Study On Method For Eradication Pathogen Of Citrus, Cloning Of Omp In Candidatus Liberibacter Asiaticus And The Protein Characters

The main Citrus virus diseases and viroid diseases are Citrus tatter leaf virus, Citrus tristeza virus, Candidatus Liberibacter asiaticus, Citrus Exocortis Viroid and so on. The study mian focus on CTLV, CTV and HLB. According insect, grafting and other pathogens, virus disease would long-distance and large-scale spread. It has caused serious economic losses, but there is lack of effective treatment and prevention methods still now. Therefore, to study the effective toxin-free method and antiviral reagents combined with modern molecular detection methods will producing significant economic value for citrus disease detection. The outer member protein(OMPs) existed in bacteria has been recognized as a candidate protein studying for interspecific and intraspecific variation. OMPs plays a key role in many gram-negative bacterial pathogenesis and involves in the adaptive response, Because the outer membrane easily is recognized by the host immune system, possiblely OMP can prepare for vaccines. In this study, we designed primers to clone omp gene from subject is the citrus greening bacteria according to a published omp gene sequences Candidatus Liberibacter asiaticus, the last purpose of the reaserch is to express the target protein, obtain high purity and high bioactivity purifiled product and prepare Monoclonal antibody.The mai results as followed:1. Taoye Cheng infected CTLV treated50d with phytotherapyand thermotherapy(37℃,10.11.3—11.1.20), the virus concentration is reduced. Set three groups:i. Single treated with Andrographis paniculata2g/trains; ii. Single treated with Phellodendron amurense cortex phellodendri,2g/plant; iii. Co-treatmnet and each1g/strains.The extracts were irrigated by root irrigation every5days. Continue the treatment50d (11.4.19—6.9) in the same way, the detection rate of CTLV is8.7%.2. Qianyang seedless ponkan and Fukumoto infected CTLV treat with thermotherapy (39℃2d,35℃2d) and phytotherapy50d (2011.10.18-12.7), the detoxification rate of CTLV is100%. But the mortality of Qianyang seedless ponkan is too high, the survival rate is11.1%.3. Detecting Candidatus Liberibacter asiaticus from Citrus and periwinkle, and succeed in cloning the omp gene from genone DNA extracted from periwinkle leaves, the gene was2346bp.4. Constructing recombinant vector pET-22b-omp, but there in no expression of the target protein in three kinds of strain. We predicted the structure and function of protein, truncated561-781aa in C-terminal and the fragment named ompC. Successed in cloning and expressing the truncation proteint wich has high expression in strain BL21(DE3) plysS. Screening the optimal effectors for its expression, induced with0.6mM IPTG and then cultivated over8h in28℃, the truncated protein is part of the soluble.