The Cloning And Function Research Of Cytochrome B5 Gene In Typha Angustata Bory Et Chaub

In order to clone genes responsible for degradation of Trichloroethylene(TCE),, which has been broadly used as a solvent in industry and caused a serious problem of environmental pollution, we chose cattail (Typha angustata Bory et Chaub) as a starting material since it can grow well in a high level of TCE. We isolated the total RNA from treated cattail by TCE, and then made a cDNA library. The double strand cDNAs were further synthesized by SMART PCR synthesis kit and used for making a cDNA library which can express in E. coli. we got some clones tolerant to TCE by screening the library in the presence of TCE. Through plasmid isolation, sequencing and alignment, one of the isolated clones(XP141) contained a putative cytochrome b5 gene. The predicted protein, we found contains a transmembrane domain at its C-termial and a heme domain at its N-termianl. This result indicated that cytochrome b5 may be involved in the degradation of TCE.In order to confirm the function of this cytochrome b5 gene(XPb5) on TCE resistance, we constructed the vector-pYES2-XP141 which can express in yeast, our result showed that yeast transformed with XPb5 were more resistant to TCE than those transformed by empty vetor pYES2In order to find out whether cattail XPb5 can function in cytochrome P450's metabolizing TCE, we constructed some vectors, such as pCW-XPb5 , pCW-CYP1A2/NPR and pCW-XPb5/CYPlA2/NPR which can express in E. coli. Until now, the expression of those vectors in E.coli is under doing.To examine the function of XPb5 in plant resistance to TCE, we transformed the gene into Arabidopsis and tobacco plants, respectively. We have completed the screening of transgenic plants. Detailed dissection of transgenic plants is on going.