A PCR product of 383 bp corresponding to pnl2 gene (clpnl2 fragment) was ligated into the pCR 2.1 vector and introduced into E. coli TOP 10 strain from the TOPO TA Cloning kit (Invitrogen). Genomic DNA Selleckchem Palbociclib library construction and screening Partial Sau3AI digestion of genomic DNA from race 1472 was used to construct a genomic library in Lambda DASH II/BamHI according to manufacturer’s instructions
(Stratagene). Screening was performed using 15 × 104 UFP with three rounds of hybridization filters and the homologous Clpnl2 fragment, which was 32P-radiolabeled using the Radprime DNA Labeling System Life Technologies Kit (Tech-Line). Molecular cloning of the Clpnl2 full-length cDNA and expression analyses The cDNA was amplified by RT-PCR as specified by the manufacturer. SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) was used to prepare cDNA from total RNA. PCR was performed using the upstream primer Pnl67 (5′-ATGAAGTCTACCATCTTCTCCG-3′) and downstream primer Pnl1569 (5′-TTAGATCTTGCGAAACCGGC-3′) designed from the RG-7388 DNA Clpnl2 genomic sequence of C. lindemuthianum. The PCR incubation
mixture was heated at 94°C for 5 min in a thermocycler (Eppendorf Master Cycler Gradient, Brinkmann, Westbury, NY), followed by 30 cycles of denaturation for 20 sec at 94°C, annealing for 30 sec at 54°C, extension for 1.5 min at 72°C and then by a final extension for 7 min at 72°C. A PCR product of 1,140 bp obtained from total RNA of race 1472 induced with pectin for 4 h and corresponding to the Clpnl2 gene, was ligated into the pCR 2.1 vector (Invitrogen) and three clones were selected and sequenced. The 5′ end of cDNA was amplified by 5′RACE as specified by the manufacturer
(5′RACE System for Rapid Amplification of cDNA Ends, Invitrogen), with total RNA from race 1472 induced for 4 h with 92%-esterified pectin, using the selleck inhibitor specific reverse primers Pnl1249 (5′-GTA GTT GTT GAC GAC GTG GAC G-3′) and Pnl975 (5′-CGA TGT GCT GGC GGC CG-3′). The amplification products were cloned and five clones were selected and sequenced. For expression analysis, total cDNA (1140 pb) was amplified with specific primers Pnl67 and Pnl1569 in the same conditions DNA ligase described above using total RNA of mycelia from both races induced with 92%-esterified pectin or cell walls from P. vulgaris for 2, 4, 6, 8, 10 and 12 h. For expression analysis, cDNA obtained from cells grown under different conditions was also amplified by PCR using oligonucleotides prepared from ribosomal 18S RNA as a control (5′- TTAGCATGGAATAATRRAATAGGA-3′and 5′-ATTGCAATGCYCTATCCCCA-3) [38]. The PCR incubation mixture was heated at 94°C for 3 min, followed by 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 56°C, extension for 1 min at 72°C and then a final extension for 10 min at 72°C.