TGTCACA motif is a novel cis-regulatory enhancer element involved in fruit-specific expression of the cucumisin gene.
Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a beta-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides -234 and -214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATATGATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions -254 and -215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.
Yamagata
Hiroshi
H
Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Nada, Kobe, 657-8501 Japan. yamagata@kobe-u.ac.jp
Yonesu
Kiyoaki
K
Hirata
Ayako
A
Aizono
Yasuo
Y
D001483:Base Sequence; D003001:Cloning, Molecular; D028462:Cucumis / Q000201:enzymology* / Q000235:genetics; D018744:DNA, Plant; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D018744:DNA, Plant; D009687:Nuclear Proteins; D012697:Serine Endopeptidases; C041541:cucumisin