Introduction
The transition from skotomorphogenesis to photomorphogenesis is an extremely rapid and complex process. Several developmental changes occur: decrease in the rate of the stem elongation, apical hook straightening, and the initiation of the synthesis of the chlorophyll pigments. In angiosperm, the cotyledons are white until they reached the sunlight to be coloured in green by chlorophylls while in the gymnosperm, cotyledons are capable of greening in the dark \cite{Solymosi_2010}. Obviously, this step of greening in angiosperm is regulated by a transduction signal that allows the activation of genes. Recent research has shown that genes from the nucleus and from the plastid have an impact on this transition (Pfannschmidt, et al.) and the development of the chloroplast. Yet the mechanism is poorly understood, we already know what the essential element for this transition are. The shift from plastid state to the complex chloroplast state is regulated by a plastid-encoded RNA polymerase (PEP). The PEP core works with 12 small PEP-associated proteins (PAP).
Conforming to their functional and structural units, we can hypothesize that PAPs can be divided into three major groups. Group 1 comprises PAPs involved in DNA/RNA metabolism (PAP1, 2, 3, 5, and 7), group 2 includes proteins involved in redox regulation (PAP6 and 10) and ROS protection (PAP4 and 9) and group 3 includes proteins with unknown function (PAP8, 11, and 12) \cite{Steiner_2011} \cite{Kindgren_2015}\cite{Pfannschmidt_2015}. The understanding of this system requires isolation of each gene to be able to work on each part of the complex.
Here we focus on the purification and amplification of the PAP 1, 2, 3 and 11 genes. In this article, we will however only explain in detail the steps needed to obtain PAP1 and use those results to discuss the outcome of this difficult process.
Materials and Methods
Fragment of interest amplified by PCR
Amplification of the DNA fragments of interest via PCR. The PCR machine is programmed for a cycle composed of 5 steps : denaturation, hybridization, polymerase passage, phusion passage, A tailing, recovery) at different temperatures for each steps (98°C, 98°C, 52°C, 72°C, 72°C, 12°C respectively). Denaturation separates of the double strand of DNA. Hybridation lets the primer bind so elongation can begin. Polymerase and Phusion passage allows elongation by each respective enzyme. Polymerisation by the phusion protein allows for DNApolymerase and Phusionprotein (add dNTP to part that DNApolymerase did not polymerase). A tailing adds dATP at the 3’ ending of the strands to facilitate plasmid insertion. The first three steps are repeated 39 times in order to obtain a high number of fragments.
Directed mutagenesis
Modification of restriction enzyme sites on DNA of interest make later incorporation in cells possible. This mutagenesis is done by adding specific primers that are engineered to add the mutation. Small fragments are obtained and are separated from the rest of the genome by SDS page? Ligation and amplification by PCR is performed.
Ligation
Ligation of the fragment of interest on ---- vector via ligase enzyme. The fragments and vectors are added to a tube with T4 DNA ligase and specific buffer solution, and incubated all night. These vectors contain a region coding for antibiotic resistance and the LacZ gene which will later help with colony selection.
Bacterial transformation
Insertion of the fragment ligated to the vector in bacteria. In a tube, the plasmid solution is mixed with bacteria : the transformation is done via a heat shock of 45 seconds at 42°C and returned quickly in ice. After 20 min of incubation, the resulting liquid is spread in petri dish filled with Luria Berthani medium (rich medium made of peptides, vitamins and minerals) and antibiotic. The bacteria are incubated one night at 37°C.
White Blue screen
This screen allows for selection of correctly inserted transformants who will be able to produce and amplify the sequence of interest (Fig. 1)