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. 2021 Aug 23;12:717952. doi: 10.3389/fpls.2021.717952

Figure 1.

Figure 1

Generation of transplastomic tobacco plants expressing RGNNV capsid protein (RGNNV-CP). (A) Physical map of the target region in the plastid genome (WT ptDNA) and schematic diagram of the chloroplast transformation vector, pRGNNV1 (construct not drawn to scale). Synthetic DNA covering the codon-optimized RGNNV-CP (RGNNV-CPpt) and the subsequent 3' non-cooding regioon (3' NCR) derived from the RNA2 genome of red-spotted grouper NNV (RGNNV) is driven by the tobacco plastid psbA promoter and its 5'-UTR (PpsbA), and the transcript is designed to be stabilized by the 3'-UTR of tobacco plastid psbA (TpsbA). The selectable marker gene aadA is under the control of the tobacco plastid ribosomal RNA operon promoter (Prrn). The transgenes are targeted to the intergenic spacer region between trnI and trnA in the tobacco plastid genome. The location of the probe used in Southern blot analysis is shown as a black bar. The wild-type and the transgenic chloroplast genomes give rise to hybridization signals corresponding to 2.3 kb and 5.3 kb XmnI-XmnI DNA fragments, respectively. (B) Southern blot analysis of two independent transgenic lines (RGNNV1-1 and RGNNV1-2). Total cellular DNA was digested with XmnI and subjected to hybridization analysis with a DIG-labeled probe shown in (A).