Cloning And Functional Analysis Of The Cucumisin Gene Promoter In Cucumis Melo L.

Cucumisin , an extracellular subtilisin-like serine protease, is expressed at high levels in the fruit of melon(Cucumis melo L. )and accumulates in the juice of melon. The cucumisin promoter region between positions -310bp and -1 relative to the transcriptional initiation site was amplified from melon(Cucumis melo L.cv Hetao) genomic DNA by polymerase chain reaction(PCR) and cloned into pUC19 vector. Sequence analysis showed that this sequence is identical with published sequence and contains characteristic motif of typical fruit-specific promoter, such as : TATA-box, CAAT-box, G-box, I-box-like and enhancer element. A plant expression vector pPZP-CGN was constructed containing the cucumisin 310bp promoter region, the coding sequence of p-glucuronidase(GUS) as a reporter gene and NOS terminator of pPZP221G. The melon cultivar Hetao was transformed with pPZP-CGN by pollen tube pathway transformation method. Melon transformed seeds were harvested after the 40th day after pollination. PCR amplifications analysis showed GUS was integrated into the genome of some seeds. Subsequently, the seeds of the melon fruits, which had high positive rate by PCRdetection, were cultivated in a greenhouse. Self-pollination was done during anthesis. Fluorescence analysis of GUS activity in transgenic melon plants revealed that the promoter were able to direct GUS expression at high levels in fruits , but not in leaves, stems and roots almost. It was determined that the region from transcription start site to upstream -310 bp is responsible for not only high level expression in fruits but also for directing fruit-specific expression. The cloned cucumisin promoter region may be useful for genetic engineering of melon fruit.