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研究生:黃和章
研究生(外文):Ho-Chang Huang
論文名稱:青花菜於熱逆境下抗氧化酵素活性分析與基因選殖
論文名稱(外文):Activity analysis and gene cloning of antioxidant enzyme in Broccoli under heat stress
指導教授:林冠宏林冠宏引用關係
指導教授(外文):Chun-Hung Lin
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:144
中文關鍵詞:青花菜熱逆境高溫抗氧化酵素超氧化物歧化酶抗壞血酸過氧化酶過氧化氫酶即時聚合鏈反應快速增幅cDNA末端
相關次數:
  • 被引用被引用:6
  • 點閱點閱:261
  • 評分評分:
  • 下載下載:13
  • 收藏至我的研究室書目清單書目收藏:1
台灣處於熱帶及亞熱帶地區,夏季高溫是青花菜栽培不易、產量不高的原因之一,青花菜受高溫傷害的程度與品種、生長期、器官、溫度、高溫逆境期長短有關。高溫逆境易引起植物蛋白質變性、氨毒害、膜傷害、光合作用下降、呼吸作用升高造成饑餓傷害等。本研究欲瞭解青花菜耐熱‘欣樺1208’與不耐熱‘優秀’二品種於不同溫度(25, 30, 35, 40℃)不同處理(0, 1, 3, 6, 12, 48 及72小時)下,植株生長、外部形態及葉片膨壓、電解質滲漏、抗氧化系統之差異與變化,評估酵素活性可否成為蔬菜品質與產量之生理指標,並將此酵素選殖出來。
以actin為internal control,利用real-time PCR分析在高溫逆境下抗氧化酵素mRNA之相對表現量。利用RACE法擴增其抗氧化酵素基因cDNA full length末端,經選殖入載體並定序後,至NCBI網站比對此cDNA full length序列,以確定此段基因。
結果顯示高溫造成青花菜產生外部形態的變化,葉片出現下垂、枯萎、萎縮、捲曲等症狀。而葉片膨壓、電解質滲漏無法明顯區別出耐熱與不耐熱品種。‘優秀’ 之CAT活性於熱逆境下增加較多,‘欣樺1208’APX活性於熱逆境下增加較多。
mRNA表現量‘優秀’於40℃處理6小時後,APX及CAT表現量皆大幅增加。‘欣樺1208’APX於35℃及40℃處理24小時後有所增加,40℃處理6小時後CAT表現量皆大幅增加。經RACE擴增出SOD、APX和CAT序列,將定序結果整理,送至NCBI blastn比對後,與阿拉伯芥SOD序列有93%、阿拉伯芥APX序列有81%、芥菜(Brassica juncea) CAT序列有99%以上的相似度,推論RACE擴增出之片段分別為SOD、APX和CAT cDNA序列。 本研究已將青花菜之CAT 及ubiquiitin全長ORF序列選殖出來。
Taiwan is located in the tropical and subtropical regions. Production of broccoli is limited to a large extent by climates with high temperatures. The growth and development of broccolis are strongly influenced by heat stress that can be initiated by oxidative stress. To cope with this stress, plants have developed a series of defense mechanisms and detoxification systems. The objectives of our work were to study the changes of antioxidant system in broccoli under 25, 30, 35 and 40℃ for 0, 1, 3, 6, 12, 48, and 72 hours, and identify any antioxidant system linked to heat-tolerance traits under heat stress. Heat-tolerant variety “SF1208 “ and heat-susceptibility variety “Green Magic“ were used to measure the antioxidant enzymes, turgor pressure and electrolyte leakage under the growth chamber. The response of target gene expressions and regulations under the stress were determined using real-time PCR. A full-length target cDNA was amplified using RACE technique followed by cloning it into the vector for sequencing, and comparing it with any known sequences in the NCBI-blastn.
The result shows that the increased catalase and ascorbate peroxidase activity provides the broccoli plants with increased tolerance. The turgor pressure and electrolyte leakage were not able to significantly identify the differences between genotypes. Both of the APX and CAT gene expressions were not well consistent to their activities. The sequences of SOD, APX and CAT were 93% and 81% identical to A. thaliana , as well as 99% to B. juncea, respectively. We have clones both catalase and ubiquiitin full length cDNA sequences (open reading frame) in broccoli.
目錄

表目錄 ……………………………………………… II
圖目錄 ……………………………………………… III
中文摘要 ……………………………………………… V
英文摘要 ……………………………………………… VI
壹、前言 ……………………………………………… 1
貳、前人研究 ……………………………………………… 2
参、材料與方法 ……………………………………………… 11
一、材料 ……………………………………………… 11
二、生理指標測定 ……………………………………………… 11
三、蛋白質萃取 ……………………………………………… 12
四、酵素測定 ……………………………………………… 13
五、數據分析 ……………………………………………… 14
六、萃取RNA ……………………………………………… 15
七、引子設計 ……………………………………………… 17
八、引子測試 ……………………………………………… 18
九、快速增幅cDNA 末端 ……………………………………………… 19
十、Gene Cloning ……………………………………………… 30
十一、親緣分析 ……………………………………………… 33
十二、即時聚合鏈反應 ……………………………………………… 34
肆、結果 ……………………………………………… 38
一、生理指標 ……………………………………………… 38
二、抗氧化酵素活性 ……………………………………………… 40
三、引子之cDNA序列 ……………………………………………… 44
四、RACE ……………………………………………… 44
五、親緣分析 ……………………………………………… 45
六、Real-time PCR ……………………………………………… 46
伍、討論 ……………………………………………… 50
陸、結論 ……………………………………………… 64
柒、表附錄 ……………………………………………… 66
捌、圖附錄 ……………………………………………… 78
玖、參考文獻 ……………………………………………… 134


