Chloroplast Target Exogenous Protein Expression In Chlamydomonas Reinhardtii

It was found that Chlamydomonas reinhardtii could accumulate energy storage compounds and some proteins in chloroplast happened to be degraded or modified under nitrogen stress in the previous studies.The changes of the proteins thus would affect the overall carbon flow distribution,and contribute to the accumulation of energy storage compounds.Based on the existing information,this study focused on the key enzyme of photosynthetic carbon fixation,glyceraldehyde 3-phosphate dehydrogenase?GAP3?,and the key enzyme of lipid metabolism phospholipid:diacylglycerol acyltransferase?PDAT?.Therefore,the aim of this work was to build the expression system of the two proteins and investigate the physiological and biochemical changes of the transformed strains using the Hsp70A-RbcS2 promoter.Two recombinant expression vectors,GAP3 and ScPDAT with chloroplast transport peptide?cTP?were constructed in vitro and expressed in C.reinhardtii cc-137,and realized the overexpression of GAP3 and PDAT,and the transformed strains were named pChlamy₁-GAP3?GAP3??pChlamy₃-GAP3?Scpdat respectively.Besides determining the expression of GAP3 and PDAT protein,the growth,chlorophyll fluorescence,fatty acids,carbohydrates total proteins and other physiological and biochemical indexes were investigated.The results are as followed:?1?In shake flask culture with TAP medium of GAP3,pChlamy₃-GAP3 and wild type,no differences were found between the growth and the chlorophyll fluorescence of the two transformants and the wild type.But,compared to the wild type,the contents of carbohydrates and total protein of the was two transformants were significantly increased,and the maximum rates of carbon fixation were 84.2 mg L^-1 day^-1 and 91.8 mg L^-1 day^-1 of GAP3 and pChlamy₃-GAP3.?2?In shake flask culture with high salt medium,no significant difference in the growth rate of GAP3 was found compared to the wild type.And the overall level of chlorophyll fluorescence of the transformed GAP3 was lower than that of wild type.When cultured in the photobioreactors with TAP medium,the growth rate of GAP3 was basically consistent with the wild type.But the total protein content was significantly higher than that of wild type.?3?Scpdat and cc-137 were cultured using TAP medium in the shake flasks.Comparing the growth curves,chlorophyll fluorescences and western blot verifications of the two strains,it was found that the expression of Scpdat protein was associated with its growth phase.?4?The accumulation of total fatty acids and TAG in Scpdat increased by 22.51%and31.15%respectively,among which C16 series fatty acids were the most influential.A new method for the study of the expression of chloroplast localization proteins in C.reinhardtii was established,which provides a valuable reference for the biological research on enhancing the accumulation of the storage compounds in C.reinhardtii.