E coli dh5α competent cell

PCR primers were designed by primerselect 7.0 (DNASTAR Inc, Madison, WI) and synthesized (Sangon Biotech, Shanghai, China). rs72755295 and rs4149909 surrounding regions (~1.5 kb) were amplified with Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA) and primers shown in Table S1. Thermocycling conditions for routine PCR was as follows: 98 ℃ for 30 s; 35 cycles of 98 ℃ for 10 s, 68 ℃ for 30 s, 72 ℃ for 45 s, and finally 72 ℃ for 2 min. The PCR product and pGL3-promoter vector (Promega, Madison, WI) were digested by MluI and XhoI (NEB), purified by GeneJET Gel Extraction Kit (Thermo Fisher Scientific) and ligated by T4 DNA ligase (NEB) according to the manufacturer’s manual. The recombinant plasmids were transformed into E.coli DH5α competent cells (Takara, Dalian, China), cultured, and then extracted by TIANpure Midi Plasmid Kit (Tiangen Biotech, Beijing, China). After sequencing, the plasmids with corresponding alleles were generated by Q5 Site-Directed Mutagenesis Kit (NEB) and primers in Table S1. Before transfection, all plasmids were sequenced to rule out artificial mutations and verify the haplotype orientation.

The promoter regions of PpDAM and PpFT2 were isolated using a Genome Walking Kit (Clontech) according to the manufacturer’s protocols. The primers for amplification of PpFT2 were designed based on the complete cDNA sequence of PpFT2a [GenBank: AB571595]. The primers are listed in Supplementary Table S11 in Supplementary File 3 at JXB online. PCR products were analysed on 1% agarose gels. For each reaction product, a single fragment was recovered from the gels and purified using a DNA purification kit (Takara). The fragment was then ligated into the pMD18-T vector, transformed into E. coli DH5α competent cells (Takara), and then sequenced (Sangong, Shanghai, China).

The blastula and hatching larva stages were selected for sampling. These two stages are the representative period of M. nipponense embryonic development. The fertilized egg, cleavage, and blastula stages of the embryo all belonged to the cell division phase, from single cell to multicellular. Hence, the blastula stage with more cells was selected as the representative sample stage. The embryo enters the tissue differentiation and organogenesis stage since the gastrula stage, and fully differentiated larvae were chosen as another representative sample stage. In a mixed embryo mutation analysis, since prawn embryos are very small (5–600 μm) in diameter, the unhatched injected embryos were tested by mixing 30 of them in a group. In a single embryo mutation analysis, injected larvae after hatching were used as templates individually for detection. DNA extraction and PCR methods were performed as described in Section 2.4. The PCR product was purified using the DiaSpin DNA Gel Extraction Kit (Sangon, China), then ligated to the pMD18-T vector (Takara, Japan), and transformed into E.coli DH5α competent cells (Takara, Japan; 9057). Thirty positive clones were selected by blue–white spot screening for sequencing by Shanghai Sangon Company (Sangon, China).

Primer pairs used in this study are listed in Table 3. The oacC-1 primer pair was used for oacC gene detection. The oacC-2 primer pair was used for oacC gene function analysis. The oacC-3 and oacC-4 primer pairs were used to amplify regions up and downstream of oacC in serotype 6 isolates. Oligonucleotide primers were synthesized by Sangon Biotech (Shanghai). PCR amplifications were performed using a TaKaRa PCR Amplification Kit (Takara, Japan) following a standard protocol. PCR products amplified from strain 51579 using the oacC-2 primer pair were purified and cloned into the T-vector pMD20T (TaKaRa, Japan), which carries an additional T at both 3′ terminus and can complement the A base of the PCR product, to generate the pSQZ5 expression plasmid. The recombinant plasmids were first transformed into commercial E. coli DH5α competent cells (TaKaRa, Japan), and then into S. flexneri strains tested, using a standard protocol [30 ]. The transformants were selected on LB plates supplemented with ampicillin (100 μg ml⁻¹) and further confirmed by PCR amplification of the oacC gene.

Sixteen clone libraries of actinobacterial 16S rRNA genes were constructed using the pMD18-T Vector Cloning Kit and E. coli DH5α competent cells (Takara, Dalian) following the manufacturer′s instructions. The positive clones from each library inoculated on MacConkey agar with ampicillin (100 μg/ml) were randomly picked and sequenced using M13F (−47) primer on ABI 3730xl capillary sequencers (Applied Biosystems, USA).