Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21942
標題: 大量表現miR160a與miR167b對水稻生長發育之影響
The effect of miR160a and miR167b on rice growth and development
作者: 鄭至忠
Cheng, Chih-Chung
關鍵字: rice:microRNA:OsARF
水稻:microRNA:OsARF
出版社: 分子生物學研究所
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摘要: microRNA為一群小片段且不會轉譯出蛋白的RNA,其大小約為19-25核醣核酸,microRNA主要能抑制並調節其目標基因的表現,在植物的研究中發現,microRNA能調節細胞分化生長、開花時間、荷爾蒙與逆境的反應等等。除此之外,也有研究指出某些microRNA具有組織特異性,為了了解水稻中microRNAs的表現位置與特性,前人利用microarray技術對水稻microRNA進行初步檢測,在眾多水稻microRNA中,發現miR160a主要表現在水稻孕穗時期的穗組織中,當水稻抽穗後表現就下降且消失。經序列分析後發現,OsARF18與OsARF22可能為miR160a所調控的目標基因,miR160a分別能互補在兩基因第二個exon上。real-time RT-PCR結果顯示OsARF18與OsARF22表現於所有偵測的組織中,但是在miR160a表現多的孕穗時期穗組織中表現量較低。為了瞭解miR160a在水稻的生理與生長所扮演的角色,利用農桿菌轉殖技術得到含有大量表現miR160a的水稻轉殖株,經real-time RT-PCR結果可知OsARF18與OsARF22表現受到抑制,除此之外也利用RNA Ligase-Mediated 5’RACE(Rapid Amplification of 5’ cDNA Ends)確定OsARF18與OsARF22的mRNA會受到miR160a切割,因此證明了其為miR160a所調控的基因。進一步觀察發現轉殖株並無明顯的生長異常,除了在發芽的時期有輕微發芽延遲的現象,在植株進入種子黃熟時期,也發現轉殖株的葉片較早黃化。第二個研究的microRNA為miR167b,在先前microarray的結果發現,其主要表現在第100天劍葉與下位葉中,同樣的經由序列比對分析,其可能的目標基因為OsARF25,在大量表現miR167b的轉殖株中雖然可以看出OsARF25的mRNA受到抑制,卻無法利用RLM 5’RACE直接的証明OsARF25為miR167b的目標基因,且受到miR167b的調控。在這些轉殖株中,大量表現miR167b造成植株生長異常的現象發生,其植株較矮,分蘗少且呈現扇形狀的生長。本研究已經初步證明了microRNAs所調控的目標基因,也發現大量表現microRNAs對植物所造成的影響,但是microRNA影響植物生長與發育的詳細情形,未來必須做更深入的探討。
MicroRNAs (miRNAs), a group of small noncoding RNAs, are approximately 19-25 nucleotides in length. These miRNAs down-regulate their target genes at the post-transcriptional level. In plants, miRNAs regulate their target genes expression mostly by guiding mRNA cleavage. Many reports indicate that miRNAs are involved in cell differentiation, cell growth, organogenesis, flowering time, hormone response, stress response, antibacterial resistance in plants. Recent studies demonstrated that expressions of some miRNAs are tissue-specific suggesting that those miRNAs play important roles in plant development and growth. In order to explore tissue-specific miRNA expression profile in rice, we have established a rice miRNA microarray. Our preliminary microarray results showed that expression of miR160a is mostly in booting panicle. From sequences analysis, we identified that the second exon of rice OsARF18 and OsARF22 genes contains complementary sequence of miR160a and predicted that it can be regulated by miR160a. Real-time RT-PCR analysis revealed high expression of OsARF18 and OsARF22 in all tested tissues except booting panicle which showed high level of miR160a. To explore the biological function of miR160a, a construct containing precursor of miR160a was obtained and used for transforming rice. Transgenic rice over-expressing miR160a could suppress the expressions of OsARF18 and OsARF22 genes. The RLM-5'RACE assay demonstrated that OsARF18 and OsARF22 genes are the targets of miR160a and their targeted-cleavage sites were identified. However, all transgenic rice did not reveal any obvious changed in phenotype except a slightly delay of seed germination and earlier leaves yellowing during the late ripening stage. A second miRNA, miR167b that used in this study was detected mainly in flag leaf and in the leaf below the flag leaf by microarray assay. Similar transgenic approach to that of miR160a was applied for miR167b, and results showed that the expression of OsARF25, a gene with a complementary sequence to the mature miR167b, was suppressed in transgenic plants expressing miR167b. While the targeted-cleavage site of OsARF25 by miR167b can not be identified, these transgenic rice plants revealed dwarf and less tillers. However the mechanisms of miRNAs involved in regulating plant growth and development required further studies.
URI: http://hdl.handle.net/11455/21942
其他識別: U0005-1908200817082900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1908200817082900
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