| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 91 === In order to obtain the promoters and coding sequences of polyubiquitin genes, two genomic clone lBUq20 and lBUq137 isolated from banana (Musa spp. Hsien Jin Chiao, AAA group) were sequenced and characterized. According to the result of sequence analysis, the open reading frame in lBUq20, designated as Mh-UBQ3, encodes 229 amino acids with a calculated molecular mass of 25.7 kD. Mh-UBQ3 contains three contiguous direct repeats of the protein coding region in polyprotein conformation. The deduced amino acid sequence of every ubiquitin monomer is identical to each other and to ubiquitins from other plants. Southern bolt analysis indicated that Mh-UBQ3 from banana belongs to a low-copy gene family. The length of Mh-UBQ3 upstream promoter is about 2.8 Kb. It contains a putative intron of 547 bp immediately followed by the translation start codon. The predicted TATA box is located at 620~ 626 bp upstream from the translation start site. Several conserved responsive elements related to salicylic acid, ABA, auxin, drought and low temperature could be found in Mh-UBQ3 promoter. The other genomic clone, lBUq137, contains an open reading frame, named Mh-UBQ4, encoding 533 amino acids whose molecular weight is 59.7 kD. Mh-UBQ4 consists seven head-to-tail repeats of the ubiquitin monomer. Few of the deduced amino acid sequences among repeat units, however, are not conserved. Based on the result of Southern hybridization, Mh-UBQ4 belongs to a single copy gene in banana genome. Upstream promoter sequence of Mh-UBQ4 about 2.8 Kb was obtained by polymerase chain reaction using GenomeWalkerTM DNA Kit. A possible intron of 496 bp in Mh-UBQ4 is immediately 5’ upstream to the translation start codon. Putative TATA box is located at 595~ 601 bp upstream from the translation start site. Elements related to salicylic acid, ethylene, heat shock, wounding, and elicitor could be found in Mh-UBQ4 promoter.
According to the results of Northern blot analysis, Mh-UBQ3 whose hybridized signal corresponding to 0.8 Kb is expressed in leaf, bract, ovary, pistil, suspension cells, and banana fruit. Most of the Mh-UBQ3 mRNA accumulated after the onset of the ripening process, especially from stage 2 through stage 7. Furthermore, expression of Mh-UBQ3 was suppressed either in pulp treated with IAA or in peel treated with low temperature. No mRNA was detected in pulp treated with low temperature. Using Mh-UBQ4 gene-specific probe, a 1.7 Kb transcript was identified whose abundance was found in bract, pistil, suspension cells, and banana fruit. However, the expression of Mh-UBQ4 was lower than Mh-UBQ3.
The promoter activity of banana polyubiquitin gene Mh-UBQ2, Mh-UBQ3, and Mh-UBQ4 were investigated by transient assay using particle bombardment. The results of transient expression in petal discs from Phalaenopsis showed that both activities of Mh-UBQ2 and Mh-UBQ3 were higher than CaMV 35S promoter, but the activity of Mh-UBQ4 promoter was less than seven tenth of CaMV 35S promoter. On the contrary, CaMV 35S promoter represent the highest acitivity of all tested promoters in tobacco leaf discs. This result indicated that expression of polyubiquitin gene promoters from banana were suppressed in dicot expression system. GUS activity for construct containing Mh-UBQ4 promoter is non-detectable, but for constructs containing Mh-UBQ3 and Mh-UBQ2 promoters is 3.7- to 29-fold greater than construct containing CaMV 35S promoter in banana peel discs.
|
|---|
