Cloning And Characterization Of Resistance Gene Analogs And Related Genes In Brassica Oleracea Var.italica Planch

The objective of this paper was to study the cloning and characterization of disease resistance related genes, beta-1,3-glucanase gene and resistance gene analogs in Brassica oleracea var.italica planch1.Cloning and Characterization of Beta-1,3-glucanase Gene cDNA from Brassica Oleracea L. and Construction of Its Plant Expression Vector.A pair of degenerating primers was designed according to the conserved regions of Beta-l,3-glucanase Gene from other plant species and a cDNA fragment was isolated from Brassica Oleracea L using RT-PCR. Full-length cDNA with 1277bp was acquired by 5'RACE and3'methods. Its ORF encodes a polypep tide of 352 amino acids. The sequence was accepted and released by GenBank (accession number EF484879) and named as BObg. The putative protein of this gene had an isoelectric point of 7.35 and a calculated molecular weight of 43.3KD. Alignment and phylogenetic tree showed it had high homologies with Beta-1, 3-glucanase Gene from other plant species. Semi quantitative RT-PCR analysis indicated this gene was up-regulated expressed at 48h after downy mildew was inoculated, while it was down-regulated expressed in the plant without inoculation and other phases post inoculation. It was subsequently cloned into plant expression vector PBI121 and located between 35S promoter and NOS terminator.2.Isolation and Characterization of Resistant Gene Analogs from Brassica oleracea var.italica planchThe degenerated primers were designed according to the conservative domain of disease resistant genes in plants, and the genomic DNA of Brassica Oleracea L.were amplified. Four resistant gene analogs were obtained which contain the NBS-LRR domain. The deduced amino acid sequences have high homology scores compared with those of resistance genes or resistance gene analogs from other plants in GeneBank. A cluster analysis was performed based on the deduced amino acid sequences with several resistance genes from other plants, these RGAs can be classified into four different major groups. These RGAs can further be used as probes for cloning of resistance gene in Brassica Oleracea L.