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基于宏基因组学的转基因棉田土壤微生物功能多样性分析
引用本文:马世宏,高国庆,刘标,崔中利,曹慧.基于宏基因组学的转基因棉田土壤微生物功能多样性分析[J].安徽农业科学,2010,38(36):20559-20561,20599.
作者姓名:马世宏  高国庆  刘标  崔中利  曹慧
作者单位:南京野生植物综合利用研究院,江苏南京,210042;南京农业大学农业部农业环境微生物工程重点开放实验室,江苏南京,210095;南京农业大学农业部农业环境微生物工程重点开放实验室,江苏南京,210095;环境保护部南京环境科学研究所,江苏南京,210042
基金项目:环保公益性行业科研专项,江苏省自然科学基金项目
摘    要:目的]研究转基因棉田土壤微生物的功能多样性。方法]采用直接法提取转基因棉根际土壤微生物总DNA,用限制性内切酶BamHI进行部分酶切,回收2.0~4.0 kb大小的片段插入到pUC19/BamHI脱磷载体上,转化到DH5α高效感受态细胞,构建宏基因组文库。随机挑取10个克隆序列测序,并通过NCBI预测可能存在的开放式阅读框和进行序列比对分析。结果]该文库的外源插入片段平均长度为2.4 kb,90%以上克隆都含有插入片段,10个序列中存在着多种功能的开放式阅读框,包括一些水解酶、脱氢酶、脂解酶、修饰酶、抗生素及与电子传递链相关的酶类。结论]该研究可为研究转基因棉大面积野外种植以后的生态安全性提供参考。

关 键 词:转基因棉  宏基因组文库  未培养微生物

Analysis of Functional Diversity of Microbes in the Bt Cotton Plot Based on Metagenomics
Institution:MA Shi-hong et al (Nanjing Institute for Comprehensive Utilization of Wild Plants; Nanjing; Jiangsu 210042);
Abstract:Objective] The research aimed to study the functional diversity of microbes in the Bt cotton plot.Method] The total DNA of microbes in rhizospheric soil of Bt cotton was extracted by the direct extraction method,and was partially digested by Bam HI.The fragments between 2.0 to 4.0 kb were recovered and inserted into pUC19/Bam HI vector,then transferred into DH5α,to construct the metagenomic library.DNA sequencing was conducted on 10 clones,then predicated maybe existing open reading frame(ORF) by NCBI and analyzed the sequence.Method] The results showed that average length of inserted fragments was 2.4 kb and over 90% clones had inserted fragments.Ten sequences had various functional ORF,including some hydrolase,dehydrogenase,lipases,modification enzyme,anti-biotic,and enzymes related to the electron transport chain.Conclusion] The study can provide the reference for researching the ecological safety after the Bt cotton planting in wild field in large scale.
Keywords:The Bt cotton  The metagenomic library  The functional uncultureable microorganism
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