《植物生理学报》 2013, 49(2): 195-199

不同的固定方法对激光显微切割分离油松胚珠雌配子体的影响

王英旗, 姚阳, 陈彬丽, 张淑静, 李芳, 郑彩霞*

北京林业大学生物科学与技术学院, 北京100083

通信作者：郑彩霞；E-mail: zhengcx@bjfu.edu.cn；Tel: 010-62337717

摘　要：

为了精确分离油松胚珠雌配子体, 获得单一种类的细胞, 进一步研究油松胚珠发育分子调控的时空特异性, 本文利用激光显微切割技术(LCM)对2年生油松胚珠雌配子体进行了微切割; 比较了冷丙酮(4 ℃)、卡诺固定液、FAA固定液固定及冻藏处理4种不同方法对冰冻切片的影响。结果表明, 冻藏处理结合冰冻切片可以保证雌配子体的完整性, 采用该方法获得了不同时期油松胚珠雌配子体, 为后续油松胚珠雌配子体发育相关分子机制的研究提供了实验材料。

关键词：油松胚珠; 雌配子体分离; 冰冻切片; 激光显微切割

收稿：2013-01-04   修定：2013-01-30

资助：国家自然科学基金(31170563)和教育部高等学校博士学科点专项科研基金(20110014110014)。

Effects of Different Fixing Methods on Isolation of Ovules in Female Gameto-phyte of Pinus Tabulaeformis Carr. by Laser Capture Microdissection

WANG Ying-Qi, YAO Yang, CHEN Bin-Li, ZHANG Shu-Jing, LI Fang, ZHENG Cai-Xia*

College of Biological Science and Technology, Beijing Forestry University, Beijing 100083, China

Corresponding author: ZHENG Cai-Xia; E-mail: zhengcx@bjfu.edu.cn; Tel: 010-62337717

Abstract:

In order to separate the ovules in female gametophyte accurately, acquire single cell type and further study the molecular mechanisms of the ovules in female gametophyte during development, laser capture micro-dissection (LCM) was used to isolate the ovules in female gametophyte of 2-year old Pinus tabulaeformis. And the effects of four methods, including cold acetone fixation (4 ℃), Carnoy’s fixation, FAA fixation and frozen storage, on frozen sections were compared. The results showed that frozen storage combined with frozen sec-tions could ensure the intact morphology of female gametophyte, and obtain the female gametophytes from dif-ferent developing stages. The method could provide experimental materials for investigating the molecular mechanisms of the development of ovules in female gametophyte of Pinus tabulaeformis.

Key words: ovule of Pinus tabulaeformis; female gametophyte separation; frozen section; laser capture micro-dissection