| Citrus is an important economic fruit, however, the disease influent the product seriously. One of which is Huanglongbing that is a destructive disease in the world, which can lead citrus to reduction even dead. Due to having not got the stable pure stain and lack of high effective bactericides, people mainly rely on uprooting and burning diseased trees from quarantine area, or enhancement of quarantine regulations in non-quarantine area to prevent the extension of the disease. It is an important measure to use the disease-resistant cultivar and improve plant disease prevention to control plant diseases. Although the Rutaceae plant is the main host of Citrus Huanglongbing, however, Degree of sensitivity or tolerance of the host to the disease has obvious discrepancy during the different species. So using separation disease resistance gene or breeding has the important meaning to prevent and control this kind of disease. Resistance breeding first need has disease-resistant germplasm resources or disease-resistant genes. Now, the researchers are studying the breeding to resistant to disease, although there is not any resistant citrus species. There are very few disease resistance materials in the current cultivars. The key problem to breed resistant species is to find the resistance gene from citrus relatives. One aim of plant breeding is to improve the resistance ability to disease. With the development of molecule technology, a lot of resistant genes have been cloned from much different plants through positional cloning and transposon tagging. This paper aimed to clone RGAs from Rutaceae plant to apply some basic resource.The study is consisted of two parts: (1) Degenerate primers designed according to the conservative NBS (nucleotide binding site) domain contained in some plant disease resistance genes were used to isolate resistance gene analogs (RGAs) from genomic DNA of two spices tolerant to HLB such as Citrus maxima and Murraya paniculata. Then biology information analysis of sequences obtained was done to reveal the diversity of plant. (2) The resistance gene analogs were amplified from cDNA of Citrus maxima using degenerated primers based on the conserved regions of NBS (Nucleotide Binding Site) - LRR (Leucine Rich Repeat) domain from plant resistance genes cloned and the Quantitative Real-time PCR was used to check the expression of 5 RGAs in sweet orange after infected with HLB. According to the reference reported, 5 degenerate primers designed based on conserved NBS (nucleotide binding site) motifs contained in some plant resistance genes were used to isolate analogous sequences called resistance gene analogs (RGAs). After analysis of RFLP, about 43 resistance gene analogs (RGAs) were cloned from several spices related to HLB using PCR method with primers above-mentioned. The BLASTN searches revealed that 43 DNA fragments showed different sequence homology with known plant R-genes or deposited RGAs. 12 sequences could be translated to polypeptides without stop codons. These RGAs and 12 deduced amino acids were analyzed by Clustalx and DNAMAN software. Sequence analysis showed these RGAs owned the fllowing conserved domains: P-loop, Kinase-2a and GLPLAL, which was conserved in NBS-LRR type disease resistance gene. The 12 RGAs shared 37.39%-43.43% identity with the resistance genes of Tobacco N,Flax L6,Arabidopsis thaliana RPS2,RPS5,RPP8,RPM1. Gene duplication followed by sequence divergence were proposed as the mode for the evolution of a large number of distantly or closely related RGA genes in plants, and this mode may play a role in the generation of new resistance specificity. In addition, using PCR amplification from cDNA of Citrus maxima, after RFLP, we choose 6 clones to sequence. Analyses of DNAMAN reveals that 5 fragments belong to RGAs were successfully obtained. We examined their expression after infection with HLB pathogen by Quantitative Real-time PCR. The result showed that expression of 5 RGAs had the characteristics of constitutive expression. The 5 RGAs shared 19.71%-42.86% identity with the resistance genes of Tobacco N,Flax L6,Arabidopsis thaliana RPS2,RPS5,RPP8,RPM1. And all the RGAs belong to non-TIR-NBS-LRR with a tryptophan at the end of kinase-2a.The expression levels of 5 RGAs were significantly elevated by the Quantitative Real-time PCR. The result showed that the expression of 5 RGAs in sweet oranges changed differently after graft with HLB pathogen. GJ20 and GJ31 have a big expression in the second sample in the group of experiment. GJ28 expressed improved in the forth sample of experiment group and constantly expressed during other months before and after grafting pathogen. GJ35 expressed declined on the whole. GJ10 decresed in the third month after grafted. The present study shows that RGAs are abundant in plant genome. The RGA cloned in this experiment may provide potential use for fruit tree disease breeding and important gene cloning. At the same time, the identification of sweet orange RGAs induced by pathogens should provide valuable information for cloning related resistance genes about HLB.
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