摘要
采用国产硅胶GF254(60型)代替进口Glass bead的改良方法抽提土壤微生物DNA,然后设计了一对扩增木聚糖酶基因片段的新简并引物,对抽提的土壤微生物DNA进行PCR扩增。扩增片段连接pMD18T载体,转化大肠杆菌,重组片段通过酶切进行RFLP分析后,测序分析得到10个木聚糖酶基因片段。对所得片段翻译的氨基酸序列进行BLAST分析表明有8个片段与来自放线菌的木聚糖酶具有较高的同源性,2个与假单胞菌的木聚糖酶具有较高的同源性。所得10个木聚糖酶片段的氨基酸序列同源性比较显示,第27个氨基酸均为天冬酰胺(N),暗示这些来自土壤微生物DNA的基因片段编码耐碱的木聚糖酶。通过构建系统进化树,发现扩增的木聚糖酶片段之间的相似性均在70%以上。
An improved method was used to extract soil microbial DNA by indigenous silica gel instead of imported glass beads. A new degenerate PCR-primer pair P1/P2 was designed to amplify directly xylanase gene fragments from soil microbial DNA. PCR products were ligated with pMD18T vector. Transformants were got by transforming ligation product into E. coll. Ten different recombinants that were confirmed by RFLP were sequenced. The sequence analysis shows that all of the ten fragments encode partial xylanase. The BLAST of deduced amino acid of fragments show that eight out of the ten were high in sequence homology with xylanase from Actinomycetes, two with Pseudomonas. Asn27 was found in all partial xylanase based on the alignment of deduced amino acid sequences, which means that all xylanase fragments were alkali-tolerant. Phylogenetic tree shows that all xylanase fragments were positive and over 70% of similarity.
出处
《土壤学报》
CAS
CSCD
北大核心
2006年第2期295-299,共5页
Acta Pedologica Sinica
基金
国家高技术863计划(2001AA214161
2002AA227011)
湖北省自然科学基金项目(2005ABA201)资助
