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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 生物科技學研究所 > 依資料類型分類 > 碩博士論文 >  阿拉伯芥中調控細胞分裂與配子體發育相關基因之功能性分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152588

標題: 阿拉伯芥中調控細胞分裂與配子體發育相關基因之功能性分析
Functional analysis of genes regulating cell division and gametophyte development in Arabidopsis
作者: 許巍瀚
Hsu, Wei-Han
Contributors: 楊長賢
Chang-Hsien Yang
生物科技學研究所
關鍵字: 阿拉伯芥;細胞分裂;開花;配子體
Arabidopsis;cell division;flowering;gametophyte
日期: 2012
Issue Date: 2013-11-18 10:44:15 (UTC+8)
Publisher: 生物科技學研究所
摘要: 植物如何針對已分化之細胞進行細胞分裂的調控與抑制,而非在分生組織的細胞進行仍未被明確地了解與研究。 類Yippee (Yippee-like, YPEL) 基因家族是一群能在所有真核生物細胞中找到的保守性基因。然而在植物中YPEL 基因尚未被研究及報導。本研究針對阿拉伯芥中YPEL家族的基中一個成員,AtYIP1 基因進行選殖與功能性分析。在啟動子融合GUS基因的分析中發現AtYIP1的mRNA持續性的表現在分生組織細胞以外的所有的組織中,並且在已不分裂的細胞中,AtYIP1蛋白質是不斷的在進行降解。以35S啟動子進行AtYIP1 RNAi的轉殖植物中的性狀可見植物的生長速度變快、體型變大、細胞數及同時期的葉片數較多。相對的也發現大量、異源性的表現AtYIP1基因在植物中會造成生長狀況顯著性的被抑制。從組織解剖分析的結果也發現性狀強烈的35S::AtYIP1轉殖株中異變的原因是在於失去幼芽及根系的細胞分裂能力。由上述的結果顯示AtYIP1在阿拉伯芥中扮演著抑制者的角色去阻止細胞進行分裂。此項假說由後續的二項試驗支持:菸草BY-2細胞株以及轉染進行大量表現flag標定的ATYIP1蛋白質的人類HEK 293T腎胚胎細胞株都發生細胞生長被抑制的現象。進一步的發現AtYIP1蛋白能夠結合在DNA上去抑制已分化細胞不正常的進行分裂活動。我們的結果發現一個能在動植物皆能造成抑制細胞分裂生長的植物基因。 (第一章)
阿拉伯芥的AGL13基因是一個MIKC類型MADS-box基因家族的AGL6的系群當中。在過往實驗室的研究中AGL13基因的表現在花粉及胚珠從起始到成熟階段皆能被專一性的測得。異源性的表現AGL13 RNAi構築體可發現阿拉伯芥的轉殖株,其花器的花粉及胚珠皆造成缺陷進而造成無法受孕、稔實的現象。在本次研究中35S::AGL13 RNAi/qrt1-2轉殖突變株的四分體融合花粉中出現數目各半的二種不同花粉。第一種與一般的野生型植株相同,但第二種的花粉外型較小呈現扁平或皺縮的形狀。由此可推論花粉在減數分裂期間若帶有35S::AGL13 RNAi的片段會造成發育停滯的狀況。另外像野生型的花粉也進行了花粉沾附及授粉試驗。由結果發現AGL13基因不僅控制花粉發育也控制著絨氈層進行花粉壁形成的功能。融合了抑制序列的AGL13:SRDX大量的在植物中表現會出現與減少AGL13 RNA內含量的植株性狀更加強的結果。進一步的實驗也發現AGL13:SRDX與SEP2:SRDX的融合表現植株,花器會因A、B及C群MADS-box基因表量被抑制而造成相同發育上的缺失。目前已確認AGL13能與B、C群MADS-box蛋白結合,並形成蛋白複合體,進而調控花粉發育及絨氈層形成下游基因的表現。(第二章)
最後的部分主要說明架構了一系列不同系統的雙元素表達戴體,用來進行植物分子選殖及功能性分析。全系列的戴體皆共用同一份限制酵素截切位點序列,並含有不同啟動子組合變化的、融合標簽蛋白和篩選標記基因。這些戴體可應用在不同的實驗分析技術,包括:異源性表現、RNA靜默、螢光標簽融合、熱處理表現、單子葉作物轉殖、顯性負抑制、顯性正向活化及橫越性表現分析。新的戴體能增加實驗室內不同的構築程序,並且能使實驗分析的設計能以標準系統化的程序進行。(第三章)
How to suppress the cell division in the differentiated cells but not the meristematic cells is largely unknown in plants. The family of Yippee-like (YPEL) genes has been found in various eukaryote species. However, no study on YPEL genes has been reported in plant species. In this study, an Arabidopsis YPEL gene AtYIP1 was characterized. The promoter::GUS assay indicated that AtYIP1 mRNA was constitutively expressed in all tissues except meristematic cells and the AtYIP1 proteins were degraded constantly in cells without further division. Hastened growth and increased size and cell number of leaf were observed in 35S::AtYIP1 RNAi plants. By contrast, growth in the 35S::AtYIP1 plants ectopically expressing AtYIP1 was significantly inhibited. The anatomical analysis revealed that the severe 35S::AtYIP1 mutant phenotype is primarily due to the lack of the cell division in both shoot and root apical meristem. These results revealed a