APORTES DE LA EDUCACIÓN INTERCULTURAL A LA EDUCACIÓN PREESCOLAR

The psbA gene is a main component in photosynthesis. The light-regulated gene expression of psbA is conducted by phosphorylation and redox potential that modulate the interaction of a complex of RNA binding proteins (RBPs) and psbA 5’UTR. A working model of psbA gene regulation has been postulated (Trebitsh et al., 2000). The role of the tobacco

psbA 5’UTR has been previously investigated by in vivo analyses via chloroplast transformation (Eibl et al., 1999; Staub and Maliga, 1994a). Those investigations did not supply detailed information about the cis-acting elements for the stability and translation of

psbA in chimeric transcripts. By use of an in vitro chloroplast translation system, three cis-

acting sequences essential for psbA translation have been determined (Hirose and Sugiura, 1996), whereas other elements required for mRNA stability were inevitably disregarded. Thus, the in vivo analysis via chloroplast transformation appears to be the most suitable approach to simultaneously define the cis-acting determinants for mRNA stability and translation in higher plants as well as in Chlamydomonas (Higgs et al., 1999; Mayfield et al., 1994; Nickelsen et al., 1999). Accordingly, it is exploited in this study that aims to characterise the cis-acting elements of tobacco psbA 5’UTR correlated to the modulations of mRNA stability and translation efficiency. For this purpose, serial modifications of the psbA

5’UTR were created and used as the 5’ leader sequence of reporter uidA gene in a set of plastid transformation constructs. By comparing the Gus activities and chimeric uidA mRNA levels in the corresponding transplastomic plants, the cis elements of psbA 5’UTR conferring the particular regulatory function could be determined.

The full-length (85nt) tobacco psbA 5’UTR existing in plasmids pUC16SpsbA5’uidA- rbcL3’ or pUC16SpsbA5’uidApsbA3' is shown in figure 3.16 (page 75). From the 5’end to the 3’ end of this 5’UTR, some cis-elements might be of regulatory significance, including the

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poly(A) sequence (AAAAA), stem-loop (SL), SD-like RBS (GGAG), internal AUG codon and AU-Box (UAAAUAAA). Furthermore, the 5’end structural sequences of the rbcL 5’UTR were presumed to convey high mRNA stability signals to the psbA 5’UTR. Based on these concerns, the following modifications of the psbA 5’UTR were introduced.

In plasmid pUC16SpsbA5’uidArbcL3’, the reporter uidA is flanked by psbA 5’UTR and rbcL 3’UTR as the leader/trailer and driven by promoter Prrn in the framework of plasmid pUC18. This plasmid was used as the template for Pfu DNA amplification to modify the psbA 5’UTR through a strategy as indicated in figure 3.7. Two common primers were used in all PCR-based mutagenesis of psbA5’UTR, in which one primer pUCBASE-Fw (5’- CTGCGTTATCCCCTGATTCTGTG-3’) was based on the sequence of pUC18, and another primer GusSnaBI (5’-TCACACAAACGGTGATACGTAC-3’) was according to the coding region of uidA gene. For each psbA 5’UTR modification, a pair of primers was designed with introduction of specific restriction enzyme sites (termed as E or E* in all cases). One generally named as PsbAMod-Re(E) together with primer pUCBASE-Fw could be used to generate the PCR fragment PucPsbAmod (450~530bp) containing Prrn and 5’ part of psbA

5’UTR, another as (E/E*)PsbAMod-Fw could couple with primer GusSnaBI to amplify the fragment PsbAmodGus (420~500bp) containing 3’ part of psbA 5’UTR and partial uidA gene. Both PCR products with a part of psbA 5’UTR were cloned into plasmid pUC18 at proper sites by blunt end ligation. Then, the psbA 5’UTR was regenerated within the merged insert PucPsbAmodGus by digestions with enzymes (E or E*) and the related cloning operations. The partial uidA was further substituted with intact uidA from plasmid pUC16SpsbA5’uidA- rbcL3’ by digestions with NcoI and another appropriate enzyme. The resultant plasmid resembled pUC16SpsbA5’uidArbcL3’, but with a modified psbA 5’UTR. Finally, the fragment comprising Prrn, either form of psbA 5’UTR (wt or modified), uidA and tobacco

rbcL 3’UTR (Prrn-psbA5’-uidA-rbcL3’) was isolated by SmaI (blunt end enzyme) and SacII, then replaced the tobacco rbcL 3’UTR of vector pKCZ cut by Eco47III (blunt end enzyme) and SacII to create the plastid transformation construct containing wt or mutated psbA 5’UTR. The detailed process for each psbA 5’UTR modification and the generation of the final corresponding construct is individually described as below. In most cases, the versatile plastid transformation vector pKCZ digested by Eco47III and SacII with the removal of tobacco rbcL

3’UTR served as the frame in a general term of pKCZ (Eco47III+SacII) to take in the uidA

cassette. However, few constructs were obtained by alternative pathways. As the controls, plastid constructs with wt psbA 5’UTR were also included. It is necessary to note here that the plasmid pUC16SpsbA5’uidArbcL3’ applied for PCR-based mutagenesis of psbA 5’UTR had

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a variant form of wild-type tobacco psbA 5’UTR termed as ori*, which was resulted from its own PCR cloning in the previous work where the small poly(A) sequence at the 5’ end was slightly changed into ‘ACTAA’ by two nucleotide alterations. Therefore, another control construct with original psbA 5’UTR termed as ori was specially required to examine the consequence of this small modification. For those constructs containing the psbA 3’UTR and wild-type or original psbA 5’UTR, this psbA 5’UTR could be distinctly marked as Ori*-P or

Ori-P.

Figure 3.7: The process to modify psbA 5'UTR and create plastid transformation constructs containing reporter uidA gene and various forms of psbA 5'UTR

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