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標題: | Small RNAs與一氧化碳在甘藷傷害防禦機制之功能性分析 Functional studies of small RNAs and carbon monoxide in wounding responses of sweet potato(Ipomoea batatas cv. Tainung 57) |
作者: | Jeng-Shane Lin 林振祥 |
指導教授: | 鄭石通 |
關鍵字: | 甘藷,傷害,small RNA,木質素,過氧化氫,一氧化碳,ERK, Sweet potato,Wounding,small RNA,Lignin,Hydrogen peroxide,carbon monoxide,ERK, |
出版年 : | 2013 |
學位: | 博士 |
摘要: | 植物因為根固著性的關係,造成植物常需要面對外在環境改變所造成的威脅,譬如高鹽、乾旱、冷與傷害等。由於植物無法藉由逃避來因應,因此植物發展出多種可調節的系統以面對各種環境變化和逆境衝擊,進而啟動防禦機制。傷害逆境會影響植物的生長,並且增加微生物病原菌感染植物的機會,因此植物會調節釋放訊息傳遞物質或者調控防禦相關基因的表現,來保護自己。
近年研究發現small RNAs (sRNAs)扮演著基因表現調節者的重要角色,sRNAs是一段約20-24個核苷酸的小分子核醣核酸,不轉譯出蛋白質,而且主要區分為miRNA與siRNA。sRNAs是藉由調控與其序列互補相似基因(此為目標基因),而來參與多種生物反應,但現今對於sRNAs在植物遭受傷害逆境的調控研究還是很少。在本研究中發現一個已知的miRNA與一個新的sRNA會參與調控甘藷(Ipomoea batatas cv. Tainung 57)傷害逆境的防禦反應,他們分別為miR828與small RNA-8105 (sRNA8105)。在甘薯葉片遭受到傷害後,miR828與sRNA8105會大量的表現。此外我們利用cDNA cloning或生物資訊法來釣取和預測目標基因,結果得知IbMYB和IbTLD可能是miR828的目標基因;IbMYB1可能是sRNA8105的目標基因。而IbMYB、IbTLD和IbMYB1的表現量在甘薯葉片遭受傷害後會被抑制。此外我們也利用small RNA在目標基因上的剪切點分析、small RNA與目標基因於菸草進行暫時性表現之分析、以及甘藷轉殖植物之研究,來確認miR828與IbMYB、miR828與IbTLD、以及sRNA8105與IbMYB1之間的關係。而在大量表現miR828或sRNA8105前驅物的轉殖甘藷中,我們發現IbMYB和IbMYB1的表現量分別在這兩種轉植株中被抑制,進而影響phenylpropanoid pathway的代謝流(metabolic flux)趨向木質素(lignin)的生合成。在大量表現miR828前驅物的轉殖株中,過氧化氫(H2O2)的含量也會因IbTLD表現量的降低而受到影響。此外甘薯葉片在遭受到傷害時,木質素與過氧化氫的含量也會增加。因此,在甘藷遭受傷害的反應中,miR828與sRNA8105會被誘導表現,來抑制他們的目標基因,而使得木質素與過氧化氫的含量提升,進而被分別用來強化細胞壁的強度與扮演訊息傳遞分子的角色,以達到修復與防禦的功能。 除了sRNAs外,訊息傳遞分子也在逆境防禦上扮演著重要的角色。近期研究也指出一氧化碳(Carbon monoxide)是傳遞生理訊息的分子之一。在植物中,一氧化碳是heme oxygenase (HO)分解heme而釋放出的產物,而進一步參與植物的生長發育與逆境的調控。然而一氧化碳對於植物在傷害逆境的調控所知有限。甘薯葉片在遭受到傷害時,一氧化碳的濃度與IbHO的表現量都明顯地下降。傷害誘導相關基因Ipomoelin (IPO)的表現會受到一氧化碳和與HO活性相關的藥劑影響。一氧化碳會提升ascorbate peroxidase、catalase與peroxidase的活性,進而使得過氧化氫的含量大幅度的下降;此外一氧化碳也會抑制傷害所誘導的ERK磷酸化。過氧化氫與磷酸化的ERK皆是傷害所誘導IPO表現的重要因子。因此,在誘導IPO表現的訊息調控機制中,一氧化碳是扮演著負調控者的角色。 綜合結果得知,在甘藷傷害中,sRNAs的表現量與一氧化碳的含量會被調節,以達到防禦機制的啟動。傷害會誘導miR828與sRNA8105的表現,而使得他們的目標基因被抑制;此外傷害也會抑制IbHO的表現,而使得一氧化碳的含量降低。如此一來phenylpropanoid pathway的代謝流、抗氧化酵素的活性、以及ERK的磷酸化都因此受到影響,緊接著植物傷害防禦機制會被啟動,包含木質素的生合成與傷害誘導相關基因IPO的表現,而來保護或修復自己。 Plants are immobile organisms and are always exposed to various environmental stresses such as salt, drought, cold, and wounding. Hence, they develop various defense systems to survive from biological threats. Wounding affects plant growth and facilitate pathogen infection. In wounding responses, plants produce a wide variety of defense-related hormones and second messengers to unlock the defense-related regulatory networks. Genes related to defense systems and wound healing also express to protect plants from attack by herbivores and pathogens. Small RNAs (sRNAs) have been recently considered as the most important elements in regulation of gene expression. Most sRNAs are 22-24 nucleotide (nt) non-coding RNAs, and mainly divide into microRNAs (miRNAs) and small interfering RNAs (siRNAs). sRNAs play important roles in various biological processes via the regulation of their targets. However, little