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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  異位表現OsMADS34及OsCP7基因導致水稻穗生長異常之探討

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/151743

標題: 異位表現OsMADS34及OsCP7基因導致水稻穗生長異常之探討
Rice gene functional analysis of panicle aberrant development in ectopically overexpressed OsMADS34 and OsCP7
作者: 黃建富
Huang, Jian-Fu
Contributors: 陳良築
Liang-Jwu Chen
分子生物學研究所
關鍵字: 早開花;半胱胺酸蛋白酶;早開花;節間縮短;種子黑斑
rice;T-DNA;overexpression;MADS-box;cysteine protease;internode
日期: 2013
Issue Date: 2013-11-07 12:20:22 (UTC+8)
Publisher: 分子生物學研究所
摘要: 實驗材料來自Taiwan Rice Insertional Mutants library (TRIM library)中的T-DNA突變株M52048。M52048活化水稻第三號染色體上的OsMADS14、OsMADS34和OsCP7基因,造成矮株、早開花、節彎曲和穗無法完全抽出之特異性狀。OsMADS14和OsMADS34皆屬於MADS-box基因家族轉錄因子,可調控水稻開花機制及花器生長發育,OsMADS14在光週期調控下,可促使水稻開花,其機制已被釐清;OsMADS34可調控花器分生組織的型態、穗及穎花外桴的發育;OsCP7為putative cysteine protease,在水稻扮演的功能角色仍未明朗。利用大量表現OsMADS34和OsCP7的方式研究其基因功能。
  Ubi:OsMADS34有早開花和穗無法完全抽出等性狀,穗無法完全抽出主要為節間縮短所致,雖然第三節間較長仍無法彌補第一節間過短所造成的影響,過去認為水稻在乾旱逆境下會有穗無法完全抽出之性狀,透過Microarray和miRNA mocroarray分析得知OsMADS34的確可調控幾個與乾旱相關的基因和miRNA,因此假設OsMADS34可能參與水稻體內賀爾蒙調節和逆境相關反應。實驗利用賀爾蒙處理來改善Ubi:OsMADS34穗無法完全抽出之性狀,但結果卻不如預期,進一步分析GA處理的確有促進節間的延長,但主要為促進第二節間和第五節間延長,並沒有延伸第一節間的長度,因此沒有改善穗抽出之性狀。透過分析第一節間的GA代謝相關基因和細胞壁重建相關酵素基因的表現,發現Ubi:OsMADS34大量提升EATB基因的表現,可抑制多數GA生合成基因的表現,同時也減少OsXTH19和OsXTH28的表現,造成節間延長被抑制的現象,然而大量表現OsMADS34導致節間縮短的機制仍需更進一步的研究。
  OsCP7擁有典型的cysteine protease結構,signal peptide、prodomain和protease domain,為一主要表現在水稻幼苗及葉片的基因,但不表現於穎花中,利用CaMV 35S啟動子大量表現OsCP7卻無法成功得到穩定轉殖株,僅有的兩株轉殖株在移出培養基後便迅速死亡,加上實驗室前人亦無法利用玉米Ubiquitin啟動子大量表現OsCP7於水稻,推測過度表現其基因皆會造成水稻死亡;而以OsCP7上游1.6 Kb當作啟動子,構築OsCP7:OsCP7轉殖株,OsCP7在轉殖株各時期組織中均被大量表現,包含穗的部分,造成的性狀有矮株、略晚抽穗和種子黑斑且不稔實等。根據2011年二期作分析,證明此性狀與OsCP7:OsCP7構築是完全相關,大量表現OsCP7影響花粉的發育,造成穎花的穎片於抽穗後開始發生黑斑,且曝露於陽光面的穎片較早產生斑狀,接著影響種子糊熟的過程,累積大量的ROS造成大面積細胞死亡現象,致使轉殖株種子稔實率極低,稔實的種子重量也較輕,而經過確認有Knockdown OsCP7基因的35S:OsCP7 RNAi轉殖株並沒有特殊性狀,推測可能為水稻中有互補功能的基因存在。
M52048 is a rice mutant with T-DNA insertion which activated flanking genes OsMADS14, OsMADS34 and OsCP7, resulting in darwf, early flowering, node bending and impaired panicle exertion. OsMADS14 and OsMADS34 belong to MADS-box gene family, can regulate the flowering time and the floral meristem development. OsMADS14 regulate the flowering time and has been well studies. OsMADS34 has been known to regulate the floral meristem development and panicle branching, OsCP7 encodes a putative cysteine protease, but the function by overexpressing OsMADS34 and OsCP7 genes remained unknown.
The OsMADS34 overexpressing transgenic rice, Ubi:OsMADS34, revealed early flowering and impaired in panicle exertion. Morphological dissection indicated that the impaired panicle exertion was caused by shortening the first and second internodes. Previous study showed drought stress inhibited panicle exertion by reducing the peduncle elongation during flowering and induced the accumulation of ABA. The present study showed some dorught-related genes and miRNAs were regulated in Ubi:OsMADS34, and therefore, hypothesized that the phenotype of Ubi:OsMADS34 may regulated by drought stress or plant hormones. However, ABA inhibitor and GA treatments in this study could not improve the first internode elongation and panicle exertion. Gene expressinon analysis indicated the GA-regulated gene, EATB, was up-regulated and cell elongation promoting genes, XTH19 and XTH28, were reduced in the first and second internodes of Ubi:OsMADS34, suggesting that the limited internode elongation may be due to the differential expression of these internode regulating genes. However, how these genes were regulated remains further elucidation.
OsCP7, like a typical cysteine protease, consist of a signal peptide, a prodomain and a protease domain, expressed in vegetative tissues but not in reproductive tissues. No OsCP7 overexpressing transgenic rice driven by ubiquitin or 35S promoters was stably obtained, though few of them could survive in the medium. Instead of these consititutive promoters, a 1.6 kb promoter fragment of OsCP7 could drive the expression of OsCP7 in rice successfully. This 1.6 kb OsCP7 promoter-driven transgenic rice, OsCP7:OsCP7, showed slightly dawrf, delayed flowering, low pollen viabillity, lower fertility seeds, and lesion-like brown spots appeared on spikelet. The lesion-like spots were first occuerd on the outer surface of spikelet when exposed to sunlight after heading. The spots could spread quickly in days and led to extensive cell death with ROS accumulation. However, using RNAi approach to knockdown the expression of OsCP7 did not show any specific phenotype. Further investigation to understand the molecular mechanism of the formation of the lesion-like spot is underway.
Appears in Collections:[依資料類型分類] 碩博士論文

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