棉粕发酵样品总DNA的提取方法

A method of DNA extraction from fermented cottonseed meal

棉粕是我国重要的饲用蛋白质原料,采用微生物发酵原棉粕脱毒是当前重要的高效技术手段。为了使用分子生态学方法监测微生物发酵棉粕的过程,优化发酵工艺,试验先以脱色缓冲液去除棉粕发酵样品中的游离棉酚及色素类杂质,继之以溶菌酶—SDS—蛋白酶K温和裂解,提取获得总DNA产量达1.8~6.4μg/g棉粕样品（湿）,DNA的提取效率达89%以上,片段大小均在23 kb左右,不经纯化即可直接进行PCR扩增和限制性酶切。以该DNA为模板扩增16S rRNA V3区片段,可用DGGE分析棉粕发酵过程中细菌群落结构的变化。该方法是一种适用于棉粕发酵样品总DNA提取的简便、可靠方法。

Cottonseed meal is one of the important forage protein raw materials.Microbial fermentation is one of the most efficient methods for detoxification.In order to monitor the microbial fermentation process and optimize the fermentation parameters of cottonseed meal,an efficient DNA extraction method was established.Decoloration buffer was used to remove free gossypol and other polyphenolic pigment from fermented cottonseed,and then lysozyme,SDS and proteinase K were combined for fragmentation.As a result,the total DNA yields ranged from 1.8 to 6.4 μg per gram of wet fermented cottonseed meal,the DNA extraction efficiency was more than 89%,and the fragment size was about 23 kb.The extracted DNA could be used directly for PCR amplification and restriction enzyme digestion without additional purification.In addition,using the extracted DNA as template,the 16S rRNA V3 fragments were amplified and then analyzed by DGGE to investigate the diversity and change of microbial community structure of the fermented cottonseed meal.Therefore,this is a simple,reliable protocol of DNA extraction from the fermented cottonseed meal.