基于土壤宏基因组文库筛选非培养木聚糖酶基因

Screening of xylanase genes from the soil metagenomic library based on uncultured method

采用物理化学法提取土壤基因组DNA，将土壤DNA纯化后采用序列筛选策略获得非微生物纯培养方式的木聚糖酶基因。序列筛选依据木聚糖酶保守序列设计简并引物，PCR扩增获得200bp的木聚糖酶核心序列，构建了约5000个克隆子的序列文库。随机挑取200个克隆子测序，获得11条来自未培养微生物的木聚糖酶核心序列。并对其中细菌源序列1～19进行研究，利用HiTAIL—PCR扩增细菌源序列1—19的木聚糖酶基因全长，分析其编码蛋白，并将其与表达载体pET-28a（＋）连接后导入E．coli表达，超声破壁后测得木聚糖酶酶活力为（19．94±0．3）U／mL。利用序列筛选获取土壤宏基因组源木聚糖酶基因的研究为筛选获得土壤未培养微生物木聚糖酶基因新资源提供了新的途径。

The physical chemistry method was used to extract microbial DNA directly from the soil. The soil DNA was purified to obtain xylanase genes based on function and sequence screening. Primers were designed for sequence screening according to conserved domains of xylanase. Various DNA segments encoding core sequence of xylanases, which were about 200bp,were obtained by PCR amplification and used for constructing a metagenomic sequence li- brary containing about 5000 clones. After that, 200 clones of sequence library were selected randomly and se- quenced. Sequence analysis of clones showed that 11 sequences encoded partial of G11 xylanases, which could be de- rived from uncultured microorganisms. The full-length xylanase gene of 1-19xynA, which was classified as sequence of bacterial source, was acquired by HiTAIL-PCR amplification. Then, analysis on the encoded protein of 1-19xynA was carried out. Finally, the full-length gene was cloned into the expression vector pET-28a for recombinant protein ex- pression. The ceils were harvested by centrifugation and subsequent ultrasonieation. It was found to display activity of （ 19.94 ± 0.3 ） U/mL for intracellular xylanase. Study on xylanase genes from the metagenomic library of soil based on sequence PCR had important value for the development of novel gene resources derived from the uncultured microbes in soil.