INTRODUCCIÓN

In the stem-loop region of the barley psbA 5’UTR, several nucleotides at the side of the stem adjacent to the 5’ terminus were speculated to be crucial for the binding of a 95kd RBP (RNA binding protein) (Mullet, unpublished data). Since the psbA 5’UTR was found to be highly conserved in plants (Kim and Mullet, 1994), the interaction of 95kd RBP with the stem-loop sequence was proposed to occur similarly in tobacco. Thus, the putative ‘core’ nucleotides (asterisked in figure 3.9 and 3.10) in the stem-loop of tobacco psbA 5’UTR were submitted to mutagenesis, termed SLM1, with additional concern that nucleotide alterations did not change the melting temperature (Mt) of the stem-loop. Mt is known to be an important aspect to evaluate the status of stem-loop that might fluctuate among the linear, hairpin, and other intermediate structural states through its interaction with cpRBPs. Changes in Mt of the stem-loop were presumed to have an influence on chloroplast gene expression. Therefore, another stem-loop mutation was also conceived, termed as SLM2 in which the GC content of the stem-loop was increased by alternating some nucleotides excepting those putative ‘core’ bases (figure 3.10). The generations of SLM1 and SLM2 were conducted as shown below 4.

4 _{First mutation of the stem-loop (SLM1)}

Two primers SLMut1-Li and SLMut1-Re were designed and synthesised (figure 3.9). Primer pair SLMut1-Li/pUCBASE-Fw was used for the generation of PCR product, Prrn-included PucSLM1, as well as another primer pair SLMut1-Re/GusSnaBI for the amplification of PCR product, SLM1Gus comprising partial

uidA gene. Both PCR products with identical size (~470bp) were further cloned into plasmid pUC18 at Ecl136II

and SmaI sites by blunt end ligation, respectively.

Because base residues 5’CTC3’ (at the 5’ end of primer SLMut1-Li) localised behind the cut-end (5’-//- GAG↓3’) of Ecl136II in pUC18 could restore the SacI site (5’GAGCTC3’), the recombinant plasmids with insert PucSLM1 was preliminarily identified by SacI digestion. Likewise, primer SLMut1-Re contained an SspI

site (5’AAT↓ATT3’) at its 5’ end, therefore the positive clones with insert SLM1Gus were primarily detected by

SspI digestion, and those with introduced SspI site distal to SacI were selected. The pUC18 DNA was dually cut by XbaI and SacI to act as the cloning frame to accept the insert PucSLM1 with the cut-ends of XbaI and SacI

(blunted), and another insert SLM1Gus with the cut-ends of SspI and SacI, from their related PCR clones. The resultant plasmid contained a merged insert PucSLM1Gus, in which the full-length of psbA 5’UTR with local nucleotide mutations in the stem was regenerated. With respect to this mutation, the stem-loop Mt was maintained, whereas four alterations of base-pairs occurred in the stem to inactivate the ‘core’ nucleotides, incidentally with an additive MunI site (5’C↓AATTG3’) (figure 3.9). Then, the partial uidA was substituted with intact uidA by NcoI and SacI digestions to form the intermediate plasmid comprising the full uidA cassette Prrn- psbA5’(SLM1)-uidA-rbcL3’ that was subsequently separated by SmaI and SacII, and introduced into vector pKCZ (EcoR47III+SacII) to create plastid transformation construct pKCZ-psbA5’(SLM1):: rbcL3’.

Second mutation of the stem-loop (SLM2)

Two primers SLMut2-Li and SLMut2-Re were synthesised, with internal nucleotide alterations for the mutation SLM2 (figure 3.10). Primer pairs SLMut2-Li/pUCBASE-Fw and SLMut2-Re/GusSnaBI were individually used to amplify the same size (~ 470bp) of PCR products PucSLM2 (Prrn-included) and SLM2Gus

Results 67

The nucleotide substitutions for SLM1 and SLM2 were marked in distinct colours (see figure 3.16, page 75).

Figure 3.9: The strategy to create stem-loop mutation (SLM1) of psbA 5’UTR. Nucleotide substitutions are indicated by arrows and ‘core’ bases are asterisked.

Figure 3.10: The strategy to create stem-loop mutation (SLM2) of psbA 5’UTR. Nucleotide substitutions are indicated by arrows and ‘core’ bases are asterisked.

The secondary structures of wild-type psbA 5’UTR and two mutants (SLM1 and SLM2) were predicted, using software programme ‘RNA Structure 2.52’ (Zuker, 1999). Their stem-loop structures near the 5’ end are demonstrated in figure 3.11.

(uidA-related) that were further cloned into plasmid pUC18 at Ecl136II and HincII site by blunt end ligation, respectively.

According to the same strategy as for SLM1, the PCR clones of PucSLM2 and SLM2Gus were preliminarily identified by SacI and SspI, respectively. The recombinant plasmids with PCR inserts in desired orientation were selected. Plasmid with insert PucSLMut2 was sequentially treated with SacI, T4 DNA polymerase and PstI, then served as the vector to incorporate the insert SLMut2Gus released from its recombinant plasmid by SspI and PstI. The resultant plasmid contained a combined insert PucSLM2Gus, in which the original size of mutated psbA 5’UTR was formed. This mutation of psbA 5’UTR was attributed to several nucleotide substitutions from A/T to G/C in the stem, and therefore increased the stem-loop Mt from dG (-11.9 kcal/mol) to dG (-17.6 kcal/mol), but the putative ‘core’ nucleotides were unchanged (figure 3.10 and 3.11). Afterwards, the partial uidA was replaced with intact uidA by NcoI and PstI to form the recombinant plasmid containing entire uidA cassette Prrn-psbA5’(SLM2)-uidA-rbcL3’ was excised by SmaI and SacII, then integrated into vector pKCZ (EcoR47III+SacII) to achieve plastid transformation construct pKCZ- psbA5’(SLM2)::rbcL3’.

Results 68

Figure 3.11: Comparison of the free energy (dG) of the stem-loop structures among the wild-type psbA 5’UTR (B) and its two mutants SLM1 (A) and SLM2 (C). Arrows indicate nucleotide substitutions

Comparing their potentials for structural maintenance, the SLM1 mutant of psbA

5’UTR had the same value of dG (-11.9 kcal/mol) (figure 3.11, A) as well as the wild-type (figure 3.11, B), indicating the nucleotide alterations of SLM1 in the stem was neutral to the structural state. However, the value(dG) of mutant SLM2 was considerably increased up to – 17.6 kcal/mol, due to a higher GC content in the stem resulting from nucleotide changes (figure 3.11, C), implicating the conformation of the stem-loop structure in mutant SLM2 was comparatively stable.