The Impact Of Starch And Glycolipids Biosynthesis-related Genes On Lipid Synthesis In Chlamydomonas Reinhardtii

Micro algae can use photosynthesis convert light energy into chemical energy,and the storage of micro algae organisms are mainly in the form of starch and lipid,store lipid mainly as triglycerides(TAG),and exists in the form of oil droplets in the cytoplasm.The starch in the form of amyloid exist in plasmid.Current awareness on fat metabolism and its regulation in microalgae cells is also very simple,there is an urgent need to enhance lipid metabolism and formation mechanism of oil droplets in the studies and explore in the metabolic pathway of how these types of energy storage materials are transformed,and provide theoretical basis for the breeding of high-yielding oil from microalgae.Chlamydomonas reinhardtii is a eukaryotic unicellular green algae and its genetic background is clear.Chlamydomonas reinhardtii as a model organism,explore the gene affecting the accumulation of lipids in Chlamydomonas,whose results can be achieved for the genetically engineered microalgae improving fat content provides a theoretical basis.In this study,three genes STA6,SSS2 and AMYB1 of Starch metabolic pathway of C.reinhardtii and the SQD1?SQD2?MGDG synthase gene were chosen as the targets of RNA interference.The amiRNA expression vectors pmiR1162-STA6?pmiR1162-SSS2?pmiR1162-AMYB1 and pmiR1162-SQD1?pmiR1162-SQD2?pmiR1162-MGDG were constructed,based on one-step PCR process and mating-assisted genetically integrated cloning(MAGIC)method.Then,C.reinhardtii cells were transformed with the six amiRNA vectors.Higher oilseed crops,DAG is the direct precursor synthesis TAG,DAG is also synthesized glycolipids important precursor molecule,if we can prevent the DAG turned into glycolipids,synthesis may contribute to TAG.So we choose the way to interfere with the synthesis of glycolipids from DAG,observe its impact on TAG synthesis.Suffoquinovosyl diacylglycerol(SQDG)is a sulfur-containing glycolipids.The biosynthesis of UDP-sulfoquinovose(UPD-SQ)from UDP-Glc and sulfite,is catalyzed by SQD1.Then the transfer of sulfoquinovose from UDP-sulfoquinovose to diacylglycerol to synthesise Suffoquinovosyl diacylglycerol(SQDG),which is carried out by SQD2.Monogalactosyldiacylglycerol(MGDG)by UDP-galactose and DAG through MGDG synthase catalytic synthesis.The results of quantitative RT-PCR suggested that the six genes transcript level were reduced in the transgenic lines.And The results of quantitative RT-PCR suggested that SSS2 transcript level was reduced by 29%in the transgenic lines;The results of quantitative RT-PCR suggested that STA6 transcript level was reduced by 45%in the transgenic lines;The results of quantitative RT-PCR suggested that AMYBI transcript level was reduced by 41%in the transgenic lines;The results of quantitative RT-PCR suggested that SQD1 transcript level was reduced by 61%in the transgenic lines;The results of quantitative RT-PCR suggested that SQD2 transcript level was reduced by 48%in the transgenic lines;The results of quantitative RT-PCR suggested that SSS2 transcript level was reduced by 63.8%in the transgenic lines;The results of quantitative RT-PCR suggested that MGDG transcript level was reduced by 39%in the transgenic lines.The starch content and lipid content of these mutants were analyzed.The starch content were increased in the transgenic line expressing amiRNA-SQD1?amiRNA-SQD2 and amiRNA-MGDG,but reduced in amiRNA-AMYB1?amiRNA-SSS2 and amiRNA-STA6.The lipid content of all mutant algae strains-were increased.After cultivate 48h,the lipid content of C.reinharddtii(pmiR1162-SSS2)increased to 126%;the lipid content of C.reinharddtii(pmiR 1162-AMYB1)Increased to 93.7%;the lipid content of C.reinharddtii(pmiR1162-STA6)increased to 89.9%;the lipid content of C.reinharddtii(pmiR1162-SQD1)increased to 101%;the lipid content of C reinharddtii(pmiR 1162-SQD1)increased to 152%;the lipid content of C.reinharddtii(pmiRl1 62-MGDG)increased to 167%.