Pilot Studies On Black-Dot Disease Of Bagged Apple In Shaanxi And Cloning And Prokaryotic Expression Of HC-pro Gene Of SCMV

Sugarcane mosaic virus (SCMV), a member of Potyviridae and a nonpersistent aphid transmission virus, is a dangerous pathogen in maize, sugarcane and sorghum in the world, leading to significant crops losses. HC-pro gene, which is encoded by the SCMV genome, is a protein related with aphid-transmission, otherwise it takes part in many other bio-chemistry procession. In this experiment, we designed the particularity primers, and got the HC-pro gene fragment from infected corn of SCMV in Shaanxi with RT-PCR technology. Then, the HC-pro gene was prokaryotic expressed.We got the sugarcane mosaic virus isolate in shaanxi and identified the pathogen with a series of biological and electron micrograph research. Purified virus of SCMV is filamentous flexuous particles about 720～760nm long arid 13nm wide, with a typical UV spectrum of nucleoprotein and OD280/OD260 was 0.69. With the whole tissue lysate RNA purified from diseased maize leaves infected by SCMV, RT-PCR reverse transcript reaction was amplified, and we got a fragment of SCMV HC-pro gene about 1.4 kb finally. Then the fragment was cloned and sequenced. Sequence analysis shows that the complete viral genomic RNA of SCMV shaanxi isolate is composed of 1380 nucleotides. The GenBank accession number for the sequence in this paper reported is DQ667961. Compared with counterpart of other isolates in China, the nucleotides identity and aminophenol identity were Shandong isolate (91.7%/99.3%), Henan isolate (91.6%/99.3%), Beijing isolate(90.9%/98.7%),Mexico isolate (89.0%/98.9%), Zhejiang isolate (88.8%/98.0%), Guangdong isolate (83.3%/96.1%), Lingpin isolate (81.2%/95.4%), Yuhang isolate(81.0%/95.2%). The cloned HC-pro gene was ligated into pET30a expression vector for construction of pETHC. And the recombinant plasmids were transformated into E.coli BL21 for expressing. The SDS-PAGE suggested that pETWHC had the higher expression with a especial protein weight of about 57kD as similar as expected. After a serious of experiment ,we found that the different pre-culture in 37℃ lead to the difference of the pBVHC HC-pro expression level.We purified virus of SCMV, cloned the HC-pro gene of SCMV shaanxi isolate, and did a primary study of prokaryotic expression firstly. Base on this study, the identity of SCMV, the special site of aphid-transmission can been analysised, and the accepted protein in the aphids with the HC-pro can been separated and purified with gene project and molecule biology technology. All the results are very important for deep research.