repressor role for AtYIP1 in preventing cell division in Arabidopsis. This assumption was further supported by the suppression of the cells growth for tobacco cell line BY-2 and human embryonic kidney cell line HEK 293T after transfection with flag-tagged AtYIP1. Furthermore, AtYIP1 proteins were found to be able to suppress the abnormal cell division for differentiated cells by entering nucleus and bind to DNA. Our data represents a novel finding that a plant gene is able to suppress cell division and growth in both plant and animal system. (Chapter 1)
Arabidopsis AGL13 is the gene classified as AGL6 lineage of MIKC type MADS-box gene family. Our previous study indicated that AGL13 expression was specifically detected from the initiation to maturation of both pollen and ovules. Ectopic expression of AGL13 RNAi construct was found to cause sterility by inducing the production of flowers with defective pollen and ovules in transgenic Arabidopsis plants. In this study, two types of pollen in equal numbers were found in 35S::AGL13 RNAi/qrt1-2 tetrads. The first type resembles wild-type whereas the second type is reduced in size with a flat or collapsed shape, suggesting that the development of the pollen grains that carried the 35S::AGL13 RNAi was arrested during meiosis. The viability of the wild-type like pollen in AGL13 RNAi plants was tested by adhesion assay and pollination assay. These data indicated that AGL13 not only regulates pollen development but also controls the tapetum function for exine formation. The enhancement of the alteration of pollen development, the sterility of the plants and the flower organ formation in AGL13:SRDX (containing a suppression motif) transgenic plants suggested that AGL13 acts as a repressor. Furthermore, similar defects in floral organs was observed in AGL13:SRDX and SEP2:SRDX plants which was caused by the suppression of the expression for A, B and C function MADS-box genes. AGL13 could interact with B, C function MADS-box protein and form complex to regulated downstream genes expression, including the pollen developing and tapetum formation genes. (Chapter 2)
Finally, series systems of binary vectors have been generated for plant molecular cloning and functional study. All vectors share the same restriction enzyme cloning sites, and contain different combinations of promoters, fusion tags and selection markers. These vectors can be applied to the experimental assays of ectopic expression, RNA silencing, fluorescent tags fusion, heat inducible expression, monocot transformation, dominant negative repression, dominant positive activation, trans-expression. New vectors provided the additional construction procedures for the lab and the experiment assay could be designed with systematically work through standard operating procedure (SOP). (Chapter 3)
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