| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 91 === MCm-AEC3 gene in genomic clone λMCm-AEC3-16, spanning 3,825 base pairs, contains six exons and five introns with consensus GT-AG dinucleotides locating at their boundaries. The amino acid sequence of bitter gourd auxin efflux carrier MCm-AEC3 shows 44.4 ~ 74.3 % homology to auxin efflux carriers of Arabidopsis, mustard, cucumber, cotton, alfalfa, pea, white poplar and rice. The first 28 amino acids are predicted to be a putative signal peptide. Four conserved repeats exist in the polypeptide. The putative TATA and CAAT box are 207~200 bp and 298~295 bp upstream from the translation start site in MCm-AEC3. Conserved elements responsive to auxin, ethylene, abscisic acid, light, low temperature and wounding are found in the promoter region.
Northern blot analysis indicates that the expression of bitter gourd auxin efflux carrier gene MCm-AEC3 can be induced by 10-6 M IBA and 10-5 M 2,4-D. Higher concentration of IAA can induce more accumulation of mRNA, but no obvious gene expression is detected in treatment of NAA.
To investigate the promoter activities of auxin efflux carrier genes MCm-AEC1 and MCm-AEC3, p121-28 and p121-93 plasmid was constructed by fusing the β-glucuronidase (GUS) reporter gene with promoter fragments and transiently expressed in young leaves, staminate petals and fruit sections from bitter gourd. p121-28 and p121-93 are also transiently expressed in petal discs from Phalaenopsis. The activities of p121-28 and p121-93 are demonstrated. For transient expression in young leaves from bitter gourd by particle bombardment, the distance for delivering DNA coated on 1.7 μm tungsten particle is 6 cm, and the pressure of delivery is 900 psi. For transient expression in staminate petals and fruit sections from bitter gourd by particle bombardment, the distance for delivering DNA coated on 1.7 μm tungsten particle is 6 cm, and the pressure of delivery is 1100 psi. The expression of GUS in staminate petals is higher than others.
Arabidopsis plants transformed with p121-93 express GUS activity in shoot meristem, young leaf, as well as stamen. Apical bud does not grow normally in the Arabidopsis plant transformed with p131-93AS.
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