Hindiii kpni digested pptrii

The sequence of the AsojAgdL gene was identified in the whole genome sequence of A. sojae NBRC4239 (accession no. BACA00000000) using the homolog gene sequence of　A. oryzae (accession no. XP_001825390). Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific, USA) was used in PCRs, and the oligonucleotide primers used are shown in Table S1 (see J. Appl. Glycosci. Web site). To express the AsojAgdL gene under the control of the A. oryzaeTEF1 gene promoter, an expression plasmid was constructed as follows. An 0.8 kb fragment containing the TEF1 gene promoter was amplified from genomic DNA of A. oryzae RIB40 using the primer pair promoter1/promoter2. The AsojAgdL gene without the stop codon was amplified from genomic DNA of A. sojae NBRC4239 using the primer pair AgdL1/AgdL2. An 0.3 kb fragment of the α-glucosidase terminator containing 10 × His-tag and a stop codon was amplified from genomic DNA of A. sojae NBRC 4239 using the primer pair terminator 1/terminator 2. The three PCR products and the HindIII/KpnI-digested pPTRII (Takara Bio) were joined in a four-piece In-Fusion reaction using the In-Fusion HD Cloning Kit (Takara Bio). Cloning of α-glucosidase from A. niger (AnigAgdA) was similarly carried out, except that primers AgdA1, AgdA2, terminator3, and teminator4 and genomic DNA from A. niger NBRC4066 were used instead.

The agdB gene (GenBank (https://www.ncbi.nlm.nih.gov/genbank/) accession no. LX063802) was cloned from A. niger NBRC4066 and used to construct an expression vector. Phuson Hot Start II DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) was used for polymerase chain reaction (PCR) analysis. The oligo nucleotide primers used in this study are shown in Table S1 (see the J. Appl. Glycosci . website). To express agdB under the control of the A. oryzaetef1 gene (GeneBank accession no. Q9Y713) promoter, an expression plasmid was constructed as follows. The 0.8-kbp fragment of the tef1 gene promoter was amplified from the genomic DNA of A. oryzae RIB40 using primer No. 1 and 2. The agdB gene lacking a stop codon was amplified from the genomic DNA of A. niger NBRC4066 using primer No. 3 and 4. The 0.3 kbp fragment from the terminator region of agdB containing 10 × His-tag and a stop codon was amplified from the genomic DNA of A. niger NBRC4066 using primer No. 5 and 6. The three PCR products and the Hin dIII/ Kpn I-digested pPTRII (Takara Bio Inc., Shiga, Japan) were joined in a four-piece in-fusion reaction using the In-Fusion HD cloning kit (Takara Bio Inc.) and the resulting expression vector was designated pPTRII-AgdB.