The Genomic DNA Library Construction Of Erwinia Carotovora CXJZ11-01 And Cloning Of Xylanase Gene

Xylanases have wide commercial applications in industrial processes,such as feed ,paper and pulp,foodstuff and energy industry. With the development and application of the new technologies , such as molecular biology , structural biology and protein engineering , many progresses have been made in the research of structures and functions of xylanase. With the development and application of the new technologies ,many progresses have been made in the study of xylanase gene , including the molecular cloning ,the expression and secretion in homologous and heterogenous hosts , the structure and the function of xylanases and in the reconstruction of the properties of xylanases for industrial production. Research Fellow Liu Zhengchu , Institute of Bast Fiber Crops in CAAS, has taken the lead of transforming the plasmid in strain ly into the Erwinia Carotovora and obtaining a series of variant strains by means of genetic engineering. On the basis of preparatory work, we have screened the high-yield of xylanase strain Erwinia Carotovora CXJ11-01. the genomic library of Erwinia Carotovora CXJ11-01 was constructed, and the xylanase gene was cloned from the library. The xylanase gene sequencing and analysis was also completed. This study will lay a foundation of further research on the construction of xylanase high efficiency expression system and the obtaining of efficient herbaceous extraction gene engineering strains:1 By using selective culture media and DNS detection method, we screened xylanase high yielding strain CXJ11-01 from more than 300 Erwinia Carotovora variant strains. The results from comparing the dimension of the transparent zones of the secondary screening strains on the plates and determining the enzyme activity of the fermentation broth all indicated that the enzyme activity for xylanase is: CXJZ11-01>>CXJZ11-03>CXJZ11-02>T85-1.2 By using shot-gun method,the genomic DNA library of the efficient herbaceous extraction strain E.carotovora CXJZ11-01 was constructed.Using the PCR method to the positive clones, it is indicated that they included the identical xylanase genes.The xylanases gene was successfully cloned,analyzad and expressed.3 the genomic DNA library of the efficient herbaceous extraction strain E.carotovora CXJZ11-01 was constructed and its xylanases gene was successfully cloned.The gene is registered at GenBank numbered EU233656.The sequence length of the xylanases gene is 708bp,including a 642bpORF which encodes for 213AA of molecular weight of 23.36KDa.It's an extracellular enzyme classified as hydrolase 11.