Cloning And Functional Analysis Of Pathogenesis-related Protein Gene CpPR-4from Chimonanthus Praecox(L.) Link

Pathogenesis-related protein （PRs） is a number of proteins that were induced by pathogen attacks, chemical treatments or wounding,and believed to be important in protecting plants from stresses. The utilization and development of the pathogenesis-related protein is an effective way to raise the disease-resistance of the plant. The pathogenesis-related protein family are about17, respectively, PR-1to PR-17.On the basis of the construction of the cDNA library, the pathogenesis-related protein4gene, CpPR-4, was cloned from Chimonanthus praecox（L.）Link in the study. The properties of CpPR-4were analyzed using bioinformatics methods and the functions were predicted. Then plant expression vector and prokaryotic expression vector were constructed, and their functions were further proved. The main results are as follows:Full-length coding region of CpPR-4has been obtained, and properties and structure of the CpPR-4were analyzed by bioinformatics methods. The results showed that the CpPR-4encodes142amino acids with predicted molecular weight15KD and PI5.57, without introns, and it has2phosphorylation sites, two signal peptides. CpPR-4protein secondary structure was composed of α-helix （26.76%）, extended strand （26.06%）, random coil （33.10%） and some little βturn（14.08%）. The encoded protein had characteristics of DPBB₁super family according to conserved domain analysis in the NCBI.Plant expression vector pCAMBIA2301g/CpPR-4was constructed, then CpPR-4was transformed into tobacco by agrobacterium-mediated transformation and14transgenic plants were obtained. We conducted TMV infection test and tobacco frogeye leaf spot test.The test of infection of TMV virus or tobacco frogeye leaf spot showed that transgenic plants were not able to resist tobacco mosaic virus, but resist frogeye leaf spot effectively; the test of low temperature stress showed that transgenic tobacco has a certain resistance to the unfavorable environment of low temperature compared with wild-type tobacco.The prokaryotic expression vector pET-32a/CpPR-4was constructed, which was then transformed into E.coli BL21（DE3）.The fusion protein with the size of about36kD was induced and expressed via optimal conditions. The soluble analysis of this fusion protein revealed that it exists in the form of inclusion bodies.