| Cherry rasp leaf virus(CRLV) and Cherry mottle leaf virus (CMLV) are the two most important viruses infecting cherry. On susceptible cherry cultivars, CMLV cause symptoms such as chlorotic mottle-leaf pattern, distortion and necrotic spots. The symptoms of CRLV in cherry include leaf deformation, folding, narrowing, and enations on the underside of leaves along the midribs, and limbs of the infected trees might become bare near the bas es. The two viruses both result in reductions in size and quality of fruits, even more serious,they may bring a total loss of the fruit yields. In this study, antiserums specific for the two cherry viruses were prepared by modern gene technique. It is important for the cherry to control the two viruses’incidence and prevalence.The recombinant expression vectors were transformed and induced by IPTG in Escherichia coli BL21(DE3) Plys. The fusion proteins were purified by GST-compatible column, then were used to prepare the antiserums specific for CMLV and CRLV respectively.With the above antiserums, the incidence of CMLV and CRLV in Jining was investigated by ID-ELISA. In the investigated nurseries7.14%of the samples were infected with CMLV,7.14%with CRLV, and3.57%with both of them. In the work titer1:1000, the antiserums have a specific response to induced proteins and infected leaves by the Western blot methods. The plants were gathered from the city of Jining in Shandong province, they were defined to be infected with CMLV and CRLV by RT-PCR, Western blot and ID-ELISA assay. The leaves with viruses were used to determining the titer of the antiserums. The titers were respectively1:4096,1:4096and1:512.The ORFs encoding VP24,VP25of CRLV and CP of CMLV were successfully amplified by RT-PCR, and cloned into pMD18-T, then the target genes were determined by sequencing. The ORF encoding VP24of CRLV Jining isolate from Shandong is579bp, encoding a21kDa protein with192aa, and shares100%similarity with the Canada isolate at nucleotide sequence leval. The ORF encoding VP25of CRLV Jining isolate from Shandong is657bp, encoding a23.8kDa protein with223aa, and shares100%similarity with the Canada isolate at nucleotide sequence leval. The ORF encoding CP of CMLV Jining isolate from Shandong is579bp, encoding a21kDa protein with192aa, and shares100%similarity with the Canada isolate at nucleotide sequence leval. |
