【作者】 马莉莉；

【导师】 宗浩； 宋培勇；

【作者基本信息】 四川师范大学 ， 细胞生物学， 2010， 硕士

【摘要】 从贵州石漠化重灾区安顺、毕节和黔西南等地采集土样,设计比较了TENS-乙酸铵直接提取法、SDS高盐直接提取法、间接提取法和试剂盒提取法对石漠化环境土壤DNA的提取效果。以试剂盒提取的DNA为模板,采取TA克隆策略,对6份土样的宏基因组DNA进行了16S rDNA扩增,胶回收PCR产物,和pMD-18T载体链接,转化宿主菌DH5α,通过蓝白斑试验筛选阳性克隆,提取质粒,随机挑取23个阳性克隆的质粒送生物公司测序,将测序结果登录NCBI,利用BLAST工具在GeneBank中查找相似性序列,找出与其相似性最高的菌种。利用ClusterW2进行在线多序列比对,构建出系统发育树。相似性比对结果以及系统发育分析揭示在贵州石漠化这一特殊生境中,微生物种类有限,除了三个在GeneBank中还没有的菌种外,几乎全为柠檬酸杆菌,优势菌为柠檬酸杆菌。更多还原

【Abstract】 The soil samples were collected from rocky desertification areas, Anshun,Bijie in GuiZhou province and the southwest of GuiZhou .The extraction effects of the desertification environment soil DNA were designed and compared among TENS-ammonium acetate direct method, SDS high-salt direct method, indirect method and kit extraction method .The template of follow-up experiments was soil DNA extracted by kit extraction method .The follow-up experiments as follows: metagenomic DNA of soil samples extracted from 6 soil samples was amplified by 16SrDNA; PCR products were purified from agarose gel and linked into pMD-18T vector and transformed into host strain DH5α; positive clones were selected through blue and white blots test; plasmids were extracted; 23 positive clones plasmids were selected randomly and sequenced by biology company ,the sequenced results compared with the similar sequences searched by BLAST tool in the GeneBank on NCBI to get the highest similar bacteria .Finally, phylogenetic tree was constructed by using ClusterW2 on-line multiple sequence alignment. The similarity comparison results and phylogenetic analysis reveal that microorganisms are limited in the special desertification environment of Guizhou. They are nearly Citrobacter except three bacterias not to be found in the GeneBank .So the dominant bacteria is Citrobacter.更多还原