Identification Of Apple Stem Pitting Virus And Prokaryotic Expression Of Its Coat Protein Gene

Apple stem pitting virus(ASPV) is worlds-wide distributed in apple and pear-growing areas.It usually latently infects apple and pear trees without visible symptoms.However,it can cause the tree decline and reducethe yield and quality of fruits when the rootstock is sensitive to it.In present,the cultivation of virus-free varieties is the effective way to reduce the losses caused by ASPV.The virus detection is the critical step for obtaining virus-free germplasm.In this study,three ASPV isolates P-HH and P-3-2-67 from pear and G-L3 from grapevine were detected and identified by biological indexing and PCR.Comparisons of their biological characteristics,particle morphology under electronic microscopy and sequences suggested that the isolate G-L3 from grapevine should be ASPV.A prokaryotic expression vector of CP gene of ASPV from Pyrus pyrifolia was constructed and the CP gene was expressed in Escherichia coli(E.coli) successfully.Antiserum against the recombined fusion protein was prepared by the immunonization into two rabbits.The results are summarized as follows.1.The isolates P-HH and P-3-2-67 from pear trees and G-L3 from grapevine displayed obvious symptoms after mechanical inoculation on herbaceous indicators Nicotiana occidentalis '37B':P-HH displayed hollowed necrosis spot,vein necrosis and crinkle symptoms;P-3-2-67 displayed black spot,vein necrosis and branch etiolating symptoms;G-L3 displayed chlorotic spot,black spot and necrosis symptoms.After TC-RT-PCR(Tube capture-reverse transcription PCR) amplification,PCR products of the three isolates all showed the 316 specific fragments on 6%PAGE.Further purification of viruses and electron microscopy showed that the viral particles morphology of the three isolates were the same,which belonged to flexuous filamentous virus particles.Then we can get that the isolates P-3-2-67,P-HH and G-L3 from pear and grapevine presumably were ASPV.2.The 316bp were amplified from Nicotiana occidentalis '37B' infected ASPV inoculation of P-HH,P-3-2-67 and G-L3 by TC-RT-PCR.The PCR products of three isolates of P-HH,P-3-2-67 and G-L3 were cloned,sequenced.Analysis of sequence revealed that the amplified fragments of the three isolates shared 98.4%of nt identity with P1 from Pyrus pyrifolia by our lab,shared 93%of nt identity with Quince isolated from Pyronia veitchii and shared only 77.8%of nt identity with N1 isolated from Cydonia oblonga.The nucleotide sequences among the three isolates also showed some differences.The similarities of P-HH and P-3-2-67,P-HH and G-L3,G-L3 and P-3-2-67 were 99.1%,99.1%and 99.4%,respectively.This is the first report on a virus isolate (G-L3) from grapevine showed a high similarity with ASPV.3.Base on the sequence of previously cloned CP gene of isolate 6-1-13 from Pyrus pyrifolia,a pair of primers with two enzyme-digestion sites at two ends was designed for the expression of the gene.The complete CP gene was amplified from a clone containing the CP gene of isolate P1.A prokaryotic expression vector of the CP gene(pET-ASPV-CP) was constructed by inserting the amplified products into a vector pET-28a(+).The vector was transformed into BL21(DE3).The expression products under the inducing of IPTG were analyzed by SDS-PAGE and Western-blot.Results showed the coat protein gene was expressed in BL21(DE3),and its molecular weight was about 45 kDa.Polyclonal antibody against the protein was prepared by immunizing rabbits with the purified expression protein,and its titer was 1:1 000 in indirect ELISA test.Western-blot showed that the prepared antibody can react with the recombinant protein.