Pmd19 t

The construction of prokaryotic expression of EmSAG was conducted as previously described [20 (link)]. Briefly, the EmSAG-encoding sequence (GenBank: XM_013482011.1) was amplified by PCR. EmSAG-specific primers were utilised for the PCR assays: SAG1 (forward primer: 5'-CGC GGA TCC GAC ACA ATC TCC AGC CCT-3'; BamHI restriction sites underlined) and SAG2 (reverse primer: 5'-ATT GCG GCC GCT CAA ATG AGA ACA GAT GCG-3'; NotI restriction sites underlined) with E. maxima cDNA as a template. The amplification products of EmSAG cloned in pMD-19T (TaKaRa, Dalian, China) resulted in the formation of recombinant plasmid pMD-19T-EmSAG. Subsequently, the EmSAG gene was inserted into the pET-32a (+) (Novagen, Madison, WI, USA) frame of expression vector system and confirmed by endonuclease digestion. Following sequence analysis (Invitrogen Biotech, Shanghai, China) and verification, the positive clones were confirmed as pET-32a/ EmSAG.

First, the transcription factor-binding sites for all full-length MtGST promoter sequences were predicted on the website http://alggen.lsi.upc.es (accessed on 10 November 2022) to avoid disrupting the integrity of the binding sites when the promoters of different fragments were cloned. The 5’ loss fragments of each MtGST promoter were amplified from the full-length sequences of the MtGST promoters using TaKaRa Ex Premier™ DNA Polymerase (Takara, Dalian, Liaoning, China). All primers were designed by Primer 5 software and were listed in Table 1. Each forward primer sequence and reverse primer sequence were added with Nhe I and Xho I restriction enzyme cleavage sites, respectively. Each 5’ loss fragment of MtGST promoter was ligated to a TA clone vector pMD-19T (Takara, Dalian, Liaoning, China), and the correct clone product was obtained by sequencing. The PGL4.10-Basic vector and the pMD-19T with the 5’ loss fragment were digested with Nhe I and Xho I. Then, the 5’ loss fragment of MtGST promoter was ligated to the PGL4.10-Basic vector using T4 DNA ligase (Takara, Dalian, Liaoning, China), and the ligation product was transformed into E. coli cells. The plasmid DNA was purified from E. coli cells for subsequent cell transfection experiments.

Isolates were cultivated on ISP 3 medium at 28°C for 2 weeks, and then preliminarily identified according to their phenotypic characteristics including the characteristics of colonies on plates, color of aerial and substrate mycelium, and production of diffusible pigment. Genomic DNA was extracted from isolates using lysozyme-SDS-phenol/chloroform method (Maniatis et al., 1982 ). PCR amplifications for the 16S rRNA gene sequence were performed using the universal bacterial primers, 27F and 1492R (Lane, 1991 ). The reaction conditions were 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 1 min, with a final extension at 72°C for 10 min. The PCR products were purified and cloned into the vector pMD19-T (Takara) for sequencing. The almost full-length 16S rRNA gene sequences (∼1500 bp) were obtained and neighbor-joining phylogenetic tree was constructed using the molecular evolutionary genetics analysis (MEGA) software version 7.0 (Kumar et al., 2016 (link)). Bootstrap replication (1000 replications) was used to assess the topology of the phylogenetic tree (Felsenstein, 1985 (link)). The obtained gene sequences were deposited in the GenBank database under accession numbers KP784763-KP784808, KX777578-KX777633, KX977397-KX977399, and KR261651-KR261652, respectively.

The complete Cap gene of PCV4 was amplified with Cap-forward and Cap-reverse primers, as seen in Table 1. It was then cloned into the vector pMD-19T (TaKaRa) and designated as pMD-19T-Cap. The recombinant plasmids were sequenced by Nanjing Tsingke Biotechnology Co., LTD and the concentration determined using an ND-2000c spectrophotometer (NanoDrop, Wilmington, USA). The copy number of pMD-19T-Cap was calculated as the number of plasmids in copies/μL = 6.02 × 10²³ × plasmid concentration (ng/μL)/genome length (bp) × 10⁹ × 660, then serial 10-fold dilutions from 1 × 10⁵ to 1 × 10¹ copies/μL were created and stored at −20°C for subsequent assays.

The Tryptic Soy Broth (TSB) medium and Tryptic Soy Agar (TSA) medium were purchased from Becton, Dickinson and Company (USA). The Oxford cup and Gram Stain Kit were purchased from Guangzhou Heyue Biotechnology Co., Ltd. (China). Endospore Stain Kit was purchased from Solarbio Company (China). TIANamp Bacteria DNA Kit was purchased from TIANGEN Biotech Co., Ltd. (Beijing, China). The Premix Taq™ (LA Taq™ Version 2.0 plus dye) and pMD19-T were purchased from Takara (Dalian, China).