臺灣博碩士論文加值系統

光和溫度影響蝴蝶蘭開花時間，然而有關其分子調控途徑的研究甚少。目前對於光週期調控開花的部分，僅在阿拉伯芥有較深入的研究，故本論文以夏天開花之大葉蝴蝶蘭為材料，初步針對其光週期調控開花的上游基因GIGANTEA（GI）進行選殖與分析。利用基因同源性設計引子進行5’及3’RACE後，成功選殖到PbGI cDNA，全長3,480 bp，可轉譯成1,159個胺基酸。經序列分析後，得知PbGI在序列長度、親源演化分析及胺基酸組成上，皆與OsGI較為相似。利用多重序列排比分析得知PbGI與其它物種之GI之間有大於69%的保守性，並且與AtGI和OsGI相同有5個預測的穿膜區域，故推測為一穿膜蛋白質。集群與對應分析結果顯示，PbGI的RSCU值與AtGI最為相似。經基因套數分析後得知PbGI為單一複本數，與HvGI和OsGI相同。而基因表現量分析的結果則顯示PbGI與AtGI和OsGI同樣受光與生理時鐘調控且常態表現，且在花朵、氣根與葉片中亦能偵測到其表現量，其中以花瓣和成熟葉片表現量為較高。綜合前述，可推測PbGI應為GI同源基因。另外，利用主動誘變技術研究蝴蝶蘭開花時間的部分，從30株轉殖株觀察到1株開花時間延遲且外表形態改變的蝴蝶蘭，經由分析有可能為pTAG-8單一插入或是其他原因所致，其所影響之因素值得未來進行深入探討。

Although light and temperature affect the flowering time in Phalaenopsis, the molecular mechanism of flowering in such floral crop remain unclear. Possible flowering-time pathways are mainly studied in Arabidopsis. To have a better understanding on the photoperiodic flowering of Phalaeanopsis, this studies started with a circadian controlled gene GIGANTEA(GI) in Phalaenopsis bellina. According to the gene homology and 5’ and 3’ RACE, the full-length of PbGI (3,480 bp) has been cloned and bioinformatically analyzed. The deduced amino acid sequences, composition and phylogenic analysis indicated that PbGI is more closed to rice GI (OsGI) and shared more than 69% of similarity with other GI homologues. PbGI is predicted to have five transmembrane regions like other GI homologues, meanwhile the cluster and correspondence analysis of its RSCU values are more similar to AtGI’s. By Southern blots and real-time PCR analysis, it was found that PbGI, a single copy gene like HvGI and OsGI, is controlled by light conditions and can express constantly in almost all tissues, especially in mature leaves and petals. Therefore, PbGI must be a GI homolog. On the other hand, an activation-tagged strategy was used to study the flowering time in Phalaenopsis, and one of the 30 transgenic Phalenopsis was observed with a delayed flowering phenotype. Such change might caused by a single insertion of pTAG-8 or other factors that remained to be studied.

