臺灣博碩士論文加值系統

在本實驗室前人的研究中，可以知道小球藻Chlororella 211-8b具有耐乾旱的特性。曾(2000)的研究中甚至發現小球藻亦有耐熱的特質。曾(2000)提及小球藻經過46.5 ℃熱處理1小時後，再以17μmol．m-2．s-1照光培養，會導致葉綠素的分解而產生白化現象，但經過一段時間後又會恢復綠化而重新生長;避光處理的小球藻則沒有此白化現象。另外，當光照強度變弱，小球藻進入白化的速率也變慢;而照光過程中避光一段時間，時間越長，進入白化的比例也越少。由以上可以知道：小球藻葉綠素的分解與光照有關，而給予避光處理能提供它修復的機會。
本實驗的目的是希望進一步探討熱處理後白化過程的生理、生化特徵以及細胞熱耐受性與白化的關係。我們發現葉綠素分解的過程有分階段性，在前4小時的變化很小，然後是快速的降解，在12小時內便完全分解。然而整個過程中偵測到部分的葉綠素分解物，推測其分解機制不像是高等植物的酵素分解過程。另外在分解過程中，與葉綠素結合的蛋白質複合體於4小時後開始分解，直到8小時便分解的十分完全，故推測蛋白質結構不穩定是第二階段葉綠素快速分解的原因。在顯微鏡下的觀察，可發現隨著光照時間加長，藻細胞內的葉綠體顏色越來越淡，但是12小時之後，仍有一些淡綠色，並未全變白。如使用弱紅光（≧630nm，4.6 μ mole．m-2．s-1）照射處理，發現葉綠體有內縮為一半的情況，但在白光照射處理時，這種情況卻較少見到，故推測葉綠體縮為一半的變化是白化的過程之一，但為時很短。我們也探討熱處理的溫度對細胞光合作用的傷害，當熱處理的溫度大於等於46.5 ℃時，光合作用活性在20分鐘內就降為0，而溫度逐步調降為45 ℃、44 ℃時，光合作用消失的時間越往後延，43 ℃時只稍微下降，40 ℃則不受影響。將上述各溫度處理過的小球藻，再經由光照培養，可發現光合作用消失的時間越快，細胞越早進入白化階段，傷害也越大。當光合作用活性被抑制住後，再持續給予光能的話，極可能使細胞進入氧化逆境而導致細胞本身的修復系統崩潰，而進行白化。

In the previous study,we know that Chlorella pyrenoidosa has a characteristicof heat-tolerance.Tzeng (2000)even suggested that cells of Chlorella pyrenoidosa were subjected to heat stress of 46.5℃ for 1h. It found that the heat-treated cells lost all the chlorophyll when they were cultured again in the growth medium.The culture started to grow and regained its green color several days later.By using flow cytometer ,it was found that the disapperence of pigment seemed to proceed in two distinguishable steps .When the Chlororella culture had been kept in the dark after the heat -treatment, the chlorophyll content of cells remained unchanged for up to 5 days.It seemed that darkness protects chlorophyll from degradation.It was found that at a light intensity lower than 4μmolm-2s-1,chlorophyll degradation was slowed down.It concluded (1)Light is needed to trigger the chlorophyll degradation process.(2)After heat stress,light is another stress to the recultured cells ,the lower light intensity the less damage.(3)When culture is kept completely in the dark,the cells may repair the stress due to heat stress.
In this study,we want to further study the physiology and biochemistry of whiting cells and the relationship between heat-tolerance and chlorophyll degradation.It was found that the heat-treated cells lost all the chlorophyll in two distinguishable steps.The first stage starts immediately as soon as the cells were recultivated .The second stage of chlorophyll degradation would start about 4h after recultivation.In the degrading pathway,we can detect chlorophyll partial degrading intermediates. During the chlorophyll degradation,some thylakoid menbrane proteins with specific molecular weight disappeared.(24kD;30kD;50kD). 30kD and 50kD would not be degradated until 4h after reculivation, and 24kD disappered at 8h after recultivation.We suggested the degradation of chlorophyll might be due to the specific thylakoid menbrane proteins degradation.Under microscopy chlorophyll might be seen in the white cells . But when the Chlorella were cultivated under red light
condition(≧630nm ,4.6μmolm-2s-1),the chloroplast would turn to be half . Under the confocal microscopy scanning microscopy ,we could observe clear fluorescence image of chloroplast。
We also measured the photosynthetic activity of Chlorella during heat stress.It was found that photosynthesis was inactivated after 20 minutes of 46.5℃ heat treatment.While lowering temperature to 40℃,the photosythesis would little affected by heat. We found that the more rapidly disappeared of photosynthesis,the more green cells turned to white cells.
We suggested that heat stress(46.5℃ for 1h ) make photosynthesis activity disappear rapidly,and the more rapidly photosynthesis disappears ,the more green cells turned to white cells.And continuous illumination might cause oxdative stress in cells and break up the protection system of the cells ,leading green cells turn to white cells.

目錄
摘要…………………………………………………….1
Abstract…………………………………………………3
前言……………………………………………………..5
實驗材料與方法………………………………………17
結果……………………………………………………25
討論……………………………………………………33
圖目……………………………………………………40
參考文獻………………………………………………66

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