大豆胚軸細胞核內組蛋白的分離及二氯苯氧乙酸(2,4-D)對其磷酸化作用的影響

Abstract

本研究乃是將發芽三天的大豆白化幼苗用20×19 □ □—D (2,4-dichlorophenoxyacetic acid)(pH□)，處理24小時後，分別由對照組及處理組幼苗的下胚軸來分離細胞核內的組蛋白，以探討2,4-D對組蛋白的電泳膠質類型及磷酸化作用的影響。(1)首先，先判定大豆組蛋白的電泳膠質類型，發現大豆組蛋白的電泳膠質類型和碗豆組蛋白相似，但與牛胸腺組蛋白則具有移動性的差異。大豆的五種組蛋白在polyacrylamide gel上的移動性大小是 H4>H3>H2b/H2a>H1。(2)分別引用鹽酸，硫酸，醋酸所萃取出的組蛋白之電泳膠質類型相似，但所抽出的五種組蛋白在比例上有所差異。而且，以醋酸萃取組蛋白時，效果較差，組蛋白在polyacrylamide gel上的解析□不好，且所抽出的組蛋白含有較多的非組蛋白鹼性蛋白質。而以鹽酸或硫酸來萃取組蛋白時，效果較好，且所抽出的組蛋白所含雜質較少。(3)經2,4-D處理後，組蛋白的電泳膠質類型不變。但H1a與H1b的比值變大，且所抽出的組蛋白含有較多的非組蛋白鹼性蛋白質。(4)比較由核仁及細胞核所抽出的組蛋白，由結果顯示二者的電泳膠質類型相似，但由核仁所抽出的組蛋白含有較多的非組蛋白鹼性蛋白質，這些蛋白質可能是核醣體蛋白質。(5)結合在組蛋白上的磷酸含量會因2,4-D的處理而增加□□□2,4-D處理24小時後，組蛋白的磷酸令量最高(由□□□增至0.26％)。(6)由32P-orthophosphate標示磷3H-leucine標示蛋白質合成的放射性同位素追綜實驗發現，2,4-D會促進H1的磷酸化作用，且由3H-leucine的標示顯示蛋白質的新合成和磷酸化作用並不一致。 故大豆白化幼前經2,4-D處理後，雖組蛋白的電泳膠質類型不變，但H1a與H1b的比值改變，且H1的磷酸化作用增強。顯然2,4-D促進核醣核酸的合成可能和基因的調節作用有關。

Three-day old so bean seedling were □ □ □ M2, 4-D (pH6 . 0) for 24 hrs. Histones were□ from nuclei or nucleoli of hypocotyls and □ socted to polyacrylamide gel electrophoresis to □ □ effect of 2,4-D on histone modification. For identification of soybean histones, histones □ calf thymus and peas were used as references. The □ of migration for soybean histones on acetic acid □ polyacrylamide gel was H4>H3>H(subscript ab)/H(subscript)2a>H1, simila□ of histones from peas. Histones extracted with 0.25HCl, O.4N H2SO4 □ □acia1 acetic acid were separated on 12.5 □ SDS □lyacrylamide gel by electrophoresis. The electro-□oretic ge1 Pat□ of histones extracted with various □ids we re similar, but the ratios of five groups of histones were different. Histones extracted with glacial acetic acid had more nonhistone basic proteins and showed more bands on polyacrylamide gel. Histone extracted from nucleoli had more nonhistone hasic proteins than that extracted from nuclei. It appears that nonhistone basic proteins are ribosomal proteins. □ did not affent the electophoretic ger pateem of □ histones, but afferted the patio of □ □ □. The pherphorus content of histones increased as a result of 2,4□ □ up to 24 hr, peaking at 24 hr. □ P-orthophosphate for studying phosphory lation of histones during the initial 8 hr treatment of 2,4-D We □ that phosphorylation of H1 histone was enhanced and pho-phorylated H1 histone did not coincide with the hewly synthesiged H1 histone bascd on a(superscript 3) H-leucine □ study. The quantitative changes of H1 histone and the embancement or H1 phosphorylation. by 2,4-D treatment □that the effect of 2,4-D on enhancement of rANA synth□ may partly be due to phosphorylation of H1 histone as well as increase in RNA polymerase activity.