Up-and Downregulation Of Pepc2 Gene Expression In Two Transformed Mutants Of Chlamydomonas Reinhardtii

Phosphoenolpyruvate carboxylase(PEPC)is able to fix CO2 in higher plants,but the understanding of PEPC function in eukaryotic algae has been little,especially of “bacteria” type PEPC(PEPC2).In order to explore the function of PEPC2 and construct high lipid engineering mutant,in this study,the expression of PEPC2 was inhibited,to enhance the lipid content.Chlamydomonas reinhardtii pepc2(Crpepc2)has five conserved sequences(CSs),and two active sites(H and K),a 639 bp fragment of initiative CS? was cloned,which also include an active site H.Two nucleus expression vectors: pSP124s-HR-reve-Crpepc2 and pSP124s-HRforw-Crpepc2,and two chloroplast expression vectors: pASapl-forwCrpepc2 and p ASapI-reve-Crpepc2 were constructed for C.reinhardtii,then,we try to explore the response to C,N metabolism in C.reinhardtii after regulating PEPC2 expression through nucleus and chloroplast transformations.In the first chapter,after nucleus transformation physiology and biochemistry of C.reinhardtii mutants were searched.The data showed that: when Crpepc2 was downregulated,higher photosynthetic efficiency and lower dark respiration characterized the reverse mutant,mayde it relate to the reduced Ik and increased Ic.On the contrary,the upregulation of Crpepc2 generated higher dark respiration and lower photosynthetic efficiency,which may refer to the elevated Ik and declined Ic.Also,PEPC activity was related to the accumulation of protein and lipid.Higher PEPCase activity and enhanced protein biosynthesis characterized the forward mutant;furthermore,the inhibition of PEPCase prompted lipid content elevating in reverse mutant.In addition,suppression of Crpepc2 gene enhanced the mutants' tolerance to low temperature and pH,and the unsaturated fatty acids level is increased in the reverse mutant.Based on these data,a hypothesis has been presented as follows: PEPC may be a ligase,which balances carbon and nitrogen metabolisms.Downregulation of PEPCase enhances photosynthetic and relieves the competition of PEP,which accelerates lipid biosynthesis.Moreover,Upregulation of PEPCase elevates respiration and alters the photosynthate partitioning,resulting in the accumulation of protein.Therefore,PEPC is able to affect not only photosynthesis and carbon metabolism,but also Ncompounds and energy metabolism in C.reinhardtii.In the second and third chapter,the 639 bp fragment was transformed C.reinhardtii(cc-400)chloroplast,and searched their physiology and biochemistry.Although chloroplast transformation started later than nucleus,it has high transformation and expression efficiency,and the directional transformation in chloroplast had been resolved,so C.reinhardtii chloroplast transformation method may improve the production of metabolites.First,Crpepc2 expression was measured with RT-qPCR,and until now,Crpepc2 gene expression in chloroplast had not been reported.Second,it has been observed that PEPC can regulate the allocation of photosynthate,so overexpressed PEPC promoted protein synthesis and suppression of PEPC promoted lipids synthesis.It is interested that the regulation of PEPC had little influence on mutants' growth rate.In conclusion,the data of physiologiy and biochemistry following chloroplast transformation was similar to following nucleus transformation.C.reinhardtii "bacteria" type PEPC seems to be regulate protein and lipid accumulation,and did not affect their growth rate.The improvement of protein content was useful for exogenous protein elvation,and increased lipid content was useful for constructing high oil engineering algal.Comparing with the nucleus transformation following features of chloroplast transformation: the lipid content of reverse chloroplast mutant increased 70.32%,and that of reverse nucleus mutant increased 39.24%.Compared RT-qPCR and Western blot results,we found that chloroplast transformation method had greater effect of PEPC2 inhibition,so the lipid content enhanced more,so did fatty acid,especially unsaturated fatty acid.In the aspect of photosynthetic parameters: the light saturation point of reverse nucleus mutant was increased 19.06%,and that of reverse chloroplast mutant was enhanced 29.49%;the light compensation point of reverse nucleus mutant was decreased 26.09%,and reverse chloroplast mutant was reduced 51.31%.The respiration of forward nucleus mutant was increased 19.77%,and that of forward chloroplast mutant was enhanced 32.44%;also,reverse nucleus mutant was decreased 21.22%,and reverse chloroplast mutant was reduced 28.21%.The change of protein content of forward nucleus mutant was up-regulated 26.99%,and that of forward chloroplast mutant was increased 37.03%.Above data showed that: whether nucleus mutants or chloroplast mutants,their physiological and biochemical changes showed similar trend,while chloroplast transformation showed greater variation tendency,protein and lipid content all change more.Both two transformation methods demonstrated that PEPC2 affect the photosynthesis,meanwhile our hypothesis was also support by with chloroplast transformation.Fossil fuel as non-renewable energy,will be exhausted in decades,biological energy,especially microalgae biodiesel has the advantages of clean,rich sources and wide application,and it has become one of the most promising alternative choice.Unfortunately both screening high lipid content algae strains and improving lipid content at physiological,biochemical levels,are difficult to get a balance between the growth rate and lipid content,and it is difficult to apply in the production,and reduce the production costs.The past 40 years,many efforts have been done,however the industrialization has still been difficult to realize.Therefore genetic engineering on improving lipid production has been paid more attention.Our reverse mutant may make contribution to high oil content engineering algae.