Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92112
標題: 利用高通量定序法鑑定並分析冰花 miRNA 與鹽逆境反應之探討
Identification and analysis of ice plant (Mesembryanthemum crystallinum) conserved and novel microRNAs by high-throughput sequencing and their association with salt stress response
作者: Chih-Pin Chiang
姜智彬
關鍵字: 冰花;ice plant miRNA
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摘要: 
Ice plant (Mesembryanthemum crystallinum L.) is a facultative halophyte that high degree of salt tolerance can be induced at a specific developmental stage. Several levels of regulation, including genetic/epigenetic, transcriptional/post-transcriptional, and translational/post-translational, are involved in the complex process of stress induction. High-throughput sequencing was used to identify miRNA profiles in ice plant. Sequences of poly (A)+ RNA and small RNA (sRNA) from 3-day-old seedlings (control) and 200 mM NaCl-treated 6 h (salt) were obtained and analyzed. Total 3,089,326 and 3,157,610 redundant clean reads of 18- to 25-nt sRNA, representing 1,106,388 and 1,118,741 unique sRNA sequences were generated from control and salt libraries, respectively. The 24-nt sRNAs were the most abundant sRNA and exhibited the highest sequence diversity, consistent with the profiles obtained by other plant sRNA. miRBase annotation of ice plant sRNA revealed 143 conserved miRNAs belonging to 38 families. Among them, miR166 was the most abundant family and miRNA156/157 consisted of the largest numbers. The hairpin precursor structures of total 19 conserved mcr-miRNAs belonging to 12 miRNA families and 24 novel mcr-miRNAs were identified according to strict criteria. Most of them paired with the star sequences, with two exceptions. Minimal folding free energy index of most mcr-miRNA candidates were greater than 0.85, a threshold value to distinguish miRNA precursor and other structural RNAs. Three conserved mcr-miR156 candidates were salt-induced and two of them were highly abundant in both control and salt libraries, indicating the conserved miR156/157 family was highly expressed at the early stage of development and was responsive to salt stress. The further investigation for the expression of mcr-miRNA and potential target gene would help understanding of miRNA regulation in response to salt stress in ice plant. Two strategies, poly(A)-tailing and stem-loop RT-PCR, were used to evaluate the expression profiles of conserved and novel mcr-miRNA in control and 200 mM NaCl-treated seedlings. Poly(A)-tailing RT-PCR was able to detect the expression profile of mcr-miRNA families. Five miRNA families, namely miR159, miR162, miR168, miR171, and miR396,were stress-responsive and their potential target gene families exhibited reciprocal expression patterns under salt stress. Furthermore, stem-loop RT-PCR was performed to detect individual miRNA expressions using optimalized miRNA-specific forward primers. Nine pairs of salt-responsive individual mcr-miRNAs/target genes including mcr-miR156-1/SPL6, mcr-miR159-1/MYB33, mcr-miR162/DCL1, mcr-miR164/NAC100, mcr-miR168/AGO1, mcr-miR168/CYP76C4, mcr-miR169-2/NFYA1, mcr-miR396/FBP, and mcr-miR403/AGO2 showed reciprocal correlation patterns. Degradome analysis validated mcr-miR164, mcr-miR166-1, mcr-miR169-1, mcr-miR169-2, and mcr-miR403 specifically cleaved the transcripts of target genes. Target predictions using ice plant transcriptome revealed mcr-miRNAs function in transcriptional regulation, chromosome remodeling, ion homeostasis, photosynthesis, protein degradation, signal transduction, hormone-related, and redox reaction. High-throughput sequencing is powerful technique to identify miRNA profile and stem-loop real-time PCR is highly specific method for miRNA quantification. Characterization of mcr-miRNAs using these two strategies is useful in exploring the unique mechanism of salt tolerance in ice plant.

