- 學位論文
阿拉伯芥miR164在長時間熱逆境下對NAC1轉錄因子的調控
miR164 regulates NAC1 transcription factors in Arabidopsis under long-term heat stress
摘要
MicroRNA是不轉譯為蛋白質，其長度約為21~24個核苷酸的小分子RNA，藉由剪切mRNA或抑制轉譯的方式調控目標基因的表現，在植物的生長與發育中扮演重要的調控因子，也參與在植物面對生物與非生物逆境的反應中。然而，目前miRNA調控熱逆境反應的相關研究與其他逆境相較並不多。在先前的研究中利用微陣列分析在高溫處理下表現量有明顯變化的基因群，找出可能被熱逆境調控的miRNA。我們發現長時間熱逆境下miR164會被誘導上升，而其目標基因群NAC轉錄因子的表現量則被抑制。而在miR164三個前驅物中，miR164a受高溫處理誘導有最高的表現量，且僅有miR164a突變株在熱逆境下產生葉片黃化的外表型。為了進一步研究miR164與其目標基因在熱逆境下的關係，利用miR164a前驅物與miR164目標基因的轉殖株測試其對高溫環境的耐受性，結果發現無法表現miR164a前驅物的突變株葉綠素含量在熱逆境下大幅減少，且長時間熱處理下植株的存活率也顯著降低。miR164目標基因NAC1與ANAC092的表現量在熱逆境下受miR164a所抑制，然而nac1與anac092突變株的葉綠素含量卻沒有明顯變化。我們推測NAC1與ANAC092可能直接導致葉綠素的分解，而導致miR164a突變株植株在熱逆境下產生不耐熱的性狀。.nac1與anac092突變株以及miR164a前驅物大量表現轉殖株於五個葉綠素分解基因中表現量皆較野生型為低，而ANAC092大量表現轉殖株則於SGR與PPH結果中表現量較野生型高。由以上實驗結果得知，在熱逆境下植物中miR164的表現量增加，抑制目標基因NAC1和ANAC092的表現，減緩了下游葉綠素分解基因的表現而增加植株在熱逆境下的存活率，顯示miR164a對NAC1和ANAC092的調控影響了植物對熱的耐受性。
並列摘要
MicroRNAs (miRNAs) are non-coding small RNAs approximately 21~24 nucleotides long that regulate target gene expression by mRNA cleavage or translational repression. Many miRNAs play important regulatory roles in plant growth and development, and also participate in plant responses to biotic and abiotic stresses. However, heat-responsive miRNAs have not been studied well in plants in comparison to other stress-responsive miRNAs. In previous study, microarray analyses were used to examine the fold changes of gene profile under heat stress to find heat-responsive miRNAs. We found that miR164 was induced and the transcription of its target genes NACs, encoding transcription factors, were reduced under long-term heat stress. Moreover, miR164a was increased most by heat treatment in three miR164 precursors, and only the transgenic plants with the mutated miR164a precursor had chlorosis phenotype. To further study the relationship of miR164 and its target genes under heat stress, transgenic plants modifying miR164a precursor or miR164 target gene expression were tested for thermotolerance. The chlorophyll contents of T-DNA insertion lines with no miR164a precursor expression were highly reduced under heat stress, and the survival rates were also lower than that of wild-type. In addition, the expression levels of target gene NAC1 and ANAC092 were repressed by miR164a, while the chlorophyll contents of nac1 and anac092 mutant lines were not significantly changed. The differences of mir164a mutants and target mutants in phenotype and chlorophyll content may result from chlorophyll degradation, hence the expression of several genes involved in chlorophyll degradation pathway were measured. All chlorophyll degradation genes tested were down-regulated in nac1 and anac092 mutant lines and miR164a overexpression line, and SGR and PPH were induced in ANAC092 overexpression lines. Taken together, under heat stress the expression miR164 was induced, its target genes NAC1 and ANAC092 were inhibited, and the expression of chlorophyll degradation genes was reduced in order to increase the survive rate of plants. Our results suggest that miR164a regulation of NAC1 and ANAC092 may be crucial for plant thermotolerance.