Molecular Characterization Of Antifungal Genes In Brassica Campestris Ssp. Chinensis

The PCR primers were designed on the conservative domain encoding known antifungal genes, a set of antifungal genes were obtained from Brassica campestris ssp. chinensis resistant material Suzhouqing by the homology-based cloning technique, which will do a great helpful to the transgenic research, and further to enhance host resistance to fungal pathogens. Meanwhile, by using cDNA-AFLP approach, a TDF which encoding a novel chitinase gene was isolated from fungal pathogen infected non-heading Chinese cabbage leaves, RT-PCR and rapid amplification cDNA end (RACE) technology were then used to clone the full-length cDNA clone. Further analysis showed that the novel chitinase gene showed lower identies with other known chitinase genes.1. Molecular Characterization of A full-length cDNA of chitinase gene in Brassica campestris ssp. chinensisAccording to the consensus domain of rape chitinase gene, and by using RT-PCR and RACE technology, a full-length cDNA of chitinase gene named Bcchi was cloned from Brassica campestris ssp. chinensis cultivar Suzhouqing. The cDNA clone Bcchi, contained 1068 bp nucleotide, has been submitted to the DDBJ database under accession number AB302323. The longest open reading frame (ORF) in Bcchi encoded a polypeptide of 268 amino acids, and the N-terminal signal peptide was comprised of the first 24 amino acids. The molecular weight (Mw) of deduced amino acids is 8.7 kDa with an isoelectric point (pI) of 8.28. Bcchi showed the highest homology to rape ChB4 (EMBL accession number CAA43708) and Chinese cabbage CHB4 (DDBJ accession number BAF35569) with 100% and 99% identity, respectively. The identity to Arabidopsis thaliana endochitinase (GenBank accession number AAB64047) is 88%, and the identity to other plant chitinases is from 59% to 78%. Southern-blot analysis indicated that there was more than one copy of Bcchi gene in Brassica campestris ssp. chinensis genome. Real-time quantitative PCR and northern blot analysis revealed that Bcchi transcript was rapid accumulated in the infected leaves upon inoculation with P. parasitica. Tissue-specific expression analysis showed that 24 h post-inoculation the highest expression tissue of Bcchi mRNA was leaves. These data revealed that Bcchi might be involved in plant resistance against fungal pathogen infection.2. Molecular Characterization of A full-length cDNA of plant defensin gene in Brassica campestris ssp. chinensisAccording to the consensus domains of plant defensin genes, and by using RT-PCR and RACE technology, a full-length cDNA of chitinase gene named BcAF was cloned from Brassica campestris ssp. chinensis cultivar Suzhouqing. The cDNA clone BcAF, contained 1068 bp nucleotide, has been submitted to the DDBJ database under accession number AB302324. The longest open reading frame (ORF) in BcAF encoded a polypeptide of 79 amino acids, and the N-terminal signal peptide was comprised of the first 29 amino acids. The molecular weight (Mw) of deduced amino acids is 8.56kDa with an isoelectric point (pI) of 8.15. BcAF showed the highest homology to rape AFP3 (GenBank accession number AAB03224) and radish AFP3 (EMBL accession number CAA65984) with 100% and 96% identity, respectively, and the identity to other plant defensins is from 82% to 88%. Southern-blot analysis indicated that there was more than one copy of BcAF gene in Brassica campestris ssp. chinensis genome. Real-time quantitative PCR and northern blot analysis revealed that Bcchi transcript was rapid accumulated in the infected leaves upon inoculation with P. parasitica. Tissue-specific expression analysis showed that 12 h post-inoculation the highest expression tissue of BcAF mRNA was leaves. These data revealed that BcAF might be involved in plant resistance against fungal pathogen infection.3. Molecular Characterization of A full-length cDNA of Pathogenesis-related Protein 4 gene in Brassica campestris ssp. chinensisAccording to the consensus domain of Chinese cabbage PR4 gene, and by using RT-PCR and RACE technology, a full-length cDNA of PR4 gene named BcPR4 was cloned from Brassica campestris ssp. chinensis cultivar Suzhouqing. The cDNA clone BcPR4, contained 617 bp nucleotide, has been submitted to the DDBJ database under accession number AB325873. The longest open reading frame (ORF) in BcPR4 encoded a polypeptide of 140 amino acids, and the N-terminal signal peptide was comprised of the first 21 amino acids. The molecular weight (Mw) of deduced amino acids is 15.5kDa with an isoelectric point (pI) of 8.13. BcPR4 showed the highest homology to Chinese cabbage (GenBank accession number AF528181) with 100% identity, and the identity to other plant chitinases is from 63% to 75%. Southern-blot analysis indicated that there was more than one copy of BcPR4 gene in Brassica campestris ssp. chinensis genome. Real-timequantitative PCR (qPCR) analysis revealed that BcPR4 transcript was rapid accumulated inthe infected leaves upon inoculation with SA and P.parasitica, with the highest expressionlevel at 24h and 48h, respectively. These data revealed that BcPR4 might be involved inplant resistance against fungal pathogen infection.4. Molecular Characterization of A Novel Chitinase Gene in Brassica campestris ssp.chinensisA gene involved in a fungal pathogen infection in non-heading Chinese cabbage (Brassica campestris ssp. chinensis) was isolated by a cDNA-amplified fragment length polymorphism (cDNA-AFLP) approach. The gene was conducted on mRNAs from Peronospora parasitica infected plant leaves. Among changes observed throughout the infection process, a cDNA fragment not detected in the control plant leaves, but present in Peronospora parasitica infected plant leaves after 12h was further studied. The full-length cDNA was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) technology. Sequence analysis showed the gene was a typical chitinase gene which included an open reading frame (ORF) predicting a 278-amino-acid polypeptide of 30kDa with an isoelectric point (pI) of 8.92. Nucleotide sequence and deduced amino acid sequence analysis revealed a lower homology with other chitinase proteins, designated Bcenchi (DDBJ accession number AB369105). Southern blot analysis showed that the gene was composed by a multiple gene family. Real-time quantitative PCR (qPCR) analysis carried out on mRNAs from leaves at different time points post-inoculation with two fungal pathogens (Peronospora parasitica and Alternaria brassicicola) and two signal transduction chemicals, salicylic acid (SA) and methyl jasmonate (MeJA). Results showed Bcenchi must be involved in plant resistance against fungal pathogens infection.