土壤宏基因组的提取及基于免培养技术分析细菌16S rDNA  

Preparation of Metagenome from Soil and Analysis on the Sequenceof a Bacteria 16S rDNA by Culture-independant Method

作　　者：方光伟[1] 洪雪梅[2] 蔡丽希[1] 彭锟[1] 林毅[1] 

机构地区：[1]华侨大学生物工程与技术系,福建泉州362021 [2]华侨大学信息科学与工程学院,福建泉州362021

出　　处：《江西农业大学学报》2005年第4期505-507,共3页Acta Agriculturae Universitatis Jiangxiensis

基　　金：福建省自然科学基金项目(B0510011);福建省青年科技人才创新项目(2003J024)

摘　　要：采用CTAB-SDS-冻融法和玻璃粉吸附法提取土壤宏基因组(metagenome),经琼脂糖凝胶电泳后用回收试剂盒对其进行纯化。以细菌16SrDNA通用引物从宏基因组中扩增出V8和V9两个高变区,回收纯化后与克隆载体pMD18-T连接,转化大肠杆菌DH5α,随机挑取一个阳性克隆菌进行序列测定,经BLAST分析表明该序列所代表的是一个不动杆菌属(Acinetobacter)细菌。本研究结果为分析土壤微生物种群结构和直接从土壤中分离功能基因奠定了基础。Two simple and rapid methods, which were CTAB -SDS -freezing -thawing and glass powder absorbing, were applied for metagenome preparation from soil samples. Ultraclean 15 DNA Purification Kit was used to purify the crude metagenome extract prepared by CTAB -SDS -freezing -thawing. PCR amplification for bacteria 16s rDNA by universal primers was achieved from the two metagenome preparations. The products were purified, then ligated into pMD18 -T vector, and transformed into E. coli DHSα. One positive clone was sequenced. Blast analysis showed that the sequence was part of the 16S rDNA of Acinetobacter. This study provides foundation for microbial diversity analysis and direct cloning of functional genes from soil samples.

关 键 词：土壤宏基因组 提取 16S RDNA 免培养 

分 类 号：S154.3[农业科学—土壤学]