JIA-2019-11

2552 LI Liu et al. Journal of Integrative Agriculture 2019, 18(11): 2549–2560 obtained sequences were assembled into a contiguous sequence at a standard of over 99.9% similarities at each of the overlapped regions. 2.4. Sequence analyses Sequences were aligned using Clustal W 2.0 with default settings, and imported into the MEGA 7.0.14 Program. Multiple nucleotide sequence alignments were performed using the MUSCLE algorithm implemented in the MEGA 7.0.14 Program (Kumar et al . 2016). Phylogenetic trees were inferred by using the Maximum Likelihood method packaged in the MEGA7.0.14 Program with 1000 bootstrap replicates. The sources and GenBank accession numbers of the genomic sequences of ASPV isolates and two AGCaV isolates referred from the GenBank database (www. ncbi.nlm.nih.gov) were listed in Appendix A. The putative epitopes of ASPV coat protein were analyzed with a Kolaskar and Tongaonkar method (1990). Surface plot and antigenic index analysis packaged in the DNAStar Software (DNAStar, Madison, WI, USA). 2.5. Expression of ASPV CP genes in E. coli and Western blot assay The CP gene of isolate ASPV-LYC was inserted into prokaryotic expression vector pET-28a(+) (Novagen, Madison, WI, USA) for recombinant protein production. Meanwhile, three recombinant prokaryotic expression plasmids containing CP genes of isolates HB-HN6, HB-HN9 and YN-MRS17 (Ma et al . 2016) and the antisera raised against their recombinant coat protein (rCP) (unpublished data) were included in the serological analysis in this study. The recombinant plasmids were denoted as pET-LYC, pET-HN6, pET-HN9, and pET-MRS17 and transformed into E . coli BL-21 (DE3) pLysS competent cells. Protein production was done by adding 0.5 mmol L –1 isopropyl-β- D-thiogalactoside (IPTG) into 2-h pre-incubated bacterial culture and inducing at 28°C for 6 h in Luria-Bertani (LB) medium containing 50 mg L –1 kanamycin. The expressed protein was evaluated by 12% SDS-PAGE. Gels were stained with 0.25% Coomassie blue G250 solution. For Western blot, total proteins from induced cells were separated on 12% SDS-PAGE and electro-transferred onto PVDF membranes. Membranes blocked with 5% (w/v) skim milk in PBST (0.01 mol L –1 PBS, 0.05% Tween-20, pH 7.4) were individually incubated with antibodies of HN6, HN9 and MRS17. Alkaline phosphatase-conjugated goat anti- rabbit IgG diluted at 1:5 000 (Sigma, Germany) was used as the secondary antibody. Antigen-antibody reactions were visualized by incubation in the substrate solution containing 0.35 mg mL –1 nitroblue tetrazolium (NBT) and 0.18 mg mL –1 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Amresco, USA). 2.6. Biological characterization One-year-old P . betulifolia seedlings were used as root stocks. Each plant was double-grafted (Siebert and Engelbrecht 1981) with a bud from a one-year-old shoot of Pyronia veitchii as an indicator and a bud-chip of ‘Chili’ infected by ASPV-LYC as an inoculum. The inoculation test was triplicated. Three plants mock-inoculated using healthy pear buds as inocula and buds of P. veitchii as indicators were used as negative controls. Symptoms were visualized following sprouting of new leaves during the growing season for three successive years. 3. Results 3.1. Characterization of the genome of ASPV-LYC The primary RT-PCR detection using two sets of ASPV- specific primers ASPV247-F/ASPV247-R and 370A/370B showed that four out of the five ‘Chili’ samples were positive for ASPV. Sequencing for amplified products of ASPV CP gene showed that theASPV isolates from ‘Chili’ were highly divergent by having low nucleotide sequence similarities (less than 70%) for their partial CP gene with that of other known ASPV isolates, indicating the ASPV isolate from ‘Chili’ might be a novel molecular ASPV variant. Here, it was named asASPV-LYC based on its geographic and host origins. Thereby, one sample was used for amplifying the complete genome of the ASPV-LYC. The genome of ASPV-LYC (accession no. MG763895.1) was 9273 nucleotides (nts) long, excluding the poly(A) tail at 3´ end (Table 2). The genomic organization was similar to previously reported ASPV isolates (Jelkmann 1994; Adams et al . 2012). ORF1 (60 to 6 614 nt) encoded a 247-kDa polymerase. The ORF1 contained all domains conserved in RNApolymerases of members in the family Betaflexiviridae (Jelkmann 1994; Zhang et al . 1998; Martelli et al . 2007; Morelli et al . 2011; James et al . 2013). These domains included a methyltransferase at aa 43 to 356, an AlkB-like domain (aa 764 to 854) related to the 2-oxoglutarate- and Fe(II)- dependent oxygenase superfamily, a cysteine protease (aa 1 098 to 1 194) homologous to the ovarian tumour (OTU) gene of Drosophila spp., a peptidase (aa 1 200 to 1 287) belonging to the C23 Merops family, a helicase (aa 1 378 to 1 636) and an RNA-dependent RNA-polymerase (RdRp, aa 1 764 to 2 170). Near the C-terminus of the RdRp (aa 2 021 to 2 056), there was the core motif TG(x) 3 T(x) 3 NT(x) 22 GDD conserved in the members of genus Foveavirus (Martelli et al . 2007). ORF2 (672 nts, 6 684 to