is known of the wounding-responsive sRNA. In this study, a known miRNA, miR828, and a novel sRNA, sRNA8105, were identified to participate in the wounding responses of sweet potato (Ipomoea batatas cv. Tainung 57). The expression of miR828 and sRNA8105 was largely induced in the wounding response. Two miR828 target candidates, IbMYB and IbTLD, and a sRNA8105 target candidate, IbMYB1, were obtained by cDNA cloning and bioinformatics analysis, respectively. Expression of IbMYB, IbTLD, and IbMYB1 was repressed after wounding. The interactions of miR828-IbMYB, miR828-IbTLD, and sRNA8105-IbMYB1 were confirmed by cleavage site mapping, Agrobacterium-mediated transient expression assay, and transgenic plant studies. Transgenic sweet potatoes overexpressing miR828 precursor gene and sRNA8105 precursor gene, which repressed IbMYB and IbMYB1, respectively, could affect the metabolic flux toward the biosynthesis of lignin in the phenylpropanoid pathway. miR828 overexpression plants also affected H2O2 contents via reducing IbTLD expression. Thus, miR828 and sRNA8105 were induced to repress their targets expression in wounding responses. Subsequently, lignin and H2O2 contents were elevated for reinforcing the strength of cell wall and playing a signal messenger in wounding defense of sweet potato, individually. In addition to sRNAs, signal transducers also play important roles in wounding defense mechanisms. Carbon monoxide has been recently considered as a messenger involved in various physiological properties, and it is produced by heme oxygenase (HO) in plants. However, the role of CO in wounding response is still poor understood. In sweet potato leaves, wounding could significantly reduce the endogenous CO contents and the IbHO expression levels. Expression of wound-inducible gene ipomoelin (IPO) was affected by the addition of CO and chemicals related to HO activities. The addition of CO also affected the activities of ascorbate peroxidase, catalase, and peroxidase, further largely decreasing H2O2 content. In addition, wounding-induced ERK phosphorylation was prohibited by CO. H2O2 and the phosphorylated ERK are important signals in IPO induction. Thus, CO may negatively regulate the signal transducers involved in IPO induction after wounding. Conclusively, sRNAs and CO participate in the defense system involved in wounding. Wounding could increase the expression of miR828 and sRNA8105 to repress their target genes. Wounding also decreased the expression of IbHO to reduce CO production. Subsequently, metabolic flux of the phenylpropanoid pathway, activities of antioxidant, and phosphorylation of ERK were affected. Defense systems including lignin biosynthesis and IPO induction were then switched to protect and heal plants, respectively. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17682 |
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顯示於系所單位: | 植物科學研究所 |
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