Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22272
標題: 水稻 T-DNA 插入突變體 M52048 分析及其活化的三個基因 OsMADS34、OsMADS14 及 OsCP7之功能研究
Characterization of the T-DNA insertion mutant M52048 and functional study of three activated genes OsMADS34、OsMADS14 and OsCP7
作者: 李咨胤
Li, Tzu-Yin
關鍵字: mutant line
水稻
rice
early flowering
cysteine proteinase
早開花
半胱胺酸蛋白酶
出版社: 分子生物學研究所
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摘要: A rice T-DNA insertion mutant M0052048 showing extreme early flowering, bending tillers and impairment in panicle exertion was isolated from the Taiwan Rice Insertion Mutant (TRIM) library. This mutant contained a copy of the T-DNA tag inserted in the chromosome number 3 at the 64,867 bp position of OSJBa0032E21 BAC clone where the expression of 048-3 (OsMADS34), 048-4 (OsMADS14) and 048-7 (putative cysteine proteinase, OsCP7) genes were activated by the 35S enhancer located in the T-DNA. The OsMADS14 and OsMADS34 are MADS box-containing transcription factors that have important roles in plant growth and development and furthermore, OsCP7 is a putative cysteine proteinase that may be involved in the program cell death, pollen and xylem maturation, embryogenesis and flowering time in plants. In order to understand the functions of these activated genes and their contribution to the mutant phenotype, transgenic rice over-expressing each of these genes were created and expression profiles of these genes in various rice plant tissues were analyzed. RT-PCR analysis revealed that OsMADS14 and OsMADS34 expressed mainly in panicles but not necessarily in vegetative tissues, with the exception of a relatively high expression level of OsMADS14 in the 90-day-old leaf; For OsCP7, no clear expression signals were detected in all tested tissues. Transgenic rice Ubi:MADS14 showed constitutive expression of OsMADS14 in leaf tissue and revealed extreme early (63.6 days vs 121.3 days in TNG67) flowering and tiller bending phenotypes. The transgenic rice Ubi:MADS34 showed constitutive expression of OsMADS34 and revealed impairment in panicle exertion and slightly early (110 days vs 121.3 days) flowering compared to TNG67. For the overexpression study of OsCP7, no transgenic plant with ubiqutin promoter construct (Ubi:CP7) was obtained, thus a 1.6 kb promoter region from native OsCP7 gene was used to replace the ubiqutin promoter and several ectopically-expressed OsCP7 transgenic rice plants were obtained. These OsCP7 transgenic rice plants showed increased levels of OsCP7 mRNA and protein and higher cysteine protease activity compare to that of TNG67. In addition, these plants had reduced height, approximately 88% of TNG67, and revealed brown lesions on the surfaces of most spikelets that were neither observed in the TNG67 nor in M0052048. In summary, the present study suggests that the activation of OsMADS14 and OsMADS34 genes contributes to early flowering, bending tillers and impairment in panicle exertion phenotypes in the mutant M0052048. However, the effect of OsCP7 activation in mutant M0052048 and the function of OsCP7 in rice plants are still not clear and further investigations will be needed to answer these questions.
M52048 為 T-DNA 插入的水稻(Oryza sativa L. cv. Tainung 67)突變株。其具有早開花、穗抽出不完全及分蘗彎曲等多重外表性狀。前人研究發現此突變株的 T-DNA 是以順向的方式插入於水稻的第三對染色體編號 OSJNBa0032E21 BAC 的 64,867 bp 位置,導致上游 048-3(OsMADS34)、 048-4(OSMADS14)及下游 048-7(OsCP7, putative cysteine proteinase)三個基因的活化表現。OsMADS14 及 OsMADS34 兩基因皆為含有 MADS-box 的轉錄因子,對植物生長發育上扮演重要角色。OsCP7 則隸屬於 C1A cysteine proteinase,可能參與了計畫性細胞死亡,以及植物的開花時間、花粉及胚的形成、木質部生合成…等。為了探討三個被活化的基因在水稻中所扮演的功能及與突變株 M52048 的相互關係,本研究以取得三基因個別大量表現的轉殖株與不同時期及組織中三基因表現情形的檢測來進行分析。RT-PCR 結果顯示:OsMADS14 與 OsMADS34 主要表現於生殖生長組織的稻穗上,而在營養生長組織中,只有 OsMADS14 可在 90 天葉片上檢測到較高的表現量。而 OsCP7 則無法在檢測的組織中偵測到明顯的表現量。OsMADS34 大量表現的轉殖株具有些微早抽穗(110 天 vs 121.3 天)、穗抽出異常及高節位根的形成,而大量表現 OsMADS14 的轉殖株具有極早抽穗(63.6 天 vs 121.3 天)及導致分蘗有彎曲之現象。兩者的外表性狀與 T-DNA 突變體 M52048 相符。另外,因為無法得到大量表現 OsCP7 的轉殖株,於是以 OsCP7 原生性啟動子置換 Ubiquitin 啟動子以得到異位表現轉殖株。這些 OsCP7P:OsCP7 轉殖株具有較高的 OsCP7 基因之 RNA、蛋白表現量及具有較高的 cysteine proteinase 活性。由外表性狀的分析,異位表現 OsCP7 會導致植株些微矮化,且在穎花上有類似病斑的性狀,然而此性狀卻無法從突變株 M52048 中觀察到。由以上研究顯示,T-DNA 突變株 M52048 的早開花、分蘗彎曲及穗抽出不完全等外表性狀為活化OsMADS14 及 OsMADS34 所導致而成,然而 OsCP7 與突變株 M52048 的相互關係及 OsCP7 的功能則仍需進一步的探討。
URI: http://hdl.handle.net/11455/22272
其他識別: U0005-2408201113452900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2408201113452900
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