臺灣博碩士論文加值系統

在植物中已知有九種賀爾蒙，參與調節植物代謝、生長、以及發育過程；細胞分裂素為一種腺嘌呤衍生物，在腺嘌呤的第六個氮上有不同的取代基。細胞分裂素在植物中扮演許多不同生理功能角色，如：促進細胞分裂、頂端優勢、根部生長與分枝、葉綠素的生合成及葉的老化作用等。目前阿拉伯芥中有三個被確定的細胞分裂素受體AHK2、AHK3及AHK4/CRE1，皆屬於複合型的組胺酸激酶受體，其中組胺酸激酶受體在原核生物或植物中被認為能夠啟動二元訊息傳導系統。綠竹具有生長迅速的特性，在多雨的夏季開始大量發筍，豐沛雨水可能會造成生存條件變化，如：滲透壓的改變。酵母菌當中的SLN1被認為與感應滲透壓變化相關，在互補 (complementary) 實驗當中，將CRE1轉入酵母菌SLN1剔除突變株，外加細胞分裂素可以拯救致死突變株。我們想要瞭解綠竹中可能的細胞分裂素受體所扮演的生理角色，本論文主要目標即為綠竹筍細胞分裂素受體cDNA之選殖與分析。利用非放射線標定不同組織差異性表現片段DIG-GA作為探針，由王愛玉博士實驗室提供之綠竹筍cDNA庫當中，篩選出可能為細胞分裂素受體之基因。在第一次的篩選，由106個clone中篩出四個正反應株，以聚合酶鏈鎖反應進行初步確認後，挑選兩個正反應株進行後續篩選。經過四次篩選確認，最後選殖出可能為細胞分裂素受體之cDNA，命名為BPCRE。序列分析結果顯示BPCRE包含3578 bp，與水稻中可能為組胺酸激酶的蛋白質、及玉米的組胺酸激酶之cDNA分別有96%及90%的相似度。然而，BPCRE缺乏5’端非胺基酸轉譯區域及一小部份的胺基酸轉譯區，且包含一段40 bp大小、可能為intron的片段。BPCRE所轉譯出的蛋白質可能包含有三個 α 螺旋構形的疏水性穿膜區域、一個CHASE功能區塊、一個組胺酸激酶功能區塊及一個receiver功能區塊。利用北方雜合法分析BPCRE在不同生長時期及不同組織部位竹筍表現情形，發現BPCRE之mRNA幾乎完全只表現於竹筍頂端分生組織，與差異性表現分析的結果可互相呼應；此外，由於頂端分生組織具有持續細胞分裂之特性，這暗示著BPCRE在此過程中扮演重要的角色。

At least nine plant hormones have already been identified to regulate the metabolism, growth, and development in plants. Among them, cytokinins are a group of molecules derived from adenine with N6-substitution. They played diverse physiological roles in cell division, apical dominance, root growth and branching, chloroplast biogenesis, and leaf senescence. Three hybrid-type histidine kinase receptors, AHK2, AHK3 and AHK4/CRE1, have been identified in the perception of cytokinins in Arabidopsis, where HK is generally employed in two-component signaling of plant and bacteria. We are interested in the physiological functions played by putative cytokinin receptors in bamboo. Bamboo shoots possess the characteristics of fast growth in the rainy season. One possible effect of excess water that changes the growth condition of bamboo is the change of osmolarity. CRE1 from Arabidopsis can rescue the yeast lethal SLN1 knock-out mutant, which is originally discovered to sense the osmolarity change. The aim of this thesis was to clone and analyze the cDNA encoding cytokinin receptor homologs from bamboo cDNA library. A fragment encoding a putative CRE, selected using differential display by Dr. A. Y. Wang’s lab, was used as a probe to screen the cDNA library. From 106 clones, we have identified 4 positive plaques that can be detected by the DIG-GA probe. The positive clones were isolated and identified by PCR. Two of them were selected for subsequent rounds of screening. After four rounds of screening, one putative cytokinin receptor-like gene, BPCRE, was identified. Sequence analysis of BPCRE revealed that there were 3578 nucleotides in the cDNA insert with 96% and 90% similarity to the putative histidine kinase of rice and of maize, respectively. However, the cloned cDNA sequence seems lacking the 5’-UTR and part of the coding region and containing an extra intron-like fragment with 40 nucleotides in length. This 40 nucleotide chain is in the position corresponding to the first intron in rice genomic DNA. A putative protein sequence translated from BPCRE without the 40 nucleotides shows more than 90% identity to Oryza sativa putative histidine kinase protein. The encoded protein was composed of three α-helix hydrophobic regions, one CHASE domain, one histidine kinase domain, and one receiver domain. The expression pattern of BPCRE in different stages and in different regions of bamboo shoots was investigated by Northern blotting. BPCRE was tissue-specific expressed in the apical meristems of the tip of bamboo shoots. This result agreed with the observation gained from differential display. Since the apical meristem of the tip of bamboo is known for its rapid growth, the enriched expression of this putative cytokinin receptor, BPCRE, implies that it plays an important role in this process.

目錄 Ⅰ
縮寫表 Ⅲ
中文摘要 Ⅳ
Abstract Ⅵ
第一章 緒論
1 植物賀爾蒙─細胞分裂素 (Cytokinin) 1
2 細胞分裂素對植物之生理作用 1
3 細胞分裂素之訊息傳導 3
3.1 二元傳導系統 (Two-component system) 3
3.2 細胞分裂素訊息傳導路徑 5
3.3 細胞分裂素受體之結構特性 8
4 研究材料與研究目的 9
第二章 材料與方法
1 實驗材料 12
2 實驗藥品 13
2.1 一般化學試劑 13
2.2 酵素 13
2.3 培養基 14
2.4 探針標定藥品 14
3 重要儀器設備 14
4 實驗方法 15
4.1 cDNA庫之篩選 15
4.1.1 探針之設計 15
4.1.2 Plating 16
4.1.3 λ ZAP II cDNA庫之轉印 17
4.1.4 雜合反應 (Hybridization) 18
4.1.5 梯度嚴苛度漂洗 (Stringency washing) 18
4.1.6 酵素連結免疫反應與鹼性去磷酸酶呈色反應 18
4.2 正反應株之挑選與鑑定 19
4.2.1 胞內噬質體裁切 (In vivo excision) 20
4.2.2 質體快速檢定法 21
4.2.3 小量質體DNA之純化 (Plasmid minipreparation) 21
4.2.4 限制酶切割分析 24
4.2.5 洋菜膠體電泳 24
4.3 北方雜合分析法 (Northern blotting analysis) 25
I
目錄
4.3.1 全部RNA之抽取 25
4.3.2 RNA樣品定量 27
4.3.3 甲醛洋菜膠體電泳 27
4.3.4 北方轉印法─毛細管轉移法 28
4.3.5 北方雜合反應 30
4.4 其他重要實驗方法 31
4.4.1 聚合酶鏈鎖反應 (Polymerase Chain Reaction) 31
4.4.2 以T-A cloning保存PCR片段 32
4.4.3 質體DNA之轉形 (Transformation) 33
第三章 結果與討論
1 利用聚合酶鏈鎖反應製備DIG標定核酸探針 35
1.1 部份細胞分裂素受體cDNA之選殖 35
1.2 使用差異性表現基因片段為模版製備核酸探針 36
2 綠竹筍細胞分裂素受體cDNA序列之選殖 37
3 BPCRE蛋白質一級結構分析及cDNA序列分析 38
4 Phylogenetic分析 39
5 北方雜合分析結果 40
第四章 結論與展望 41
參考文獻 45
圖與表 48

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