臺灣博碩士論文加值系統

TLDc是在2002年被發現含有Tre2/Bub2/Cdc16 (TBC) 與lysin motif (LysM) 的蛋白質功能區塊，TLDc相關的蛋白質可能與由過量活性氧化物 (ROS) 所引起之氧化逆境的防禦有關，而植物中ROS (包含H2O2與O2-) 的含量則會受到鹽逆境或是其他許多環境的改變而增加，但是，目前對於TLDc相關蛋白的調控機制尚不清楚。在本研究中，利用會大量表現甘藷 (Ipomoea batatas) TLDc基因 (IbTLD) 的轉基因菸草來觀察IbTLD在鹽逆境與耐受力所扮演的角色。在鹽逆境下，TLD轉殖株 (TLD-1與TLD-2) 具有較野生型 (W38) 更好的發芽率、葉綠素含量與根長表現。除此之外，利用Gene Ontology (GO) term分析RNA sequencing的結果，進而了解TLDc相關的下游基因調控網路，結果顯示在鹽逆境下轉殖株有許多與逆境反應、DNA保護和修復的基因會受到逆境所誘導。在RT-qPCR也顯示轉殖株中的NtFeSOD3.2、NtAPX3、NtNUDT10L與NtNUDT9L的表現量都比W38還要高。在一般情況與鹽逆境下，轉殖株的抗氧化酵素抗壞血酸過氧化酶 (ascorbate peroxidase, APX) 活性較W38的還要高，且在鹽逆境下，轉殖株的過氧化氫 (H2O2) 與丙二醛 (malondialdehyde，MDA) 含量都較W38的還要低。在轉殖株中，鹽逆境所導致的DNA ladder也有受到降低。因此推測，IbTLD可能會調控許多逆境相關基因的表現來清除ROS以及保護DNA，進而提高植物對於鹽逆境的適應力。

TLDc domain, a novel protein functional domain containing Tre2/Bub2/Cdc16 (TBC) and lysin motif (LysM) domain, was identified in 2002. TLDc-related protein might be involved in the defense of oxidative stress caused by excessive reactive oxygen species. The amount of ROS, including H2O2 and O2−, in plants can be changed upon the different external environments including salt stress. However, the regulatory mechanism of TLDc-related protein is still unknown. In this study, transgenic tobaccos overexpressing Ipomoea batatas TLDc gene (IbTLD) were created to characterized the roles of the IbTLD in tolerance of salt stress. Under salt stress, transgenic lines showed better germination rates, chlorophyll contents and root lengths than wild type (W38) . In addition, RNA sequencing (whole transcriptome shotgun sequencing) incorporating with Gene Ontology (GO) term analysis was used to investigate TLDc-related downstream genes network. Results revealed several genes involved in responses of stress and protection and repair of DNA were influenced. Under normal and salt stress conditions, quantitative reverse transcription PCR also presented expression levels of NtFeSOD3.2, NtAPX3, NtNUDT10L and NtNUDT9L in transgenic lines were elevated compared to those in W38. The transgenic lines showed higher ascorbate peroxidase (APX) activity than W38 under normal and salt stress conditions compared to W38. Besides, transgenic lines also showed lower hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents under salt stress conditions. The salt-induced DNA ladder in transgenic lines was also deceased. Therefore, IbTLD might regulate the expression of stress related genes to mediate scavenging of ROS and protecting of DNA. Further, the abilities of adaptation to salt stress were enhanced.

