Development of Novel Types of Plastid Transformation Vectors and Evaluation of Factors Controlling Expression

Abstract

Two new vector types for plastid transformation were developed and uidA reporter gene expression was compared to standard transformation vectors. The first vector type does not contain any plastid promoter, instead it relies on extension of existing plastid operons and was therefore named "operon-extension" vector. When a strongly expressed plastid operon like psbA was extended by the reporter gene with this vector type, the expression level was superior to that of a standard vector under control of the 16S rRNA promoter. Different insertion sites, promoters and 5'-UTRs were analysed for their effect on reporter gene expression with standard and operon-extension vectors. The 5'-UTR of phage 7 gene 10 in combination with a modified N-terminus was found to yield the highest expression levels. Expression levels were also strongly dependent on external factors like plant or leaf age or light intensity. In the second vector type, named "split" plastid transformation vector, modules of the expression cassette were distributed on two separate vectors. Upon co-transformation of plastids with these vectors, the complete expression cassette became inserted into the plastome. This result can be explained by successive co-integration of the split vectors and final loop-out recombination of the duplicated sequences. The split vector concept was validated with different vector pairs.

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…The necessity of a promoter within our transgene insert, and thus the sequence homology that would be associated with the additional promoter, was therefore avoided. So called "operon-extension vectors" that allow the placement of transgenes downstream of an endogenous promoter and coding sequence have been previously described using the reporter gene uidA encoding GUS and the selectable marker aadA providing resistance to spectinomycin (Herz et al, 2005). To our knowledge, our efforts are the first time this strategy has been used for the expression of four transgenes.…”

“…A set of vectors for plastid transformation was designed to target the insertion of transgenes encoding enzymes of the PHB biosynthetic pathway behind the native psbA promoter and its coding sequence. This so-called operon-extension vector (Herz et al, 2005) reduces the repetition of plastidial sequences that may lead to unwanted rearrangements in the plastome by eliminating the need for a promoter in the inserted DNA that may possess homology to regions in the host's plastid genome. The tobacco psbA coding sequence was used as a left flank, and the 3# UTR of psbA, the trnH gene, and a partial sequence of ribosomal protein L2 (rpl2) was used as a right flank within the plastid transformation vectors to promote homologous recombination into the selected site.…”

“…thiolase (phaA) and synthase (phaC; Schembri et al, 1995) and the Bacillus megaterium reductase (phaB; McCool and Cannon, 1999) were chosen from the set of available PHB biosynthetic pathway gene sequences, since their GC content is similar to that of the tobacco plastome and codons with a low frequency of use in the tobacco plastome are either absent or rarely occur. The 5# UTR of gene 10 from bacteriophage T7 (Kuroda and Maliga, 2001) and short (fewer than 56 nucleotides) spacer elements of plastidial origin (Herz et al, 2005) were used upstream of the individual transgenes to optimize expression. Construct pCAB, containing the PHB pathway genes and the aadA gene conferring resistance to spectinomycin, was prepared using this strategy.…”

“…This apparent colocalization has also (Yanisch-Perron et al, 1985). Abbreviations and notes are as follows: left flank of plasmids pUCaadA, pCA, and pCAB contains the complete coding sequence of psbA (D1 protein of PSII), left flank DNA is homologous to the reverse complement of nucleotides 536 to 1,597 of the tobacco plastome (Herz et al, 2005); aadA, gene encoding aminoglycoside 3#-adenyltransferase from E. coli providing spectinomycin/streptomycin resistance (Svab and Maliga, 1993); right flank of plasmids pUCaadA, pCA, and pCAB contains 3# UTR of psbA, trnH (tRNA-His), and part of rpl2, right flank DNA is homologous to the reverse complement of nucleotides 155,398 to 155,943 and 1 to 530 of the tobacco plastome (Herz et al, 2005); 5# UTR T7g10, 5# UTR of gene 10 of bacteriophage T7 (Kuroda and Maliga, 2001), DNA homologous to nucleotides 22,904 to 22,969 of the bacteriophage T7 genome (EMBL accession no. V01146; Dunn and Studier, 1983); phaC As , gene encoding PHB synthase from Acinetobacter sp., homologous to nucleotides 2,351 to 4,123 of the Acinetobacter sp.…”

“…L37761; Schembri et al, 1995); rps19/rpl22 spacer, DNA homologous to nucleotides 86,353 to 86,399 of the tobacco plastome, contains an intergenic region between rps19 and rpl22 (Herz et al, 2005); phaA As , gene encoding thiolase from Acinetobacter sp., homologous to nucleotides 4,206 to 5,384 of the Acinetobacter sp. PHA biosynthetic gene locus; 5# UTR rbcL, 15 nucleotides of the 5# leader sequence of the gene encoding the large subunit of Rubisco, homologous to nucleotides 57,580 to 57,594 of the tobacco plastome (Svab and Maliga, 1993); psbD/C spacer, DNA homologous to nucleotides 35,463 to 35,517 of the tobacco plastome, contains the sequence region upstream of the gene encoding PSII 44-kD protein (psbC), which is overlapped by the coding region of the psbD gene encoding the PSII D2 protein (Herz et al, 2005); phaB Bm , gene encoding acetoacetyl-CoA reductase from B. megaterium, homologous to nucleotides 4,758 to 5,501 of the B. megaterium PHA gene cluster (EMBL accession no. AF109909; McCool and Cannon, 1999).…”