Apple stem pitting virus (ASPV) is the type species of the genus Foveavirus, the family Flexiviridae (Martelli and Jelkmann, 1998). ASPV has a worldwide distribution and often remains symptomless. It has been demonstrated that pear corky pit, pear stem pitting, pear vein yellow, pear yellow etc. were caused by ASPV (Jelkmann et al., 1992; Jelkmann, 1994; Deng and Wang, 2002). ASPV was found worldwide and frequently occurs in combination with other latent virus such as Apple chlorotic leafspot virus (ACLSV) and Apple stem grooving virus (ASGV) (Lenoe et al., 1998). This kind of co-infection is the main cause of a decrease in yield quality and quantity, and even leads to tremendous economic losses.

The genome of ASPV is a single-stranded positive-sense RNA with molecular weight of 3.6×10⁶ Da and encompasses 44~48 kD coat protein (CP). Most recently, there were only four complete genomic sequences of ASPV isolates had been deposited in GenBank, the accession numbers were NC_003462 (Jelkmann, 1994), AB045371 (Yoshikawa et al., 2001), EU095327 and FR694186, respectively. Many ASPV CP gene sequences from different regions have been reported and the analyses have been demonstrated that the sequences of CP gene were highly variable amongst isolates (Schwarz and Jelkmann, 1998; Nechinov et al., 1997; Li et al., 2010a). ASPV content was low in plant tissues, and difficulty to separation and purification. Jelkmann (1994) prokaryotic expressed the ASPV CP gene and preparation of ASPV antiserum, but not used for field detection. Recently, there was not prepared high efficiency antiserum for ASPV. In this study, the complete CP gene of ASPV Ya isolates provided additional baseline data for further study of ASPV differentiation and molecular variation, and prokaryotic expression vector of Ya pear provided some insights for preparation recombinant polyclonal antibody of ASPV and further study of ASPV molecular biology.

1 Results and Analysis
1.1 Cloning and Sequencing of ASPV CP gene
Taken the phloem of pear (Y) which were infected with ASPV as materials to extract total RNA, first strand cDNA synthesis was obtained by reverse transcription using specific primer, a 1 392 bp fragment was amplified by PS/PA primers as shown in Figure 1. The purified DNA fragments were ligated into the PMD19-T vector and transform into E. coli DH5α. The positive clones were confirmed by PCR and restriction enzyme digestion and designated Y/PMD-T before sequencing (Figure 2).

Figure 1 Detection of ASPV CP gene by RT-PCR Note: M: D514A 200 marker; 1: Y RT-PCR product; 2: Negative control

Figure 2 Identification of recombined plasmid by PCR and restriction enzyme Note: M: D514A 200 marker; 1~3: Identification recombined plasmid by PCR; 4~9: Identification recombined plasmid by restriction enzyme

1.2 Analysis of ASPV isolates
The complete CP gene of Y isolate, which has been deposited in the GenBank and allocated the accession numbers of JF946774, was consisted of 1 194 bp and potentially encoded a protein of and molecular mass (Mr) of 42 kD, consisting of 397 amino acids. The base compositions of CP gene were found to be adenine 27%, cytosine 25%, guanine 24%, and uracil 24%, respectively.

To include a wider range of isolates and thus obtain more representative results in the phylogenetic analysis of ASPV, we further analyzed the complete CP gene sequences of 28 ASPV isolates including the determined in this research. Y isolate CP gene at aa sequences showed approximately 70% identities with the CPs of ASPV isolates (Table 1).

Table 1 Accession number and geographical origin and identities of CP amino acid sequences of ASPV isolates

Phylogenetic analysis of the CP genes of 28 ASPV isolates at aa sequence revealed three groups. All ASPV isolates from apple were clustered to group I, whereas those of pear were clustered to group â…¡ which included nine isolates (except NC_003462, apple) and Y isolate was clustered to group â…¢ (Figure 3). Furthermore, group I and group II both can be further divided into five sub-groups. However, the formation of these sub-groups had not certain regularity.

Figure 3 Phylogenetic tree constructed from the complete CP gene at aa sequences of 28 ASPV isolates

1.3 Cloning and construction of the ASPV-CP-Y/PET
A predicted 1 194 bp product containing the entire CP gene of ASPV was amplified using PCR from Y isolate. The sequence result of the resulting clone CP-Y/PMD was consistent with the sequence of previously study. The constructed CP-Y/PMD-T was digested with Salâ… and Ncoâ… , and the insert was ligated into PET-28a vector precut with the same enzymes. The resulting ASPV-CP-Y/PET-28a was verified by PCR and restriction enzymes analysis (Figure 4).

