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b'Chloroplast gene expression is predominantly regulated at the posttranscriptional\\nlevels of mRNA stability and translation efficiency. The expression of psbA, an important\\nphotosynthesis-related chloroplast gene, has been revealed to be regulated via its 5\\u2019-\\nuntranslated region (UTR). Some cis-acting elements within this 5\\u2019UTR and the correlated\\ntrans-acting factors have been defined in Chlamydomonas. However, no in vivo evidence with\\nrespect to the cis-acting elements of the psbA 5\\u2019UTR has been so far achieved in higher plants\\nsuch as tobacco. To attempt this, we generated a series of mutants of the tobacco psbA 5\\u2019UTR\\nby base alterations and sequence deletions, with special regard to the stem-loop structure and\\nthe putative target sites for ribosome association and binding of nuclear regulatory factors. In\\naddition, a versatile plastid transformation vector pKCZ with an insertion site in the inverted\\nrepeat region of the plastid genome was constructed. In all constructs, the psbA 5\\u2019UTR (Wt or\\nmodified) was used as the 5\\u2019 leader of the reporter gene uidA under control of the same\\npromoter, Prrn, the promoter of the rRNA operon. Through biolistic DNA delivery to tobacco\\nchloroplasts, transplastomic plants were obtained. DNA and RNA analyses of these\\ntransplastomic plants demonstrated that the transgenes aadA and uidA had been correctly\\nintegrated into the plastome at the insertion site, and transcribed in discrete sizes. Quantitative\\nassays were also done to determine the proportion of intact transplastome, the uidA mRNA\\nlevel, Gus activity, and uidA translation efficiency. The main results are the following:\\n1) The insertion site at the unique MunI between two tRNA genes (trnR-ACG and trnNGUU)\\nis functional. Vector pKCZ has a large flexibility for further DNA manipulations\\nand hence is useful for future applications.\\n2) The stem-loop of the psbA 5\\u2019UTR is required for mRNA stabilisation and translation. All\\nmutants related to this region showed a 2~3 fold decrease in mRNA stability and a 1.5~6\\nfold reduction in translation efficiency. The function of this stem-loop depends on its\\ncorrect sequence and secondary conformation.\\n3) the AU-box of the psbA 5\\u2019UTR is a crucial translation determinant. Mutations of this\\nelement almost abolished translation efficacy (up to 175-fold decrease), but did not\\nsignificantly affect mRNA accumulation. The regulatory role of the AU-Box is sequencedependent\\nand might be affected by its inner secondary structure.\\n4) The internal AUG codon of the psbA 5\\u2019UTR is unable to initiate translation. An\\nintroduction of mRNA translatability from this codon failed to direct the translation of\\nreporter uidA gene, overriding the mutation of the AU-Box.\\n5) The 5\\u2019end poly(A) sequence does not confer a distinct regulatory signal. The deletion of\\nthis element only insignificantly affected mRNA abundance and translation. However,\\nthis mutation might slightly disturb the conformation of the stem-loop, resulting in a\\nmoderate decrease in translation efficiency (~1.5 fold).\\n6) The SD(Shine-Dalgarno)-like RBS (ribosome binding site) of the psbA 5\\u2019UTR appears to\\nbe an indispensable element for translation initiation. Mutation of this element led to a\\ndramatically low expression of the uidA gene as seen by Gus staining.\\n7) The 5\\u2019end structural sequence of the rbcL 5\\u2019UTR does not convey a high mRNA\\nstabilising effect to the psbA 5\\u2019UTR in a cycling condition of the light and the dark. Their\\ndistinct roles appear to be involved in darkness adaptation.\\nFurthermore, with respect to the overall regulatory function of the psbA 5\\u2019UTR, two\\nmodels are proposed, i.e. dual RBS-mediated translation initiation, and cpRBPs-mediated\\nmRNA stability and translation. The mechanisms for mRNA stabilisation entailed by the rbcL\\n5\\u2019UTR are also discussed. Direct repeat-mediated transgene loss after chloroplast\\ntransformation and other aspects related to the choice of insertion site and plastid promoter\\nare also analysed.'