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研究生:王崇華
研究生(外文):Chung-Hua Howard Wang
論文名稱:水稻葉片老化的分子層次探討
論文名稱(外文):Molecular Studies on Rice Leaf Senescence
指導教授:陳淑真陳淑真引用關係
指導教授(外文):Shu-Chen Grace Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:82
中文關鍵詞:葉片老化人為暗處理誘導之葉片老化扣除雜交北方墨點轉印分析法
外文關鍵詞:Leaf SenescenceDark-induced leaf senescenceSubtractive HybridizationNorthern Blot Analysis
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  • 被引用被引用:3
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葉片老化為植物生長發育過程的一個重階要段。於此時期,葉片整個生理、生化代謝,發生急劇變化,逐漸導致葉片之黃化、枯萎。
為瞭解葉片老化的分子調控機制,本研究利用連續黑暗處理,誘導全株水稻綠苗葉片老化,以 PCR-Select cDNA Subtraction 的方法希望能找到葉片老化早期表現之基因,再以北方墨點轉印分析法來確定該基因表現時期,及 transcript 大小。
我們初步找到45個不同大小之 cDNA 片段,利用 Reverse Northern Analysis 認為有25個 cDNA 片段在葉片老化時,其表現量有增加。針對其中在黃、綠葉表現差異甚大之片段進行 Northern Blot Analysis,找到4個葉片老化相關基因株,分別為CH 16、CH 36、CH 69 及CH 70。
利用 FASTA 及 BLASTX 進行相似性比對,發現CH 16 為 isocitrate lyase,是glyoxylate cycle 內主要酵素,transcript 大小為2.7 kb,在暗處理2天後的水稻幼苗葉片中見其表現量迅速增加;CH 36為acetylornithine aminotransferase,是arginine 生合成與代謝之重要酵素,transcript 大小為2 kb,在暗處理2天後的水稻幼苗葉片見其開始表現;CH 69 為seed imbibition protein,參與種子萌芽之蛋白質,transcript 大小為2.8 kb,在暗處理的水稻幼苗葉片中,見其持續表現;CH 70為polygalacturonase isoenzyme 1 之 beta subunit, 是分解細胞壁pectin的主要酵素,transcript 大小為1.5 kb,在暗處理2至3天的水稻幼苗葉片表現量最高。
Leaf senescence is an important stage in plant development. During this stage, leaf cells undergo distinct metabolic and structural changes that lead to leaf yellowing and death. We adopted dark-induced leaf senescence in rice seedlings as the experimental model to study the molecular mechanism of rice leaf senescence. Using the method of PCR -Select cDNA subtraction, we designed to isolate the senescence-associated genes ( SAGs ) that express during the early stage of leaf senescence .
We have isolated 45 different cDNA fragments from cDNA subtraction. Only 25 cDNA fragments showed increased signals when probed with yellow leaf cDNA in Reverse Northern Analysis. We finally obtained 4 positive cDNA clones including CH 16, CH 36, CH 69 and CH 70 which were proved to be leaf senescence-specific by Northern blot hybridization.
Using FASTA and BLASTX program for homology search, CH 16 is isocitrate lyase, the key enzyme of glyoxylate cycle. CH 16 showed enhanced expression of 2.7 kb transcript after 2 days of dark treatment. CH 36 was found to be ornithine / acetylornithine aminotransferase involved in arginine metabolic pathway, which showed enhanced expression of 2 kb transcript also after 2 days of dark treatment. CH 69 was found to be seed imbibition protein involved in seeds germination, which showed constant expression of 2.8-kb transcript after 1-6 days of dark treatment. CH 70 was found to be beta-subuint of polygalacturonase isoenzyme 1 - the key enzyme for degrading cell wall pectin, which reached maximal level of expression of 1.5 kb transcript 2-3 days of dark treatment.
In the future, we will examine the regulation of these senescence- associated genes using natural senescent tissues from the field. It will lead to our further understanding of the leaf senescence mechanism.
內容 頁次
中文摘要 1
Abstract 2
前言 3-8
實驗材料 9-11
實驗方法 12-23
實驗結果 24-28
討論 29-32
參考文獻 33-43
圖表 44-72
附錄 73-77
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