Escherichia coli trans1 t1 cells

Total RNA was extracted as described above, and the cDNA was synthesized from 1 μg RNA using PrimeScript^® RT reagent Kit With gDNA Eraser (TaKaRa, Japan). Sixteen pairs of primers for obtaining pre-miRNA sequences were listed in Supplemental Table S6. Polymerase chain reaction (PCR) contained 2.5 μL cDNA, 2 μL mix solution of target gene primers, 2.5 μL 10× TransTaq^® HiFi Buffer II, 2 μL dNTPs (2.5 mM), 0.3 μL TransTaq^® HiFi DNA Polymerase (TransGen, China), 15.7 μL of ddH₂O. And the amplification was performed under the following conditions: 94 °C denaturation for 3 min, 35 running cycles of 94 °C for 30 s, 55.2 °C for 30 s, 72 °C for 30 s, and an elongation cycle of 72 °C for 10 min. PCR products were separated by 3% agarose gel electrophoresis, and the incised gels were purified using a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 (TaKaRa, Japan). The extracted products were cloned into pEASY^TM-T5 Zero vector (TransGen, China) and transformed into competent Escherichia coli Trans1-T1 cells (TransGen, China). The recombinant plasmids were sent Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) to sequence.

The partial cDNA sequences of HcToll6 and HcToll7 were retrieved from our previous hepatopancreas transcriptome database (unpublished). In this experiment, 3′ and 5′ RACE were performed by using a Clontech Advantage^® 2 PCR kit from Takara (Japan) with two pairs of specific primers (HcToll6-F:5′-TGGATAAACGATGTGCTAAGCGACCCC-3′, HcToll6-R: 5′-CTGTGAAGACCACGCAAATAGAGACGGAAC-3′; HcToll7-F: 5′-GAGAGAACTTGGACAGGAAAGGGGGCT-3′, HcToll7-R: 5′-CGTATCGGCAGGTCGCCAAGGGTAAC-3′) to obtain the full-length cDNAs of HcToll6 and HcToll7. The amplification products were purified using a DNA gel extraction kit (Shanghai Generay Biotech Co., Ltd., Shanghai, China), inserted into the pEasy-T3 vector, and transformed into Escherichia coli Trans1-T1 cells (TransGen Biotech, Beijing, China). The putative clones were identified by PCR with M13F and M13R primers. The selected clones were sequenced by a commercial company (Springen, China).

Escherichia coli Trans1-T1 cells (TransGen Biotech, Beijing, China) and Pichia pastoris GS115 (Invitrogen, CA, U.S.A) were used as gene cloning and expression hosts, respectively. The P. pastoris–E. coli shuttle expression vector pPIC9-lacb was previously constructed in our laboratory (Zhang et al. 2002 ). Media, including minimal dextrose (MD) medium, minimal methanol (MM) medium, and yeast peptone dextrose (YPD) medium, were prepared according to instructions in the Pichia expression kit (Invitrogen). Fermentation basal salts (FBS) medium and Pichia trace metal (PTM) complied with Pichia fermentation guidelines (Invitrogen).