Fbpase, Ald And Tpi Cyanobacteria Genetic Engineering And Its Regulation On Photosynthesis

By selecting Fructose-1, 6-bisphosphatase (FBPase), Fructose-1, 6-bisphosphate Aldolase (ALD) and Triosephosphate isomerase (TPI) as our targeted enzymes, which they are continuous catalysis from the Calvin Cycle and by transferring them into cyanobacteria to research regulation of photosynthesis, it has great and far-reaching significance to further understand regulation rules of photosynthesis and to get demanding plants of human being according to these rules to change. At the same time, it may be a way to effectively control or decrease the daily increasing concentration of atmospheric CO2. Aimed at this, we focus on transferring one, two and three targeted enzymes genes into cyanobacteria, then to research them how to regulate cyanobacteria photosynthesis and thus a series of studies have been carried out as follows: First, FBPase gene was cloned by PCR, and ligated with expression vector pRL-439, then ligated into the downstream of promoter PpsbA of shuttle vector pDC-08, to form shuttle expression plasmid, pDC-fbp, the plasmid was transferred into Anabaena sp. PCC7120 by tri-parental conjugation to gain the transgenic Anabaena sp. PCC7120. The expression levels of FBPase in the transgenic Anabaena sp. PCC7120 were 16.8 percent higher than that of wild-type cells. Moreover, its enzyme activity was 1.34-fold higher.A co-expression system of tandem genes coding for ALD and TPI was designed and ligated into the downstream of promoter PpsbA of shuttle expression vector pRL-489, to form shuttle expression plasmids, pRL-AT1 and pRL-AT2, those two plasmids were transferred into Anabaena sp. PCC7120 by tri-parental conjugation to generate the respective transgenic Anabaena sp. PCC7120. The transgenic Anabaena sp. PCC7120 containing pRL-AT2, which contains a dimmer of the tandem gene co-expression operator, shows higher protein expression levels and enzyme activity. The expression levels of FBPase and TPI in the transgenic Anabaena sp. PCC7120 were 58.9 percent and 53.3 percent higher than that of wild-type cells, respectively. Moreover, their enzyme activity was 2.59-fold higher.A co-expression system of tandem gene coding for ALD, TPI and FBPase was designed and ligated with expression vector pRL-439, then ligated into the downstream of promoter PpsbA of shuttle vector pDC-08, to form shuttle expression plasmid, pDC-ATF, the plasmid was transferred into Anabaena sp. PCC7120 by tri-parentalconjugation to get the transgenic Anabaena sp. PCC7120. The expression levels of ALD, TPI and FBPase in the transgenic Anabaena sp. PCC7120 were 22.8 percent, 20.9 percent and 6.2 percent higher than that of wild type cells, respectively. Moreover, their enzyme activity was 1.96-fold higher.Under atmospheric conditions with 360 p.p.m.CO2, the transgenic Anabaena sp. PCC7120 enhanced photosynthetic efficiency, increased oxygen-releasing activity, raised CO2 assimilative efficiency and heightened growth characteristics. Net photosynthesis, true photosynthesis and CO2 assimilative efficiency after transferring FBPase gene into Anabaena sp. PCC7120 were 17.2 percent, 22.5 percent and 18.1 percent higher than those of wild-type cells, respectively. Furthermore, the transgenic Anabaena sp. PCC7120 enhanced their growth characteristics. Net photosynthesis, true photosynthesis and CO2 assimilative efficiency after transferring a dimmer of ALD and TPI tandem genes into Anabaena sp. PCC7120 were 45.5 percent, 41.6 percent and 36.8 percent higher than those of wild-type cells, respectively. Furthermore, the transgenic Anabaena sp. PCC7120 heightened their growth characteristics. Net photosynthesis, true photosynthesis and CO2 assimilative efficiency after transferring ALD, TPI and FBPase tandem genes into Anabaena sp. PCC7120 were 31.3 percent, 38 percent and 33.2 percent higher than those of wild-type cells, respectively. Furthermore, the transgenic Anabaena sp. PCC7120 increased their growth characteristics.From the assay of absorption spectrum under room temperature, its data indicated that compared with wild-typ...