Study On Importing Of Coat Proteins Of Three Viruses Spread By Aphids Into Intact Choroplasts In Vitro

Turnip mosaic virus (TuMV), soybean mosaic virus (SMV) and sugarcane mosaic virus (SCMV) which belong to Potato virus Y group are worldwide spread mainly by aphids in non-persistent mode under natural conditions. They are the chief viruses of Brassicaceae, Leguminous and Poaceae families, respectively.Viruses were isolated from the leaves of cabbage, soybean and maize which had typical symptoms, and then identified and purified by RT-PCR and sequence determination methods. The nucleotide sequences of the isolate from leaves of cabbage in Nanjing shared a less homology with TuMV-CP isolated from India,Hebei and Beijing, but over90.0%homology with those from other areas. The nucleotide sequences of the isolate from leaves of soybean in Hongze shared a less homology with SMV-CP isolated from China, but over90.0%homology with those from other areas. The JY-SCMV-CP,JY-HC-Pro,JY-Vpg and JY-NIb nucleotide sequences of the isolate from leaves of maize in Jiangyan shared a less homology with MDMV,SrMV and JGMV, but over90.0%homology with those from other areas.Therefore, viruses isolated from the leaves of cabbage, soybean and maize were identified as TuMV, SMV and SCMV, respectively. And then purification was carried out. The ratio of A260/A280using UV absorption spectrum scaning was1.17,1.15and1.30, which indicated the concentration of TuMV, SMV and SCMV was13.70,11.68and2.51mg/mL, respectively. Three antiserums, Tull, S21and SC31, were produced using purified TuMV, SMV and SCMV, respectively, and the corresponding diluted times werel:512000,1:204800and1:25600.Referred to Banerjee’s method, the systems of TuMV, SMV and SCMV importing into intact chloroplasts in vitro were established, effects of the incubation time, the concentration of TuMV-CP,SMV-CP and SCMV-CP on import efficiency were studied and its species speciality. Results showed TuMV-CP, SMV-CP and SCMV-CP was rapidly imported into isolated chloroplasts of vegetables, soybean and maize. In the test the lowest incubation times of TuMV-CP,SMV-CP and SCMV-CP importing into the chloroplasts were all5min, the amounts of TuMV-CP,SMV-CP and SCMV-CP imported into the chloroplasts were not increased with extending the incubation time from15min. Besides, the relation between the amounts of TuMV-CP,SMV-CP and SCMV-CP imported into the chloroplasts and the amounts of TuMV-CP,SMV-CP and SCMV-CP added in the import system was significantly positive correlation.With reference to the experimental laboratory of TMV-CP and two kinds of wheat soil borne mosaic virus CWMV-CP and BaYMV-CP chloroplasts isolated transmembrane transport research results, we found infection of import time and the amount of CP to import efficiency for TuMV-CP,SMV-CP and SCMV-CP were the same. The amount of CP imported into the chloroplasts was independent of the import time, if the time preponderate over15min. Besides, the relation between the amount of CP imported into the chloroplasts and the amount of CP added in the import was significantly positive correlation, and had a certain correlation with the coat protein of the size the lowest concentrations of TuMV-CP,SMV-CP and SCMV-CP in the import system were54.80μg/mL,49.05μg/mL and50.20μg/mL. The establishments of TMV-CP,SMV-CP and SCMV-CP importing into intact chloroplasts in vitro could supply foundation for studying the pathogenesis of Potato virus Y group members.