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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭石通(Shih-Tong Jeng) | |
dc.contributor.author | Yun-Wei Kuo | en |
dc.contributor.author | 郭芸瑋 | zh_TW |
dc.date.accessioned | 2021-07-10T22:02:24Z | - |
dc.date.available | 2021-07-10T22:02:24Z | - |
dc.date.copyright | 2018-12-13 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-12-12 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77447 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T22:02:24Z (GMT). No. of bitstreams: 1 ntu-107-D00b42012-1.pdf: 8143263 bytes, checksum: 3d7d748d0b9fd562614cf31bfbd29bae (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 誌謝…….……………………………...…………………………………………………Ⅰ
摘要…….……………………………...………………………………………………Ⅱ Abstract………………………………………………………………………..…….Ⅲ Abbreviation……………………………………………………………..…..………..Ⅺ Introduction……………...………………………………………………………...…...1 Defense responses of wounding in plant…….………………………………......….1 Plant miRNAs biogenesis and function…………………………………………......2 Roles of miRNA in plant defense response…………………………….……......….4 Plant miR408……………………………………………………………..…......….5 Targets of miR408………………………...…………………………………......….8 Research objectives…………………………....………………………………………11 Materials and Methods………………………………………………………........…..12 Plant materials, growth conditions and stress treatments……….………..……...…12 Small RNA library construction, sequencing and processing……….……………..12 Transcriptome sequencing and de novo assembly………………………..….….…13 Prediction and validation of miR408 precursor and target genes……………......…13 Small RNA blot assay………...……………………………………………….…..15 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis...….……….16 Isolation and sequence characterization of IbKCS, IbPCL, and IbGAUT genes…………………………………………………………………...………….17 Mapping of miR408 cleavage sites……………….……………………….……....17 Constructs and plant transformation…………….…………………………………18 Agrobacterium-mediated transient expression in tobacco…….………...…………19 Insect bioassay with Spodoptera litura……………………………….……….…..20 Chlorophyll and chlorophyll fluorescence measurement………………..……...…20 Water loss rate measurements...…………………………………………….….….21 JA measurement………………………………………………..……………..…...22 Scanning electron microscopy (SEM) analysis……………………………..……..22 Transmission electron microscopy (TEM) analysis.…………………….………...22 Results………………………………………………………………………………….24 Identification of miR408 during the wounding response……………..………...….24 Isolation and validation of miR408-targeting mRNAs in sweet potato……....……27 Overexpression of miR408 reduces the resistance of plant against insect feeding………………………………………………….…………………………29 Involvement of miR408 in JA response…………………...……...…………….…31 Effects of miR408 on water loss rate, plant growth, and chlorophyll degradation, and ROS production……………………………………….…….………..……….32 Scanning electron microscopy (SEM) observations…….…………………..……..33 Transmission electron microscopy (TEM) observations…….………….………....35 IbRLK, another potential target of miR408.………………………………….……35 Spatial expression of pre408, miR408 and miR408 targets in sweet potato.………………………………………………………….………………….36 Expression of miR408 and miR408 targets in STTM408 transgenic plants.…………….…………………………………………...…………………...37 Discussion...……………………………………………………………………………38 MiR408 in plants…………………………………………….………....………….38 Targets of miR408 in sweet potato………………………..……………….………39 Functions of sweet potato miR408 in the mechanical and herbivore wounding responses…………………………………………………………………………..42 The functions of miR408 targets in the sweet potato……………………..………..47 Conclusion…………………………………………………………..…………...…….49 Figures………………………………………………………………...……………….50 Fig. 1. The length distribution of small RNAs in the unwounded and wounded sweet potato leaves………………………………………………………...……….....…50 Fig. 2. Expression patterns of miR408 precursor (pre408) and miR408 in response to wounding.……………………………………………….……………………...51 Fig. 3. Clusters of miR408 precursors (MIR408) in different plant species.…...…..53 Fig. 4. Expression patterns of miR408 target genes upon wounding and insect feeding.………………………………………………………………………...