從甘藷老化葉片利用扣減雜交及RACE-PCR選殖得到一個全長cDNA，Blast序列比對分析與植物天門冬胺酸蛋白酶包括馬鈴薯及番茄有72％至73％的序列相似性。因此命名為甘藷天門冬胺酸蛋白酶SPAP，其open reading frame含有1,515個核苷酸 （504氨基酸），編碼的蛋白質含有DTG及DSG組成的catalytic site以及一段約100胺基酸所構成的plant specific insert （PSI）。甘藷天門冬胺酸蛋白酶SPAP基因表現從L1未展開的幼葉至L3成熟葉片逐漸增加，當葉片老化至中期及末期時其表現量也持續減少，另外L3成熟葉片亦比塊根、根及莖有較高的表現量。RT-PCR及西方墨點法分析亦顯示ethephon（益收生長素）處理的葉片也會劑量正相關的暫時性誘導甘藷天門冬胺酸蛋白酶SPAP表現量增加。Ethephon處理會誘導甘藷葉片老化以及H2O2含量增加，外加純化的天門冬胺酸蛋白酶SPAP融合蛋白可加速 ethephon 誘導的甘藷葉片老化以及H2O2含量增加的速率。利用anti-SPAP多株抗體、天門冬胺酸蛋白酶抑制劑 pepstatin A、及95℃加熱使SPAP融合蛋白失去活性等方式可拮抗SPAP融合蛋白的作用，顯著減緩其加速ethephon誘導的甘藷葉片老化以及H2O2含量增加的速率。另外單獨外加天門冬胺酸蛋白酶SPAP融合蛋白並無顯著加速黑暗處理甘藷葉片老化以及H2O2含量增加的速率。因此根據上述的實驗結果結論甘藷天門冬胺酸蛋白酶SPAP為ethephon可誘導的基因，其生理功能可能參與於ethephon誘導的甘藷葉片老化以及H2O2含量增加有關，且需與ethephon可誘導的交互作用因子（interacting components）一起協力作用。

A full-length cDNA was isolated from sweet potato senescent leaves with PCR-selective subtractive hybridization and RACE PCR. Blast analysis showed that it exhibited high nucleotide sequence identity (ca. 72-73%) with plant aspartic proteases including potato and tomato, and was named as sweet potato aspartic protease SPAP. Its open reading frame contained 1515 nucleotides (504 amino acids), and encoded a putative aspartic protease with a catalytic site composed of DTG and DSG and a plant specific insert constituted of ca. 100 amino acids in length. Gene expression of sweet potato aspartic protease SPAP increased gradually from L1 immature leaves to L3 mature leaves, and then continuously decreased from intermediate L4 to late L5 leaf senescent stages. In addition, L3 mature leaves also exhibited higher expression level than that of storage roots, roots and stems. Ethephon treatment also caused temporary enhancement of sweet potato aspartic protease SPAP dose-dependently detected by RT-PCR and Western blot hybridization. Ethephon treatment promoted sweet potato leaf senescence and H2O2 elevation. Exogenous purified SPAP fusion protein significantly accelerated the ethephon-mediated promotion of leaf senescence and H2O2 elevation. The SPAP fusion protein-mediated effects could be remarkably antagonized by anti-SPAP antibody, aspartic protease inhibitor pepstatin A, and 950C boil treatment of the purified SPAP fusion protein, which significantly alleviated the effects of exogenous SPAP fusion protein on the acceleration on ethephon-mediated promotion of leaf senescence and H2O2 elevation. In addition, no significant variation on the promotion of leaf senescence was observed between dark treatment and treatment with exogenous SPAP fusion protein alone. These results conclude for the first time that sweet potato aspartic protease SPAP is ethephon-inducible, and likely participates in ethephon-mediated promotion of leaf senescence and H2O2 elevation. Sweet potato aspartic protease SPAP may require interacting components for effective promotion of ethephon-mediated leaf senescence and H2O2 elevation in sweet potato.

目錄
頁次
誌謝………………………………………………………….…………..ii
中文摘要…………………………………………………………….....iii
圖次……………………………………………………………………...x
英文摘要……………………………………………………………….iv
壹、緒論………………………………………………………………..1
甘藷………………………………………………………………..1
葉片老化…………………………………………………………..2
乙烯………………………………………………………………..3
天門冬胺酸蛋白酶（Aspartic protease）……….…..……………4
貳、實驗材料………………………………………………………….8
甘藷（Ipomoea batatas（L.）Lam）……………………………8
G14 ………………………………………………………………..8
參、實驗方法………………………………………………………….8
甘藷天門冬胺酸蛋白酶（aspartic protease）SPAP之基因選殖及定性分析…………………………………………………………..8
天門冬胺酸蛋白酶SPAP基因選殖及生物資訊學分析………....8
天門冬胺酸蛋白酶SPAP生長發育及組織專一性表現分析........9
SPAP重組表現載體構築與融合蛋白表現、純化及抗體製備…..9
不同濃度ethephon處理甘藷成熟葉片…………………………10
甘藷天門冬胺酸蛋白酶SPAP融合蛋白處理……...…………..11
外加SPAP融合蛋白處理甘藷成熟葉片……….………………11
外加SPAP融合蛋白及ethephon處理甘藷成熟葉片….………11
外加SPAP融合蛋白、anti-SPAP抗體及ethephon處理甘藷成熟
葉片………………………………………………………………11
外加失活之SPAP融合蛋白及ethephon處理甘藷成熟葉片…12
外加SPAP融合蛋白、pepstatin A及ethephon處理甘藷成熟葉
片…………………………………………………………………12
扣減雜交…………………………………………………………13
差異性cDNA選殖………………………………………………19
Rapid amplification of cDNA ends（RACE）…………………..22
SPAP融合蛋白純化……………………………………………..25
葉綠素含量測量…………………………………………………25
光合作用效率測量………………………………………………26
3,3-Diaminobenzidine（DAB）染色分析………………..……..26
H2O2含量測定…………………………………………………...26
RT-PCR…………………………………………………………..27
Total RNA extraction……………………………………………..27
Reverse transcription……………………………………………..28
PCR………………………………………………………………29
Primer 設計……………………………………………………...29
PCR 流程………………………………………………………..29
西方墨點法………………………………………………………30
肆、結果………………………………………………………………33
甘藷天門冬胺酸蛋白酶SPAP的分子選殖及生物資訊學分析.33
甘藷天門冬胺酸蛋白酶SPAP在不同葉片生長發育階段及組織專一性的表現……………………………………………………33
甘藷天門冬胺酸蛋白酶SPAP基因表現受乙烯所誘導………35
甘藷天門冬胺酸蛋白SPAP融合蛋白的誘導與純化………….36
Ethephon誘導甘藷葉片老化與劑量呈正相關…………………37
天門冬胺酸蛋白酶SPAP融合蛋白加速ethephon誘導的甘藷葉片老化速率………………………………………………………37
Anti-SPAP抗體減緩天門冬胺酸蛋白酶SPAP融合蛋白加速ethephon誘導甘藷葉片老化的作用……………………………38
外加失活的天門冬胺酸蛋白酶SPAP融合蛋白不會加速ethephon誘導甘藷葉片老化速度……………………………….39
天門冬胺酸蛋白酶抑制劑 pepstatin A減緩外加SPAP融合蛋白加速ethephon誘導中甘藷葉片老化作用………………………40
單獨外加天門冬胺酸蛋白酶SPAP融合蛋白沒有顯著增加黑暗處理下甘藷葉片老化速率………………………………………42
伍、討論……………………………………………………………….43
陸、參考文獻.…………………………………………………………48

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