Dna gel extraction kit

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partial cDNA sequences of HcToll6 and HcToll7 were retrieved from our previous hepatopancreas transcriptome database (unpublished).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).

We have identified some candidate salt-responsive genes in S. salsa using the full-length cDNA over-expressing gene (FOX)-hunting system^{53 (link)}. The SsMT2 gene was one of those genes identified. Seeds of S. salsa plants were collected from an alkaline soil area in Northeast China and germinated on MS medium^{54 (link)} at 28 °C under 2000 Lux irradiation with a 16 h light/8 h dark photoperiod in an illuminated incubator. Total RNA was isolated from 4-week old seedlings using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized from l µg of the total RNA with Prime-Script Reverse Transcriptase (Takara, Tokyo, Japan) using an oligo (dT) primer. MT cDNA sequence from FOX-hunting system was obtained and open reading frame (ORF) was found by blasting in the NCBI database. A transcript fragment was amplified by PCR from the cDNA with the forward primer (5′-ATGTCTTGCTGTGGTGGTAACTGTGG-3′) and reverse primer (5′-TCATTTGCAGGTGCATGGGTTG-3′), which were designed from the MT ORF sequence. The PCR product was purified from agarose gel using the DNA Gel Extraction Kit (Generay, Shanghai, China) and cloned into plasmid pMD18-T (Takara, Tokyo, Japan) and sequenced. A new gene was designated as SsMT2 and its ORF nucleotide sequence and protein sequence was deposited to GenBank database (MF447531).