Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97773
標題: 阿拉伯芥 YPEL 基因 AtYIP1 對抑制細胞分裂調控之功能分析研究
Functional analysis of yippee-like gene AtYIP1 in suppressing cell division in Arabidopsis thaliana.
作者: 郭晉維
Chin-Wei Kuo
關鍵字: 阿拉伯芥;細胞分裂;細胞週期;Arabidopsis;cell division;cell cycle;yippee;YPEL
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摘要: 
類yippee基因家族為真核生物細胞中一段保守序列區域,能與鋅離子鍵結並主要表現在細胞核,且與癌細胞複製相關。實驗室前人的研究結果指出,過表達yippee基因家族中的AtYIP1會在植株分生組織以外的各部位中皆有表現。與野生型阿拉伯芥相比,此類轉殖株生長矮小。而強烈性狀的表現株,根細胞與葉細胞可能出現不可逆轉的性狀。另一方面,AtYIP1表達RNA干擾的轉殖株能加快生長速度與提早開花。此外分析阿拉伯芥pAtYIP1::GUS轉殖株GUS之表現,顯示AtYIP1 mRNA的高表現量,但是蛋白質表現量顯著減少。因此推論AtYIP1是藉由持續的蛋白降解來達到精細的轉譯後調控。本研究首先藉由重新轉殖AtYIP1過表達與靜默基因構築載體來重現了擬南芥轉殖株的表現型。進一步的實驗發現在較矮小的35S::AtYIP1轉殖株中,AtYIP1基因的表現量較高。另一方面在不影響其他同源YPEL基因的表現下,藉由RNAi專一性抑制AtYIP1基因表現的植株個體較大。接著由植株葉片長與寬的記錄,作出轉殖植株生長的狀況統計,並估算AtYIP1基因轉殖株之間的相對生長速率。不同於AtYIP1過表達的轉殖株,將AtYIP1靜默只會加速生長速率。在葉片早期可以見到更多的細胞分裂與更大的細胞,但是葉片成熟後,細胞數與細胞大小將與野生型相近。此外在AtYIP1基因過表達與靜默的轉殖株T2子代性狀也有相近的結果。將35S::AtYIP1轉殖入Dr5::GFP的轉殖株,綠色螢光蛋白顯示出生長素在根部表現正常的濃度梯度分布,並顯示清楚的靜止中心位子。這可以證明AtYIP1基因表現量的多寡,只與根尖細胞的層數相關。實驗發現AtYIP1基因靜默與過表達會造成根部分生組織細胞生長加速或減緩的情形。藉由以上實驗確認AtYIP1對於細胞在分裂上的調控功能。有趣的是在過表達AtYIP1的強性狀轉殖株根尖分生組織細胞所觀察到的性狀,與SUMO E3 ligase相關基因研究報告所提到的hpy2突變株性狀相當相似,兩基因之間可能具有上下游的調控關係。

Yippee-like gene (YPEL) family are conserved proteins in all eukaryotic cell, contain a zinc-binding domain and localized in the nucleus, which inhibits cancer cell replication. In the previous study, ectopic expression of Arabidopsis yippee-like gene 1 (AtYIP1) significantly inhibited the growth of the leaf and root in transgenic Arabidopsis. In contrast, AtYIP1 RNA interference line grows faster and blooms earlier than wild-type plants. These results indicated that AtYIP1 prevents cell division and growth rate in Arabidopsis. A promoter::GUS assay indicated that AtYIP1 mRNA was highly expressed in all tissues except meristematic cells. However, the AtYIP1 proteins were constantly degraded in non-dividing cells. This result indicates that AtYIP1 is possibly controlled by post-translational regulation. In this study, further functional analysis confirmed the phenotypes observed in AtYIP1 overexpression and silencing Arabidopsis. We found that a high expression level of AtYIP1 was strongly associated with the small plant size in 35S::AtYIP1 Arabidopsis. In contrast, the more down-regulation of AtYIP1 expression, the larger plant size was produced in AtYIP1-RNAi Arabidopsis. The length and width of the leaves in transgenic plants were measured and the exquisite statistics of different transgenic lines were set up, and draught the relative growth rate between AtYIP1 transgenic plant. The result indicated that the silence of AtYIP1 only speeds up the growth rate. More cell division and larger cell size were observed in AtYIP1-RNAi than in wild-type plants in the early stage of leaf development. However, similar cell number and cell size were observed in mature leave of the both. Besides, AtYIP1 overexpression and silencing of T2 plants showed similar phenotypes to their respective T1 plants. When 35S::AtYIP1 was introduced into Dr5::GFP transgenic plants, green fluorescence protein indicating the normal auxin gradient loop of root and demarcation site of the quiescent center was observed. Silencing or overexpression AtYIP1 will hasten or delay the growth rate of root meristem cell. In conclusion, we have proved the cell division suppressing of AtYIP1. Interestingly, the strong strait of overexpression AtYIP1 transgenic plant shows a similar feature with the hpy2 mutant plant, which is reported to be a SUMO E3 ligase related gene. It may have some association between them.
URI: http://hdl.handle.net/11455/97773
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