| Gray mould caused by Botrytis cinerea is one of the important world diseases, and always results in significant economic losses. Resistance of different Arabidopsis ecotypes and genotypes to some important fungal pathogens were studied, the phenotype, cell histology and the biochemical mechanism of Arabidopsis against infection of B. cinerea was discussed. Genetic linkage analysis and mapping of resistance gene to B. cinerea were also analysised. Two resistance-related genes were cloned. The recombinant proteins of one cDNA expressed in E. coli. It encodes a protein with resistance to Botrytis cinerea. Our work paved way to clone of resistance gene and genetic improvement.All the 11 Arabidopsis ecotypes were inoculated by 40 isolates of fungal pathogens. 19 isolates of fungal pathogen appeared different symptoms on the leaves of different Arabidopsis ecotypes, among them 10 isolates were B. cinerea. Isolate BC18 of B. cinerea and Col-0 ecotype, Ws-0 ecotype, her ecotype were chosen for further study. Col-0 ecotype and Ws-0 ecotype were incompatible to isolate BC18 of B. cinerea, her ecotype were compatible to it. After inoculation, Col-0 ecotype appeared immunity symptoms, while Ws-0 ecotype appeared HR response.To identify the resistance of Col-0 ecotype to isolate BC18 of B. cinerea, Cell histology of Col-0, Ws-0 and Ler ecotypes after being inoculated by high concentration of B. cinerea spores were observed, Results indicated that Col-0 ecotype could be infected by B. cinerea, and Col-0 ecotype appeared HR response after being inoculated with high concentration of B. cinerea, It also suggested that Col-0 ecotype appeared high resistant response to B. cinerea. We analyzed changes of some defensive enzymes activities and MDA content after being inoculated by B. cinerea. We found that activities of POD, CAT, PPO, PAL and MDA content of Arabidopsis changed regulatory after being infected by B. cinerea. But activity of SOD seems no relationship with Arabidopsis against being infected by B. cinerea.Resistance Col-0 ecotype and susceptible Ler ecotype had been crossed. According responses of F1 and F2 population, we identified that two genes mediated resistance of Arabidopsis against infenction of B. cinerea. Through the method of map-based cloning,we found that two disease-resistant genes located on the fourth and fifth chromosome respectively. The result showed that one disease-resistant gene was linked to DNA markers CCR1 and DHS1 on the fourth chromosome of Arabidopsis with genetic distances of 1.2cM and 1.6cM to CCR1 and DHS1 respectively. The other disease-resistant gene was linked to DNA markers CA72/NGA151 and NGA106 on the fifth chromosome with genetic distances of 1.4cM and 2.4cM to CA72/NGA151 and NGA106 respectively. In our research, we temporarily designated two resistance genes as BC1 and BC2.SSH method was utilized to study the differential expression genes in different Arabidopsis ecotypes after being inoculated by B. cinerea. Results of homology indicated that genes related to substance transmission in cell membrane, cell signal transduction, and protein synthesis, reverse transcription were induced after inoculation. Among them, AM-2 fragment which having GAF conserved domain might relate to ethylene signal transduction in Arabidopsis. We speculated that it might relate to resistance of Arabidopsis against infection of B. cinerea.DDRT-PCR method was also utilized to study the differential expression genes in different Arabidopsis ecotypes after being inoculated by B. cinerea. Total 49 differentially expressed cDNA fragments were obtained, among them 11 cDNA fragments were cloned and sequenced. A33-2 fragment displayed high homology (98%—99%) to TAC clone K9I9 of Arabidopsis chromosome 5 genomic DNA and protein binding AT5G67480 transcript variant, A3 3-3 fragment displayed 98% homology to NADH-dehydrogenase subunit 4L of Arabidopsis, M7-1 fragment displayed 98% homology to phosphatidylinositol transfer-like protein IV ( Secl4p protein) of Arabidopsis. Six fragments displayed low homology to known protein in GenBank, and the other two fragments were new sequences in GenBank. We speculated that five cDNA fragments (A33-2, A33-3, J49-1, J7-1 and M7-1) might relate to disease-resistance of Arabidopsis against infection of B. cinerea.Two special primers of A33-2 fragment are designed for the 5' and 3' RACE (Rapid amplification of cDNA ends). We obtained full cDNA length of A33-2 fragment. A33-2 fragment contained one intact ORF which length was 119bp. The putative protein contains 372 amino acids, molecular weight was 12.50401 kD and isoelectric point was 4.75. Through the method of Southern blotting, we found that A33-2 sequence was single copy in Arabidopsis genomic DNA. It located between 26948165bp and 2695015lbp on the fifth chromosome of Arabidopsis. Result of homologic indicated that A33-2 sequence identified to AT5G67480 gene, which encoded binding protein or transcription regulator. Length of AT5G67480 gene was 1987bp and contained four introns. It has two geneic modes, for example AT5G67480.1 and AT5G67480.2. Among them AT5G67480.1 was theclassical mode of AT5G67480. This gene contained BTB/POZ domain of TAZ zinc protein famliy. Its cDNA has been cloned and the recombinant proteins expressed in E. coli. It encodes a protein with resistance to Botrylis cinerea. Through the method of RACE, we also obtained flanking sequence of A33-3 fragment. The RACE product contained one intact ORF. It was identified as NAD4L gene that encoded NADH dehydrogenase subunit 4L. NAD4L gene belongs to ATMG00650 gene and locates in the mitochondria of Arabidopsis Col-0 ecotype. NAD4L gene plays a role in cellular respiration and electronic transfer.
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