| Cabbage (Brassica oleracea L. var. capitata) is an important vegetable crop of Cruciferous crops and is cultivated world-wide. Black rot, caused by (Xanthomonas Campestris pv. Campestris, Xcc) is one of most serious diseases which is a limiting factor of cabbage production and quality. Thus, development of the cabbage varieties resistant to black rot is becoming more and more important. In order to screen out the resistant germplasm and find out relative biochemical mechanisms and resistance genes,31 cabbage varieties at seedling stage were tested by different inoculating methods, bacterium concentrations and seedling stages. Two varieties C7 and C26 were infected with Xcc to discover the relationship between the resistance to cabbage black rot and the PAL (phenylalanine ammonia-lyase) activity, accumulation of phenolics and lignin. Sequence-related amplified polymorphism (SRAP) technology is a simple and efficient marker system that can be used for map construction, gene tagging, genomic and cDNA fingerprinting, and map-based cloning. The major results were as follows:1. Study on the identification method of black rot-resistant in cabbage seedling and evaluation of cabbage germplasmThrough using different inoculating methods, bacterium concentrations and seedling stages, we developed a set of rapid and accurate methods to evaluate cabbage black rot resistance during seedlings. The suspensions with 1.0×108 colony forming units/mL is the best concentration when it was sprayed to the leafs of seedlings with four to five leaves. At the same time,31 cabbage varieties at seedling stage were identified for the resistance to black rot. The result showed none of the tested varieties were of immune response. The resistance of different varieties of cabbage was different. Among them, high resistant varieties accounted for 3.22% of the total varieties; middle resistant varieties accounted for 38.72%; tolerant varieties accounted for 16.13%; susceptible varieties accounted for 22.58% and high susceptible varieties accounted for 19.35%.2. Study on phenolics in cabbageIn order to discover the relationship between the resistance to cabbage black rot and the PAL(phenylalanine ammonia-lyase) activity, accumulation of phenolics and lignin, C26 and C7, two cabbage cultivars of different disease resistant capacity were treated with 1.0×108 colony forming units/mL. The result showed, comparing to inoculating distilled water cabbage leaves (CK), the PAL activity in both of them increased significantly after 1-2 days, for C26 it reached the highest level in 2 d and decreased rapidly after 5 days, while the PAL activity of C7 reached the highest level in 2 and 4 days, and then decreased. The phenolics and lignin contents of two varietyes inoculated with Xcc increased rapidly and reached the highest level in 3 d. After 7 days, the phenolics and lignin contents in C26 decreased rapidly, but the contents in C7 still maintained higher lever.3. Expression analyses of genes related to black rot in cabbageThe resistant variety C7 was sprayed with 1.0×10cfu/mL Xcc(Xanthomonas Campestris pv. Campestris) while the other group was sprayed distilled water as usual (Control, CK). Leaves were collected in 1 d,3 d, and 5 d after Xcc and distilled water treatment. The mixture of an equal amount total RNA came from above two groups as two gene pools. Different expression of the genes was analyzed by SRAP technique. The results showed:using 60 pairs of primers, about 685 cDNA fragments in size between 100～750bp were amplified, and got averagely 11 bands per primer combination, and there were 24 polymorphic loci in these two pools. After they were sequenced and the Blast program was used to find out homology with reported genes. Sequence alignment indicated that 6 out of these 9 TDFS showed homologies to certain genes from 43% to 59% in NCBI, while the other three showed no significant homology with reported gene. Based on their homologies, these genes were assumed to NADH dehydrogenase, vegetative cell wall protein, DELTA-VPE and unknown functional proteins. |
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