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对微分离的大豆单染色体的克隆及其PCR产物的原位杂交分析

周奕华, 胡赞民, 王兰岚, 陈正华

中科院遗传研究所.北京 100101

摘要

通过玻璃针分离法从大豆(Glycine max L.)根尖细胞中期分裂相中显微分离出一条染色体,经Sau3A人工接头介导的两轮PCR后,将其第二轮扩增产物克隆到质粒载体上,构建了单染色体质粒文库。经分析,该微克隆文库包含约200 000个重组子。随机挑选178个重组子进行鉴定,证明该文库的插入片段主要介于200-1 800bp之间,平均大小830bp;其中,中、高拷贝重复序列占44%,单、低拷贝序列占56%。微分离染色体体外扩增产物的原位杂交分析表明它们来自于大豆基因组,然而却未能将其只标记在该条微分离的染色体上。

关键词： 微分离; 微克隆; 单染色体; 大豆

Cloning A microdissected chromosome of soybean and in situ analysis of the chromosome PCR products

Zhou Yihua, Hu Zanmin, Wang Lanlan, Chen Zhenghua

Institute of Genetics,Chinese Academy of Sciences.Beijing 100101

Abstract

A single chromosome was microdissected from the metaphase chromosomes of soybean by using a glass needle. After two rounds of Sau3A linker-adaptor-mediated PCR, the second-round PCR products from the microdissected chromosome were cloned into plasmid vector to generate a chromosome-specific microclone library, which included approximately 200 000 recombinant clones. 178 randomly selected clones were estimated to be ranging in size from 200-1 800bp, with an average size of 830bp, of which approximately 44% were high/medium copy clones, while 56% were low/unique copy clones. In situ hybridization of the chromosome PCR products to the mitotic metaphase spreads showed that the PCR products were hybridized to the genomic soybean DNA, but the labeling was not restricted to the microsissected chromosome.

Key words: Microdissection;Microcloning;A single chromosome;Soybean

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引用本文：

周奕华, 胡赞民, 王兰岚, 陈正华. 对微分离的大豆单染色体的克隆及其PCR产物的原位杂交分析[J]. 生理学报 2000; 52 (3): .

Zhou Yihua, Hu Zanmin, Wang Lanlan, Chen Zhenghua. Cloning A microdissected chromosome of soybean and in situ analysis of the chromosome PCR products. Acta Physiol Sin 2000; 52 (3): (in Chinese with English abstract).