摘要
本文以春兰(Cymbidiumgoeringii(Rchb.f.)Rchb.f.)为研究试材,对植物基因组DNA提取方法中的若干影响因子诸如药品、试剂配制方法、DNA取材部位、蛋白质去除次数、DNA析出时间、提取样品水浴时间等进行了比较分析,试图找出春兰基因组DNA提取的最佳方法。结果表明:采用进口的CTAB提取效果比国产的CTAB要好,特别适用于花药的提取,蛋白质去除效果很好;SDS提取缓冲液配制方法很重要,不同的SDS缓冲液配制方法,其提取效果差异较大。春兰花苞的DNA提取得率较叶片高,但是叶片DNA的纯度则相反,叶片较花苞高。在提取花苞基因组DNA过程中,增加氯仿/异戊醇抽提次数,对去除DNA中蛋白质、多糖及其它杂质有一定的作用,但是对DNA的损失也是很明显的。从实验效果来看,氯仿/异戊醇抽提次数以2次为宜。提取基因组DNA在异丙醇沉淀时,采取挑取絮状沉淀的方法,也可减少蛋白质、多糖等其它杂质的影响,从而提高DNA纯度。无论是叶片还是花苞材料,采用异丙醇沉淀5min,一般析出的以核酸基因组DNA为主,蛋白质为次,核酸析出量在80%以上。继续沉淀(15min)的产物中DNA含量明显较低,而且沉淀的纯度相对不高,因此异丙醇沉淀时间可以缩短到10min之内以提高DNA的纯度。使用CTAB法提取DNA时,水浴时间至少需要30min,过短的水浴时间会造成DNA得率的下降,同时过长的水浴时间,如超过1h,则可能会引起DNA的降解,或者最后产物中增加其他杂质,因此,适宜的水浴时间当为40￣60min。
Isolation ofgenome DNA in Cymbidium goeringii （Rchb.f.） Rchb.f. was studied in this paper. Some influence factors on extraction of plant genome DNA, such as drugs, the reagent confecting method, material parts of obtaining DNA, the times of getting rid of protein, the time of separating out DNA, water bath time of the sample, and so on had compared and analyzed, it was tried to find out the best extraction method ofgenome DNA in Cymbidium goeringii （Rchb.f.） Rchb.f.. The result indicated that the import CTAB （Cetyl-dimethylethyl-ammonium bromide, BBI, USA） was better than the homemade CTAB （Cetyl-trimethyl-Ammonium Bromide, Shanghai, China） and it was suitable specially for extraction genome DNA of flower on orchid and was effective on getting red of protein. It was very important about SDS buffers confected method, and its effect was much different by different SDS buffer in DNA isolation. DNA percentage of flowers was higher than leaves in orchids, but the reverse was DNA purity. It had the certain function to remove the protein, the polysaccharide and other impurities by increasing chloroform/isoamyl alcohol extraction times in genome DNA isolation process, but it was also very obvious to genome DNA loss. According to the experimental effect, it was suitable for chloroform/isoamyl alcohol extraction to take two times. When the genome DNA was separated out by isopropyl alcohol, it may also reduce pro tein, polysaccharide and so on other impurities with fishing for precipitates, and thus increased the DNA purity. Generally, genome DNA was main parts which accounted for above 80% in deposit by isopropyl alcohol in 5 minutes, and the protein was next, regardless of the leaves and flowers. However, genome DNA was lower in next precipitates than before, and the purity is not high relatively, so the isopropyl alcohol precipitation time may reduce 10 minutes in order to enhance DNA purity. It took at least 30 minutes with water bath when the genome DNA was isolated by CTAB. It would reduce DNA percentage with excessively short water bath time, at the same time it would decomposed genome DNA or increased other impurities with long water bath time such as above 1 hour. So it was suitable to take 40-60 minutes with water bath.
出处
《分子植物育种》
CAS
CSCD
2006年第1期135-142,共8页
Molecular Plant Breeding
基金
本项目得到宁波市科技合作基金（2003D10023）
宁波市农科院院长基金资助.
作者简介
通讯作者，zdsun．cn@163．com