致謝 i
摘要 ii
Abstract iii
目次 iv
表目次 vi
圖目次 vii
前言 1
1. 植物與非生物逆境以及氧化逆境 1
2. Reactive oxygen species (ROS) 活性氧化物 3
3. TLDc功能區塊 (TLDc domain) 5
4. 次世代定序技術中RNA sequencing的應用 5
5. 研究目的與方向 6
材料與方法 7
1. 菸草 7
2. H2O2處理 7
3. 鹽處理 7
4. 次世代定序 (next-generation sequencing，NGS) 7
5. Gene ontology (GO) term 分析 7
6. 引子對設計 7
7. RNA萃取 8
8. 反轉錄聚合酶鏈式反應 (RT-PCR) 8
9. 聚合酶鏈式反應 Polymerase chain reaction 8
10. 即時聚合酶鏈式反應 Real-time polymerase chain reaction (RT-qPCR) 9
11. 發芽率 (germination rate) 測定 9
12. 存活率 (survival rate) 測定 9
13. 根延長 (root elongation) 測定 10
14. 細胞色素含量測定 10
15. 抗氧化酵素 (APX、CAT) 萃取 10
16. 抗氧化酵素POX萃取 11
17. 抗氧化酵素SOD萃取 12
18. H2O2含量測定 12
19. 丙二醛 (MDA) 含量測定 12
20. DNA 萃取 13
21. DNA保護測定 13
結果 14
1. 確認轉基因菸草之IbTLD基因表現 14
2. 內源性菸草之NtTLD基因表現 14
3. 鹽逆境對於菸草外表型的影響 14
4. RNA sequencing中基因表現篩選與Gene ontology-term分析 15
5. 轉基因菸草中逆境反應相關基因的表現量分析 16
6. 抗氧化酵素活性 17
7. 鹽逆境對菸草植株內MDA以及H2O2含量的影響 17
8. IbTLD對DNA保護的影響 18
討論 19
1. 鹽逆境下TLDc與種子萌芽、存活率、根長以及細胞色素含量的關係 19
2. TLDc與氧化逆境所誘導的抗氧化逆境基因分析 19
3. TLDc、鹽逆境與H2O2以及MDA的關係 21
結論 23
圖表 24
表一、本論文所使用引子 24
表二、受到誘導而表現量上升基因 25
表三、其他物種之TLDc 26
圖一、IbTLD轉錄表現量分析 27
圖二、NtTLD轉錄表現量分析 28
圖三、鹽逆境對種子發芽率的影響 29
圖四、鹽逆境對存活率的影響 30
圖五、鹽逆境對根長的影響 31
圖六、鹽逆境對葉綠素與類胡蘿蔔素含量的影響 32
圖七、RNA-sequencing以及Gene Ontology-term網路分析 33
圖八、逆境反應基因轉錄表現量分析 34
圖九、鹽逆境對抗氧化酵素活性的影響 35
圖十、鹽逆境對H2O2含量的影響 36
圖十一、鹽逆境對丙二醛 (MDA) 含量的影響 37
圖十二、逆境反應基因轉錄表現量分析 38
圖十三、轉殖株中因鹽而導致之DNA ladder檢測 39
圖十四、IbTLD在遭受氧化逆境與鹽逆境時的調控反應 40
參考文獻 41

Allen RD, Webb RP, Schake SA, 1997. Use of transgenic plants to study antioxidant defenses. Free Radic Biol Med 23, 473-479.
Apel K, Hirt H, 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55, 373-399.
Bartsch M, Gobbato E, Bednarek P, et al., 2006. Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7. Plant Cell 18, 1038-1051.
Bolwell GP, 1999. Role of active oxygen species and NO in plant defence responses. Curr Opin Plant Biol 2, 287-294.
Clark D, Durner J, Navarre DA, Klessig DF, 2000. Nitric oxide inhibition of tobacco catalase and ascorbate peroxidase. Mol Plant Microbe Interact 13, 1380-1384.
Creissen G, Firmin J, Fryer M, et al., 1999. Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress. Plant Cell 11, 1277-1292.
Delledonne M, Xia Y, Dixon RA, Lamb C, 1998. Nitric oxide functions as a signal in plant disease resistance. Nature 394, 585-588.
Delledonne M, Zeier J, Marocco A, Lamb C, 2001. Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci U S A 98, 13454-13459.
Devireddy AR, Zandalinas SI, Gomez-Cadenas A, Blumwald E, Mittler R, 2018. Coordinating the overall stomatal response of plants: Rapid leaf-to-leaf communication during light stress. Sci Signal 11.
Doerks T, Copley RR, Schultz J, Ponting CP, Bork P, 2002. Systematic identification of novel protein domain families associated with nuclear functions. Genome Res 12, 47-56.
Domoki M, 2018. Oxidative stress tolerance and plant development: the functional characterization of the "oxprot" gene.
