Isolation And Characterization Of Cysteine Protease From Bursaphelenchus Xylophilus And Radopholus Similis

Cysteine proteases (CP) are so-named due to the function of a core catalytic cysteine, which mediates protein hydrolysis. Cysteine proteases expressed in worm's esophagus gland and intestine cells are the main "digestive enzymes" of nematodes. Previous studies indicated that cysteine proteases play key roles in tissue and cellular invasion, moult, embryogenesis nutrient acquisition, host protein processing, as well as immunoevasion. Lots of animal and plant diseases have been shown to be closely related to cysteine proteases, which increases the concerns about their biological and enzymatic characteristics research. Up to now, most studies have been focused on cysteine proteases from animal-parasitic nematodes and free-living nematodes. Recently, some reports proved the importance of cysteine proteases in the sedentary plant-parasitic nematodes. And there is no report yet about cysteine proteases in the migratory plant-parasitic nematodes. In this study, we isolated the full-length cDNA of cysteine proteases from Bursaphelenchus xylophilus and Radopholus similis which both belong to the migratory plant-parasitic nematodes. Moreover, we successfully expressed the recombinant proteins in E. coli, which provides the foundation for further analysis of the enzymatic activity and understanding of the protein function. This study also suggested potential solutions for anti-plant-parasitic nematodes drug design and disease control. The main results were as follows:1. Cysteine proteases gene cloning: Full-length cDNA of cysteine proteases from the migratory plant-parasitic nematodes B. xylophilus and R. similis were obtained using degenerate PCR primers designed from the flanking region of highly conserved motifs and rapid amplification of complementary ends (RACE). Two full-length cDNA of cathepsin L-like cysteine proteases were cloned from B. xylophilus, named Bx-CPL1 and Bx-CPL2, with 1220 bp and 1161 bp in size, and the GenBank numbers are EU651860 and EU659123, respectively; a 1388 bp full-length cDNA and a 498 bp fragment of cathepsin L-like cysteine proteases, and a 1112 bp full-length cDNA of cathepsin S-like cysteine proteases were cloned from R. similis, named Rs-CPL1, Rs-CPL2 and Rs-CPS respectively. The GenBank numbers of Rs-CPL1 and Rs-CPS are EU659124 and EU659125, respectively. Rs-CPL2 hasn't be submitted to GenBank. The bioinformatic and phylogenetical analysis demonstrated that all of the obtained cDNA sequences have the conserved characteristics of cysteine proteases family.2. Full-length DNA clone: The two full-length DNA of cathepsin L-like cysteine proteases genes of B. xylophilus were shown to comprise two introns and three exons, which provides basic information of gene structures of cysteine proteases genes.3. Prokaryotic Expression of recombinant proteins: The coding region of Bx-CPL1 and Bx-CPL2 for the mature cathepsin L were subcloned into an expression plasmid pET-28a(+). And an expression system in E.coli BL21(DE3) was successfully developed to overproduce the recombinant protein Bx-CPL2, but the recombinant Bx-CPL1 had a low level expression. Prokaryotic expression of the recombinant proteins established essential base for further studies at the protein level, such as purification of recombinant proteins, western blot and enzymatic activity assay.