Regulation of pepc gene expression in Anabaena sp. PCC 7120 and its effects on cyclic electron flow around photosystem I and tolerances to environmental stresses.

Since pepc gene encoding phosphoenolpyruvate carboxylase (PEPCase) has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregulated (forward) or downregulated (reverse) pepc gene in Anabaena sp. PCC 7120. Results from real-time quantitative polymerase chain reaction (RT-qPCR), Western blot and enzymatic analysis showed that PEPCase activity was significantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I (PSI) activity was inhibited, as reflected by Y(I), and Y(NA) was elevated, and dark reduction of P700(+) was stimulated, indicating enhanced cyclic electron flow (CEF) around PSI in the reverse mutant. Additionally, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.

Recombinant expression and functional analysis of a Chlamydomonas reinhardtii bacterial-type phosphoenolpyruvate carboxylase gene fragment.

To investigate the function of a bacterial-type phosphoenolpyruvate carboxylase (PEPC2) derived from photosynthetically-grown Chlamydomonas reinhardtii, a fragment of the pepc2 gene was cloned and expressed in Escherichia coli. After optimal induction for 6 h, PEPC activity in the reverse mutant was lower than wild type (0.9 vs. 1.7 U/mg protein), and soluble protein was also lower than wild type (119 vs. 186 mg/g dry wt). In contrast, the total lipid content was increased from 56 (in wild type) to 71 mg/g dry wt, despite the growth rate being slightly diminished. The changes in PEPC activity, soluble protein and total lipid in the forward mutant were the opposite (2.4 U/mg, 230 mg/g, and 44 mg/g dry wt, respectively). Together, these data indicate that PEPC may function as a metabolic pivot in the regulation of protein and lipid accumulation in this alga.

Homologous comparisons of photosynthetic system I genes among cyanobacteria and chloroplasts.

It has now believed that chloroplasts arose from cyanobacteria, however, during endosymbiosis, the photosynthetic genes in chloroplasts have been reduced. How these changes occurred during plant evolution was the focus of the present study. Beginning with photosystem I (PSI) genes, a homologous comparison of amino acid sequences of 18 subunits of PSI from 10 species of cyanobacteria, chloroplasts in 12 species of eucaryotic algae, and 28 species of plants (including bryophytes, pteridophytes, gymnospermae, dicotyledon and monocotyledon) was undertaken. The data showed that 18 genes of PSI can be divided into two groups: Part I including seven genes (psaA, psaB, psaC, psaI, psaJ, ycf3 and ycf4) shared both by cyanobacteria and plant chloroplasts; Part II containing another 11 genes (psaD, psaE, psaF, psaK, psaL, psaM, btpA, ycf37, psaG, psaH and psaN) appeared to have diversified in different plant groups. Among Part I genes, psaC, psaA and psaB had higher homology in all species of cyanobacteria and chloroplasts. Among Part II genes, only psaG, psaH and psaN emerged in seed plants.

[Studies on relationship between the expression of hTNF-alpha gene and photosynthesis in Anabaena sp. IB02].

The effects of illumination on growth of Anabaena sp. IB02 and hTNF-alpha expression were studied. Photosynthetic activity, PS I and PS II activity of Anabaena sp. IB02 were assayed. Illumination enhanced the growth of Anabaena sp. IB02 and hTNF-a expression. Some relations were observed between hTNF-alpha expression and ture photosynthesis activity, PS I, PS II activity of Anabaena sp. IB02. Significant differences of the photosynthetic activity of host were detected simultaneously when hTNF-a expressed: the respiration rate increased (-68%), the light saturation point descended (+66%), all these suggested that the metabolic charge of host were increased and grow faster than wild type under low illumination.

[Bioinformatics studies on photosynthetic system genes in cyanobacteria and chloroplasts].

This study compared homology of base sequences in genes encoding photosynthetic system proteins of cyanobacteria (Synechocystics sp. PCC6803, Nostoc sp. PCC7120) with these of chloroplasts (from Marchantia Polymorpha, Nicotiana tobacum, Oryza sativ, Euglena gracilis, Pinus thunbergii, Zea mays, Odentella sinesis, Cyanophora paradoxa, Porphyra purpurea and Arabidopsis thaliana) by BLAST method. While the gene sequence of Synechocystics sp. PCC6803 was considered as the criterion (100%) the homology of others were compared with it. Among the genes for photosystem I, psaC homology was the highest (90.14%) and the lowest was psaJ (52.24%). The highest ones were psbD (83.71%) for photosystem II, atpB (79.58%) for ATP synthase and petB (81.66%) for cytochrome b6/f complex. The lowest ones were psbN (49.70%) for photosystem II, atpF (26.69%) for ATP synthase and petA (55.27%) for cytochrome b6/f complex. Also, this paper discussed why the homology of gene sequences was the highest or the lowest. No report has been published and this bioinformatics research may provide some evidences for the origin and evolution of chloroplasts.

Cloning and expression of metallothionein mutant alpha-KKS-alpha in Anabaena sp. PCC 7120.

The mouse metallothionein (mMT) mutant alpha-KKS-alpha has a higher capacity for binding heavy metals than wild type mMT. The mMT mutant alpha-KKS-alpha gene was placed under the control of the strong promoter PpbsA to generate the intermediate vector pRL-alpha-KKS-alpha. pRLalpha-KKS-alpha was then linked with the plasmid pDC-08 to construct shuttle expression vector pDC-alphaKKS-alpha. This expression vector was transformed into Anabaena sp. PCC 7120 using triparental conjugative transfer. After antibiotic selection (ampicillin and kanamycin), transgenic Anabaena was identified by PCR and Western blotting. The expression level of the mMT mutation alpha-KKS-alpha reached 7.4 mg/g dry cells weight, as detected by ELISA, and heavy metal resistance of the transgenic Anabaena was significantly improved.

Co-expression of Triosephosphate Isomerase, Fructose-1, 6-bisphosphate Aldolase and Fructose-1, 6-bisphosphatase in E.coli.

To establish a way to control or to decrease the daily increasing concentration of atmospheric CO(2), metabolically engineering Cyanobacteria was taken for the improvement of its efficiency of photosynthetic CO(2) fixation. As a preliminary stage of this study, three genes coding for three important Calvin cycle enzymes, i.e. triosephosphate isomerase (TPI), fructose-1, 6-bisphosphate aldolase(FBP aldolase),and fructose-1, 6-bisphosphatase(FBPase), respectively, have been cloned into one plasmid, pTrcFAT, which is controlled by promoter trc. Successful co-transcriptional expression of these three genes resulted inhigh yields of these enzymes under the induction of 0.25 mmol/L IPTG. Bioassay showed that the expressed enzymes from one liter of culture could directly catalyze DHAP conversion into 700 &mgr;mol of fructose-6-phosphate (F-6-P) per one minute. Furthermore, in order to introduce the three genes co-expression system into Cyanobacteria, a shuttle plasmid between E.coli and Cyanobacteria was constructed using plasmid pTrcFAT and a shuttle vector pDC-8, forming ashuttle plasmid pDCFAT-2 containing a dimer of the three genes co-expression operator. Successful co-expression in E.coli of pDCFAT-2 with higher full activity has been obtained. This shuttle was used to transform of Cyanobacteria Synechococcus sp. PCC 7942, and a few positive colonies were obtained.