Research On Promoters And Expression And Regulation Involved In Genes Of Lyase In Anabaena PCC 7120

Lyase plays an important role in the prosess of most cyanobacteria phycobiliproteins synthesis.alr0617-encoded CpcS1 and all5339-encoded CpcT1 have been shown to have lyase activity.Genes cpcS2 (all5292) and cpcT2 (alr0647), which are high homologous with cpcS1 and cpcT1, were confirmed without catalytic activity.Tn addition, these two genes only express in the nitrogen-dificiency conditions, in the normal cases they do not express.Construction ofâ–³alr0617 deletion mutant can choose gene knockout approach. Through molecular biology approach, alr0617 and its upstream and downstream were amplified by PCR. alr0617 was deleted and replaced by spectinomycin and was linked to another vector pRL271 which containing sacB gene.Then the vector was to be transformed to the Anabaena PCC 7120 wild-type strain by conjugation. Resistance and sucrose were used for screening transformants. Since cyanobacteria contain multiple copies in the genome, the strains obtained are single exchange mutants. Compared with wild-type cells,â–³alr0617 have the same ability against the pressure of nitrogen deficiency , but quite more sensitive to sucrose.This paper also researched on cpcS2 (all5292) and cpcT2 (alr0647) promoter fragments. By choosing 5 ' upstreams different lenths of cpcS2 (all5292) and cpcT2 (alr0647) and fusing with gene gfp in vitro, then transferring into cyanobacteria cell by conjugation. Through the observation of GFP fluorescence to identify the transcriptional activity of promoter fragments. The results showed that :in the normal medium BG11 and nitrogen deficiency medium BG110, 1100bp fragment which is upstream of cpcS2 have not promoter activity,2000bp fragment have weak promoter activity; 1300bp and 2600bp fragments of the cpcT2 have strong promoter activity, 680bp was relatively weak. In the BG110 medium there are a large number of heterocysts produced, yet ,in the heterocysts there are not GFP can be detected.