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1. Effects on rice growth and development by overexpressing miR160a and miR167b 2. A quick method for detecting miRNA target genes
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Genome Res 18, 1456-1465.||摘要:||
MicroRNA（miRNA）是一群大小約21nt的單股RNA，在生物體中會藉著與目標基因mRNA配對而對目標基因mRNA進行剪切，或是抑制轉譯作用來達到調控基因表現的目的。目前已知miRNA在植物的生長發育扮演著重要的角色，包括藉著抑制某些轉錄因子來調控下游基因的表現，Auxin response factor（ARF）就是其中一群轉錄因子。水稻中已經有許多miRNA被發現，但只有少數被深入研究。本研究室過去發現大量表現miR160a和miR167b的水稻轉殖株（35S::miR160a和35S::miR167b）會影響目標基因ARF之表現，以RLM-5’RACE證實miR160a會對目標基因OsARF18與OsARF22 mRNA進行剪切而抑制其表現量。本研究觀察到在ABA的處理下，轉殖株35S::miR160a幼苗根部生長被抑制的情形較Wild-type嚴重，顯示大量表現miR160a可能增加了植株幼苗根部對ABA的敏感度。另外還觀察到從孕穗期開始，轉殖株的分蘗角度較大，顯示大量表現miR160a可能改變了植株的株型。miR167b經前人以軟體比對發現其目標基因可能為ARF25，但在轉植株35S::miR167b中兩者並無明顯消長的情形。本研究檢測轉植株35S::miR167b五個獨立株系後代的miR167b及ARF25表現量，結果顯示miR167b表現量越高的轉殖株，其ARF25表現量越低，可見ARF25很有可能是miR167b的目標基因。對轉殖株進行性狀觀察，發現其中三個miR167b表現量為Wild-type 4~7倍的品系，性狀與Wild-type相較之下無明顯差異，但另外兩個miR167b表現量為Wild-type 9~11倍的品系，則有分蘗數較少且分蘗角度較大的性狀，顯示miR167b表現量的增加或ARF25表現量降低可能與此性狀有關。另外，本研究以miR160a對OsARF18的調節作用為依據，擬建構miRNA調節其目標基因表現的檢測方法，但目前尚未獲得明確及穩定之結果。
MiRNA (miRNAs), a group of single-stranded small RNAs, are about 21 nucleotides in length. These miRNAs regulate their target genes by mRNA cleavage or transcriptional repression. MiRNAs play an important role in plant growth and development, including down-regulating transcription factors to operate the transcript levels of downstream genes. Auxin response factors (ARFs) are one of the transcription factor families which are regulated by miRNAs. There have been many miRNAs found in rice, but few of them have been carefully investigated. It has been shown that transgenic rice over-expressing miR160a could suppress the expressions of OsARF18 and OsARF22 genes, and their targeted-cleavage sites has been identified by RLM-5'RACE assay. Further treatment with exogenously added ABA showed more severe inhibition on seedling roots growth in miR160a over-expression transgenic rice than those of the wild-type. In addition, a wider tiller growth angle since the booting stage was observed in miR160a over-expression transgenic rice suggesting a regulatory role of miR160a in rice architecture development. Another rice miR167b has been studied to analyze its effects on plant growth and target gene OsARF25 by creating miR167b over-expression transgenic rice. The expression levels of miR167b and its putative target gene OsARF25 of five transgenic rice lines were analyzed. Results showed that the higher the expression levels of miR167b in the transgenic rice lines, the lower the expression levels of OsARF25 were detected, suggesting that OsARF25 is the target gene of miR167b. A phenotype with dwarf and wider tiller growth angle was observed in two independent transgenic lines that expressed approximately 9 to 11-fold of miR167b than that of the wild-type, while the other three lines that expressed approximately 4 to 7-fold of miR167b revealed the same phenotype as the wild-type. How can this phenotype be attributed by the different expression levels of miR167b and OsARF25 require further investigation. In this study, an approach to establish a quick miRNA target genes screening method using the known relationship between miR160a and OsARF18 was also performed, but was not successful. Further investigations to increase the sensitivity and stability of the assay method are required.
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