Production Of Monoclonal Antibodies Against Turnip Mosaic Virus And Virus Genomic Variation

1. Preparation of monoclonal antibodies to Turnip mosaic virus and itsapplication for the virus detection 1. 1 Identification of the pathogen of mosaic disease of Brassica juncea Var.multiceps in ZhejiangA virus isolate NBXC was collected from Brassica juncea Var. multiceps in Ningbo, Zhejiang Province. The isolate could infect eight tested host plants. Many filamentous virions were found in electron microscopy with length of about 740 nm. CP and HC-Pro genes of isolate NBXC were then amplified by immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR), and the amplified products were as expected with the length of 0.85 kb and 1.5 kb, respectively. The PCR products were cloned and determined. CP gene of NBXC was composed of 864 nucleotides encoding a polypeptide of 288 amino acids; HC-Pro gene was composed of 1374 nucleotides encoding a polypeptide of 458 amino acids. CP gene of NBXC has 90.0-98.3% nucleotide and 95.8-99.3% amino acid sequence identities with other reported Turnip mosaic virus (TuMV) isolates, while HC-Pro gene of NBXC has 82.2-96.9% nucleotide and 95.6-99.3% amino acid sequence identities with other reported TuMV isolates. The results of indirect ELISA test indicated that 83.6% of the collected samples were infected by TuMV, and all the samples were not infected by Cucumber mosaic ivirus (CMV) and Tobacco mosaic virus (TMV). The above tests results indicated that TuMV is the major pathogen of virus disease in Brassica juncea Var. multiceps in Zhejiang province.1 . 2 Preparation of monoclonal antibodies to Turnip mosaic virus and its applicationfor the virus detectionFour hybridoma cell lines capable of secreting monoclonal antibodies (MAbs) against TuMV were carried out by fusing mouse myeloma cells (SP2/0) with spleen cells taken from the BAL B/C having been immunized by the TuMV particles. The four MAbs could specifically react with TuMV. The ELISA titres of ascitic fluids of the four MAbs rangedfrom in 10-5~10-6. The result of Western-blot showed that 4 MAbs could react specifically with the 34 KD coat protein subunit of TuMV. TAS-ELISA was set up with polyclonal and MAbs for TuMV detection, and the MAbs could successfully detect 21.9 ng purified TuMV or virus in plant sap at 1:5120 dilution. TAS-ELISA was then used for detection of TuMV and the virus was detected in seven crops.1. 3 Application of TuMV monoclonal antibodies for identification of the resistanceof Brassica juncea Van multiceps to TuMVThe resistances of 57 Brassica juncea Var. multiceps cultivars to TuMV were identified by artificially inoculated with TuMV in greenhouse and field. The inoculation results showed that 7 cultivars were resistant to TuMV, 22 were tolerant, 28 were susceptible and no one was highly resistant to TuMV among all cultivars. In general, thin leaf cultivars were more resistant than wide leaf cultivars and mosaic leaf cultivars. TAS-ELISA using the produced monoclonal antibodies was used for detection of TuMV in inoculated leaf of cultivars, 46.55%, 91.38%, 100% and 100% of cultivars were found to be infected by TuMV, respectively, when tested at 10, 15, 20 and 30 days after inoculation. OD405 values in TAS-ELISA of most cultivars were increased after inoculation. TAS -ELISA could effectively applied in identification of the resistance of Brassica juncea Var. multiceps to TuMV with high sensitivity and accuracy.2. Variability and evolution of CP and HC-Pro gene of TuMV isolatesThe coat protein (CP) genes of of 15 TuMV isolates from different provinces, China, were determined. Phylogenetic analyses of the coat protein nucleotide and amino acid sequences revealed that all TuMV sequences fell into two distinct groups. TuMV isolates HZLB 1 and HZLB2 (all from Raphanus) were similar and placed in a distinct but smaller group, while other 13 isolates (almost from Brassica spp.) were placed in the largest group. Both nucleotide and amino acid sequence identities of CP genes between HZLB1 and HZLB2 were 99.3%, while those identities of other 13 isolates were 96.8-99.5% (nucleotide) and...