I.Gene mapping of genes involving DNA methylation II.Identification of AtRH57-interacting proteins using yeast two-hybrid system

第一章 參與DNA甲基化之基因定位 基因沉默發生原因可分成PTGS(post-transcriptional gene silencing)以及TGS(transcriptional gene silencing)。PTGS主要是由RNAi所造成而TGS是藉由DNA甲基化或組蛋白修飾所影響。先前實驗室利用已知參與基因沉默的突變株ros1為背景，利用T-DNA插入建立雙重突變株庫，在ros1的背景下篩選能回復的突變株。該突變株發生突變的基因即可能為影響基因沉默的重要因子。目前已篩選出兩個突變株rgs838-1及rgs3956-1。由於rgs838-1及rgs3956-1突變株中受到T-DNA插入而破壞的基因並非真正影響基因沉默的位置。因此需藉由基因定位(gene mapping)方式進行突變點的定位。利用SSLP、INDEL與CAPS等不同類型分子標誌來定位選殖。經突變株的定位分析，rgs838-1可能的突變位置已縮小至3號染色體CAPS分子標誌PERL0615887 與PERL0616720之間; rgs3956-1可能的突變位置也縮小至2號染色體CAPS分子標誌PERL0307259與PERL0308446之間。未來將增擴大篩選族群及找到新的分子標誌來定位rgs838-1及rgs3956-1的突變位點。 第二章 利用酵母菌雙雜合系統篩選與AtRH57相互作用的蛋白質 rh57-1是對葡萄糖高敏感之突變株。AtRH57為DEAD box RNA 解螺旋酶一員。AtRH57主要表現於細胞核與核仁中，推測可能和核糖體的生合成有關。為了進一步釐清AtRH57扮演的角色，利用酵母菌雙雜交系統(yeast-two-hybrid system)篩選與AtRH57進行交互作用蛋白質。將AtRH57構築於酵母菌表達載體內，在此同時，以三週齡全株阿拉伯芥構築cDNA基因庫並得到3×106個轉形株(transformant)。以AtRH57作為釣餌蛋白質篩選阿拉伯芥cDNA基因庫，有12顆菌落可在缺乏histidine培養基中生長且具有β-galactosidase活性。最後篩選到1個可與AtRH57專一性交互作用的蛋白質，此蛋白為protein N-terminal glutamine amidohydrolase (At2g41760)。將完整此蛋白質與AtRH57進一步測試，並沒有專一性的交互作用。由於第一次沒有成功篩選到與AtRH57有交互作用蛋白質，所以再次進行一次大規模酵母菌轉形。這此酵母菌轉形效率比第一次高出三倍，以AtRH57作為釣餌蛋白質篩選阿拉伯芥cDNA基因庫，有276顆菌落可在缺乏histidine培養基中生長且具有β-galactosidase活性。最後篩選到8個可與AtRH57專一性交互作用，蛋白為OBE1 (AT3G07780)、MED31 (AT5G19910)、Aluminium induced protein with YGL and LRDR motifs (AT4G27450)、Haloacid dehalogenase-like hydrolase (HAD) superfamily protein (AT5G36790)、ETC2 (At2g30420)、proline-rich family protein (AT2G40070)以及2個unknown function (AT5G15120、AT1G25400)。這些可能與AtRH57相互作用的蛋白質將進一步鑑定。Chapter 1 Gene mapping of genes involving DNA methylation Gene silencing is divided into two types: PTGS (post-transcriptional gene silencing) and TGS (transcriptional gene silencing). PTGS is elicited by RNAi (RNA interference) while the TGS is caused by either DNA methylation or histone modification. It is known that ros1 causes transcriptional gene silencing. In order to screen ros1 suppressed gene, a T-DNA-mutagenized ros1 population was generated and screened. Two mutants rgs838-1 and rgs3956-1 were identified, but T-DNA mutated genes did not suppress silencing. Suggesting that gene silencing is caused bu other gene. Therefore, gene mapping was used to identify the gene that suppressed silencing in rgs838-1 and rgs3956-1 mutants. SSLP, INDEL and CAPS markers were used for the positional cloning. Gene mapping localized rgs838-1 to chromosome 3 in between the CAPS markers PERL0615887 and PERL0616720 while rgs3956-1 to the bottom of chromosome 2 in between the CAPS markers PERL0307259 and PERL0308446. Further gene mapping is needed done by enlarging population and finding new molecular