熱休克對大豆白化幼苗去氧核糖核酸——組蛋白與核糖核酸——核糖體蛋白協調合成的影響

Abstract

本研究仍是將在28℃發芽三天的大豆白化幼苗分別以28℃，41℃，45℃各3小時或40℃ 2小時至45℃ 2小時等四種不同溫度處理，萃取DAN，RNA，核糖體蛋白與組蛋白，以探討熱休克對上述化合物合成的影響與經休克後，在28℃回復合成的能力，並同時觀察DNA—組蛋白與RNA（2RNA）—核糖體蛋白合成間是否具有互相協調的現象。 大豆白化幼苗的生長溫度自28℃升至45℃時，DNA, RNA，核糖體蛋白與組蛋白的合成，會受到抑制。41℃ 3小時的處理組，DNA合成量?對照組(28℃)的35％，RNA?49％經處理後放回28℃的幼苗在第30小時時DNA與RNA的合成均可完全回復正常。45℃ 3小時的致死溫度處理組，DNA與RNA合成分別?對照組之15％與9％。經處理後放回28℃的幼苗，無法回復正常合成能力，且幼苗會逐漸萎縮死亡。40℃ 2小時至45℃ 2小時的處理組，雖經2小小時的45℃致死溫度處理，但由於40℃ 2小時的先處理，使DNA, RNA合成可在放回28℃後的第36小時時完全回復正常。顯示致死溫度前的熱休克有助於大豆幼苗克服因致死溫度所帶來的傷害。 經41℃ 3小時處理的大豆幼苗，3H-Leucine併入核糖體的量?對照組的67％，40℃—45℃?51％，45℃?2％，其中41℃與40℃—45℃處理組可能由於有熱休克蛋白(Heat Shock Proteins簡稱HSPs)的結合，使百分比相對提高。以冰醋酸萃取核糖體蛋白，並以電泳分析，發現在41℃與40℃—45℃處理下，除band 5外，大部分核糖體蛋白的合成會受到抑制，但熱休克下有二條特殊的蛋白質帶（此處稱?a，b band）出現於電泳圖中，其中b band在電泳中的移動性大於a band，且41℃處理組a，b band的含量較40℃—45℃處理組?多，而45℃處理組無此二band的合成。經處理後放回28℃的幼苗，隨著在28℃時間的增加，a, b band與band 5含量會逐漸降低，而其他核糖體蛋白的合成會逐漸增加，至第24小時時，a band已不覆存在，而b band只有在41℃處理組中仍出現，band 5與其他核糖體蛋白的合成，此時也已回復到正常的含量。同一時間RNA合成在41℃與40℃—45℃處理組分別?對照組的95％與73％，顯示核糖體蛋白合成與RNA合成間有互相協調的現象，當RNA合成量增加時核糖體合成也相對增加。至於熱休克下所合成的a, b band是否即?HSPs或?經休克後改變的核糖體蛋白，仍待進一步實驗證實。 大豆白化幼苗經41℃ 3小時處理後，組蛋白合成?對照組的60％，40℃—45℃?58％，而45℃?4％。經休克後放回28℃的幼苗，第24小時時41℃處理組的組蛋白合成已完全回復，（?對照組的109％），40℃—45℃?78％，45℃?5％。同一時間41℃ DNA合成?對照組的80％而40℃—45℃只有50％，顯示DNA合成與組蛋白合成間可能具有互相調合的現象。當DNA進行回復時組蛋白合成也相對會回復，且所回復的量與DNA回復的量有關。取等量放射活性組蛋白作電泳分析發現40℃—45℃與41℃處理組除Hla合成量較穩定外，其他組蛋白的合成均有降低的現象，且不同組蛋白間的降低量均相似，顯示組蛋白間的合成彼此可能也具有互相調合的現象，當某一組蛋白含量降低時，其他也相對降低，使得細胞內組蛋白間能維持一定的比例值。

Three-day etiolated soybean seedlings were treated with different temperature regimes and studies the effect of heat shock on synthesis of DNA, RNA, ribosomal proteins and histones and the ability of recovery at 28℃ after each temperature treatment. When soybean seedlings were shifted from a normal growth temperature of 28℃ up to 40℃ or 45℃, there was a dramatic inhibition in DNA, RNA, ribosomal proteins and histones synthesis. After incubation at 41℃ for 3 hours, the synthesis of DNA, RNA, in the seedling were inhibited 65% and 60%. The synthesis of DNA and RNA can recover to the normal level on hour 30 and 36 at 28℃. After incubation at lethal temperature (45℃) for 3 hours, the synthesis of DNA and RNA were only 15% and 9% of the control and this inhibition can't recover at 28lC. However, a pretreatment at 40℃ for 2 hours before lethal temperature (45℃) treatment, the synthesis of DNA and RNA were completely recovered on hour 36 at 28℃. The amounts of 3H-Leucine incorporated to ribosomal fractions were 67%, and 2% of the control in 41 C, 40℃-45℃ and 45℃ treated seedlings. Among those treatment, there might have some HSPs associated with rebosomes, which cause the percentage increase in 41℃ and 40℃-45℃ treated seedlings. After extracting ribosomal proteins by glacial acetic acid and electrophoresised by acetic acid urea gel, we found almost all ribosomal proteins synthesis are inhibited by heat shock except band 5 and appeared two special protein bands (band a and band b) in 40℃-45℃-and 41℃-treated seedlings. Protein bands b has higher mobility than a band in electrophoresis and 40℃-treated seedlings have more a. b bands than 40℃-45℃-treated seedlings. During the recovery at 28℃, the synthesis of a. b bands and band 5 decreased, but the synthesis of rivosomal proteins increased. After recovery at 28℃ for 24 hour, band a almost disappeared and only band b retained in 41℃-treated tissue, band 5 and other ribosomal proteins synthesis had returned to normal level. At the same time, RNA systhesis was 95% and 73% in 41℃ and 40℃-45℃ treated seedlings. From those results suggest that they have coordinated synthesis between RNA and ribosomal proteins. The systhesis of histones in 41℃, 40℃-45℃ and 45℃ treated seedlings were 60%, 58% and 4% of the control. During the recovery at 28℃, 41℃ treated seedlings will completely recover of histones systhesis on hour 24, but 40℃-45℃ treated seedlings still not yet. On the same time, DNA systhesis of 41℃, 40℃-45℃ and 45℃ treated seedlings was 80% and 12% of the control. Suggest there might have coordinated systhesis between DNA and histones. After taking equal amounts of radioactive histones were analyzed by acetic acid urea acrylamide gel electrophoresis, indicate that almost all histones synthesis except Hla were inhibited by heat shock. From the results shown in this experiment indicated that there are a coordinate relationship between RNA - ribosomal proteins and DNA - histones synthesis.