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批量分离玉米活性精细胞方法的某些改进

徐恒平, 曹宗巽

北京大学生命科学学院.北京 100871

摘要

目前,从玉米花粉中分离活性精细胞时,若用半乳糖作低渗介质,虽然得率较高,但是花费较大;若以蔗糖为介质,花粉胀破率(约50%)与精细胞得率(17%左右)均较低。而且,所用的Percoll不连续密度梯度离心,对于大批量分离操作繁琐。通过4种不同方法对玉米花粉的胀破效果的比较,发现经BKS35温育后再以蔗糖低渗缓冲液处理,花粉胀破率可提高到95%以上。该花粉胀破液经过滤,用30%Percoll溶液离心分离精细胞,得率达23.4%。用这种改进的批量分离方法,6h内可从100g新鲜花粉中获得约1.2×10~(8)生活精细胞。它不仅提高了产率,以满足玉米精细胞表膜特异蛋白生化分析的需要,而且还简化了操作,更具有经济节省等优点。

关键词： 纯系玉米; 批量分离; 生活精细胞; Percoll不连续密度梯度离心

An improved procedure for the mass isolation of viable sperm cells from pollen of Zea mays L.

Xu Hengping, Tsao T.H.

College of Life Sciences,Peking University.Beijing 100871

Abstract

Mass isolation of viable sperm cells from maize pollen was further studied to increase the yield of sperm cells. Four different approaches on pollen bursting and sperm collection were tried. Results showed that pollen bursting rate could reach as high as 95% with BKS35 preincubation followed by buffered osmotic shock solution (in sucrose 0.44 mol/L, Mes 2 mmol/L and 0.1%BSA, pH 6.7). After examining the isolation effeciency of 4 different Percoll solutions, it is found that the higher sperm yield (ca. 23.4%) could be obtained using 30% Percoll in buffered sucrose solution. Using the above modified two-step osmotic shock followed by 30% Percoll centrifugation, about 1.2×10~(8) viable sperm cells could be obtained from 100 g fresh pollen sample within 6 h.

Key words: Zea mays L.;Inbred line;Mass isolation;Sperm cell yield

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引用本文：

徐恒平, 曹宗巽. 批量分离玉米活性精细胞方法的某些改进[J]. 生理学报 1997; 49 (3): .

Xu Hengping, Tsao T.H.. An improved procedure for the mass isolation of viable sperm cells from pollen of Zea mays L.. Acta Physiol Sin 1997; 49 (3): (in Chinese with English abstract).