Du M, Zhao J, Tzeng DTW, et al., 2017. MYC2 Orchestrates a Hierarchical Transcriptional Cascade That Regulates Jasmonate-Mediated Plant Immunity in Tomato. Plant Cell 29, 1883-1906.
Durand M, Kolpak A, Farrell T, et al., 2007. The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins. BMC Cell Biol 8, 13.
Falace A, Filipello F, La Padula V, et al., 2010. TBC1D24, an ARF6-interacting protein, is mutated in familial infantile myoclonic epilepsy. Am J Hum Genet 87, 365-370.
Faust F, Schubert S, 2017. In vitro protein synthesis of sugar beet (Beta vulgaris) and maize (Zea mays) is differentially inhibited when potassium is substituted by sodium. Plant Physiol Biochem 118, 228-234.
Fedurayev PV, Mironov KS, Gabrielyan DA, et al., 2018. Hydrogen peroxide participates in perception and transduction of cold stress signal in Synechocystis. Plant Cell Physiol.
Finelli MJ, Sanchez-Pulido L, Liu KX, Davies KE, Oliver PL, 2016. The Evolutionarily Conserved Tre2/Bub2/Cdc16 (TBC), Lysin Motif (LysM), Domain Catalytic (TLDc) Domain Is Neuroprotective against Oxidative Stress. J Biol Chem 291, 2751-2763.
Fonseca JP, Dong X, 2014. Functional characterization of a Nudix Hydrolase AtNUDX8 upon Pathogen Attack Indicates a Positive Role in Plant Immune Responses. PLoS ONE 9.
Foyer C, Descourvieres P, Kunert K, 1994. Protection against oxygen radicals: an important defence mechanism studied in transgenic plants. Plant, Cell & Environment 17, 507-523.
Foyer CH, Noctor G, 2005. Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17, 1866-1875.
Ghosh S, Mitra S, Paul A, 2015. Physiochemical studies of sodium chloride on mungbean (Vigna radiata L. Wilczek) and its possible recovery with spermine and gibberellic acid. ScientificWorldJournal 2015, 858016.
Gill SS, Tuteja N, 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48, 909-930.
Heath RL, Packer L, 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125, 189-198.
Huang XS, Luo T, Fu XZ, Fan QJ, Liu JH, 2011. Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobacco. J Exp Bot 62, 5191-5206.
Jabs T, Dietrich RA, Dangl JL, 1996. Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 273, 1853-1856.
Jebara S, Jebara M, Limam F, Aouani ME, 2005. Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress. J Plant Physiol 162, 929-936.
Ji T, Li S, Huang M, et al., 2017. Overexpression of Cucumber Phospholipase D alpha Gene (CsPLDα) in Tobacco Enhanced Salinity Stress Tolerance by Regulating Na (+) –K (+) Balance and Lipid Peroxidation. Front Plant Sci 8.
Kangasjarvi S, Lepisto A, Hannikainen K, et al., 2008. Diverse roles for chloroplast stromal and thylakoid-bound ascorbate peroxidases in plant stress responses. Biochem J 412, 275-285.
Kim BG, Waadt R, Cheong YH, et al., 2007. The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis. Plant Journal 52, 473-484.
Kruszka K, Pacak A, Swida-Barteczka A, et al., 2014. Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley. Journal of Experimental Botany 65, 6123-6135.
Kumar RR, Pathak H, Sharma SK, et al., 2015. Novel and conserved heat-responsive microRNAs in wheat (Triticum aestivum L.). Funct Integr Genomics 15, 323-348.
Kumari J, Udawat P, Dubey AK, Haque MI, Rathore MS, Jha B, 2017. Overexpression of SbSI-1, A Nuclear Protein from Salicornia brachiata Confers Drought and Salt Stress Tolerance and Maintains Photosynthetic Efficiency in Transgenic Tobacco. Front Plant Sci 8, 1215.
Larkindale J, Knight MR, 2002. Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol 128, 682-695.
Lin JS, Kuo CC, Yang IC, et al., 2018. MicroRNA160 Modulates Plant Development and Heat Shock Protein Gene Expression to Mediate Heat Tolerance in Arabidopsis. Front Plant Sci 9, 68.
Lin JS, Lin CC, Lin HH, Chen YC, Jeng ST, 2012. MicroR828 regulates lignin and H2O2 accumulation in sweet potato on wounding. New Phytol 196, 427-440.
