Journal: Plant Molecular Biology
Article Title: Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments
Figure Lengend Snippet: A schematic representation of a double homologues recombination event between transformation vector (pCCATCH) and chloroplast genome ( a ). The sequence of the cat CH gene, under control of the C. merolae chloroplast promoter P dna K, (marked as white, curved rectangle ) of the dna K gene, was integrated into the chloroplast molecule at the selected position between the rpl 32 - psb A genes. The solid black rectangles represent the algal chloroplast sequence, also present in the transformation vector, flanking the cat CH cassettes and functioning as facilitators of double homologues recombination. b Verification of the insertion of the cat CH cassette into the rpl 32- psb A region of the chloroplast molecule via PCR and subsequent agarose gel electrophoresis of the PCR products. The PCR reaction was performed on the total DNA, isolated from the wild type ( WT ) and stable chloroplast transformant lines (transformed by B biolistic bombardment or P PEG method). Two sets of primers were used: 5UTF-5UTR amplifying a 4612 bp long product (marked as x) and 3UTF-3UTR amplifying a 3655 bp long product (marked as y). These two PCR products encompass the regions between cat CH gene and fragments from beyond the upstream or downstream homologous region in the transformation vector. The presence of the cat CH was confirmed by a PCR reaction with catCHStuI-catCHKpnI primers pair amplifying a 672 bp long product (marked as z). To exclude the possibility of unwarranted, random integration of the vector or its continuous presence in the cell, a fragment of the plasmid ori region was amplified with the pABB20R-pABB20L primer pair, yielding a 3420 bp long product, only in case of the vector control, thereby confirming the absence of any residual plasmid (marked as v). M molecular marker. c Southern blot hybridization of PCR products generated by the 5UTF-5UTR and 3UTF-3UTR primer pairs with the CATCH probe (being the PCR product generated with catCHStuI-catCHKpnI primers pair). The total DNA, isolated from the stable chloroplast transformants with the integrated pCCATCH, obtained via biolistic bombardment ( B ) or PEG method ( P ) was used as a templates for PCR. The correct integration of the transformation vector was confirmed by the size of apparent bands (4612 bp and 3655 bp, see the upper part ) and additionally by Southern blot hybridization with cat probe (the lower part ). d The Southern blot hybridization of BglII-digested total DNA with the psb A probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with BglII restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 8 µg of DNA. The DNA was transferred onto a nitrocellulose membrane and hybridized with psbA probe. The transformant-derived psbA containing band was calculated to have the length of 4.2 kbp. The WT control fragment was calculated to form a 2.5 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared psb A probe. The semiquantitative results showed two bands (pointed out with arrows ) in the P and B lines, representing the wild type ptDNA band and 1.8 kbp larger (containing P dnaK cat CH cassette) recombined ptDNA band. The presence of both types of plastids demonstrated heterogeneity of the obtained transformant lineages. e The Southern blot hybridization of EcoRI-digested total DNA with the cat CH probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with EcoRI restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 5 µg of DNA. The cat containing band was calculated to have the length of 10 kbp. Additionally, a positive control was added by digesting the transformation plasmid with EcoRI and AatII. This fragment was calculated to range ~10 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared 32 P enriched probe. The developed film shows no signal in the WT wells and one dominant signal in the transformed cells lines, confirming that the transformation vector had successfully recombined in a unique and single locus of the plastid without any unwarranted, illegitimate recombination with the genome or the plastid. The positive control is overexposed as the molar contribution of the cat sequence in the transformation vector is great deal higher than in the plastid
Article Snippet: Further, the DNA was digested with BglII restriction enzyme (Thermo, USA), cutting the plastid in the proximity of 3′ and 5′ ends of the psbA gene.
Techniques: Transformation Assay, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Amplification, Marker, Southern Blot, Hybridization, Generated, Derivative Assay, Positive Control