Diversity Of Xylanase Genes In Greater Khingan Mountains Soil And Cloning And Expression Of Xylanase Gene

In recent years, energy shortages and environmental pollution has become a global problem that can’t be ignored, development and utilization renewable resources is the most promising way to solve these problems, and the degradation of xylan and utilization is one of best solution. Xylanase divided into GH10 and GH11 glycoside hydrolase family. At present, there are many methods of gene cloning, expression and protein purification xylanase in relevant research, but these studies mainly focus on pure culture of microorganisms, and a lot of soil microbial xylanase gene related research also is less, in this study we analysis microbial xylanolytic enzymes on genetic diversity, gene cloning and expression, is expected to be a more comprehensive understanding of the gene, and could obtain the coding of xylanase enzyme with highly activity, the purpose of the gene can be used in practice.Greater khingan mountains forest surface soil are rich in lignocellulose and contain a large number of xylan degradation enzyme producing microorganisms. Collected in the area of forest soil on the surface, this paper respectively using high-throughput sequencing analysis of microbial species and glycoside hydrolase GH10and GH11 genetic diversity, some of them may be new xylanase gene cloning and expression. The main research results and conclusions are as follows:1. microbial species diversity:16S rDNA sequencing include 27273 sequence, Sphingomonas species belong to the first advantage bacterium, accounting for 6.75% of the total, followed by Porphyrobacter groups and Bradyrhizobium groups, the total of 5.58% and 4.3% respectively. Alpha index, shannon diversity index is 7.002407, the ACE index is 14704.052, Chao 1 index was 10049.676.2. xylanase gene diversity:metagenome DNA from soil in the amplification GH10 and xylanase gene fragment of GH11 family, get 1144 GH10 xylanase fragment sequence and 2712 GH11 xylanase fragment sequence. Sequence alignment analysis showed that GH10 family the highest similarity in the range of 35%-9O%, the Phylogenetic is divided into eight branches, dominant genera is sorangium; GH11 family the highest similarity in the range of 48%-94%, Phylogenetic divided into 11 branches, dominant species is Streptomyces.3. Gene clone and expression:from environmental samples using IPCR cloned from xynH6 gene, and in E. coli BL21 (DE3) of expression, the results showed that xylanase enzyme activity was 11 U/ml.The above results show that the greater khingan mountains surface soil microbial species diversity and xylanase gene diversity is highly, and some of the xylanase gene sequences has high novelty; The genetic diversity of soil GH10 xylanase and novelty than GH11. This research for the greater hinggan mountains forest soil diversity of microbial xylanase gene and use to provide foundation and basis.