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为了建立桑树根围土壤微生物群落结构的分子生态学试验技术体系,采取不同的细胞裂解方法及DNA沉淀方法直接提取桑树根围土壤微生物总DNA,并以采用试剂盒提取的桑树根围土壤微生物总DNA为参照,筛选出一种能有效提取高纯度、多样性土壤微生物总DNA的方法:以十六烷基三甲基溴化铵(CTAB)、溶菌酶、蛋白酶K、十二烷基磺酸钠(SDS)为裂解液,经反复冻融裂解细胞,在DNA提取液中添加聚乙烯吡咯烷酮K30(PVP K30)、牛血清蛋白(BSA)、CaCl2去除腐殖酸,并结合聚乙二醇8000(PEG8000)沉淀和纯化DNA。该方法简便、快速,获得的桑树根围土壤微生物基因组DNA分子长度在23 kb以上,土壤鲜样的微生物DNA产率为11.2μg/g,且纯度较高,D(260 nm)/D(230 nm)=1.42,D(260 nm)/D(280 nm)=1.37,可直接用于PCR扩增及变性梯度凝胶电泳(DGGE)分析。
In order to establish a molecular ecology experimental technology system of mulberry rhizosphere soil microbial community structure, different methods of cell lysis and DNA precipitation were used to directly extract total microbial DNA of mulberry rhizosphere soils. DNA as reference, a method of extracting total DNA from soil microorganisms with high purity and diversity was screened out. CTAB, lysozyme, proteinase K, dodecyl sulfonic acid Sodium (SDS) was used as lysis solution to lyse cells by repeated freeze-thaw cycles. Polyvinylpyrrolidone K30 (PVP K30), bovine serum albumin (BSA) and CaCl2 were added to the DNA extract to remove humic acid. Polyethylene glycol 8000 (PEG8000) to precipitate and purify the DNA. The method was simple and rapid, and the length of genomic DNA of mulberry rhizosphere soil was above 23 kb. The yield of soil microbial DNA was 11.2 μg / g and the purity was higher. The D (260 nm) / D (230 nm) nm) = 1.42, D (260 nm) / D (280 nm) = 1.37, which can be directly used for PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis.