植物体内的亚细胞组分分离1

植物体内的亚细胞组分分离

步骤:准确称取植株鲜样0.5000 g,加入20 mL提取液(0.25 mmol/L 蔗糖+50 m mol/L Tris HCl缓冲液(pH 7.5)),研磨匀浆,用尼龙纱布过滤,滤渣为细胞壁部分;滤液在600 r/min下离心10min,沉淀为细胞核部分;上清液在2000 r/min下离心15 min,沉淀为叶绿体部分;上清液在10000 r/min下离心20 min,沉淀为线粒体部分;上清液为含核糖体的可溶部分,每组两次离心,全部操作在4下进行。

植物亚细胞组分的分离

步骤:准确称取鲜样0.5000 g,加入20 mL提取液[0.25mol·L-1蔗糖+50 mmol·L-1 Tris-HCl缓冲液(pH7.5)+1mmol·L-1二硫赤鲜糖醇,研磨匀浆,匀浆液在冷冻离心机300×g下离心30 s,沉淀为细胞壁组分;上清液在2000×g下离心15 min,沉淀为细胞核和叶绿体组分;上清液在10000×g下离心20 min,沉淀为线粒体组分;上清液为含核糖体的可溶组分。全部操作在4℃下进行。

Fractionation of Leaves and Roots.Leaves and roots were homogenized using a mortar and pestle in a medium containing

0.25 M sucrose,50 mM Tris-HCl(pH 7.5),and 1 mm dithioerythritol.All steps were performed at 4 C.The resulting brei was strained through eight layers of cheesecloth,and liquid was

expressed from the residue.The residue was washed twice with the grinding medium.The pooled washes,together with the first filtrate,were centrifuged at 300g for 30 s.The resulting pellet combined with the residue of the cheesecloth filtration contained mainly cell walls and cell wall debris and was designated as cell wall fraction(I).The supernatant of the first centrifugation step was then centrifuged at 20,000g for 45 min to sediment cell organelles.The pellet was taken as organelle fraction(II).The resultant supernatant solution referred to as soluble fraction(III)was employed in subsequent characterization studies as described.Fractions I and II were analyzed for their Cd content.The supernatant,fraction III,was further fractionated by gel filtration ，using Sephadex G-100,G-25,and G-10(Pharmacia Fine Chemicals, Uppsala,Sweden).An aliquot of fraction III was applied to a column(100 x 2.6 cm)of G-100 using 50 mm Tris-HCl(pH 7.5) and 0.1 mM dithioerythritol as eluting buffer.Fractions containing Cd were pooled and chromatographed on calibrated G-25(roots) or G-10(leaves)columns(70 x 2.3 cm).Fractions were collected on an LKB Ultrarac fraction collector.The effluents were monitored at 280 nm with a Gilford spectrophotometer 2400 S.The activities of the glutamate dehydrogenase and malate dehydrogenase were determined according to references 9 and 23.