| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 91 === Abstract
To construct an ideal vector for expression in monocots like banana, we chose promoter of banana polyubiquitin (Mh-UBQ1), b-1,3-glucanase (Mh- BGL1) and 1-aminocyclopropane-1-carboxylate oxidase (Mh-ACO1) genes for activity assay. The reporter gene downstream of the promoter was GUS (b-glucuronidase). Moreover, nuclear matrix attachment regions (MARs) and an plant endoplasmic reticulum (ER) retention signal K/HDEL were included in the construction because the former could enhance the level of expression and reduce the variability of expression, and the latter could cause proteins to accumulate in the ER became more stable.
The expression vectors containing CaMV 35S promoter were named as pBI2N, pBI2NH, p35PGT and p35PGHT. Two groups of the related expression vectors contained Mh-UBQ1 promoter were divided into two groups, one group including p79PG, p79PGH, p79PGT and p79PGHT, and the other including p79PSG, p79PSGH, p79PSGT and p79PSGHT. Containing Mh-BGL1 promoter, pGLG and pGLGH were constructed. The expression vectors containing Mh-ACO1 promoter were designed as pMPG and pMPGH. Each expression was co-bombarded with internal control plasmid pU35STgfp into Phalaenopsis petal discs, banana suspension cells, banana peel discs and banana pulp discs for transient expression assay. Comparison of these promoters, the activity of Mh-UBQ1 promoter was the highest one in all the tested plant materials except banana pulp discs. It was 1.03-, 2.90- and 4.51-fold higher than CaMV 35S promoter in Phalaenopsis petal discs, banana suspension cells and banana peel discs, respectively. The activity of Mh-ACO1 promoter was lower and Mh-BGL1 promoter was much lower than CaMV 35S promoter in Phalaenopsis petal discs and banana peel discs. However, the expression activity of vectors containing Mh-UBQ1 promoter with changed intron-exon boundary dinucleotide was the least. The effect of HDEL on transient expression of GUS gene in all the tested plant materials except banana pulp discs was not significant. Existence of MAR in the vectors enhanced GUS expression significantly in suspension cells of banana, and reduced GUS expression in peel discs of banana.
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