目錄
中文摘要..................................................I
英文摘要.................................................II
誌謝....................................................III
目錄.....................................................IV
表目錄.................................................VIII
圖目錄...................................................IX
附錄......................................................X
壹、前言..................................................1
貳、文獻回顧..............................................3
一、 植物開花時間之研究...................................3
（一）開花時間調控之研究..................................3
（二）開花時間基因之研究..................................5
（三）GI相關之研究........................................6
二、 蝴蝶蘭開花研究......................................10
（一）蝴蝶蘭簡介.........................................10
（二）影響蝴蝶蘭開花之因素...............................10
（三）利用基因轉殖進行蝴蝶蘭開花時間之研究...............14
参、材料與方法...........................................16
一、試驗材料.............................................16
（一）大葉蝴蝶蘭.........................................16
（二）轉殖基因蝴蝶蘭.....................................17
二、試驗方法.............................................17
（一）PbGI之選殖.........................................17
1. 總量RNA之萃取.........................................17
2. PbGI退化性引子之設計及製備............................18
3. 反轉錄聚合酶連鎖反應..................................19
4. 聚合酵素連鎖反應......................................19
5. PCR產物之回收及純化...................................20
6. 接合反應..............................................21
7. 勝任細胞製備..........................................21
8. 大腸桿菌轉型作用......................................22
9. 小量質體DNA萃取 .......................................22
10. 基因片段之定序分析...................................23
11. 快速增幅cDNA5’和3’末端.............................23
（二）PbGI DNA序列與其所轉譯之蛋白質序列之分析...........26
1. 序列比對..............................................26
2. 蛋白質序列分析........................................27
（三）PbGI表現量分析.....................................32
1. 不同光照條件..........................................32
2. 組織特異性............................................32
3. 即時定量聚合酶連鎖反應................................32
（四）南方墨點法分析.....................................33
1. 染色體DNA之萃取.......................................33
2. 探針製備..............................................34
3. DNA製備...............................................35
4. DNA轉漬...............................................36
5. 雜合反應..............................................36
6. 呈色顯影..............................................37
肆、結果與討論...........................................38
一、PbGI之選殖與分析.....................................38
（一）PbGI之選殖.........................................38
（二）PbGI序列之生物資訊分析.............................40
1. 序列分析..............................................40
2. 蛋白質序列分析........................................42
二、PbGI表現量之分析.....................................47
（一）PbGI不同光照表現量之分析...........................47
（二）PbGI組織特異性表現模式之分析.......................49
三、PbGI複本數之分析.....................................50
四、基因轉殖蝴蝶蘭之分析.................................51
（一）外表型分析.........................................51
（二）T-DNA嵌入套數分析..................................52
伍、結論.................................................54
一、PbGI為GI同源基因.....................................54
二、PbGI受生理時鐘調控且常態表現.........................55
三、利用主動誘變技術研究蝴蝶蘭開花時間...................56
四、未來研究.............................................56
陸、參考文獻.............................................85

表目錄
表1. NCBI資料庫中已發表GI全長物種之相關資訊摘要..........58
表2. PbGI基因選殖及基因表現量使用引子一覽表..............60
表3. PbGI與其它物種GI之DNA及蛋白質序列相同度和相似度比對表61
表4. PbGI與不同物種GI之胺基酸所佔比例....................62
表5. PbGI與不同物種GI之RSCU值............................63
表6. PbGI與不同物種GI之胺基酸屬性所佔比例................66

圖目錄
圖1. 光週期影響開花時間之GI相關基因遺傳路徑示意圖........67
圖2. 大葉蝴蝶蘭之外觀型態................................68
圖3. PbGI基因選殖使用引子之相對示意圖....................69
圖4. 利用PCR以及5’& 3’RACE技術增幅PbGI之DNA電泳圖......70
圖5. PbGI與不同物種GI全長之多重序列排比..................73
圖6. PbGI與AtGI和OsGI預測之穿膜區域......................74
圖7. 不同物種GI胺基酸組成之雷達分佈圖....................75
圖8. 不同物種GI胺基酸組成之集群與對應分析................76
圖9. 不同物種GI序列RSCU值之集群與對應分析................77
圖10. PbGI親源演化樹狀圖.................................78
圖11. 不同物種GI胺基酸屬性組成之雷達分佈圖...............79
圖12. 不同光照條件PbGI表現量之探討.......................80
圖13、PbGI組織特異性表現量之探討.........................81
圖14. 利用南方墨點法分析大葉蝴蝶蘭GI之套數...............82
圖15. 主動誘變基因轉殖蝴蝶蘭轉殖株之外觀性狀.............83
圖16. 利用南方墨點法分析主動誘變轉殖基因蝴蝶蘭T-DNA之套數84

附錄
附錄1. 阿拉伯芥開花時間調控路徑模式......................97
附錄2. 農桿菌EHA105/pTAG-8質體構築示意圖.................98
附錄3. 農桿菌花粉轉殖所使用的蝴蝶蘭品種..................99
附錄4. pGEM®-T Easy Vector質體構築示意圖................100
附錄5. 5’and 3’RACE原理示意圖.........................101
附錄6. 南方墨點法之毛細管法轉印核酸裝置示意圖...........102
附錄7. pJET1.2/blunt質體構築示意圖......................103

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