CDNA Cloning And Lipid Metabolism Regulation Research On Chlamydomonas Reinhardtii Acyl-coA Synthetase

Microalgae biodiesel becomes the research focus for such advantages as environmental protection, recyclables and the short growth cycle of raw materials. But the low lipid content in natural microalgae and the high cost of lipid extraction have seriously affected industrialization process of the microalgae biodiesel, So there is great significance to improve the lipid content and the secretion of fatty acid of microalgae with the regulation of the relevant genes about the synthesis and metabolism of lipid by genetic engineering.Acyl-coenzyme A synthetase(ACS) is widely involved in fatty acid metabolic reactions, and even participate in the secretion of fatty acids. This study predicted two Chlamydomonas reinhardtii cr ACS genes from NCBI, and cloned their c DNA by RT-PCR, analyzed their encoding protein conserved domain and phylogenetic tree.The function of cr ACSs in C.reinhardtii and YB525 were also analyzed. At last, the study regulated those two ACS genes expression by antisense RNA technology in C. reinhardtii, and further explored the two cr ACSs function in vivo, ultimately confirmed the two cr ACSs involved in Fatty acid metabolism and promoted fatty acid secretion. Specific results are shown as follows:The work predicted two C. reinhardtii ACS genes, and cloned their c DNA for the first time, named cracs1 with the length of 2 004 bp and cracs2 with the length of 2016 bp.cr ACS1 and cr ACS2 contain two conserved protein-coding regions: AMP-binding region and FACS signature, and the phylogenetic tree analysis indicated that cr ACSs share high homology with Arabidopsis thaliana LACs, indicating that cr ACSs belong to long chain ACS.In vitro function exploring results showed that the cr ACS1 and cr ACS2 can activate exogenous fatty acid, the expression of cr ACS1 and cr ACS2 in yeast showed the two cr ACSs recovered the function of activation of fatty acids in Saccharomyces cerevisiae YB525(a ACS deficient yeast strain).cr ACS1 and cr ACS2 preferred to activate long-chain fatty acids, the cr ACS1 only preferred to activate long-chain fatty acid C18:1 and C16:1 while the cr ACS2 preferred to activate long-chain fatty acid C16:0,C18:0,C16:1 and C18:1. This indicatedcr ACS1 and cr ACS2 have some activation of ACS and belong to acyl-coenzyme A synthetase family.The work also constructed a antisense cracs1 transgenic strains(q-15) and a antisense cracs2 transgenic strains(p-13).The real time PCR detection of ACS m RNA expression in the antisense ACS transgenic strains showed that: the m RNA relative expression levels of target gene in each transgenic algae has reduced, the antisense cracs1 transgenic strains declined53.7%, and cracs2 declined 23.67%. There is a certain correlation between cracs1 and cracs2.Antisense cracs2 fragments could promote cracs1 expression, indicating that there may be functional compensation mechanisms between cracs2 and cracs1; And the cracs2 antisense fragment could lead to a decline cracs1 expression level in p-13.The GC-MS detection of fatty acid content within or outside of C. reinhardtii transgenic strains showed that: Fatty acid content in the transgenic cells increased significantly and achieved the extracellular fatty acid secretion phenomenon. The fatty acid content in q-15 and p-13 cells increased 45% and 55%respectively, while the extracellular secretion of the fatty acid Phenomenon was also detected,the excellar fatty acid content was 13.413mg/109cells(antisense cracs1), 16.89 mg / 109cells(antisense cracs2) respectively.All of the results show that the two cloned C. reinhardtii ACSs have certain relations with fatty acid synthesis and metabolism, antisense cracs1 and cracs2 regulation not only increased the fatty acid content in C. reinhardtii cells, but also could promote the secretion of lipid. The function exploring of C. reinhardtii ACS will lay a foundation for the study of microalgae ACS, but also provides a basis for lipid synthesis and metabolism of C. reinhardtii.The study of cr ACSs not only provides a reference for the regulation of microalgae fatty acid secretion, but also makes a contribution to promoting the development of microalgae biodiesel.