Figure 4 Identification of recombined plasmid by PCR and Restriction enzyme Note: M: D514A 200 marker; M1: SM1243 marker; 1~3: Identification by PCR; 4: recombined plasmid; 5: Identification of recombined plasmid by double restriction enzyme

1.4 Expression and SDS-PAGE analysis
The recombinant expression plasmid ASPV-CP-Y/PET-28a was transformed into E. col BL21 (DE3). To obtain a highly expressed level of ASPV CP gene protein, we tried optimizing expression conditions by using different IPTG concentrations (0 mM, 0.5 mM, 0.8 mM, 1.0 mM, respectively) and different incubation times (0 h, 1 h, 2 h, 3 h, 4 h, 6 h, respectively). The results showed that the expression level of synthesized ASPV-CP-Y/PET at 37℃ and with the increased time of incubation, and the expression proteins was increased, too. While the different concentrations of IPTG showed insignificantly influence of expression. A 42 kD fusion protein was highly expressed after induced at 37℃ for 4 h with 1 mM IPTG (Figure 5).  

Figure 5 SDS-PAG of expression products of ASPV CP gene Note: M: D523A marker; 1: Vector control; 2: Total proteins from BL/ASPV-CP-Y/PET-28a uninduced by IPTG; 3~7: Total proteins from BL/ASPV-CP-Y/PET-28a induced by IPTG for 1 h, 2 h, 3 h, 4 h, 6 h, respectively

2 Discussion
In this research, we reported the complete genomic sequences of Chinese ASPV isolates Y, and the complete CP gene consisted of 1 194 nucleotides and encoded a polypeptide of 397 amino acid (aa), which has been deposited in the GenBank and allocated the accession numbers of JF946774. Comparison of the amino acid sequences of Ya pear CP gene with other ASPV isolates showed approximately 70% similarity. Sequencing and analysis of ASPV isolates revealed that the virus had complex sequence variants among different isolates. Figure 3 showed that ASPV isolates at aa sequence demonstrated three groups. All ASPV isolates from apple were clustered to group I, whereas those of pear were clustered to groups â…¡ (except NC_003462) and the Ya pear were clustered to group â…¢. The isolates in group I and group â…¡ mostly were from Poland and China, and the two groups showed that there were no clear geographical differentiations between ASPV isolates and slight related to host, and this result was similar to Li et al (2010a). Furthermore, group I and group II both can be further divided into five sub-groups. However, the formation of these sub-groups had not certain regularity. According to the classification of the ASPV CP gene, we suggested that the variation of ASPV isolates were closely related to the host and ecological environment. Therefore, it is necessary to consider the geography, climates, as well as other part of sequences information of genomic to analyze the variation and evolutionary relationships of ASPV.

Recently, the antiserum for effective detection of ASPV had not been produced. Hou et al., (2005) fast detection of ASPV in Pyrus pyrifolia and clone and the prokaryotic expression of its coat protein gene. Li et al., (2010b; 2010c) found that the complete CP gene of ASPV was expressed in Escherichia coli using gene recombinant method, and the antiserum was produced after the rabbit was immunized with purified protein and production of antiserum of ASPV based on antigenic epitopes technique, but only partial positive samples could be detected with the antiserum. Nucleotide sequence of different isolates of ASPV CP genes have a high variation, and lead to existence difference serological of ASPV, and thus influence the detection results. We generally believed that amino acid sequence variations of ASPV CP gene may cause epitope variants, and then were divided into different ASPV serotypes. Therefore, in order to improve the accuracy of ASPV detection, it is necessary to preparation of different ASPV antiserum. In this study, we have successfully constructed ASPV CP gene into prokaryotic expression vector PET-28a, and specific expression of a 42 kD fusion protein was achieved by the inducing of 1 mmol/L IPTG. At present, we are carrying out to preparation antiserum using prokaryotic expression vector of ASPV CP gene from Ya pear.

3 Materials and Methods
3.1 Virus source
Ya pear samples were collected from Korla pear in Shayidong commercial orchard of Korla, Xinjiang, China. This isolate was designed as Y. All these symptomatic leaves and phloem were stored at 4℃.

3.2 Chemicals and reagents
Taq DNA Polymerase was purchased from Gongdong Dongsheng Biotech (China); dNTPs, Ribonuclease inhibitor, IPTG were purchased from Shanghai Sangon (China); M-MLV Reverse Transcriptase, T4 DNA ligase are from Fermentas (USA); Ncoâ… , Salâ… , PMD19-T, DNA Marker D514A and Protein Molecular Weight Marker D523A were all purchased from TakaRa (China); TIANprep Mini Plasmid Kit and TIANgel Midi purification Kit from TIANGEN (China); others were all analysis purity made in China. Expression vector PET-28a, E. coli DH5α and BL21 (DE3) as preserved strains were stored at Biotechnology Laboratory of Horticultural Department, Agriculture College, Shihezi University, China.

