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標題: | 利用阿拉伯芥noa1突變株在傷害下研究訊息調控因子 Study of target genes in noa1 Arabidopsis thaliana by wounding |
作者: | Sheng-Chih Fan 范盛之 |
指導教授: | 鄭石通(Shih-Tong Jeng) |
關鍵字: | noa1,一氧化氮,傷害,阿拉伯芥,miR156,轉錄調控因子, noa1,nitric oxide,wounding,Arabidopsis,miR156,transcription factor, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 為了研究一氧化氮 (NO)在阿拉伯芥傷害下所扮演的訊息傳遞角色,使用傷害處理過的野生型 (WT)與nitric oxide synthase 1 (noa1)突變型阿拉伯芥cDNA進行基因晶片分析,並經過gene spring軟體篩選,將noa1與WT基因表現量達到兩倍以上差異的轉錄調控因子挑選出來進行後續的實驗。從不處理傷害的 noa1/WT晶片中挑選出表現量下降,且在處理傷害下的noa1/WT晶片中表現量下降幅度更大的轉錄調控因子。希望能由晶片的資料中找出經由傷害處理後在WT中基因表現量會受到誘導上升,而在noa1中不會被誘導的轉錄調控因子。經由real-time polymerase chain reaction (real-time PCR)的檢測,找到NAC domain containing protein 96 (ANAC096)、DREB and EAR motif protein 4 (RAP2.10)、WRKY DNA-binding protein (WRKY) 55.1與WRKY55.2等基因具有與晶片資料相符合的基因表現量趨勢。為了確定那些轉錄調控因子是否在傷害下經由NO訊息傳導而誘導表現量上升,而使用了NO釋放劑sodium nitroprusside (SNP)去處理WT與noa1植株,結果顯示ANAC096、WRKY55.1、和WRKY55.2在noa1植株處理SNP後,其基因表現和WT單純傷害後相似,則可以證明這些轉錄調控因子的表現量在傷害後會經由NO的訊息調控所誘導。在另一部分的實驗,將篩選出的轉錄調控因子,與Arabidopsis Small RNA Project網路資料庫比對後,挑選出11個與已知miRNA有連結的目標基因。經過real-time PCR檢測後,確認了四個目標基因的表現量可能與NO相關聯,其中Squamosa promoter binding protein-like (SPL) 3, SPL4和SPL5與晶片上的表現量趨勢符合,而miR156已知可以調控SPL基因,SPL基因與miR156與生長時期轉換和開花調控有關。利用real-time PCR與北方墨點法確認precursor form miR156a與mature form miR156的表現量。然而miR156的表現量卻顯示與NO不相關,經過傷害後的植株顯示出開花時間延後。實驗結果發現傷害處理下SPL基因與miR156與NO不相關,但是傷害可能透過其他的機制去影響開花時間。 To study nitric oxide signaling upon wounding, Arabidopsis gene chips were hybridized with cDNA probes from wild-type (WT) Arabidopsis and mutant with noa1 (nitric oxide synthase 1) after wounding. Genes, whose expression levels were different from two folds between WT and mutants, were selected, and, especially, those encoding transcription factors were chosen for further study. The putative transcription factors were chosen from the microarray data whose values were reduced in noa1/WT wounding chip compared to those in noa1/WT non-wounding chip. Hence, genes encoding the transcription factors which were up regulated by wounding in WT plant but not in noa1 mutant were screened for further study. The real-time PCRs were used to confirm the gene expression profiles in microarray, showing the expression patterns of transcription factor NAC domain containing protein 96 (ANAC096), DREB and EAR motif protein 4 (RAP2.10), WRKY DNA-binding protein (WRKY)27, WRKY55.1 and WRKY55.2 were the same as those in microarray. To study the transcription factors induced by nitric oxide through wounding, NO donor sodium nitroprusside (SNP) was used to treat WT and noa1 Arabidopsis. SNP treatment can recover the expression levels of genes encoding ANAC096,WRKY55.1, or WRKY55.2 on noa1 mutant plant after wounding, indicating these transcription factors were involved in the nitric oxide signal pathway induced by wounding. In another part of study, the putative miRNAs for NO-related transcription factors were suggested by Arabidopsis Small RNA Project database, indicating 11 target transcription factors have corresponding miRNAs. The real-time PCRs were used to confirm the gene expression in microarray, showing the expression patterns of transcription factor Squamosa promoter binding protein-like (SPL) 3, SPL4 and SPL5 were the same as those in microarray. It has been known that some of the SPL genes can be regulated by miR156. The expression levels of precursor and mature miR156a, analyzed by real-time PCR and Northern, respectively, upon wounding are different. Moreover, the expression pattern of mature form miR156 was not related to nitric oxide. SPL gene and miR156 have been known to play important roles in regulating phase transition and flowering time. Hence, plants wounded repeatedly showed delay flowering. Conclusively, the effect of wounding on SPL genes and miR156 is not mainly dependent on nitric oxide, but wounding affects SPL gene, miR156 and flowering time by a mechanism needed for further study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17191 |
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顯示於系所單位: | 植物科學研究所 |
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