Molecular Cloning, Heterologous Expression And Characterization Of Cutinase Gene From Puccinia Striiformis F.Sp. Tritici

Yellow rust, caused by Puccinia striiformis f. sp. tritici, is a kind of serious disease on wheat worldwide. To do research on the early stage of pathogen penetration is important to find novel ways in controlling this devasting plant disease.Cuticle covers the aerial parts of plants and forms the first barrier to plant pathogens. Cutinase, which can degrade cuticle of host plants, is a kind of very important enzyme for some plant pathogens in the process of adhesion and penetration. In the previous study of our lab, we had established cDNA library of germinated urediopores of Puccinia striiformis f.sp. tritici and identified some ESTs were similar to cutinase sequences from other true fungi. To further research on the role of cutinase played in the P. striiformis-wheat interaction, the author cloned and analyzed the cutianse gene. In addition, polycolonal antibody specific to P.striiformis cutinase had been prepared. All the above work has laid sound foundation for further research on P.striiformis cutinase.1. PSCUT1 cloning. The cDNA library of P.striiformis had been screened and the whole length of cutinase gene had been cloned.2. Bioinformatics analysis of PSCUT1 sequence. The full length cDNA sequence of PSCUT1 was analysed using NCBI and EXPASY. The full length cDNA of PSCUT1 is 1129bp, which has an ORF (open reading frame) of 735bp, 5'-UTR (Untranslated Region) of 92bp and 3'-UTR of 302bp. PSCUT1 consists of 244 Amino Acids (AAS). Since the first 19 AAS of PSCUT1 have the typical feature of a signal peptide and it is likely that PSCUT1 enters the secretory pathway.3. Transcription analysis of cutinase gene in the P.stiriformis infection had been done by way of real-time PCR. The result shows that PSCUT1 mainly expresses in the early stage of pathogen penetration. At 6hpi, PSCUT1 had significant expression and at 24hpi its expression reached the maximum.4. P.striiformis cutinase had been expressed successfully in E.coli.ORF of PSCUT1 was cloned into pGEM T-easy vector and PSCUT1 -pGEM-T recombinant was obtained, PSCUT1-pGEM-T recombinant was digested with Xhoâ… and BamHâ… , and then, the ORF of PSCUT1 was inserted into pET-32a, PSCUT1-pET-32a expression vector was obtained after sequencing. PSCUT1-pET-32a recombinant was transferred into E.coli strain BL21 (DE3). After IPTG induction, a 44kDa fusion protein was expressed and after optimization, large scale expression was done.5. Antibody against P.striiformis cutinase had been prepared and the specificity of antibody had been demonstrated by Western blot. Fusion protein was purified by Triton Urea and His-Trap affinity columns. After SDS-PAGE, the purity of the purified fusion protein could be used in preparing antibody. By immulizing rabbits the antibody was prepared and the specificity of the antibody was demonstrated by Western Blot, which indicated that the antibody could be used in immunogold analysis.The full length cDNA sequence and specific antibody against PSCUT1was obtained in this study, which is very useful in continous study of the role of PSCUT1 in the interaction of wheat and P. striiformis.