Development Of New Germplasm Resistant To Canker Disease By Transforming PthA-nls And Its ScFv Genes Into Citrus

Citrus canker disease caused by Xanthomonas axonopodis pv.citri.(Xac.),affects almost all of the citrus species and cultivars in the world causing severe damages to the citrus industry in most citrus producing areas worldwide,and China is one of the most severely damaged countries.All cultivars of citrus are susceptible to canker and highly susceptible when being artificially inoculated,implying that no function genes resistant to canker resistance have been identified in citrus,pthA is essential for the pathogen to cause hyperplastic canker symptoms on citrus.The carboxyl terminal portion of pthA encodes three nuclear localization signals(NLSs) that are critical for PthA function and for localization to the host cell nucleus.NLSs were found in all members of Xanthomonas avr/pth gene family.The avr/pth gene encodes signals affecting plant cell programs including "programmed death",as avr/pthA gene products can elicit resistance,it can be used to enhance bacterial disease resistance in plants.In this research,the COOH terminal of pthA,encoding three nuclear localizing signals,was cloned into a plant expression vector system and transferred into sweet orange via Agrobacterium-mediated transformation.Meanwhile,antibody technique and genetic transformation can overcome problems associated with conventional breeding and provide new approaches for disease resistance.A number of antibody genes have been introduced into plant cells,and the transgenic plants expressed the transgenes.These recombinant antibody molecules can be effective in insect or disease resistance.In this study,monoclonal antibody of the anti-recombinant PthA-NLS was prepared and the related ScFv gene was transferred into sweet orange, in order to confer resistance in citrus to canker disease.The main results were as follows:1.50 Xanthomonas axonopodis pv.citri isolates were obtained from diseased materials of several citrus cultivars collected from Hengyang,Chenzhou and other areas of Hunan province.The isolated samples was identified by PCR assay using two pairs of specific primers,i.e.JYF5/JYR5 designed from published sequence encoding a conserved hypothetical protein gene in genome of the bacterium and P8/P9 designed from published sequence of pthA(U28802).The accurate and rapid molecular diagnosis technique was established and all the isolates were verified by PCR.The obtained pathogen isolates offered important materials for the subsequent studies.2.pthA was amplified by long-PCR and net-PCR from the plasmid of Xanthomonas axonopodis pv.citri and cloned into pBI121 plant expression vector.The recombinant plasmid named pBI-pthA was identified by restriction digesting and sequencing.The result of sequence analysis showed that pthA' had 99.8%similarity with that of pthA in Genbank. 3.The COOH terminal of pthA,encoding three nuclear localizing signals,was amplified by PCR from the plasmid of Xanthomonas axonopodis pv.citri.,then the sense and antisense sequences were inserted in plant expression vectors,the sense recombinant plasmid named pBI-nls and the antisense one named pBI-a-nls.After identified by restriction digesting and sequencing,the pthA-nls and the pthA-α-nls genes were transferred into sweet orange respectively via Agrobacterium mediated transformation.Successful integration were confirmed by PCR and southern blotting, 9 transgenic pthA-nls gene and 12 transgenic pthA-α-nls gene sweet orange plants were abtained.Disease resistance assays in vitro indicated that the transgenic pthA-nls gene clones showed significantly less lesion development than the controls.And the transgenicα-nls gene sweet orange plants were susceptivity to the disease.4.NLSs of pthA was amplified by PCR and cloned into PET32a(+) vector.The recombinant plasmid named PET-NLS was identified by restriction digesting and sequencing.The recombinant fusion protein was expressed in E.coli BL21(DE3) and analyzed by 12%SDS-PAGE.A 48 KD of recombinant fusion protein was purified with Ni²⁺-NTA resin.The antiserum was obtained by immunizing Balb/c mouse with the purified recombinant protein then identified by western blotting and ELISA.The result demonstrated that the antiserum could specifically bind to the recombinant protein and the PthA from Xanthomonas axonopodis pv.citri.,as well as to partially inhibit the disease development.5.Three cell lines producing monoclonal antibody,which can specifically recognize and strongly bound to pthA-NLS,were acquired.The immunoglobulin subtype of McAb was identified as mouse IgG1.6.The variable region genes were amplified by RT-PCR from the RNA of hybridoma cell line 3D10H2,then strung together by SOE-PCR(splicing by overlap extension) and the ScFv gene was constructed.After cloned into pGEM-T and pET32a(+) vector,the recombinant ScFv gene was identified by endonuclease digestion,PCR and sequencing.The sequencing result showed that the ScFv gene consists of 747 bp,including 360 bp heavy chain variable region gene,342 bp light chain variable region gene and 45 bp linker gene.The recombinant fusion ScFv protein was expressed in E.coli BL21(DE3) with IPTG induction and analysised by SDS-PAGE,a 44.5 KDa of recombinant fusion protein was obtained.7.ScFv gene was cloned into pBI121 plant expression vector,named p35S. Agrobacterium-mediated transformation was carried out to introduce p35S into sweet orange.12 kanamycin resistant plants were obtained.PCR analysis showed the integration of the ScFv gene in sweet orange genome.The expression of the transgene ScFv was detected by using RT-PCR,ScFv were expression successful in the 4 transgenic plants.