臺灣博碩士論文加值系統

在高等植物中蔗糖於光合作用組織合成運輸至沈積組織儲存，藉由蔗糖轉化脢將蔗糖水解成葡萄糖和果糖，而蔗糖轉化脢具有多種異構脢在醣類代謝和蔗糖調節上扮演重要的角色。目前已經從綠竹筍中細胞質和細胞壁中各分離出兩種不同形態蔗糖轉化脢，為了解蔗糖轉化脢在醣類代謝扮演角色，針對其基因結構進行相關探討。以綠竹筍抽取的 RNA 為模板，從不同植物的蔗糖轉化脢高保留性胺基酸設計引子進行 RT-PCR，得到 1.42 kb cDNA 片段 (BSIT-1.42)，在 BSIT1 上的序列設計引子，分別進行 3’RACE 和 5'' RACE，定序結果發現 3''RACE 產物有兩種的序列 (3-1 和 3-2) ，經由所有得到的 cDNA 序列重複區域比對並組合，得到兩種假設性全長蔗糖轉化脢cDNA，命名為 BSIT1 和 BSIT2。BSIT1 全長為，1984 bp，ORF 為 1755 bp，而胺基酸共584 個，預測其分子量 64.56 kDapI 值為 4.78。BSIT2 全長為 1968 bp，ORF 為 1749 bp，而胺基酸共582 個，預測其分子量 64.28 kDa，pI 值為 4.81。兩者推演的胺基酸對於玉米蔗糖轉化脢的同質性為 71%，與蕃茄比對為 56%，和 RIT1 比對為 54%，和 SPIT1 比對為 53%。另外由 Southern 分析，初步推論綠竹筍中 BSIT1 和 BSIT2 屬低拷貝基因。

Sucrose produced by photosynthesis in leaves is exported to the sink tissues. In higher plants, invertase (beta-D-fructofuranosidase，EC 3.2.1.26), hydrolyzes sucrose into glucose and fructose, are a group of ubiquitous plant enzymes and play important roles in carbohydrate metabolism and in the regulation of sucrose transport. Total RNA isolated from shoots of green bamboo as the template and two oligonucleotide primers, corresponding to the two conserved amino acid sequences of invertase found in other plant species, were used for RT-PCR. The resulting 1.42 kb cDNA fragment were analysed by agarose gel eletrophoresis. Primers designed based on the BSIT-1.42 were used in means of 5''/3’ RACE, two different sequences of cDNA were obtained by mean of 3’ RACE (3’-1 and 3’-2). By comparing with overlap region of cDNAs, two full-length invertase cDNAs were presumably obtained.They are designated BSIT1 and BSIT2. BSIT1 has 1984 bp containing an open reading frame of 1755 bp that encodes a polypeptide of 584 amino acids with molecular mass of 64.56 kDa and pI 4.78. BSIT2 has 1968 bp containing an open reading frame of 1749 bp that encodes a polypeptide of 582 amino acids with molecular mass of 64.28 kDa and pI 4.81. They both have high amino acid sequence homology with maize (71% identity), tomato (56% identity), rice (54% identity) and sweet potato (53%). Genomic Southern analysis first indicated that both BSIT1 and BSIT2 have low copy numbers in genomic DNA.

第一章 緒論 ………………………………………………………… 1
第一節 蔗糖與植物蔗糖的代謝……………………………………… 1
第二節 植物蔗糖之運輸途徑………………………………………… 3
第三節 蔗糖轉化脢的分類與生理功能……………………………… 5
第四節 蔗糖轉化脢的調節機制……………………………………… 6
第五節 蔗糖轉化脢分子生物學的研究……………………………… 9
第六節 本論之研究起源與目的……………………………………… 13
第二章 材料與方法………………………………………………… 14
第一節 材料…………………………………………………………… 14
第二節 RNA 之抽取與分析方法…………………………………… 14
第三節 DNA 片段的製備與純化…………………………………… 19
第四節 質體 DNA 之轉形…………………………………………… 29
第五節 DNA 的抽取………………………………………………… 32
第六節 核酸電泳分析法……………………………………………… 35
第七節 核酸的轉印…………………………………………………… 38
第八節 探針的標幟與雜交反應……………………………………… 40
第九節 DNA 序列分析……………………………………………… 44
第三章 結果與討論………………………………………………… 45
第一節 RNA 的抽取與檢定………………………………………… 45
第二節 轉化脢cDNA 之選殖……………………………………… 46
第三節 全長 cDNA 序列分析……………………………………… 50
第四節 轉化脢基因表現與拷貝數目的探討………………………… 52
第四章 結論與未來展望 ………………………………………… 53
圖與表……………………………………………………………………………54
參考文獻……………………………………………………………………… 76

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