Study The Relationshilp Between Polysaccharide Synthesis And The Three Acyl Glycerolin Synthesis In Chlamydomonas Reinhardtii Using AmicroRNA Series Technology

Chlamydomonas reinhardtii is a kind of unicellular eukaryotic green algae.Its genetic transformation of nucleus,chloroplast and mitochondrion had been implemented successfully.C.reinhardtii is a great model organism for understanding the regulatory mechanism of lipid metabolism of microalgae in depth.Starch and lipid are two main forms of stored energy in C.reinhardtii.When environmental conditions are unsuitable for growth;C.reinhardtii will accumulate starch and lipid.The interaction between the two pathways has been reported,although the regulation of carbon partitioning into starch and lipid synthesis pathways is not well understood.It is hypothesized that restraining carbon precursors into the starch synthesis pathway may result in overproduction of fatty acids and TAG.In this study,three genes STA6,SSS2andAMYB1 of Starch metabolic pathway of C.reinhardtii were chosen as the targets of RNA interference,aiming at understanding the relationship between the RNA interference of these genes and lipid of C.Reinhardtii.At the same time,dose effect and synergistic effect can be explored.The amiRNA expression vectors amiRNA-AMYB1,amiRNA-SSS2,amiRNA-STA6,amiRNA-SSS2-STA6,amiRNA-6STA6 were constructed,based on oligonucleotide annealing method,overlapping PCR process and the character of isocaudarner.Then,C.reinhardtii cells were transformed with the four amiRNA vectors by electrotransformation.The result of starch assay suggested that starch content was reduced by 44.1%in the transgenic line expressing amiRNA-SSS2,30.7%in the transgenic line expressing amiRNA-STA6,51.2%in the transgenic line expressing amiRNA-SSS2-STA6,and 63.8%in the transgenic line expressing amiRNA-6-STA6.The results of thin layer chromatography suggested that the neutral lipid content rised in the amiRNA-AMYB1 transformant and amiRNA-SSS2 transformant.