摘要
为了满足分子标记辅助育种中基因型鉴定、基因克隆、测序的需要,本研究借用棉签等具吸附功能的材料代替移液器,依次直接在2种DNA提取液中操作,建立了一种简单、快速、环保的水稻叶片DNA提取方法。结果表明,提取溶液通过PCR扩增,满足基因扩增对模板的要求;同时,对其进行了灵敏度测试,稀释107倍仍然能够扩增出目标片段;最后通过PCR、克隆载体连接等分子生物学手段,验证此方法也满足测序的需要。本方法能够在90s内完成植物叶片DNA的提取,解决了目前植物基因组DNA提取方法需要离心机等昂贵特殊设备、提取步骤繁锁、提取过程中刺激味大、提取时间长等问题,有利于大规模开展分子标记辅助育种中材料基因型的鉴定,具有简单、快速、廉价、环保等优点。
In order to meet the needs of genotype identification,gene cloning and sequencing in molecular marker assisted breeding,this study used absorptive function materials such as cotton swabs to replace the pipette and directly operated in two kinds of DNA extraction solution in turn,and established a simple,rapid and environmentally friendly method for DNA extraction in rice leaves.The results showed that the extracted solution could meet the requirement of the template by PCR amplification.At the same time,a sensitivity test was carried out and it showed that the target fragment can be amplified with DNA extraction solution diluted 10 7 times.Finally,molecular biological operations such as PCR and clone vector connection were performed to verify that this method also met the need of sequencing.This method can complete the extraction of plant leaf DNA in 90 seconds,it solves these questions that current plant genomic DNA extraction methods require expensive special equipment such as centrifuges,and extraction steps are tedious,big pungent taste in the process of extraction and long extraction time in the extraction process.It is beneficial to the identification of material genotype in molecular marker assisted breeding on a large scale,it has the advantages of simple,fast,cheap,environmental protection.
作者
陈晓军
王敬东
宋海丽
李树华
樊云芳
CHEN Xiaojun;WANG Jingdong;SONG Haili;LI Shuhua;FAN Yunfang(Agricultural Biotechnology Research Center,Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan Ningxia 750002,China;College of Biological Science and Engineering,North Minzu University,Yinchuan Ningxia 750021,China;Wolfberry Engineering and Technological Institution,Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan Ningxia 750002,China)
出处
《种子》
北大核心
2018年第11期26-29,34,共5页
Seed
基金
国家自然科学基金(31460338)
宁夏农林科学院科技创新先导资金项目(NKYJ-17-27)
宁夏农业育种专项(2013NYYZ 03)
