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以钦州市犀牛角镇晒盐池内(盐浓度>25%)的土壤样品为研究对象,对土壤混合基因组DNA的提取和纯化方法进行了深入研究,结果表明,冻融法、十二烷基磺酸钠(SDS)和蛋白酶K联合使用的提取法,能有效提高DNA得率,DNA的产量达到每克土壤样品10~15μg。交联聚乙烯吡咯烷酮(PVPP)、Sephadex G-200树脂以及大量胶回收的使用,可有效纯化DNA,纯化的DNA产量可达每克土壤样品46μg,DNA片段大小>30 kb。将纯化的DNA用B amHⅠ部分酶切后构建以pLAFR 3为载体的混合基因组文库,该文库包含15 600个克隆,外源片段DNA平均大小为18 kb。通过DNA序列测定和同源性比较,对从文库中随机挑取的10个克隆进行了序列分析,发现9个外源插入片段包含未知DNA序列。
Abstract：The research has been carried out for how to improved the yield and purification of DNA from hypersaline environmental sample.The results revealed that freeze-thaw lysis with SDS combine with proteinase K optimized both the amount of DNA extracted(The total DNA yield from hypersaline environmental sample was 10~15 μg DNA/g) and the molecular size of the DNA(maximum size >30 kb).PVPP with Sephadex G-200 resin combine with gel electrophoresis purification was found to be the best method for removeal of humic contaminants while minimizing DNA loss during the purification.Finally,the total DNA yield from hypresaline environmental sample was 46 μg DNA/g after the purification.In the end,the metagenomic library was constructed by inserting restriction fragments of the purified DNA into plasmid pLAFR3 and then transferring into DH5α after the genomic DNA was digested with the restrictions enzyme BamHⅠ partly.The library contained 15 600 positive clones and the average foreign fragment DNA was about 18 kb.Clones were analyzed by DNA sequencing and comparing with gene's identity.Among 10 randomly selected clones,9 clones contained inserting fragments which sequences had not been confirmed.
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基本信息:
中图分类号:S154.3
引用信息:
[1]孙栋,唐莉丽,王倩倩,等.高盐极端环境土壤基因组DNA的分离纯化方法研究及基因文库的构建[J].广西农业生物科学,2006(01):24-29.
基金信息:
国家863计划资助项目(2001AA214141)
2006-03-30
2006-03-30