Subategory:

Mutants

Description:

A codon-optimized Arabidopsis rbcL gene was created 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 rbcLΔ/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. This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Species:

Chlamydomonas

Locus:

rbcL, RBCS1, PF2

Chromosome:

Chloroplast,2,11

Phenotype:

Requires elevated CO₂ for photosynthetic growth