Rnaase free water

Total RNA was isolated using TRIzol agent (Invitrogen, Carlsbad, CA, USA), and each RNA sample was reverse-transcribed to complementary DNA (cDNA) by PrimeScript™ RT Reagent Kit (Takara, Dalian, Liaoning Province, China). cDNA was used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The sets of primer pairs of apoptotic regulating genes are listed in Table 1 [29 (link)]. For qRT-PCR reactions, the 25 μL reaction mixture included 2 μL cDNA, 12.5 μL SYBR Premix Ex TaqTM Ⅱ (Takara), 1.0 μL of forward and 1.0 μL of reverse primer and 8.5 μL RNAase-free water (Takara). Reaction conditions were 95 °C for 3 min followed by 44 cycles of 95 °C for 10 s, the specific melting temperature (Tm) of a primer pair for 30 s, and then 95 °C for 10 s, and 72 °C for 10 s, using a Bio-Rad IQ5 Thermal Cycler (Bio-Rad). β-actin was selected as a reference gene. The expression fold changes were calculated using the 2^−ΔΔCT method [30 (link)].

During the construction of the recombinant plasmid, the base T at position 3042 was replaced by base C to create a genetic marker to distinguish rDHAV-1 from the parental virus pDHAV-1. The transfected products were harvested at 60 hpt and were immediately used for RNA extraction as previously described. Following that, total RNA was used for RT-PCR amplification with primers BamH I-detect-F/R (Table 1) diluted in RNAase free water (TaKaRa, Dalian, China). RT-PCR/PCR was performed utilizing a one-step RNA PCR kit (TaKaRa, Dalian, China). The RT-PCR/PCR condition was 50 °C for 30 min, 94 °C for 2 min, and then 30 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min, and with a final step of 72 °C for 5 min. During the RNA extraction assay, the DNaseI Digestion kit (OMEGA, GA, USA) was used to remove the residual recombinant plasmid. The 2069 bp fragment including the genetic marker was then digested by restriction enzyme BamH I and verified by electrophoresis in 2% agarose gel.

One milliliter of biomass was sampled and centrifuged at 7440 × g for 2 min. The approximately 0.25 g pellet was transferred with an aseptic stainless steel spoon to extract the total genomic DNA using the Power Soil DNA isolation kit (MoBio Laboratories Inc., USA) according to the manufacturer’s instruction manual.
The 16S rRNA gene of the bacteria was amplified using a universal primer pair 8F/1492R49 (link)50 (link). The PCR amplification of the NC10 phylum pmoA gene was performed using primer pairs A189_b/cmo682 and cmo182/cmo568, as previously described51 (link). Briefly, the PCR mixtures (25 μL) contained 1 μL of template DNA, 1 μL of each primer, 9.5 μL of RNAase-free water (Takara, Japan), and 12.5 μL of Ex Taq premix (Takara, Japan) according to the manufacturer’s instruction manual. The PCR program consisted of an initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C (1 min), annealing (55 °C and 2 min for 8F/1492R; 60 °C and 1 min for A189_b/cmo682; 62 °C and 1 min for cmo182/cmo568) and extension at 72 °C (2 min for 8F/1492R; 1 min for A189_b/cmo682 and cmo182/cmo568), and a final extension at 72 °C for 10 min. The obtained PCR products were purified using agarose gel electrophoresis and Axygen PCR Cleanup kit (Axygen Scientific Inc., CA, USA). The detailed information regarding the PCR primers used above is given in Table S1.