摘要
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）、SeDhadex 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4～6μg，DNA片段大小〉30kb，将纯化的DNA用BamH Ⅰ部分酶切后构建以pLAFR3为载体的混合基因组文库，该文库包含15600个克隆，外源片段DNA平均大小为18kb。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。
The research has been carried DNA from hypersaline environmental sample. combine with proteinase K optimized both the hypersaline environmental sample was 10 out for how to improved the yield and purification of The results revealed that freeze-thaw lysis with SDS amount of DNA extracted （The total DNA yield from 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 4-6 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 BamH Ⅰ 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 idéntity. Among 10 randomly selected clones, 9 clones contained inserting fragments which sequences had not been confirmed.
出处
《广西农业生物科学》
CAS
CSCD
2006年第1期24-29,共6页
Journal of Guangxi Agricultural and Biological Science
基金
国家863计划资助项目(2001AA214141)
