CC-4931 rbcL-LSATNC-T391F/rbcS1-SSAT pf2 mt+
$30.00
From Robert J. Spreitzer, University of Nebraska, October 2014
Phenotype: requires elevated CO2 for photosynthetic growth
Fuqiao Xu in Spreitzer’s group created a codon-optimized Arabidopsis rbcL gene that encodes a large subunit with Chlamydomonas amino-acid sequences at the N and C termini. This Arabidopsis large subunit has 18 amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, T391V, S398A, V399C, V418A, V428T, V439R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). When the engineered gene was transformed into rbcL∆/rbcS1-SSAT pf2 mt+ (which lacks Chlamydomonas rbcL and rbcS, but expresses Arabidopsis rbcS), photosynthesis-competent transformants were recovered on minimal medium with 5% CO2 in air. Thus, this mutant expresses a functional Rubisco comprised of Arabidopsis small subunits and engineered Arabidopsis large subunits, but its growth on minimal medium with 5% CO2 is much reduced relative to rbcL-LSATNC mt+ or rbcL-LSATNC-T391F mt+. Subsequent DNA sequencing revealed that the engineered Arabidopsis large subunit has a new V391F mutant substitution (GTT-TTT), which may play a role in assembly with Arabidopsis small subunits. Western analysis indicated that the mutant strain has a normal level of Rubisco holoenzyme (Xu and Spreitzer, unpublished). This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.