3.3 Primer design
The sequences were amplified by PCR reaction with specific primers, which were designed according to the cDNA sequence of ASPV from GenBank NC_003462. Primer sequences are as follows: forward primer (PS) 5’-CCCATTAGGTTAGGGTGTAGTTGCT-3’ (nt 7829-7853) and the reverse primer (PA) 5’-ATGAAAGAAACACACACAT AGCCGC-3’ (nt 9249-9273). Forward primer (PS-E): 5’-CCATGG ATGGCTTCCGATGGTTCG-3’ and the reverse primer (PA-E) 5’-GTCGACC TTCCT GATTGATAA AAC-3’ containing the Ncoâ…  and Salâ… restriction sites (underlined) containing the complete ORF of ASPV CP gene according to the amplified sequence by the primers PS and PA. All the primers were synthesized by Shanghai Sangon Biotech and dissolved in ultrapure water to 20 pmol/µL concentration for use.

3.4 Total RNA extraction and RT-PCR
Total RNAs were extracted from phloem infected by Y using the approach described by He et al., (2004). The reverse transcription mixture contained 1.0 μL specific reverse primer and 5.0 μL of total RNA and 9.5 μL of ddH₂O. The mixture was kept at 70℃ for 5 min, and then immediately transferred to ice for 5 min. And then 2.5 μL of dNTPs (10 mM each), 5.0 μL of 5×M-MLV buffer, 1.0 μL of ribonuclease inhibitor (40 U•µL-1; Sangon, china), 1.0 μl of M-MLV reverse transcriptase (200 U/µL; Fermentas, USA) and make the total volume of 25.0 μL. The mixture was incubated at 42℃ for 1 h.

PCR reaction volumes were 20 μL, and contained 2.0 μL of 10×PCR buffer, 0.5 μL of dNTPs (each 10 mM), 2.0 μL of forward and reverse primers, 2.0 μL of cDNA, 0.2 μL (5 U/µL) Tap DNA polymerase and 13.3 μL of ddH₂O. PCR was carried out with an initial denaturation of 4 min at 94℃, followed by 32 cycles of 45s, 94℃; 1min, 72℃; and then by a final elongation step of 7 min at 72℃.

3.5 Cloning and sequencing
The amplified PCR products were gel purified and extracted using TIANgel Midi Purification Kit (TIANGEN, China). The purified DNA fragments were ligated into the PMD19-T vector (TaKaRa Biotechnology, China) following the manufacturer’s instruction, and used to transform E. coli DH5α. The positive clones were confirmed by PCR and restriction enzyme digestion and named as Y/PMD-T before sequencing. Two clones from independent PCR reactions were sequenced from both directions. 

The primers PS-E and PA-E were designed according to previous studies. We used these primers and template Y/PMD-T for PCR amplification. The purified DNA fragments were ligated into the PMD19-T vector and transformed into E. coli DH5α. The positive clones were confirmed by PCR and restriction enzyme digestion and named as CP-Y/PMD-T. 

3.6 Sequence analyses
Sequence homology analysis was conducted with DNAMAN; Phylogenetic trees were constructed by neighbor-joining (NJ), minimum evolution (ME), and maximum parsimony (MP) method in the MEGA (version 4.1) software (Mushegian and Koonin, 1993). Bootstrap analyses with 1000 replicates were performed to evaluate the significance of the interior branches. 

3.7 Construction of prokaryotic expression vectors of ASPV CP gene
The recombinant plasmid CP-Y/PMD-T was cloned into the Ncoâ…  and Salâ… restriction sites of the expression vector pET-28a (+). The insert and vector were ligated with T4 DNA ligase and then was used to transform E. coli DH5α. The correct recombinant positive clone was selected and sequencing to verify the correctness of ORF and designated ASPV-CP-Y/PET.

