Lmpact On Lipid Content Through Interfering MYB Or AP2 Gene And Methylation Preliminary Analysis Of Its Promoter In Chlamydomonas Reinhardtii

Microalgae is a kind of microorganism which has the advantages of fast growth, easy culture and higher oil content. The microalgae development will provide a valuable organisms material for new energy sources. Chlamydomonas reinhardtii is a kind of model organism that the known genome sequencing built on steady foundation for gene functional research. The genes associated with higher oil content in Chlamydomonas reinhardtii were screened by interfering the genome gene. Firstly, the high throughput transcriptome sequencing for Chlamydomonas reinhardtii cultured for 24 h under normal condition(TAP) and nitrogen starvation(TAP-N), respectively. The analysis showed that in low nitrogen level, the expression of 4493 genes were up-regulation and four of them, CrMYB7, CrMYB8, CrAP2-3 and CrAP2-4, were significant highly expressed.The DNA fragments of CrMYB7, CrMYB8, CrAP2-3 and CrAP2-4 were amplified by PCR. The interference expression vectors transformed into Chlamydomonas reinhardtii CC124 by using glass bead transformation method were constructed, namely CrMYB7-RNAi, CrMYB8-RNAi, CrAP2-4-RNAi and CrAP2-3-RNAi. The qRT-PCR demonstrated that the expression of interfered gene were significantly down-regulated. The Nile Red Staining was employed to indicate that the content of neutral lipid of CrMYB7-RNAi and CrAP2-4-RNAi strains cultured for 6h were reduced by 20%. The biomass of these two strains accumulated for days 1-7 were detected. The results showed that the transformed microalgae strains grew slower than the wild strain, but there were no difference at the logarithmic growth phase. Compared to the wild strain, the total lipids of CrMYB7-RNAi and CrAP2-4-RNAi strains were reduced by 12%-18%.In order to verify the relationship between the promoter methylation and higher expression of gene CrMYB7 and CrAP2-4, the promoter methylation site in Chlamydomonas reinhardtii CC124 under normal and nitrogen starvation conditions were detected by using bisulfite sequencing method. The results identified that a methylation site may exit inside 200 bp in the promoter regions of CrAP2-4 under the normal condition but not under nitrogen starvation. The demethylation may cause up-regulation expression of gene CrAP2-4 under nitrogen starvation.