Pmd19 t easy vector

The p35S::GFP-IgBBX constructs were introduced into tobacco epidermal cells via the Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaf method [26 (link)]. The plasmid of IgBBX for transient transformation was generated using the Invitrogen Gateway system, according to the manufacturer’s instructions. The IgBBX ORFs, lacking the stop codon, were amplified using a Phusion^®High-Fidelity PCR kit (New England Biolabs, Ipswich, MA, USA) (primers listed in Table S1), then inserted into the pMD19-T easy vector (TaKaRa, Tokyo, Japan) to allow its sequencing-based validation. Each pENTR^TM1A-IgBBX plasmid was previously subjected to the LR Clonase™ II enzyme mix (Invitrogen, Carlsbad, CA, USA) reaction to obtain GFP-fused constructs using the binary vector pMDC43, resulting in the plasmid p35S::GFP-IgBBX. For transient expression, Agrobacterium tumefaciens strain EHA105 carrying the pMDC43 and pMDC43-IgBBX was grown separately to OD600 = 0.8 and coinfiltrated with the p19 strain into leaves of five-week-old N. benthamiana. The YFP fluorescent signals were monitored 48 to 72 h after infiltration using a confocal laser scanning microscopy (LSM 700, Carl Zeiss).

In this study, we used an experimental approach combining sequence alignment based on tick hemolymph EV proteomics and previous R. haemaphysaloides transcriptome data with sequence search for the identification of tick hemolymph EV markers. The Rh14-3-3ζ, Rh14-3-3ε, RhCD9, RhTSG101, RhFerritin-1, RhFerritin-2, RhCytochrome c and RhCalnexin sequences were found in the salivary gland and midgut transcriptomes of fed R. haemaphysaloides. Complementary DNAs (cDNAs) were synthesized by HiScript III RT SuperMix (Vazyme, Nanjing, China) from the total RNA of the R. haemaphysaloides salivary gland and midgut. Gene-specific oligonucleotide primers for PCR are listed in Additional file 5: Table S1. The sequences were routinely cloned into the pMD-19 T easy vector (Takara, Dalian, China) using PrimeSTAR Max Premix (Takara), and the obtained clones were sequenced.

The full-length cDNAs of the Salmonella primary Sal-1 were obtained using the SMARTer^TM RACE cDNA amplification kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. Because the RNA template was from a prokaryotic organism and lacked a polyadenylated tail, we thus added a poly(A) tail using Poly(A) Polymerase (Takara). Then, the first-strand cDNA was synthesised using a modified oligo(dT) primer (5′-RACE CDS primer A or 3′-RACE CDS primer A), and the SMARTer^TM II A oligonucleotide was added for 5′-RACE cDNA synthesis. The gene-specific antisense primer sal-GSP1 was used for 5′-RACE amplification, and sal-GSP2 was used for 3′-RACE amplification. The cDNA was amplified using the Advantage 2 PCR kit (Clontech) with the above gene-specific primers and the Universal Primer A Mix in the SMARTer^TM RACE kit. The RACE product was electrophoresed, purified, and ligated into the pMD19-T Easy vector (Takara). The ligations were transformed into E. coli Top 10 chemically competent cells. The plasmids were extracted from bacterial cell suspensions using the E.Z.N.A.® Plasmid Mini Kit (Omega Bio-Tek, Norcross, GA) for sequence analysis.