Liu H, Tian D, Liu J, Ma G, Zou Q, Zhu Z, 2013. Cloning and functional analysis of a novel ascorbate peroxidase (APX) gene from Anthurium andraeanum. J Zhejiang Univ Sci B 14, 1110-1120.
Liu X, Huang B, 2000. Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass. Crop Science 40, 503-510.
Ma C, Burd S, Lers A, 2015. miR408 is involved in abiotic stress responses in Arabidopsis. Plant Journal 84, 169-187.
Milh M, Falace A, Villeneuve N, et al., 2013. Novel compound heterozygous mutations in TBC1D24 cause familial malignant migrating partial seizures of infancy. Hum Mutat 34, 869-872.
Miller G, Shulaev V, Mittler R, 2008. Reactive oxygen signaling and abiotic stress. Physiol Plant 133, 481-489.
Mittler R, 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7, 405-410.
Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K, 2012. AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819, 86-96.
Mullineaux PM, Exposito-Rodriguez M, Laissue PP, Smirnoff N, 2018. ROS-dependent signalling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes. Free Radic Biol Med, https://doi.org/10.1016/j.freeradbiomed.2018.01.033.
Munns R, Tester M, 2008. Mechanisms of salinity tolerance. Annu Rev Plant Biol 59, 651-681.
Oliver PL, Finelli MJ, Edwards B, et al., 2011. Oxr1 is essential for protection against oxidative stress-induced neurodegeneration. PLoS Genet 7, e1002338.
Pennazio S, 2005. Mineral nutrition of plants: a short history of plant physiology. Riv Biol 98, 215-236.
Poulat AL, Ville D, De Bellescize J, et al., 2015. Homozygous TBC1D24 mutation in two siblings with familial infantile myoclonic epilepsy (FIME) and moderate intellectual disability. Epilepsy Res 111, 72-77.
Saha P, Chatterjee P, Biswas AK, 2010. NaCl pretreatment alleviates salt stress by enhancement of antioxidant defense system and osmolyte accumulation in mungbean (Vigna radiata L. Wilczek). Indian J Exp Biol 48, 593-600.
Smirnoff N, 2000. Ascorbic acid: metabolism and functions of a multi-facetted molecule. Curr Opin Plant Biol 3, 229-235.
Tian Y, Fan M, Qin Z, et al., 2018. Hydrogen peroxide positively regulates brassinosteroid signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription factor. Nat Commun 9, 1063.
Udawat P, Jha RK, Mishra A, Jha B, 2017. Overexpression of a plasma membrane-localized SbSRP-Like Protein enhances salinity and osmotic stress tolerance in transgenic tobacco. Front Plant Sci 8, 582.
Vacca RA, De Pinto MC, Valenti D, Passarella S, Marra E, De Gara L, 2004. Production of reactive oxygen species, alteration of cytosolic ascorbate peroxidase, and impairment of mitochondrial metabolism are early events in heat shock-induced programmed cell death in tobacco Bright-Yellow 2 cells. Plant Physiol 134, 1100-1112.
Wei Q, Luo Q, Wang R, et al., 2017. A wheat R2R3-type MYB transcription factor TaODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants. Front Plant Sci 8.
Xiang Y, Lu YH, Song M, et al., 2015. Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1) Improves Salinity Tolerance in Tobacco. PLoS ONE 10.
Zhang TT, Song YZ, Liu YD, Guo XQ, Zhu CX, Wen FJ, 2008. Overexpression of phospholipase D alpha gene enhances drought and salt tolerance of Populus tomentosa. Chinese Science Bulletin 53, 3656-3665.
Zheng TC, Li S, Zang LN, Dai LJ, Yang CP, Qu GZ, 2014. Overexpression of Two PsnAP1 Genes from Populus simonii x P. nigra Causes Early Flowering in Transgenic Tobacco and Arabidopsis. Plos One 9.
Zhong SH, Liu JZ, Jin H, et al., 2013. Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis. Proc Natl Acad Sci U S A 110, 9171-9176.
Zhu JK, 2016. Abiotic stress signaling and responses in plants. Cell 167, 313-324.