以小麦根尖分生组织细胞核及染色体制备高分子量DNA

作物学报 ›› 2006, Vol. 32 ›› Issue (12): 1920-1923.

以小麦根尖分生组织细胞核及染色体制备高分子量DNA

郭东伟^1,2;佘茂云^2,3;李连城²;陈耀峰¹;陈明²;徐兆师²;马有志^2,*

1西北农林科技大学农学院，陕西杨凌712100；2中国农业科学院作物科学研究所/农业部遗传育种重点开放实验室，北京100081；3新疆农业大学农学院，新疆乌鲁木齐830052

收稿日期:2006-03-10 修回日期:1900-01-01 出版日期:2006-12-12 网络出版日期:2006-12-12
通讯作者: 马有志

High Molecular Weight DNA Preparation from Nuclei or Chromosomes of Root Meristem of Wheat

GUO Dong-Wei¹²,SHE Mao-Yun²³,LI Lian-Cheng²,CHEN Yao-Feng¹,CHEN Ming²,XU Zhao-Shi²,MA You-Zhi^2*

1 Agronomy College, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, Shaanxi; 2 Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/ Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081; 3 Agronomy College, Xinjiang Agricultural University, Urumqi 810012, Xinjiang, China

Received:2006-03-10 Revised:1900-01-01 Published:2006-12-12 Published online:2006-12-12
Contact: MA You-Zhi

摘要/Abstract

摘要：

为了克服以叶片为材提取HMW DNA的局限性，优化了一套简便、实用的提取方法。该法以细胞周期同步化处理的根尖分生组织为材，利用机械匀浆释放细胞核或中期染色体制备悬浮液，再以蔗糖密度梯度离心和流式分选技术分别分离细胞核和染色体，经去蛋白、酶切，透析获得HMW DNA。经检测，来自200条根尖（10个胶块）的HMW DNA的浓度约为4~20 ng/µL，而连接、转化后的BAC克隆平均插入片段超过100 kb。证明该法适用于提取HMW DNA以构建BAC文库。

关键词: 高分子量DNA, 蔗糖密度梯度离心, 细胞周期同步化, 染色体流式分选, BAC

Abstract:

A simple and practical method was optimized to overcome the limitations of current method by which HMW DNA was prepared from leaf powder. Root tips of synchronized meristem were homogenized to make preparing suspension containing intact metaphase chromosomes and nuclei, which were further isolated by sucrose gradient centrifugation and flow sorting, respectively. Finally, HMW DNA was prepared with deproteinization, digestion and dialysis of nuclei and chromosomes. The results showed that concentration of the HMW DNA from 200 root tips (10 plugs) was about 4–20 ng/µL (4 ng/µL in chromosomes DNA and 20 ng/µL in nuclei DNA). The average insert fragment size was more than 100 kb after linage, transformation and insert fragment examination by pulse field gel electrophoresis for HMW DNA from nuclei. The similar results were obtained by HMW DNA prepared from chromosomes. It is confirmed that the method is applicable to prepare HMW DNA for construction of BAC library.

Key words: High molecular weight DNA, Sucrose gradient centrifugation, Synchronization of cell cycle, Chromosome flow sorting, BAC

中图分类号:

S512

郭东伟;佘茂云;李连城;陈耀峰;陈明;徐兆师;马有志. 以小麦根尖分生组织细胞核及染色体制备高分子量DNA[J]. 作物学报, 2006, 32(12): 1920-1923.

GUO Dong-Wei;SHE Mao-Yun;LI Lian-Cheng;CHEN Yao-Feng;CHEN Ming;XU Zhao-Shi;MA You-Zhi. High Molecular Weight DNA Preparation from Nuclei or Chromosomes of Root Meristem of Wheat[J]. Acta Agron Sin, 2006, 32(12): 1920-1923.

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