Constructs for fungal xyn10A and xyn11B expression; Confirmation of homoplastomy of T
plants (cv. I64) and effects of T7G10 translational enhancer on accumulation levels of Xyn10A and Xyn11B. A. CEC4 was used to express fungal xylanases Xyn10A and Xyn11B in high-biomass tobacco cv. I64. The sequences of the xyn10A and xyn11B genes were cloned into the GOI position of pCEC4.The expected Rsr II-generated fragment sizes for Southern blot RFLP analysis are indicated for each construct and for the wild type (WT) plastome. B. Southern blot RFLP analysis of cv. I64 T0 transplastomic lines transformed with pCEC4-Xyn10A and pCEC4-Xyn11B to confirm homoplastomy, two clones per construct analysed. C. Phenotype of T0 cv. I64 transplastomic lines is identical to WT plants. D. Immunoblot-assisted accumulation analysis for Xyn10A and Xyn11B expressed from CEC4. Two independent primary transformants per construct were examined (lanes 1 and 2 for each protein). Extractions were performed using equal ratio of sample weight/extraction buffer volume (w/v = 1/5). Each lane contains extract equivalent to 4.0 mg of extracted leaf tissue. Untransformed WT extract was used as negative control. Known amounts (ng) of a c-myc-tagged control protein are indicated above the standard curve lanes. E. CEC5 construct (identical to CEC4, but lacking the T7g10 DB element) was used for expression of native forms of Xyn10A and Xyn11B without the T7g10 N-terminal fusion. F. Homoplastomy confirmation was carried out as described above for T0 cv. I64 transplastomic lines expressing Xyn10A and Xyn11B from CEC5 (B). G. Phenotype of T0 cv. I64 transplastomic lines is identical to WT plants. H. Accumulation analysis for Xyn10A and Xyn11B expressed from CEC5 was carried out as described in (D).