3.8 Induction expression and SDS-PAGE electrophoresis analysis of ASPV CP gene
The recombinant plasmid ASPV-CP-Y/PET-28a was transformed into E. coli BL21 (DE3), and harboring ASPV-CP-Y/PET was grown in 3 mL LB liquid medium supplemented with kanamycin (50 g/mL), 37℃ overnight and then 1: 100 diluted, vaccination into fresh 10 mL LB liquid medium with the corresponding antibiotic with shaking at 37℃ an OD₆₀₀ of 0.6~1.0. The culture was induced with IPTG (isopropyl-β-D-thio-galactoside) and the final concentration of 1 mM. The cells were grown for an additional 4~6 h to allow expression of the recombinant protein and then harvested by centrifugation at 8000 r/min for 5 min and resuspended in TE solution (pH 8.0), adding volume of 2×SDS buffer, and then the cells were boiled for 3 min, centrifuged at 12 000 r/min for 10 min. The recombinant protein was detected by SDS-PAGE using 12% polyacrylamide gel. Uninduced recombinant clone and E. coli BL21 (DE3) host cells (with and without IPTG) were used as controls. Briefly, the gel was stained with Coomassie brilliant blue R-250 for 1 h and destained in acetic acid until a clear background was seen.

Authors’ contributions
JXN was responsible for experimental design and the experiment direction; NL and JXN were responsible for data analysis, paper writing and modification. Both the authors had read the final version of this paper and agreed with the authors’ credits.

Acknowledgements
This study was supported by National Natural Science Foundation of China (30360066), the National Key Technologies R&D Program of China (2003BA546C), the Foundation Science and Technology Commission Xinjiang Production and Construction Crops, China (NKB02SDXNK01 SW) and Natural Science and Technology Innovation of Shihezi University, China (ZRKX200707).

References
Deng X.Y., and Wang G.P., 2002, Advances in research on pear viruses, Journal of Fruit Science, 19(5): 321-325

He M., Li H.S., Zhao Y., Niu J.X., and Ma B.G., 2004, A method of rapidly extracting total RNA from Korla pear, Shihezi, Journal of Shihezi University, 22(6): 474-476

Hou Y.X., 2005, Research on fast detection of Apple stem pitting virus in Pyrus pyrifolia and clone and the prokaryotic expression of its coat protein gene, Thesis for M.S., Huazhong Agricultural University, Supervisors: Wang G.P., and Hong N., pp.29-43

Jelkmann W., 1994, Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses, J. Gen. Virol., 75(7): 1535-1542 http://dx.doi.org/10.1099/0022-1317-75-7-1535 PMid:8021584

Jelkmann W., Kunze L., Vetten H.J., and Lesemann D.E., 1992, cDNA cloning of dsRNA associated with apple stem pitting disease and evidence for the relationship of the virus-like agents associated with apple stem pitting and pear vein yellows, Acta Horticulture, 309: 55-62

Leone G., Lindner J.L., van der Meer F.A., Schoen C.D., 1998, Sympotoms on apple and pear indicators after back-transmission from Nicotiana occidentalis confirm the identity of apple stem pitting virus with pear vein yellows virus, Acta Hortic., 472: 61-65

Li L.L., Dong Y.F., Zhang Z.P., Zhang Z.H., Fan X.D., and Pei G.Q., 2010a, RT-PCR detection and molecular variability of apple stem pitting virus, Acta Horticulturae Sinica, 37(1): 9-16

Li L.L., Yang H.Y., Dong Y.F., and Gao Y., 2010b, Production of antiserum of apple stem pitting virus based on antigenic epitopes technique, Acta Phytophylacica Sinca, 40(4): 357-36

Li L.L., Zhang Z.H., Dong Y.F., Zhang Z.P., Fan X.D., and Pei G.Q., 2010c, Prokaryotic expression of Apple stem pitting virus coat protein and antiserum preparation, Acta Phytophylacica Sinica, 37(4): 319-324

Martelli G.P., and Jelkmann W., 1998, Foveavirus, a new plant virus genus, Arch. Virol., 143(6): 1245-1249 http://dx.doi.org/10.1007/s007050050372 PMid:9687881

Mushegian A.R., and Koonin E.V., 1993, Cell-to-cell movement of plant viruses Insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems, Arch. Virol., 133: 239-257 http://dx.doi.org/10.1007/BF01313766 PMid:8257287

Nemchinov L., Hadidi A., and Faggioli F., 1998, PCR-detection of apple stem pitting virus from pome fruit hosts and sequence variability among viral isolates, Acta Hortic., 472: 67-73

Schwarz K., and Jelkmann W., 1998, Detection and characterization of European apple stem pitting virus isolates of apple and pear by PCR and partial sequence analysis, Acta Hortic., 472: 75-85

Yoshikawa N., Matsuda H., Oda Y., Ito T., and Yoshida K., 2001, Genome heterogeneity of apple stem pitting virus in apple trees, Acta Horticulture, 550: 285-290