….57 Fig. 5. Validations of miR408 target genes by RACE and Agrobacterium-mediated transient assays..………………………………………………………..…………59 Fig. 6. Gene expression and insect resistances of sweet potatoes overexpressing miR408.…...………………………………………………………………………61 Fig. 7. Analysis of transgenic plants.…………………….………………………...63 Fig. 8. Insect resistances of IbKCS-overexpressing, IbPCL-overexpressing and IbGAUT-overexpressing tobacco plants.……………...………………………..…64 Fig. 9. Expression patterns of pre408, miR408 and its targets after JA treatment.....66 Fig 10. JA contents in the miR408-overexpressing and WT sweet potatoes.............68 Fig. 11. The phenotypes of miR408-overexpressing and WT sweet potatoes.…......69 Fig. 12. Growth phenotypes of sweet potato plants overexpressing miR408……....71 Fig 13. Accumulation of H2O2 in WT and miR408-overexpressing sweet potatoes upon wounding.…………………………………………………………….……..72 Fig. 14. Phylogenetic tree of KCS protein family in plants.……...……...…………73 Fig 15. Scanning electron microscopy (SEM) analysis of the transgenic sweet potatoes leaf and stem surfaces.…………………………………………..……….74 Fig 16. Scanning electron microscopy (SEM) analysis of the transgenic tobaccos leaf and stem surfaces.…………………….……………….………………………76 Fig 17. Transmission electron microscopy (TEM) analysis of the leaf tissue in WT and miR408-ox sweet potato plants…………………………….…………….……78 Fig 18. The expression patterns and miR408 cleavage site of IbRLK………...........80 Fig 19. The expression of miR408 and miR408 targets in transgenic sweet potato overexpressing miR408 precursor.……………………………………….……..…82 Fig 20. Spatial expression patterns of miR408 and miR408 targets.………...……..83 Fig 21. The expression of miR408 and miR408 targets in short tandem target mimic miR408 (STTM408) transgenic sweet potato...………...…………………………85 Fig. 22. Comparisons of plantacyanin family.…………………………...….……..87 Fig. 23. Phylogenetic tree of basic blue protein (BBP) family in plants….…...…....88 Fig. 24. Comparisons of 3-Ketoacyl-CoA synthase-like gene (KCS) in plants.….....90 Fig. 25. Comparisons of galacturonosyltransferase-like gene (GAUT) in plants.....91 Fig. 26. Phylogenetic tree of GAUT protein family in plants.……………………..92 Fig. 27. A proposed model of miR408 upon wounding in sweet potato leaves…….94 Tables……………………………………………………………...…………………...95 Table1. Primers used in this study…………………….……………….…………..95 Table 2. Small RNA deep sequencings of the unwounded and wounded sweet potato leaves.……………………………………………………………………………..99 Table 3. The conserved miRNAs induced in sweet potato upon wounding by small RNA sequencings.………………………...……………………………………..100 Table 4. The conserved miRNAs repressed in sweet potato upon wounding by small RNA sequencings.……………………………………………………………….101 Table 5. Statistics of the paired-end transcriptome sequencing data from sweet potato leaves.…………………………………………………………………….103 Table 6. Abundances and fold change ratios of the conserved miRNAs and miRNA precursors in sweet potato leaves……………………………...…………………104 Table 7. Abundances and sequences of Ib-miR408-3p (Ib-miR408) and Ib -miR408-star (Ib-miR408*) in sweet potato leaves.………………………………………..105 Table 8. Mature miR408 sequences in different plants.……………………….….106 Table 9. Prediction of miR408 target genes from de novo transcriptome of sweet potato.……………………………………………………..…………….…...…..107 Table 10. Prediction of potential targets of miR408 in sweet potato (penalty score ≤ 4)………………………………………………………………………............…108 References……………………………………………………………………...…..…109 | |
dc.language.iso | en | |
dc.title | 傷害逆境之下甘藷miR408的調控機制與功能 | zh_TW |
dc.title | MicroR408 regulates wounding responses in sweet potato (Ipomoea batatas cv. Tainung 57) | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 謝旭亮,吳克強,邱子珍,陳玉琪,林振祥 | |
dc.subject.keyword | 甘藷,傷害逆境,次世代定序資料分析,miR408,KCS,PCL,GAUT, | zh_TW |
dc.subject.keyword | sweet potato,wounding,small RNA deep sequencing,miR408,KCS,PCL,GAUT, | en |
dc.relation.page | 134 | |
dc.identifier.doi | 10.6342/NTU201804333 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2018-12-12 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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