枝干树皮宏基因组DNA的提取  

The Extraction of Metagenom DNA in Branch Bark

作　　者：赵玲云[1,2] 范东颖 李燕芳[1,2] 颜霞[1,2] 黄丽丽[3,2] 

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]旱区作物逆境生物学国家重点实验室,杨凌712100 [3]西北农林科技大学植物保护学院,杨凌712100

出　　处：《生物技术通报》2016年第1期74-79,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31101476;31171796);陕西省科学技术研究发展计划项目(2013K01-45);杨凌示范区科技计划项目(2014NY-41);果树腐烂病防控技术研究与示范项目(201203034)

摘　　要：旨在得到一种适用于提取多种枝干树皮的高质量DNA的方法。参考前人提取植物DNA的经验,综合了研磨时加PVP、缓冲液洗涤、SDS与CTAB结合使用、高浓度KAc低温冻融、高浓度Na Cl条件下异丙醇沉淀、DNA完全溶解后加微量RNase A处理等操作,所得5种基因组DNA浓度介于190-970 ng/μL,纯度都很高,皆能被限制性内切酶切割,都可用作模板扩增到细菌16S r RNA基因片段,以苹果树皮DNA为模板扩增到苹果BIP和PDI基因且苹果树皮基因组DNA经测序公司检测,质量满足高通量测序法研究微生物多样性对环境宏基因组的要求。A suitable method capable of extracting high-quality metagenom DNA from a variety of branch barks was developed based on previous experiences of plant DNA extraction. The method consisted of several steps ：adding PVP while grinding materials, washing bark powder with buffer Ⅰ, simultaneously using SDS and CTAB, low-temperature frozen and then melting samples after adding high concentration of potassium acetate（KAc）, precipitating DNA using isopropanol under high concentration of Na Cl, and adding trace RNase A after DNA completely dissolved. Finally, the concentrations of genomic DNA obtained by this method ranged from 190 to 970 ng/μL with all in high purity. All DNA can be digested by restriction endonuclease and used as templates for bacterial 16 S r RNA gene amplification. The DNA of apple bark as the template was amplified into gene BIP and PDI, then the genome DNA of apple bark was detected by a sequencing firm, and the quality satisfied the requirements of environmental metagenome for studying microbial diversity by high-throughput sequencing.

关 键 词：枝干树皮 DNA提取 KAc RNASE A 高通量测序 

分 类 号：Q943.2[生物学—植物学]