Cloning And Function Research Of A Secretory_peroxidase Isoenzyme From Chimonanthus Praecox Flower

Abiotic stresses, including drought, high salinity and extreme temperatures, greatly impair the growth and development of plants, affected the productivity and quality of crops, baffle the development of agriculture.Class III family peroxidases are important protective enzymes , which are related with the resistance of the plant and regarded as one of the biomarkers of environment stresses. By exploring the mechanism that how class III peroxidases response to environmental stresses thus to strengthen the cultivation of new varieties of crops , provide the theory basis and the technical references for the isoenzymes of class III peroxidases research.Chimonanthus praecox (L.) Link has typical properties of drought and freezing tolerance. The full-length cDNA library of Chimonanthus praecox (L.) Link was constructed with the Creator SMART cDNA Library Construction Kit of clontech company and the large-scale ESTs sequencing at random was conducted.Then the sequence of classIII peroxidase was obtained and analyzed. the open reading frame (Open Reading Frame, ORF) sequence was 1047bp and encoding 348 amino acids of class III family secretory peroxidase , pI 9.63,37.6KD. By phylogenetic tree analysis, the gene was closely related to peroxidase12 from Arabidopsis thalia. This is possibley because they play a similar role in antioxidation.Bioinformatic analysis also revealved the protein's situation and activity sites.All the evidences confirm the prediction and the gene was then named CpPOD.In order to study its biological function, The prokaryotic expression vector pET-32-CpPOD was constructed, The protein induced byÉ‘-Lactose was successful expressed in BL21 (DE3) and the activity was assayed by guaiacol and H₂O₂. The polyclonal antibody against peoxidase was prepared and detected by Elisa and agar double diffusion test.The plant expression vector pTEV7::CpZPOD was also constructed and transformed into tobacco mediated by Agrobacterium. Transgentic tobacco was screened by Ms medium containing hygromycin and verified by PCR,RT-PCR,Western blot.The research also indicated that the cold tolerance and water holding capacity of transgentic tobacco was enhanced.All in all ,we are just on the threshold of a better understanding of the biological function of CpPOD, further studies are needed.