Prokaryotic Expression Of CP Genes Of Two Viruses From Pear And Molecular And Serological Diversity In Apple Chlorotic Leaf Spot Virus

Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are two important latent viruses in commercially cultivated apple and pear trees. Two viruses are usually mixed infection with Apple stem pitting virus (ASPV) and cause greatly decreasing the growth and productivity of infected trees. Sand pear (Pyrus pyrifolia) is widely grown in China. Over 80% of pear trees in China were infected by ASGV and ACLSV. In this paper, in order to establish the foundation for high-through and fast detection techniques of ASGV and ACLSV in China and study the serological diversity of ACLSV, the prokaryotic expression vectors of coat protein genes (cp genes) of ASGV and ACLSV from sand pear were constructed. The antisera against recombinant coat proteins (rCPs) expressed in Escherichia coli (E. coli) were prepared. Furthermore, the biological, molecular and serological characteristics of ACLSV isolates from sand pear were compared with other isolates from different fruit trees and the molecular and serological diversity in ACLSV was analyzed. The obtained results contributed to increased understanding of the molecular population structure of ACLSV in different hosts and its implication in serological detection. The obtained results are as followings:1. The cp genes of two ASGV isolates (714 bp,237 aa, GenBank accession numbers FJ608985 and FJ608986), P-4-1-69 and P-L2 from sand pear and white pear (P. bretschneideri), respectively, were inserted into prokaryotic expression vector pET-28a(+). The recombinant plasmids were denoted as pET-P-4-1-69 and pET-P-L2. E. coli strain BL21 (DE3) was transformed with two expression constructs. Protein productions were induced by 1 mM isopropyl-β-D-thiogalactoside (IPTG). SDS-PAGE and Western blot analyses indicated that cp genes of two ASGV isolates were expressed effectively under the induction of 1 mmol/L IPTG and molecular weights of their rCPs were approximately 31 kDa. These rCPs were used to raise antisera in rabbits. The titers of antisera were 1:512 000 for isolate P-4-1-69 and 1:32 000 or 1:64 000 for isolate P-4-1-69 in indirect-ELISA, respectively. In order to reduce the nonspecific reaction, the raised antisera were purified. These purified antibodies showed to possess high specificity to their rCPs in Western blot and native virus coat proteins in infected pear shoot in-vitro by tissue blotting immunoassay (TBIA).2. The cp gene of an isolate ACLSV (ACLSV-BD) from "Badongtongzili" pear shoot in-vitro was cloned and sequenced (GenBank accession number EJ608984). The cloned fragment consisted of 582 bp and encoded 193 aa. The cp gene was inserted into prokaryotic expression vector pET-28a(+). The recombinant plasmid pET-ACLSV-BD containing cp gene was obtained and transformed into BL21 (DE3). Protein productions were induced by 1 mM IPTG. SDS-PAGE and Western blot analyses indicated that cp gene of isolate ACLSV-BD was expressed effectively under the induction of 1 mmol/L IPTG and molecular weight of the rCP was approximately 26 kDa. The rCP was used to raise antiserum in rabbits. The titer was 1:32 000 or 1:128 000 in indirect-ELISA. The purified antibody showed to possess high specificity to the rCP in Western blot and native virus coat protein in infected pear shoot in-vitro by TBIA.3. Sixty-five sand pear samples were collected from the National Germplasm Conservation Center of P. pyrifolia (Wuhan, Hubei), western Hubei province (Enshi) and Yunnan province (Kunming). TC-RT-PCR (Tube capture RT-PCR) test indicated that 80% (52/65) sand pear samples were infected by ACLSV. Chenopodium quinoa and Nicotiana occidentalis were inoculated with extracts from sand pear leaves infected by ACLSV. The symptoms of systemic chloroctic, mottle and leaf roll on C. quinoa and systemic chloroctic, necrotic and yellow vein on N. occidentalis, were observed and caused by the complex infection of ACLSV and ASGV.4. The cp genes of ACLSV from 20 sand pear samples, two plum samples (Wuhan, Hubei) and a peach sample (Xian, Shanxi) were cloned. The plasmids containing cp genes were digested with the restriction enzymes EcoR I and Sac I. The result showed that digested-products of cp genes contained four patterns, I, II, III and IV, and I and II were predominant patterns. The digested-products of ACLSV cp genes from the same sample showed different patterns, indicating that ACLSV isolate was composed of a population of variants. The cp genes sequence comparison revealed that two variants (PP15-2 and PP15-4) from isolate PP15 showed lower identity (96.6%) and were designed two isolates. The identities were more than 99% among variants from other isolates.5. The cp genes of 22 ACLSV isolates (including isolate ACLSV-BD) from sand pear, two isolates from plum and an isolate from peach were compared (GenBank accession numbers EJ608984, GU327981-GU328004). The sequences of cp genes from 22 sand pear isolates showed a high divergence, with 87.3～100% identities at the nucleotide (nt) level and 92.7～100% identities at the amino acid (aa) level. The phylogenetic trees generated from the nucleotide and deduced amino acid sequences of cp genes, including 25 isolates from this study and 45 isolates from GenBank, showed that the analyzed ACLSV isolates fell into different clusters and all isolates from sand pear were grouped into a larger cluster (Ⅰ) which was then divided into two subclusters (A and B). Nineteen out of 22 ACLSV sand pear isolates were grouped into the subcluster A with five isolates from stone fruit tree (PE, PL1, PL2, HBP and SX/2), whereas other three sand pear isolates (PP39, PP54 and PP56) together with a pear isolate (Pear) from India, an apple isolate (ACLSV-C) from China and other isolates from Japan and India were grouped into the subcluster B. The isolate (Kuerle) from kuerle pear (P. sinkiangensis Yu) specially grown in Xinjiang province showed low identities with 22 sand pear isolates and was grouped into a cluster (Ⅱ) together with some apple and stone fruit isolates from Japan and India. The multiple alignment of coat proteins (CPs) revealed that the five amino acids combination at positions 40,59,75,130 and 184 (S40-L59-Y75-T130-L184) was presented in 25 ACLSV isolates analyzed in this study, suggesting that these isolates were belonged to B6 type. An aa residue Glutamic acid (E) at position 70 was conserved in three sand pear isolates (PP39, PP54 and PP56) and isolate ACLSV-C grouped into the subcluster B.6. Based on the divergence deduced from phylogenetic analysis, the cp genes of five ACLSV isolates (PP13, PP15-2, PP24, PP43 and PE) grouped into the subcluster A and three (PP54, PP56 and ACLSV-C) grouped into the subcluster B were selected for producing recombinant proteins for further analyses of electrophoretic mobility and serological reactivity. SDS-PAGE, Western blot and PAS-ELISA analyses demonstrated that rCPs of eight ACLSV isolates had different mobility rates and serological reactivity. The rCPs of five isolates grouped into the subcluster A showed stronger reactivity with antibodies against rCPs of a sand pear isolate ACLSV-BD (subcluster A) and virions of a Japanese apple isolate P-205 (clusterⅡ) than that with the antibody against a Chinese apple isolate ACLSV-C (subcluster B). Three isolates grouped into the subgroup B showed stronger reactivity with the antibody against ACLSV-C. The antigenic determinants of CPs from eight isolates and isolates ACLSV-BD and P-205 predicted nine epitope regions in their CPs. Six predicted epitopes showed some variations among different isolates.