遗传 ›› 2006, Vol. 28 ›› Issue (10): 1287-1293.

• 研究报告 • 上一篇    下一篇

碱性土壤微生物基因的克隆和多样性分析

胡婷婷; 蒋承建; 梁 璇; 隆文杰; 武 波   

  1. 广西大学微生物及植物遗传工程教育部重点实验室, 广西亚热带资源保护利用重点实验室, 南宁 530005
  • 出版日期:2006-10-01 发布日期:2006-10-01

Cloning and Diversity Analysis of Microorganism Genes from Alkalescence Soil

HU Ting-Ting; JIANG Cheng-Jian; LIANG Xuan; LONG Wen-Jie; WU Bo   

  1. The Key Laboratory of Microbial and Plant Genomic Engineering, the Ministry of Education of China; Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Nanning 530005, China
  • Online:2006-10-01 Published:2006-10-01

摘要: 从碱性土壤样品中直接抽提和分离宏基因组DNA, 首先构建了包含5 562个阳性克隆的碱性土壤16S rDNA文库, 随机抽取9个克隆测序后构建的系统进化树表明了碱性土壤环境微生物种群基因的多样性。纯化土壤宏基因组DNA后采用EcoRⅠ酶部分酶切处理, 我们又构建了以pGEM-3Zf(+)为载体的DNA部分文库AL01。AL01文库包含23650个克隆, 随机插入载体的外源DNA片段平均大小为3.2 kb左右, DNA文库的总容量为75.68 Mb。建库效率为从每克环境样品中获得6 000个左右的含随机外源DNA片段插入载体的克隆。采用酶活筛选策略, 我们从AL01文库中筛选到一个编号为pGXAA2011的阳性克隆携带有一个完整的碱性蛋白酶基因。蛋白酶活性检测其酶活作用最佳温度为40℃, 最适作用pH 值为9.5。另外, 我们还克隆和表达了一个新型b-葡萄糖苷酶基因unglu01, 该基因和现有数据库中的b-葡萄糖苷酶基因没有任何DNA或者氨基酸水平的同源性。将unglu01基因的ORF与表达载体pETBlue-2连接后导入宿主菌株Tuner(DE3)pLacI中, 该重组表达克隆在含柠檬酸高铁铵和七叶苷的LA平板上表现清晰的b-葡萄糖苷酶活性, SDS-PAGE电泳可以检测到29 kDa大小的目的蛋白。

关键词: 未培养微生物, b-葡萄糖苷酶, 碱性蛋白酶, 文库, 宏基因组

Abstract: The metagenomic DNAs were extracted and purified from alkalescence environmental samples directly. On the basis of the metagenomic DNA, the alkaline soil 16S rDNA library composed of 5 562 positive clones was constructed. The phylogenic tree indicated that the bacteria from the alkaline soils were bio-diversity. The metagenomic DNA library named AL01 was constructed by inserting restriction fragments of the purified DNAs into plasmids pGEM-3Zf(+) vector. This library contained 23 650 positive clones and the average foreign DNA fragments were about 3.2 kb. The length of the library covered 75.68 Mb. The efficiency of the metagenomic library was approximately 6 000 clones from 1g dry soil samples. After screening AL01 DNA library with the screening tactics of enzymes, we confirmed that a positive clone, designated pGXAA2011, contained an alkaline protease gene AP01. Enzymatic analysis proved that its reaction optimum pH was 9.5 and the optimum temperature was 40℃. Furthermore, a clone, designated pGXAG142 was screened from metagenomic DNA library, which expresses β-glucosidase. DNA sequence indicated that the potential ORF of pGXAG142, which was named unglu01, there was no DNA or amino acids identity with the known β-glucosidase genes in the Genbank. The integrated ORF was cloned into pETBlue-2 vector and was then transformed into Tuner(DE3)pLacI. The recombinant expression clone could express β-glucosidase on the screening plate clearly and the analysis of SDS-PAGE indicated that the target protein was about 29 kDa.

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