Browsing by Author "Jäppinen, Sanni"

Filamentous cyanobacteria taxa Nostocales and Stigonematales cells can differentiate into heterocysts nitrogen fixing cells when nitrogen is limiting the growth and into resting cells akinetes when nutrients decrease or the growth conditions become unfavorable for growth. Akinetes overwinter in the water sediments during the unfavorable growth time. When the growing condition improves akinetes germinate and can start a new cyanobacterial bloom. Akinete differentiation remains unclear. It is known from the literature that only a few akinete specific genes exist. First described akinete specific gene was avak. The morphological changes of akinete differentiation are known but the changes at molecular genetics level in regulation and differentiation remains unclear. The aim of this study was to design a method for akinete differentiation-related genes, avak, hepA and hap, for an Anabaena 1TU33s10 strain and to monitor the gene expression changes in a seven-week growth experiment. Primers for the differentiation related genes were designed based on the known whole genome sequence of the Anabaena 1TU3310 strain. In this study it was managed to design a quantitative reverse transcriptase polymerase chain reaction, qRT-PCR method, based on the genes involved in the akinete differentiation process. It was observed that gene expression changed when akinetes began to differentiate into the filaments. In the growth experiment II avaK-gene expression was increased 2-fold between the 14. and 30. days, and hap-gene showed 1.5 fold growth between 14. and 30. days. The number of akinetes was also increasing at the same time. In the growth experiment I heap-gene showed 1-8 fold growth between the days 21. and 27. –30. days when the number of heterocysts were also increasing. The number of akinetes was relatively low compared to number of vegetative cells which also explains the small expression fold-differences in the cultures during the experiment time when compared to expression fold-differences described in the literature. Designed method can thus also detect minor changes in gene expression. The designed and built qRT-PCR method can be used in the future for monitoring gene expression changes also for new akinete specific genes, and the method can be further optimized for screening natural water samples.