| Cirtus bacterial canker disease (CBCD), caused by Xanthomonas axonopodis pv. citri Vauterin et al. is one of the severe diseases in most cirtus species and cultivars. The pathogene have been appeared in lists of quarantine pests in many countries included in our country. Cloning resistance gene analog (RGA) is a new method for finding disease resistant genes from plants. The procedures are first amplify the resistance gene analogs with the degenerate primer that designed according to the conserved domains of the cloned resistance gene from the plant genomic DNA or cDNAs, and then adding them to linkage maps as molecular marker or making them probes to scanning genomic library in order to obtained the candidate disease resistance genes, and finally discovered a new plant resistance genes from these candidates.In this study, we first cloned some RGAs from six citrus clutivers which have diversity resistance to Cirtus bacterial canker disease, then analysis those sequences and determine it by southern blot analysis. The results are as follows:1. The result of RGAs clone A specific fragment of 500bp was cloned from genomic DNA of six citrus clutivers by PCR with degernate primers designed according to the conserved domains in the NBS region. The fragments are recycled and inserted into pGM-T vector, and then transformed into E.coli DH5α. Finally 13 recombinations were obtained and sequenced after comfirmed by colony PCR identification and restriction digestion analysis.2. The result of sequenceing and BLAST Nine sequences were obtained among those sequenced RGAs which were analyzed in homology through the procedure of BLAST in NCBI. Those nine sequences were all stick to the non-TIR-NBS-LRR type in terms of their putative amino acid sequences. Among of those nine RGAs, RGAsl-4,RGAs4-1,RGAs6-1 were similar to Citrus grandis x Poncirus trifoliate 18P32 gene (AY130787.1-AY130789.1), and the others were stick to the Poncirus trifoliata resistance protein-like protein (RGA 19) gene (AAU89644.1,AAU89653.1,AAU89647.1)3. The result of the structure and domains analysis The analysis of the eight RGAs structures and NBS domains which have the complete ORF found that they all contained the motif P-loop, kin-2, "PLAL" as well as motif RNBS-A,B,C which defined by Meyers. Comparison of putative amino acid sequence of these five RGAs with the reported genes showed that this RGAs from citrus has 29.6%-41.1% homologous with 2C-1gene,24.5%-29.3% with prf gene, 28.8%-55.5% with RPS5 gene,29.1%-32.9% with Mi-1.2 gene, and 21.1%-25.0% with N gene, While among this cloned RGAs the homologous was 26.8%-99.4%.4. It is confirmed the existence of RGA in 5 different varieties by method of PCR identification and southern blot analysis. Molecular identification of PCR results showed that all varieties for the experiment were amplified about 380bp specific target band, and southern blot analysis make sure the RGA's location and the copy number in citrus genomic DNA. The copy number in5 citrus varieties respectively is Ju Yuan with 2 copies, Chen orange with 3 copies, San Ye with 1 copy, Shen orange with 3 copies and sweet orange with 2 copies.The research of these RGAs will provide with theories and experiment data for isolating related resistance gene of citrus and develop new markers for Marker Assistant Selection. And also, it will be benefit to understand the origin and evolution of NBS-LRR type R genes.
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