Studies On The Upstream Sequence Of Phycocyanin β Subunit Gene From Arthrospira Platensis FACHB341

Six promoters in the 419 bp upstream sequence of the phycocyaninβsubunit gene of Arthrospira platensis FACHB341 were cloned in our previous work. In this study, site-directed mutagenesis is used for inducing manifold mutations in -10 and -35 boxes of promoter 3, -10 box of promoter 4, and -35 box of promoter 6, respectively. The expression level of report gene gfp measured by flow cytometry is used as the indicator of start-up intensity of the mutants.Results show that the effects of site-directed mutagenesis in different promoters are quite dissimilar: Mutants from -10 box of promoter 3 lead to obvious rise of GFP levels in all cases, mutants from -35 box of promoter 3 decline sharply, and in -10 box of promoter 4 and -35 box of promoter 6, the expression levels of some of their mutants rise, while some decline after mutagenesis. There has been no detailed analysis of the upstream sequence of phycocyanin in blue-green algae before. This work may provide some data for clarifying the principle of the promoters of cpcB, and some powerful promoters obtained in the present study may offer useful tools for genetic manipulation in blue-green algae and other organisms.As promoters are essential cis-acting elements in gene expression, their structure and function become one of the hot spot of molecular biology. But there are many problems on the actual promoter research. The functions of many promoter sequences are unknown, especially those on the promoters in algae. There has been no general report on the research evolution about algae promoters before. It is useful that finding out the insufficiency and ascertaining the direction for the promoter research in the future. It will promote the research on regulation of gene expression. This paper sketches the research tactics and general situation of plant promoters, with special emphasis on that in algae. It also reviews the investigations on the promoters of phycocyaninβsubunit encoding gene of blue-green alga Arthrospira platensis in our laboratory, and proposes the possibilities of application of algal promoter research in genetic engineering.When Arthrospira platensis FACHB341 was cultured at 25℃, 40μmol m?2 s?1 light intensity, only one promoter that was the number 1 took part in the transcriptional regulation. The change of temperature and light intensity will affect the expression of report gene gfp driven by promoter. In this study, transcriptional pattern of c-phycocyanin gene under different cultivation conditions is determined by SmartRace technology. Results show that the number and position of the transcription start site of phycocyaninβsubunit gene are invariable under neither 15℃or 25℃, nor 10μmol m?2 s?1 or 40μmol m?2 s?1 light intensity. The transcription start site G is located at -285bp upstream of the start codon. It is illuminated that the changes of cultivation conditions will not affect the transcription start site of phycocyaninβsubunit gene. Further research is needed to confirm the mechanism that temperature and light intensity affect the function of promoters.