Studies On The Structure And Function Of The Upstream Sequence Of CpcB Gene From Arthrospira Platensis

A green fluorescent protein(GFP) expression vector was constructed with the reporter gfp gene under the promotion of the upstream sequence of cpcB gene from Arthrospira platensis. Fluorescent microscope and flow cytometer analyses showed that GFP could express in E.coli, and it could be inferred from the expression level that some strong promoters might exist in the upstream sequence. And the expression of GFP was influenced by temperature, higher temperature led to higher expression level. The software and mutation analyses on the secondary structure of translation initiation region(TIR) revealed that a RNA thermosensor might account for the thermal regulation, and the same structure might exist in the TIR of cpcB gene too.Promoter prediction software analysis showed there were six putative promoters in the upstream sequence of cpcB gene, and each promoter could drive expression of GFP in E.coli after isolation. GFP level analysis showed promoter three was the strongest and the next one was promoter four. Point mutation to -35 box (TTCTCA) of promoter three showed the bases C and T were vital for the promoter strength, for their mutation could lead to serious decline of GFP level.There were six short open reading frames(ORFs) in the upstream sequence, and three of them might have encoding ability. Mutation analysis indicated the ORFs or their products might be related to regulation of phycoyanin expression. There was also an AT-rich region from -321 to -371, whose deletion caused decline of GFP, indicating its positive effect on promoter activity.Transcriptional analysis of cpcB gene was done by SmartRace technology. It was found that there was only one transcript of cpcB gene in the alga, which was from the promotion of promoter one according to the mapping of transcription start site. The partial upstream sequences and coding sequences of cpcB gene from other Arthrospira strains were also cloned. The cloned sequences were 474bp, including257bp upstream sequences and 217bp coding sequences. The upstream sequences, together with the coding sequences from FACHB438, FACHB793 and FACHB790 had not been reported before. There were 31 changes in the seven sequences, with 21 in the upstream sequences and 10 in the coding sequences, and most changes were scattered in FACHB439. The similarity of the upstream sequences of cpcB genes indicated the a common regulation mechanism might exist in different Arthrospira strains.A heterologous cyanobacterial reporting system was constructed. Through homologous recombination, gfp gene under the promotion of the upstream sequence of cpcB gene was integrated in the genome of Synechococcus sp.PCC7942, and the expression of GFP was verified by fluorescent microscope and flow cytometer analyses.In this reporting system, the influence of light intensity on expression of GFP was investigated. Results showed that expression of GFP was influenced by light intensity. In the scope of light intensity larger than lO.Oumol m"2 s"1, low light intensity improved the expression of GFP, the lower the light intensity, the higher the GFP level; but light intensity below lO.Ofimol m^s'1 might lead to a low GFP expression level. Deletion analysis of the upstream sequence of cpcB gene showed a light responsive elemet might exist in the region from -218 to-276.The influence of nutrition elements, such as S, P, N and Fe on GFP expression were also investigated. The nutrition deficiency led to decline of GFP expression in different way, the influence of S deficiency was relatively light, while that of N deficiency was relatively heavy.