markers to identify genes involving DNA methylation. Chapter 2 Identification of AtRH57-interacting proteins using yeast two-hybrid system rh57-1 is a glucose hypersensitive mutant. AtRH57 to encodes a member of DEAD box RNA helicase. Because AtRH57 protein was localized in the nucleus and nucleolus, it suggests, the protein is possibly involved in ribosome biogenesis. To further explore the role of AtRH57, yeast two-hybrid analysis was used to identify the potential proteins that may interact with AtRH57. A cDNA library was constructed from mRNA isolated from 3-week-old Arabidopsis plants and 3×106 transformants were generated from the library. Using AtRH57 as a bait to screen Arabidopsis cDNA library by yeast two-hybrid, we identified 12 colonies can grew up on minimal medium minus histidine and displayed β-galactosidase activity. Of these, only one interacted with AtRH57 is a protein named N-terminal glutamine amidohydrolase (At2g41760). However, when tested with entire protein, no specific interaction with AtRH57 occurred. Therefore, a screening with large-scale library transformation was examined. The transformation efficiency of secondary screening is three times higher than the previous screening. We identified 276 colonies that grew up on minimal medium minus histidine and displayed β-galactosidase activity. Of these, only eight interacted with AtRH57 is protein named OBE1 (AT3G07780)、MED31 (AT5G19910)、Aluminium induced protein with YGL and LRDR motifs (AT4G27450)、Haloacid dehalogenase-like hydrolase (HAD) superfamily protein (AT5G36790)、ETC2 (At2g30420)、proline-rich family protein (AT2G40070) and unknown function (AT5G15120、AT1G25400).The investigating these potential AtRH57 interacting proteins is currently in program.目次 第一章: 參與DNA甲基化之基因定位 中文摘要 1 Abstract 2 壹、 前人研究 3 一、 DNA甲基化 3 二、 RNA聚合酶IV & V 4 三、 RdDM之形成和調控 5 四、 RdDM修飾對植物的影響 7 五、 RdDM基因之篩選 9 貳、 材料與方法 12 一、 材料與生長條件 12 二、 總DNA之萃取 12 三、 DNA分子標誌之搜尋與CAPS分子標誌之設計 13 四、 SSLP、INDEL、CAPS分子標誌之聚合酶鏈反應 13 五、 DNA 電泳 14 參、 結果 15 一、 rgs838之定位 15 (一) rgs838粗定位(rough mapping) 15 (二) rgs838 精細定位(fine mapping) 16 二、 rgs3956之定位 17 (一) rgs3856 粗定位 17 (二) rgs3956 精細基因定位 17 三、 初步定位結果 18 肆、 討論 19 伍、 參考文獻 22 陸、 圖表 31 附圖 46 第二章:利用酵母菌雙雜合系統篩選與AtRH57相互作用的蛋白質 中文摘要 49 Abstract 50 壹、 前人研究 51 一、 DEAD-box RNA解旋酶 51 二、 DEAD-box RNA 解旋酶之解旋機制 52 三、 DEAD-box RNA 解旋酶之功能 53 四、 DEAD-box RNA 解旋酶與逆境 56 五、 酵母菌雙雜交(Yeast two-hybrid)原理 57 貳、 材料與方法 59 一、 酵母菌L40之轉形作用 59 二、 西方墨點法 (Western blotting) 60 三、 總體RNA萃取與mRNA純化 61 四、 cDNA之合成 63 五、 純化大量大腸桿菌質體DNA 66 六、 大規模酵母菌轉形作用(Large-scale library transformation) 66 七、 β-galactosidase活性分析 67 八、 酵母菌質體DNA萃取 68 參、 結果 69 一、 AtRH57非專一性活化的測試 69 二、 cDNA基因庫的製備 70 三、 大規模酵母菌雙雜交篩選法 71 肆、 討論 73 伍、 參考文獻 75 陸、 圖表 84 附圖 9