Cloning And Prokaryotic Expression In E.coli Of A Thaumatin-like Protein Gene From Wheat Inoculated With Puccinia Striiformis

Yellow rust, caused by Puccinia striiformis f. sp. tritici, is a kind of serious disease in wheat worldwide. Breeding resistant varieties is an efficient way in controlling yellow rust. Therefore, it is very important for breeding resistant varieties to clone resistant-related genes and to confirm their roles in the interaction of wheat and P. striiformis.Thaumatin-like protein classified as PR-5 is a kind of pathogenesis-related proteins. Many researchs had indicated that Thaumatin-like protein has anti-fungi activity. Cloning,bioinformatics analysis, semi-quantitative RT-PCR analysis and prokaryotic expression in E. coli were studied in this research.1. Cloning of tlp gene from wheat inoculated with P. striiformis.3'RACE and 5'RACE primers were designed according to an EST that is high homologous to Thaumatin-like protein of wheat, RNA was extracted from wheat leaves inoculated with P. striiformis after 0h,12h,24h,36h. CDNA prepared from the mixed RNA was used as the template of RACE. 3'and 5'sequence of tlp were obtained using PCR amplification.The full length cDNA sequence of tlp was obtained from aligning the 3'and 5'sequence.2. Bioinformatics analysis of tlp gene.The cDNA sequence and character of tlp were analysed using NCBI and EXPASY. The full length cDNA of tlp contains 810bp, which has an ORF (open reading frame) of 522bp, 5'-UTR of 66bp and 3'-UTR of 222bp. TLP consist of 173 Amino Acids (AAS). Since the first 20 AAS of TLP have the typical features of a signal peptide and there isn't any transmembrane structure of TLP, it is likely that TLP enters the secretory pathway.3. Semi-quantitative RT-PCR analysis of tlp gene.To investigate the expression level of tlp in wheat after inoculated with P. striiformis, semi-quantitative RT-PCR was carried out in compatable reaction and incompatable reaction. Not only in compatable group, but also in incompatable group, the expression level of tlp incresed in the early stage after pathogen infection.The response time was later in compatable group.4. Construction of expression vector of tlp. ORF of tlp was cloned into pGEM T-easy vector and TLP-pGEM-T recombinant was obtained, TLP-pGEM-T recombinant was restricted with Hindâ…¢and BamHâ… , and then, the ORF of tlp was inserted into pET-32a, TLP-pET-32a expression vector was obtained after sequencing.5. Prokaryotic expression of TLP in E.coli.TLP-pET-32a recombinant was transferred to an E.coli strain BL21 (DE3). After IPTG induction, a 38 kDa fusion protein was expressed. The optimum inducement condition is: 0.1mM IPTG 100rpm, 4h. The recombinant protein was expressed mainly as inclusion body. 6. Purification of TLP protein.Fusion protein was purified by Triton X-100,Urea and His-Trap affinity columns. Urea appeared to be more efficient than Triton X-100, and purification products were the best when using His-Trap affinity columns.The full length cDNA sequence of tlp and purified protein of TLP was obtained in this study, which is very useful for making multi-antibody and carrying out the study of the role of TLP in the interaction of wheat and P. striiformis.