Smater race 5 3 kit

Based on the DNA sequence of A. fumigatus SrbA (GenBank accession no.XM_744169), we identified an SREBP protein-encoding gene Pc20g05880 (GenBank accession no.XM_002563071) in P. chrysogenum. Given that the genome of P. digitatum and P. chrysogenum share high similarity [27 (link)], two pairs of specific primers sreA-a, sreA-b, sreA-c and sreA-d (Table 1) were designed according to the relatively conserved sequences of A. fumigatus sreA and P. chrysogenum Pc20g05880 after sequence alignment with ClustalW. Two approximately 1000-bp DNA fragments were amplified from genomic DNA of P. digitatum by PCR, and then cloned into pMD18-T vector (TaKaRa Biotech. Co., Dalian, China) for sequencing. Then an approximately 2000-bp DNA fragment of P. digitatum sreA was obtained after sequence-assembling. The 5’ flanking DNA sequence of sreA was amplified by genome walking using the Genome Walking Kit (TaKaRa Biotech. Co., Dalian, China) with specific primers sreA-e, sreA-f and sreA-g (Table 1). The 3’ flanking unknown DNA sequence of sreA was amplified using SMATer RACE 5’/3’ Kit (TaKaRa Biotech. Co., Dalian, China) with specific primers sreA-h and sreA-i. The DNA sequence of sreA has been deposited in GenBank under accession number KJ939329.

The total RNA from the five tissue of the oysters was prepared using an RNAprep Pure Tissue Kit (catalog No. DP431; TIANGEN, Beijing, China) according to the manufacturer’s instructions. The integrity of the purified RNA was assessed using 1% (w/v) agarose gel electrophoresis. RNA concentration was determined using a Nano Photometer Pearl microspectrophotometer (Implen, München, Germany). Reverse transcription of the total RNA to synthesize 5′/3′ rapid amplification of cDNA ends (RACE)-ready 1st strand cDNA was performed using a SMATER RACE 5′/3′ kit (catalog No. 634859; Takara, Dalian, China), and cDNA for quantitative real-time polymerase chain reaction (RT-qPCR) analysis was synthesized using an RT-qPCR kit (catalog No. RR047Q; Takara).

A pair of PCR primers (FT1A and RT1A; Table 1) was designed using Primer premier 5 (Premier Biosoft, San Francisco, CA, USA) to clone the partial cDNAs of CgFUT1. The PCR thermal cycling conditions were as follows: denaturation at 95 °C for 3 min; 35 cycles at 95 °C for 30 s, 48 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 5 min. To obtain the cDNA 5′ and 3′ ends of CgFUT1, two primers (RRT1-A and FRT1-A; Table 1) were designed for 5′ RACE and 3′ RACE, respectively. RACE was performed using a SMATER RACE 5′/3′ kit (Takara), and one 5′ end and 3′ end strip each was obtained. The PCR products were gel-purified and cloned into a pUC19 vector (Takara). Following transformation into competent Escherichia coli TOP10, the positive recombinants were identified via antiampicillin selection and PCR screening. Three positive clones were sequenced via Sangon Biotech (Shanghai, China). After sequencing, the 5′ and 3′ ends of the cDNA were ligated using DNAMAN. The full-length cDNA fragments were obtained and named CgFUT1.