Flavonoid 3’,5’-hydroxylase (F3’,5’H) 在花青素生合成途徑中扮演一關鍵酵素，催化反應產生藍色或紫色花瓣。本實驗以園藝作物美女櫻為材料，選殖其F3’,5’H 基因，以供給花色改良及花色調控之研究。根據已發表物種之F3’,5’H 基因序列，設計退化性引子，利用RT-PCR 方法選殖出預期片段，再配合5’及3’RACE (rapid amplification of cDNA ends) 策略，將全長F3’,5’H cDNA 序列選殖出，命名為VhFH1 (accesion number:AY604727)；另外利用IPCR (inverse PCR) 策略進行F3’,5’H 基因組DNA之選殖，同時將基因組DNA 序列與cDNA 序列經排序對比後發現，F3’,5’HDNA 序列具有兩個introns 及1542 bp 的ORF (open reading frame)，可轉譯成514 個胺基酸，並包含41 bp 的5’端非轉譯區域(UTR) 及58 bp 的3’UTR，且於基因上游發現可能為TATA box 序列的啟動子區域。將基因序列經NCBI 資料庫比對後，顯示其胺基酸序列與其他已發表之物種F3’,5’H序列有50-77﹪的相似度(identity) 。在預測蛋白質分析方面，VhFH1 的分子量為57 KD，等電點(pI) 為7.69，除了在N 端及C 端序列有較大差異外，其他序列部分皆有較高的相似度，並具備屬於細胞色素P450
(cytochrome P450) 家族所擁有的三個典型motifs，故推測此VhFH1 應為美女櫻F3’,5’H 基因。將此cDNA 序列構築於植物表現載體上，經轉型至大腸桿菌後，亦有RT-PCR 產物出現，未來更可轉殖於植物體上，以提供花色創新之原料。在不同品系下的美女櫻F3’,5’H 基因表現，由RT-PCR 顯示出藍色或紫色花品系之美女櫻皆具有此基因表現，而紅色花之美女櫻則缺乏，值得注意的是，在白色花品系中也有其表現，推測可能在後轉譯時期受到調控因子的抑制，才無法累積藍色色素，此現象提供了有利於美女櫻花色調控機制之研究。

Flavonoid 3’,5’-hydroxylase (F3’,5’H) is the key enzyme that catalyzesthe anthocyanin biosynthesis pathway for the expression of blue or purple flower color. The garden crops of Verbena x hybrida were used to clone its F3’,5’H gene for the investigation of flower color engineering and regulatory program. Degenerate primers were designed from the conservative regions of other published F3’,5’H genes to amplify the expectant DNA fragment . A full-length cDNA of the F3’,5’H gene designated VhFH1 (AY604727) was
cloned by the method of 5’and 3’ RACE, and its genomic DNA sequence was isolated by the IPCR strategy. Nucleotide sequence alignment revealed that VhFH1 contains two introns and a 1542 bp open reading frame encoding a polypeptide of 514 amino acid residues, and there could be a promoter
sequence with TATA box signal in the upstream of the transcription start site. The amino acid sequence of VhFH1 was compared with the previous reported F3’,5’H and showed between 50﹪and 77﹪identity with those species. The
expectant molecular mass and isoelectric point of VhFH1 protein is 57 KD and 7.69, respectively. There are three typical motifs of the F3’,5’H that belongs to
the cytochrome P450 proteins in the VhFH1 predicted protein. According to the above-mentioned conjecture, VhFH1 is a full-length cDNA of the F3’,5’H gene in the V. hybrida. This cDNA fragment was inserted into the plant expression vector pCambia 1304 and could be detected the expression in E. coli by RT-PCR and protein electrophoresis. It is practicable to transform the
horticultural plants with these vectors to create novel flower colors in the future. Furthermore, transcripts of the F3’,5’H gene were detected in the blue, purple
and white flowers but not in the red one as revealed by RT-PCR. These results are advantageous in the further investigation of regulatory factors of the
anthocyanin biosynthesis pathway in the V. hybrida.

文摘要----------------------------------------------------------------------------------1
英文摘要----------------------------------------------------------------------------------2
前言----------------------------------------------------------------------------------------3
前人研究----------------------------------------------------------------------------------6
一. 類黃酮及花青素的生化合成途徑-------------------------------------------6
二. 影響花色呈現之因子----------------------------------------------------------8
三. 影響花色的調節基因----------------------------------------------------------9
四. Flavonoid 3’,5’-hydroxylase 之介紹--------------------------------------11
五. 利用分子育種改變花色之可行性-----------------------------------------12
材料及方法-----------------------------------------------------------------------------14
一. 材料-----------------------------------------------------------------------------14
二. 方法-----------------------------------------------------------------------------15
(一) 總量RNA (total RNA) 之萃取---------------------------------------15
(二) 總量DNA (total DNA) 之萃取---------------------------------------15
(三) F3’,5’H 基因之選殖及分析--------------------------------------------16
1. F3’,5’H 退化性引子(degenerate primers) 之設計--------------16
2. 反轉錄酵素反應(reverse transcription reaction, RT) ----------16
3. cDNA聚合酵素連鎖反應(polymerase chain reaction)---------16
4. PCR 產物之回收及純化----------------------------------------------17
5. 接合反應(ligation) --------------------------------------------------17
6. 大腸桿菌的轉型作用(transformation)----------------------------17
7. 少量質體DNA 的抽取及殖入片段(insert) 之篩選------------18
8. F3’,5’H 基因片段之定序分析---------------------------------------18
9. 5’及3’ RACE 之引子設計及產物分析----------------------------19
(四) F3’,5’H genomic DNA 之選殖及分析-------------------------------191. IPCR (inverse PCR) 模板之製備---------------------------------19
2. IPCR 選殖分析-------------------------------------------------------20
3. Genomic DNA 之exon 及intron 之選殖-------------------------20
結果--------------------------------------------------------------------------------------25
一. 美女櫻F3’,5’H 基因選殖----------------------------------------------------25
二. 美女櫻F3’,5’H 之genomic DNA 選殖------------------------------------27
三. 美女櫻F3’,5’H 基因表現----------------------------------------------------30
討論--------------------------------------------------------------------------------------41
一. 美女櫻F3’,5’H cDNA 選殖方法------------------------------------------41
二. 美女櫻F3’,5’H cDNA基因確認及分析----------------------------------42
三. 美女櫻F3’,5’H genomic DNA 及cDNA 序列之分析------------------52
四. 不同美女櫻品系中F3’,5’H 之表現---------------------------------------53
五. 植物表現載體之建構--------------------------------------------------------53
六. 未來展望-----------------------------------------------------------------------56
參考文獻--------------------------------------------------------------------------------57

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