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標題: | 傷害逆境之下甘藷miR408的調控機制與功能 MicroR408 regulates wounding responses in sweet potato (Ipomoea batatas cv. Tainung 57) |
作者: | Yun-Wei Kuo 郭芸瑋 |
指導教授: | 鄭石通(Shih-Tong Jeng) |
關鍵字: | 甘藷,傷害逆境,次世代定序資料分析,miR408,KCS,PCL,GAUT, sweet potato,wounding,small RNA deep sequencing,miR408,KCS,PCL,GAUT, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 植物已經演化出許多機制因應傷害逆境,包含修復受傷的組織以及活化防禦相關的基因。MicroRNAs (miRNAs) 藉由對其目標基因進行靜默作用來調控和參與植物的生長發育以及逆境防禦反應,然而目前對於參與在甘藷傷害反應的miRNA研究仍是有限。本研究利用次世代高通量小分子 RNA 定序法建立甘藷中與傷害逆境相的關small RNAs資料庫,將傷害逆境相關的miRNAs與甘藷的轉錄組資料庫經由序列相似性比對,鑑定出甘藷中被傷害逆境所抑制的miR408及其前驅物 miR408 precursor (pre408) 和相對應之目標基因。qRT-PCR分析發現,在甘藷中miR408和pre408會被傷害逆境所抑制,而其特有的目標基因IbKCS (3-ketoacyl-CoA synthase 4)、IbPCL (plantacyanin-like) 與IbGAUT (galacturonosyltransferase 7-like) 的表現量則在傷害逆境後被誘導上升,表示在傷害逆境之下這些目標基因受到miR408的調控。茉莉酸jasmonate (JA) 也會抑制miR408和pre408的表現,但是只有IbKCS會受到茉莉酸誘導。經由剪切點分析證實miR408與目標基因之結合位點;菸草短暫表現分析再次驗證miR408會剪切並抑制IbKCS、IbPCL 與 IbGAUT的基因表現。為了進一步了解miR408在甘藷傷害逆境扮演的角色與功能,建立了大量表現miR408的甘藷轉殖株,在大量表現 miR408的轉殖株中其目標基因的表現量均會被抑制。經由功能性分析比較野生型與大量表現miR408的甘藷,結果顯示大量表現miR408的植株外表型矮小、葉片水分散失速率提高、葉綠素含量減少、光合作用效率減緩以及抗蟲能力下降。欲了解哪個目標基因參與在抗蟲防禦反應,又建構了大量表現IbKCS、IbPCL與IbGAUT 的菸草轉殖株,發現大量表現IbKCS可提升菸草對斜紋夜盜蛾的抗性。總結以上實驗結果顯示,甘藷在傷害逆境之下會藉由抑制 miR408表現量使得目標基因表現量上升進而調控不同的傷害相關反應機制,其中IbKCS與甘藷的抗蟲防禦反應有關。 Plants have evolved complex mechanisms, including healing tissue and defensive gene activation to respond promptly to wounding. MicroRNAs (miRNAs) play diverse roles in plant development and defense responses by binding to their mRNA targets based on sequence complementarity. In this study, a wound-related miR408 and its target genes in sweet potato (Ipomoea batatas cv. Tainung 57) are proposed by small RNA deep sequencing and transcriptome analysis. The expression patterns of miR408 and the miR408 precursor (pre408) were significantly repressed by wounding and jasmonate (JA). In contrast, expression of the putative target genes IbKCS (3-ketoacyl-CoA synthase 4), IbPCL (plantacyanin-like), and IbGAUT (galacturonosyltransferase 7-like) of miR408 was increased following wounding, whereas only IbKCS was increased after JA treatment. The target cleavage site mapping and Agrobacterium-mediated transient assay demonstrated that IbKCS, IbPCL, and IbGAUT were the targets of miR408. The expression of miR408-targeting genes was repressed in transgenic sweet potatoes overexpressing miR408. This result further indicated a relationship between miR408 and its target genes. Notably, miR408-overexpressing plants showed a semi-dwarf phenotype and attenuated resistance to insect feeding, while transgenic plants overexpressing IbKCS exhibited more insect resistance than plants overexpressing only the empty vector. Collectively, sweet potato reduces the abundance of miR408 upon wounding to elevate the expression of IbKCS, IbPCL, and IbGAUT. The expression of IbKCS enhances the defense system to against herbivore wounding. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77447 |
DOI: | 10.6342/NTU201804333 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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