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標題: OsGA2oxs之功能研究 I.水稻OsGA2ox2基因之功能及其T-DNA插入活化突變體M43852之探討 II. C20 OsGA2ox6三個保留性motifs之功能探討
Functional study of Rice GA 2-oxidase I. Characterization of rice GA2ox2 and its T-DNA activation mutant M43852 II. Functional study of the conserved motifs in C20 type OsGA2ox6
作者: 謝昆廷
Hsieh, Kun-Ting
關鍵字: Gibberellin;GA 2-oxidases;T-DNA
出版社: 分子生物學研究所
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GA 2-oxidases (GA2oxs) are enzymes involved in the GA catabolic pathway that hydroxylate the C-2 of active GAs or GA precursors to produce biologically inactive GAs. Ten GA2ox genes have been identified in rice and they were divided into C19-type (OsGA2ox1-4, 7-8 and 10) and C20-type OsGA2oxs (OsGA2ox5-6 and 9). The C20-type OsGA2oxs contains three conserved motifs that have not been previously studied and also a C19-type OsGA2ox2 gene which has not been characterized. The purpose of this study is to study the functions of OsGA2ox2 gene in rice and to elucidate the functional roles of the conserved motifs in C20-type OsGA2ox6. To study the function of OsGA2ox2, two T-DNA insertion mutants M43852 and M43211 with T-DNA tag inserted next to OsGA2ox2 gene were isolated and characterized. The mutant M43211 showed the same phenotype as TNG67 with no activation of OsGA2ox2, while M43852 revealed semi-dwarf with more tillers and showed increased expression of OsGA2ox2. Further experiments using transgenic rice to over-express OsGA2ox2 revealed severe dwarf yet fertile plants. The severe dwarf (35% of TNG67) phenomenon was different from the activation mutant M43852 (70% of TNG67), and the capability to produce seeds was in constrast to the C19-type GA2oxs over-expression transgenic rice plants which were mostly sterile. In addition, transgenic rice over-expressing OsGA2ox2 also resulted in increased expression of OsGA3ox2 and OsGA20ox2 genes and their dwarf phenotype could be partially recapitulated by adding GA3. Taken together, these results suggest that the OsGA2ox2 has GA 2-oxidase activity and is responsible for the dwarf phenotype observed in M43852 and over-expression transgenic rice. However, the different degrees of plant height between M43852 and over-expression transgenic rice could be attributed to the different levels of OsGA2ox2 expression. To elucidate the function of the conserved motifs in C20-type GA2oxs, 11 mutated OsGA2ox6 genes which each contain a single amino acid change in different conserved motifs were created and used to transform rice. Based on the dwarf degrees observed in the transgenic rice over-expressing different mutated genes, it can be observed that the mutant Y123A in motif I lost OsGA2ox6 activity completely, while mutants E140A, A141E and H143A in motif II lost OsGA2ox6 activity partially, whereas mutant G343A in motif III show reduced OsGA2ox6 activity. In summary, the C19-type GA2ox2 is a functional GA 2-oxidase when over-expression can reduce plant height and several critical amino acid sequences in the conserved motifs have been demonstrated to play a crucial role in determining the activity of OsGA2ox6.

GA 2-oxidases (GA2oxs)能夠對活化態GA或是GA前驅物的C-2位置進行hydroxylation,使其轉變成不活化態,為GA代謝途徑的主要酵素。過去的研究中發現水稻具有十個GA2oxs且被分類為C19類型 (OsGA2ox1-4、7-8 & 10)和C20類型 (OsGA2ox5-6 & 9),此外不論是C20類型的OsGA2oxs特有的三個保留性區域或是C19類型的OsGA2ox2,目前都沒有被研究過。因此本實驗之目的為研究水稻的OsGA2ox2之功能以及解釋三個保留性區域對於C20類型的OsGA2ox6扮演何種腳色。為了研究OsGA2xo2之功能,本實驗從T-DNA突變庫找到了兩個T-DNA插入位置在OsGA2ox2基因附近的T-DNA插入突變體M43852及M43211。M43211之外表性狀和TNG67相同且OsGA2ox2並沒有活化,然而M43852呈現半矮化、多分蘗和OsGA2ox2表現活化等情形。利用轉基因技術大量表現OsGA2ox2於轉殖株中,造成植株嚴重矮化,這樣嚴重矮化 (TNG67的35%)的性狀和M43852半矮化 (TNG67的70%)之性狀不同,另外轉殖株仍能夠結穗稔實這點與過去所探討的C19類型的OsGA2oxs其轉殖株皆無法結穗稔實不同。除此之外,轉殖株大量表現OsGA2ox2提高了OsGA3ox2和OsGA20ox2的表現量,且其株高能夠藉由處理GA3有部分回復的情形,綜合上述資料顯示OsGA2ox2可能具有GA 2-oxidase之酵素活性,因此造成M43852及轉殖株矮化之性狀,而M43852和轉殖株之間株高的差異性可能是因為OsGA2ox2表現量不同所導致。另一方面,為了解釋C20類型的GA2oxs三個保留性區域之功能,利用單一胺基酸置換製造十一個OsGA2ox6點突變構築並將其轉形至水稻,根據不同點突變轉殖株之株高,觀察到保留性區域I的Y123A失去了OsGA2ox6之酵素活性,保留性區域II的E140A、A141E及H143A喪失了部分的OsGA2ox6之酵素活性,而保留性區域III的G343A則輕微減少了OsGA2ox6之酵素活性。整體來說,C19類型的OsGA2ox2可能具有GA 2-oxidase之酵素活性,當大量表現OsGA2ox2時能夠減少植株之株高,而本實驗也從各保留性區域找到數個重要胺基酸能夠影響到OsGA2ox6之酵素活性。
其他識別: U0005-2408201112452400
Appears in Collections:分子生物學研究所

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