Pmd18 t

The PK15 cell line was obtained from the A.T.C.C. fetal bovine serum (FBS), dulbecco's modified eagle medium (DMEM) and Opti-MEM medium were obtained from Gibco). The vectors pcDNA™6.2-GW/EmGFP-miR, pMD-18T, pcDNA3.1(+), pDONR221 and pLenti6.3/V5-DEST, and the vector construction kits BLOCK-iT™ Pol II miR RNAi Expression Vector Kit with EmGFP, Packaging Mix, T4 DNA ligase, high purity plasmid extraction kit, Lipofectamine 2000 and TRIzol were purchased from Invitrogen. Synergy Brands, Inc (SYBR) premix ExTaq was purchased from Takara. Total protein extraction kit and BCA protein detection kit were purchased from Nanjing Keygen Technology. Primary antibodies–MyD88 (1:800), cluster of differentiation antigen 14 (CD14) (1:400), IFN-α (1:600), IL-1β (1:600), TLR4 (1:1000), TNF-α (1:1000) and β-actin (1:4000)–were purchased from Abcam. Second antibody (IgG-HRP, 1:3000) was purchased from Jackson. LPS was purchased from Sigma–Aldrich. Porcine IL-1β, TNF-α, IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1α and MIP-1β ELISA kits were purchased from AssayPro.

Fifteen overlapping fragments of the complete genome of HPyV6 strain BJ376 were PCR amplified (Table 2) with the Takara Ex Taq kit. The 15 overlapping fragments were then cloned into pMD18-T and sequenced (Invitrogen). The nucleotide sequence of the full-length HPyV6 genome was then compiled using BioEdit 9.0 software. The full-length HPyV6 sequence was aligned with the sequences of other HPyVs and other HPyV6 strains available in GenBank with DNAStar software. A neighbor-joining tree was constructed with MEGA 6.0.

RNA samples were isolated from the leaves of 5-day-old seedlings using plant Trizol reagent (Vigorous Biotechnology, China). 2 µg aliquot of total RNA was used for cDNA synthesis by M-MLV Reverse Transcriptase (TAKARA, Japan). The ORF primers of GmACSL2 are: GmACSL2-1F: 5′-ATGGCGACAATTCCTATCACCTAC-3′ and GmACSL2-1R: 5′ -TTACATGTATAGATTGTCTATTTGCTCCC-3′. The primers were designed by Primer Premier 5.0 [29] . The PCR conditions were as follows: 95°C for 5 min; 35 cycles of 95°C for 40 s, 58°C for 40 s, and 72°C for 2 min; and an additional step of 72°C for 10 min. The amplified products were cloned into pMD18-T vector (TAKARA, Japan) and then sequenced.
The PCR product of GmACSL2 from pMD18-T vector was digested with BamHI/Xbal, and then subcloned into vector pYES2 (Invitrogen, America) to generate the pYES2-GmACSL2 plasmid for yeast vector construction. GmACSL2 was first subcloned into the pENTR vector for subcellular localization, and then LR recombined with pK7FWG2.0 to obtain pK7FWG-GmACSL2-eGFP through gateway system according to the protocol of Invitrogen [30] (link). The Plasmids of pCXDR-SSE1-dsRed were constructed following the method described by Chen et al [31] (link). GmACSL2 was subcloned into the vector pGFPGUSPlus (pCAMBIAl305.1113) for soybean hairy root induction, with the HPTII gene as the selective marker and GUS/GFP as the reporter gene [32] (link).

H. sinensis L0106 was isolated from wild O. sinensis samples that were gingerly collected from Qinghai-Tibet Plateau in Qinghai Province. H. sinensis L0106 has been deposited at the China Center for Type Culture Collection (Wuhan, China) with accession number CCTCC M2011278. Subsequently, the transcriptome of H. sinensis was sequenced and the reads obtained were further assembled into Unigenes, and then BLASTx alignment (e < 0.00001) between protein databases and Unigenes was performed [21 (link)]. E. coli JM109 was selected as host for plasmid pMD18-T transformation (Invitrogen, Carlsbad, CA, USA), and E. coli BL21 (DE3) (Invitrogen) was selected as the host for expression of pET28a (Invitrogen). Lysogeny broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37°C with shaking (200 rpm) was used for the growth of E. coli transformants. Ampicillin, kanamycin, isopropyl-β-D-thiogalactopyranoside (IPTG), adenosine, vernine, cytidine, uridine, and thymidine standard substances were purchased from Sigma Chemical Co. (St. Louis, MO, USA).