Construction Of SSH CDNA Library And Expression Analysis Of Defense-Related Genes In Chinese Cabbage Induced By Peronospora Parasitica

Chinese cabbage [Brassica campestris L. ssp. Pekinensi (Lour.) Olsson] is an economically important vegetable crop with the largest cultivated area and highest yield in China, and become popular worldwide. Downy mildew is caused by the oomycete Peronospora parasitica, which damages plants during all stages of growth, and results in a high proportion of loss in yield and quality. Thus, for Chinese cabbage breeding, it is very important to clear the mechanism of resistance to downy mildew.In this study, a forward suppression subtractive hybridization (SSH) library was constructed from leaves induced by Peronospora parasitica using a DH line T12-19 resistant to downy mildew. Moreover,5 cDNA fragments of defense-related genes were isolated and their expression profiling was analyzed by using Real-time PCR method. This study will be further applied to reveal the possible mechanism of resistance and be the fundamental for improving downy mildew resistance in Chinese cabbage. Results are as follows:1. A forward suppression subtractive hybridization library was constructed from Chinese cabbage leaves induced by Peronospora parasitica using T12-19. Subtraction was performed using mixed cDNA synthesized from RNA of infected plants at 0,6,12,24,48 and 72 h as tester and those from control plants as driver. In total,768 recombinant clones were preserved. The recombinant rate was 87.3%. The length of insert fragments in these clones ranged from 200 to 1000 bp, mainly between 200-500 bp.2. By reverse Northern blot analysis,57 up-regulated ESTs were identified. A total of 55 high-quality ESTs were obtained, and these ESTs were assembled into 50 unigenes. ESTs similarity analysis via BLAST showed that 37 unigenes shared similarity to known sequences in GenBank. The function of the ESTs involves in metabolism, energy, transcription, protein synthesis and fate, membrane, transport, signal transduction, defense and so on. Additionally, the expression profiling of three clones was analyzed using Real time-PCR. The significant up-regulation was observed in leaves of 6 h post-inoculation for these clones. These results match the study by reverse Northern blot analysis.3. In this study,5 cDNA fragments were isolated according to the sequence of defense-related genes in Brassica campestris. The expression profiling of these clones was analyzed by Real time-PCR. The results indicated that the expression levels of pathogenesis-related protein biosynthesis genes, BRPR1 and BRPR5, were upregulated significantly 12 h post-inoculation, but BRPR2 was upregulated 6 h post-inoculation and restored to basal level at 48 h post-inoculation for these three genes. For BRPAL, encoding for phenylalanine ammonia lyase, it was upreulated at 6 h pos-tinoculation, and downregulated at 12 h post-inoculation. While, the expression of BRF3H, encoding flavanone-3-hydroxylase, a catalyze enzyme in early stage of anthocyanins biosynthesis, was downregulated at 6 h post-inoculation and restored to basal level at 48 h post-inoculation in either incompatibility interaction or control.