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標題: 冰花McSnRK1參與調控分生組織發育及開花之功能分析
Functional analyses of McSnRK1 in regulating meristem development and flowering in Arabidopsis
作者: 王偉名
Wang, Wei-Ming
關鍵字: 冰花;Mesembryanthemum crystallinum L.;分生組織;開花;阿拉伯芥;SnRK1;meristem;flowering;Arabidopsis
出版社: 生命科學系所
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SnRK1 (sucrose non-fermenting 1-related protein kinase 1)是一群與調控能量代謝及逆境訊息傳遞相關的蛋白激酶,McSnRK1即屬於此高度保留的SnRK1家族成員,參與冰花鹽逆境相關的訊息傳遞途徑,本論文使用專一表現在花部分生組織的APETALA1 (AP1)啟動子,驅動McSnRK1基因表現,製作基因轉殖阿拉伯芥,以探討SnRK1對分生組織和花部發育的影響。首先利用PCR擴增AP1::McSnRK1轉殖株中T-DNA的序列,確認T-DNA插入後,利用thermal asymmetric interlaced PCR (TAIL-PCR)進行T-DNA插入位置的鑑定,得知T-DNA插入位置在阿拉伯芥第二條染色體At2g32795之5’端非編碼區(5’ untranslated region; 5’UTR),而此基因目前被定義為功能未知。藉由T-DNA插入位置來鑑定AP1::McSnRK1同型合子轉殖株,並與鄰近T-DNA插入點之SALK_135548轉殖株性狀做比較,AP1::McSnRK1轉殖株有多葉、多花序、花序側部器官排序異常及花部發育出現變異的性狀,而SALK_135548轉殖株也有多葉和多花序的性狀。利用semi-quantitative RT-PCR和quantitative real-time PCR得知AP1::McSnRK1轉殖株在花部大量表現外源基因McSnRK1,且會影響阿拉伯芥內生SnRK1同源基因AtAKIN10及AtAKIN11的表現。進一步觀察AP1::McSnRK1轉殖株與細胞分裂素(cytokinin, CK)累積量高的amp1 (ALTERED MERISTEM PROGRAM 1)基因喪失功能突變株,發現皆有多葉的性狀,且開花時間皆明顯較野生型植株提早,顯示McSnRK1參與調控植株開花的時間。分析與分生組織發育相關的基因表現量,得知AP1::McSnRK1轉殖株及amp1突變株之WUSCHEL (WUS)、SHOOT MERISTEMLESS (STM)及Knotted Arabidopsis thaliana 1 (KNAT1)基因表現皆增加,顯示與莖頂分生組織(shoot apical meristem; SAM)發育相關基因的表現會受到McSnRK1調控,進而促進SAM的形成及發育;而轉殖株SALK_135548之STM及KNAT1基因表現並無提高,代表並非T-DNA插入位置的影響。綜合以上結果推論SnRK1參與CK訊息傳遞調控SAM的形成與發育,進而促進植物地上部器官的發育,使植株提早進入繁殖期。

SnRK1 (sucrose non-fermenting 1-related protein kinase 1) is a family of protein kinase that functions as a crucial integrator of energy and metabolic homeostasis and stress signaling. Previously our lab identified an ice plant SnRK1 (McSnRK1) that shares high homology to other plant SnRK1s and is involved in the salt stress-related signal transduction pathway. In this thesis, I use transgenic Arabidopsis plants that ectopically express McSnRK1 under the control of APETALA1 (AP1) promoter to examine the role of SnRK1 in regulating the development of meristem and flower. PCR amplification of T-DNA was used to confirm the insertion of T-DNA in transgenic lines. The T-DNA insertion site of one AP1::McSnRK1 mutant line was identified by thermal asymmetric interlaced PCR (TAIL-PCR), and the site is located at 5’ untranslated region (5’UTR) of At2g32795 in chromosome 2. The function of At2g32795 gene is currently unknown. The homozygous AP1::McSnRK1 mutants showed a large number of rosette leaves, multiple main inflorescences, abnormal phyllotaxis of lateral organs and floral structures. To eliminate the possible effect of T-DNA insertion, the phenotypes of SALK_135548, a mutant with a nearby T-DNA insertion site, was compared. SALK_135548 mutant also showed leafy and multiple inflorescences phenotypes but with normal arrangement of lateral organs and flower structure. Expression of McSnRK1 was highly detected in flowers of AP1::McSnRK1 mutants, and the expressions of endogenous SnRK1 gene AtAKIN10 and AtAKIN11 were affected by heterologous expression of McSnRK1. Comparing the phenotypes and flowering time with high-cytokinin-level amp1 (ALTERED MERISTEM PROGRAM 1) mutant, both AP1::McSnRK1 and amp1 mutants had leafy and early flowering phenotypes. The results suggest expression of McSnRK1 alters the time for flowering. Further analysis of the expression of meristem-related genes including WUSCHEL (WUS), SHOOT MERISTEMLESS (STM) and Knotted Arabidopsis thaliana 1 (KNAT1), all showed increased transcript levels in AP1::McSnRK1 and amp1 mutants. The results showed SnRK1 participates in the formation and development of shoot apical meristem (SAM). The expressions of STM and KNAT1 were decreased in SALK_135548 mutant, demonstrating that the abnormal floral development of AP1::McSnRK1 mutants was not caused by T-DNA insertion. In conclusion, SnRK1 regulates the development of SAM and floral organs through participation of cytokinin signaling.
其他識別: U0005-2307201322292700
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