4.4.1. Construction of the Recombinant Plasmid

Amplifications of PS and FO specific primers were performed by conventional PCR (as mentioned above) and touchdown PCR (the same 20-µL reaction; 95 ◦C for 2 min, 95 ◦C for 5 s, 50–60 ◦C to anneal for 30 s, 72 ◦C for 30 s, for eight cycles; 95 ◦C for 2 min, 95 ◦C for 5 s, 58 ◦C to anneal for 30 s, for 30 cycles), respectively (Supplementary Figure S1B). The bright electrophoretic strips were cut and extracted using gel pure DNA kits following the manufacturer's instructions (Magen D2111-02, China). The gel extraction solutions were concentrated to over 50 ng·µL <sup>−</sup><sup>1</sup> of cDNA using a concentrator for approximately 25 min at 1400 rpm (Eppendorf Concentrator Plus AG5305, V-AQ mode). DNA fragments were inserted into a vector and ligated overnight at 16 ◦C using the pMD19-T vector cloning kit following the manufacturer's instructions (Takara 6013, Japan). Then, 5 µL of vector DNA solution and 50 µL of *E. coli* DH 5α were blended and incubated in ice for 30 min. After heat shock at 42 ◦C for 60 s, the vectors were kept on ice for 3 min. The solution with 600 µL of liquid LB culture medium was closed using Parafilm and the culture was shaken for 60 min (37 ◦C, 200 rpm). Then, 45 µL of 5-Bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal), 10 µL of isopropyl-l-d-thiogalactopyranoside (IPTG) and 200 µL of the culture solution were smeared evenly on solid LB culture medium containing 0.4% Ampicillin (Amp) and then incubated at 37 ◦C for 14 h. We selected 1–2 white single colonies and added 300 µL of liquid LB culture medium containing 0.4% Amp for co-culture (37 ◦C, 200 rpm) for approximately 14 h until the solutions were turbid. PCR and 1% gel electrophoresis as aforementioned were used to identify the size of DNA fragment comparing with a DL2000 DNA Marker (Takara, Japan). The solutions containing the appropriate size of DNA fragments were chosen to extract plasmids using Hipure Plasmid Micro Kit following the manufacturer's instructions (Magen, China). 10 µL solutions of plasmids were used for sequencing in Biosune, China (987 bp for PS, 433 bp for FO; Supplementary Table S1).
