1. The experiment research of sub-cellular localization of glycolysis in
C.reinhardtii. By using the methods of bioinformatics, glycolytic-associated enzymes
were identified and sub-cellular targeting predicted. The results indicated that the
forward seven steps take place in the chloroplast, while the last three steps of the
pathway occur in cytosol. Therefore, we used the GFP-fusions to further verify the
sub-cellular localization of the representive glycolytic enzymes (PFKa, PFKb, TIM
and NAD+-GAPDH) to provide some experiment evidence for the above conclusion.
Our results suggested that enzymes participated in the upper part of the glycolysis
(step3, 5 and 6) were indeed localized in chloroplast. So our results partly verified the
early theoretic analyse.
2. Research of the coordinate mechnism between glycolysis and Calvin cycle in
C.reinhardtii. Considering that glycolysis and Calvin-cycle occur in the same
compartment and overlap with each other for five steps in the reverse direction. How
these two chains are regulated is one significant question still remaining. In order to
nivesigate this problem, we did the followed two aspects of works. Firstly, we
analyzed and predicted the enzymes participated in the five reverse overlapping
reactions using the methods of bioinformatics based on C.reinhardtii genomic
database. Results indicated that Step 1 is catalyzed by two copies of PFK in glycolysis,
while reversible step is catalyzed by FBP in Calvin cycle. Meanwhile, step 4 is
catalyzed by a photosynthetic NADP+-GAPDH for Calvin cycle and a NAD+-GAPDH
for glycolysis. There are two FBA isoforms (FBAa and FBAb) in chloroplast
catalyzed the step 4. As for step 5 and 7, both of them have only one chloroplastic
gene coding single enzyme that catalyzes the reversible reactions, respectively.
Secondly, real time RT-PCR was performed to get the expression profile of the
chloroplast enzymes participating in five reverse overlapping steps. The transcript
levels of PFK, FBAb and NAD+-GAPDH transcribe stably with little fluctuation in
the light/dark cycle, but the transcriptional level of FBAb is very low compare to PFK
and NAD+-GAPDH; FBP, FBAa and NADP+-GAPDH in light are significantly high
compared to in dark condition and reach the maxmain after 1 hour, implying that the
expression of these genes is regulated by light; the expression profiles of these two
enzymes TIM and PGK are similar to FBPase, NADP+-GAPDH and FBAa, which
only have some different in expression amount. So we conclude that PFK, FBAb and
NAD+-GAPDH are special catalyze glycolysis and FBP, FBAa and NADP+-GAPDH
are special catalyze Calvin cycle, they coordinate with each other by the regulation of
light. PGK and TIM was in charge of the two different pathways，which was possibly
regulated by the concentration of the substrate and the need of energy to regulate the
direction of the reaction to coordinate the two reactions.
This researches not only verifed the sub-cellular localization of glycolysis using
experiment in C.reinhardtii, but also revealed the coordinate mechanism between
glycolysis and Calvin cycle in C.reinhardtii chloroplast for the first time.
修改评论