Cloning And Expression Analysis Of Chitinase Gene PsChiI From Populus Szechuanica

The poplar leaf rust caused by Melampsora larici-populina Kleb.(abbr. MLP), causingleaves shedding and reduce the production of poplars, is one of the major diseases of poplar.Therefore, it is of great importance for developing resistant poplar varieties to researchPR-proteins and resistance-related genes from host poplar which had been infected.Chitinase classified as PR-3, PR-4, PR-8and PR-11are kinds of pathogenesis-relatedproteins in the defense mechanisms of plant resistance. In this study, the full length cDNA ofchitinase gene named PsChiI from Populus szechuanica was obtained using RACEtechnology, and analyzed by bioinformatics method. Real-time quantitative PCR was used toshow the expression of PsChiI during the leaf rust pathogenesis. The results are as follows:1.Cloning of PsChiI gene from P. szechuanica.RNA was extracted from P. szechuanica leaves inoculated with M. larici-populina after0h、12h、24h and48h. The cDNA using as the template of RACE was prepared from themixed RNA. The partial cDNA sequence was obtained according to the primers that designedbased on win8sequence, and3’RACE and5’RACE primers were designed on the basis of thepartial sequence. Finally,3’ end of663bp and5’ end of327bp were obtained respectively.2. Sequence analysis of PsChiI geneThe full length of chitinase gene from P. szechuanica was obtained from aligningsequence. The full length cDNA of the gene was1145bp, containing a5’-untanslated region(5’-UTR) of38bp, and a3’-UTR of147bp. The open reading frame (ORF) of954bpencoded319amino acid residues.The gene was isolated successfully, named as PsChiI, andits GenBank accession number was KC416180.3. Bioinformatics analysis of PsChiI gene.Physicochemical properties and structures of the PsChiI gene encoded protein werestudied by online service and MEGA5.0. The results showed that its molecular weight was35.7ku, and theoretical isoelectric point was5.03. and a signal peptide exist at the N-terminal.The encoded protein contained glycoside hydrolase family19signature sequences, andincluded cysteine-rich domain, catalytic domain and lysozyme-like superfamily domain in thestructure. Therefore, we could predict that the coding protein belonged to19family and was a member of Class Ib chitinase.4. Analysis of PsChiI gene expression by real-time quantitative PCR.The relative expression of PsChiI gene was analysed by qRT-PCR. It showed that PsChiIexpressed the highest level at48hpi during pathogenesis, approximatally7timing theexpression at0hpi control, then gradually reduced until96hpi, and equal expression with0hpi. It could indicate that PsChiI gene may be involved in the defense mechanisms of P.szechuanica against the rust fungus infection.