表目錄

表1 台灣各縣市青花菜歷年生產面積與產量 ………………………… 66
表2 台灣各氣象站月平均氣溫統計表 ………………………… 68
表3 本研究設計之universal引子序列 ………………………… 69
表4 本研究RACE所使用引子序列 ………………………… 69
表5 本研究colony PCR所使用引子序列 ………………………… 69
表6 本研究real-time PCR所使用引子序列 ………………………… 70
表7 ‘優秀’、‘欣樺1208’葉片膨壓分析 ………………………… 71
表8 ‘優秀’與‘欣樺1208’葉片膨壓分析 ………………………… 72
表9 ‘優秀’、‘欣樺1208’電解質滲漏分析 ………………………… 73
表10 ‘優秀’與‘欣樺1208’電解質滲漏分析 ………………………… 74
表11 ‘優秀’與‘欣樺1208’SOD活性分析 ………………………… 75
表12 ‘優秀’與‘欣樺1208’APX活性分析 ………………………… 76
表13 ‘優秀’與‘欣樺1208’CAT活性分析 ………………………… 77











圖目錄

圖1 植物細胞中清除ROS的途徑位置 ………………… 78
圖2 植物中清除ROS的途徑 ………………… 79
圖3 SOD、APX、CAT之存在位置 ………………… 80
圖4 RACE原理 ………………… 81
圖5 Real-time PCR 原理 ………………… 82
圖6 青花菜 Actin 序列 ………………… 83
圖7 植株處理情形 ………………… 84
圖8 SOD引子設計參考序列 ………………… 85
圖9 APX引子設計參考序列 ………………… 86
圖10 CAT引子設計參考序列 ………………… 87
圖11 設計之SOD引子擴增電泳圖 ………………… 88
圖12 設計之APX引子擴增電泳圖 ………………… 89
圖13 設計之CAT引子擴增電泳圖 ………………… 90
圖14 SOD 5’end RACE電泳圖 ………………… 91
圖15 APX 5’end RACE電泳圖 ………………… 92
圖16 CAT 5’end RACE電泳圖 ………………… 93
圖17 SOD 3’end RACE電泳圖 ………………… 94
圖18 APX 3’end RACE電泳圖 ………………… 95
圖19 CAT 3’end RACE電泳圖 ………………… 96
圖20 CAT 5’端比對相似物種設計引子之電泳圖 ………………… 97
圖21 yT&A 載體選殖區核酸序列 ………………… 98
圖22 yT&A 選殖載體圖譜 ………………… 98
圖23 Plasmid DNA以限制脢處理電泳圖 ………………… 99
圖24‘優秀’與‘欣樺1208’葉片膨壓 ………………… 100
圖25‘優秀’與‘欣樺1208’ 電解質滲漏 ………………… 101
圖26‘優秀’SOD活性 ………………… 102
圖27‘欣樺1208’ SOD活性 ………………… 103
圖28‘優秀’APX活性 ………………… 104
圖29‘欣樺1208’ APX活性 ………………… 105
圖30‘優秀’CAT活性 ………………… 106
圖31‘欣樺1208’ CAT活性 ………………… 107
圖32 Real-time PCR data ………………… 108
圖33‘優秀’ APX mRNA與Actin之相對表現量 ………………… 109
圖34‘欣樺1208’ APX mRNA與Actin之相對表現量 ………………… 110
圖35‘優秀’ CAT mRNA與Actin之相對表現量 ………………… 111
圖36‘欣樺1208’ CAT mRNA與Actin之相對表現量 ………………… 112
圖37 熱逆境造成葉片下垂、枯萎、萎縮、捲曲 ………………… 113
圖38 高溫逆境造成莖頂出現皺縮的現象 ………………… 113
圖39 藍白篩選(Blue-White screening) ………………… 114
圖40 設計之SOD引子所擴增出之SOD序列 ………………… 115
圖41 設計之APX引子所擴增出之APX序列 ………………… 116
圖42 設計之CAT引子所擴增出之CAT序列 ………………… 117
圖43 SOD RACE結果序列 ………………… 118
圖44 APX RACE結果序列 ………………… 119
圖45 CAT RACE結果序列 ………………… 120
圖46 設計之SOD primer擴增cDNA序列比對結果 ………………… 121
圖47 設計之APX primer擴增cDNA序列比對結果 ………………… 122
圖48 設計之CAT primer擴增cDNA序列比對結果 ………………… 123
圖49 RACE:SOD cDNA比對結果 ………………… 124
圖50 RACE:APX cDNA比對結果 ………………… 125
圖51 RACE:CAT cDNA比對結果 ………………… 126
圖52 青花菜CAT cDNA序列(ORF) ………………… 127
圖53 青花菜CAT cDNA序列(ORF)比對結果 ………………… 128
圖54 SOD蛋白質序列分析之親緣關係樹 ………………… 129
圖55 APX蛋白質序列分析之親緣關係樹 ………………… 130
圖56 CAT蛋白質序列分析之親緣關係樹 ………………… 131
圖57 青花菜ubiquitin ………………… 132
圖58 青花菜ubiquitin比對結果 ………………… 133
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