冰花(Mesembryanthemum crystallinum L.)為一種耐鹽模式植物,其耐鹽能力會隨著發育時期的轉變與鹽逆境的誘導而提高,目前已鑑定出特定機制在基因、轉錄與後轉錄、以及轉譯與後轉譯層次參與調控冰花耐鹽機制的誘導。在本論文中利用高通量定序法鑑定並分析冰花的基因表現圖譜,以播種後三天大的小苗為對照組以及200 mM鹽處理6小時的小苗為實驗組,進行mRNA與small RNA (sRNA)的高通量定序,定序結果在長度介於18到25個核苷酸的sRNA中,對照組與實驗組分別獲得3,089,326與3,157,610條重複序列,以及去除重複序列後得到的 1,106,388 與1,118,741條單一序列。分析核苷酸長度的分布,發現長度24個核苷酸的sRNA佔有最大的比例,並且具有最高的序列歧異度,此現象與分析其他植物的sRNA的結果相同。利用miRBase進行sRNA的身份鑑定得到143個高度保留性的miRNA,分別屬於38種miRNA家族,其中miR166家族具有最高的表現量而miR156家族則具有較多的成員。接著以嚴格的設定條件進行冰花miRNA的鑑定,共有19個高度保留性的miRNA與24個在其他物種中尚未被鑑定的miRNA,在冰花的transcriptome中可以找到具有hairpin結構的pre-miRNA,除了兩個例外,其餘均可鑑定到miRNA*的序列。在這些高度保留性以及嶄新的miRNA中,大多數的minimal folding free energy index大於0.85,此數值為區別miRNA與其他sRNA的閥值,以上參數皆可提高經由預測所得到的冰花特有miRNA的真實性。三個冰花miRNA成員在鹽逆境下具有明顯的表現量差異,其中兩個在對照組與實驗組當中都具有極高的表現量,顯示miRNA156家族在冰花發育的早期大量表現並參與鹽誘導相關基因的調控。進一步分析冰花miRNA與目標基因的表現情形,以期瞭解冰花中miRNA所調控的耐鹽機制。利用poly(A)-tailing與stem-loop RT- PCR兩種策略在對照組及鹽處理的實驗組中對冰花保留性與嶄新的miRNA進行偵測並定量分析,其中poly(A)-tailing RT- PCR可有效的偵測miRNA基因家族的表現,鑑定出五個對鹽有反應的miRNA基因家族miR159、miR162、miR168、miR171與miR396,且在鹽逆境下皆與預測之目標基因家族表現呈現相互消長的趨勢。進一步利用stem-loop RT- PCR搭配2-3核苷酸互補的最佳化miRNA專一性引子,可專一性並零敏的偵測個別miRNA的表現,一共鑑定出九個對鹽處理有反應並具有相互消長表現的個別miRNA與目標基因的組合,分別是mcr-miR156-1/SPL6、mcr-miR159-1/MYB33、mcr-miR162/DCL1、 mcr-miR164/NAC100、mcr-miR168/AGO1、mcr-miR168/CYP76C4、mcr-miR169-2/NFYA1、mcr-miR396/FBP與mcr-miR403/AGO2。由植物降解組的分析中證實mcr-miR164、mcr-miR166-1、mcr-miR169-1、mcr-miR169-2與mcr-miR403專一性的切割並調控目標基因在鹽逆境下的表現。預測冰花中受到miRNA調控的目標基因群,發現其功能參與轉錄的調控、染色體的重組、細胞內離子的平衡、光合作用、蛋白的降解、訊息傳遞、氧化還原反應與賀爾蒙相關基因的調控等生理反應。高通量定序法與stem-loop RT- PCR可大量且專一性的鑑定並分析miRNA的表現,兩種策略的搭配可有效的進一步探究後轉譯修飾參與調節冰花的耐鹽性。
URI: http://hdl.handle.net/11455/92112
Rights: 同意授權瀏覽/列印電子全文服務,2018-02-06起公開。
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