGL6 其表現量則沒有改變。顯示AGL13 在調節花粉、胚珠的起始與發育具有特定的功能。阿拉伯芥AGL13 基因的第一個內含子長600 bp,內含子中可能具有一些調控因子對於AGL13 基因的表現時間、表現組織很重要。AGL13△In1::GUS (AGL13 上游啟動子區域)只在七天大幼苗的胚根有非常微弱的GUS 表現,但長大後全株皆無GUS 活性;AGL13In1::GUS (AGL13 上游啟動子區域包含第一個內含子)則是在幼苗的頂芽與 胚根分生組織、成株次級花序的分生組織與雄雌蕊有GUS 訊號。因此推論AGL13上游啟動子扮演抑制基因表現的角色,而其第一個內含子則具備類似強化子(enhancer)的功能。目前已經知道AGL13 第一個內含子對於基因的表現是必需的,因此想進一步了解內含子中的哪些DNA 序列區段對於基因的調控是至關重要的。AthaMap 預測第一個內含子中有四個MADS-box 類轉錄因子的結合位。接續的實驗將對第一個內含子進行專一性MADS-box 結合位刪除與逐步DNA刪除,探討內含子中的哪些DNA 因子會影響AGL13 的表現。
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.
其他識別: U0005-1708201014023300
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