| Nitrogen starvation is the most effective way of promoting lipid accumulation in microalgae,but the exact mechanism is not clear.Previous studies have shown that lipid content in Chlorella and Chlamydomonas reinhardtii cells was positively correlated with the expression of copper amine oxidase gene and the enzyme activity.We proposed that copper amine oxidase gene might play a key role in regulating carbon distribution and promoting lipids accumulation in microalgae.Chlamydomonas rheinistidis is the model organism of many biological processes,and therefor was used as the material to sudy the function of AMXs(copper amine oxidase genes)in the process of lipid accumulation.Firstly,Agrobacterium tumefaciens mediated genetic transformation system for C.reinhardtiii CC425 was established.Then the AMXs mutant strains by overexpression and CRISPR-Cas9 technology were contructed to analyze the effect of AMXs on the lipid synthesis of C.reinhardtiii CC425 under nitrogen-starvation conditions.The main results are as follows:1.The genetic transformation system of C.reinhardtii CC425 was established.First the optimal concentrations of cefotaxime sodium and hygromycin were screened.Then the effect of solid co-culture and liquid co-culture on the transformation efficiency of C.reinhardtiii CC425 mediated by A.tumefaciens LBA4404 was compared.The highest transformation efficiency was achieved by 5 days liquid medium co-culture of A.tumefaciens LBA4404 and C.reinhardtiii CC425 and obtained 46.67±1.67 transformants/10~6 algal cells from solid TAP medium containing hygromycin(15?g/m L)and cefotaxime sodium(500?g/m L).Finally,the optimal reaction conditions and amplification efficiency of two-step PCR after TE cleavage and one-step PCR without TE cleavage were examined and compared.The optimal reaction conditions were:amplification with high fidelity DNA polymerase Taq 1;the cell density involved in PCR was 5×10~3 cells/m L-5×10~6 cells/m L;Before amplification,cells were boiled in TE lysis buffer for 20min(two-step PCR method),or pre denaturation for 15 min(one-step direct PCR method).The amplification efficiency of two-step PCR is better than that of one-step PCR,but the latter is more concise.2.The full length CDS of AMXs for C.rheinistidis CC425 were obtained by PCR amplification.Bioinformatics analysis showed that the protein encoded by the AMXs not only contains two typical domain of copper-amine oxidase,a Cu?amine?oxid N2 domain and a Cu?amine?oxid domain,but also contained the active site"NYE/D"of copper-amine oxidase and three histidine residues related to the binding of copper ions.3.The overexpression vector of AMX1 was successfully constructed,and the plasmid vector was transformed into C.rheinistidis CC425 by Agrobacterium mediated method with liquid-medium co-culture 5 d.After resistance screening and PCR identification,Hpt positive transformants were obtained.When AMX1 overexpressed strains O16,O38 and wild-type strains were cultured for 3 days under nitrogen deficiency,the content of neutral lipid in strains O16 and O38 was significantly higher than wild-type strains(p<0.05),which were 2.04 and 2.18 times higher than wild-type strains,respectively.Copper amine oxidase activity and AMX1 gene expression level of strain O16 and O38 was significantly higher than wild-type strains(p<0.05),Copper amine oxidase activity were 3.60 and 2.57times higher than wild-type strains,respectively,meanwhile,AMX1 gene expression level were 3.37?2.10 times higher than wild-type strains,respectively.These results indicte that AMX1 may play a key role in the lipid accumulation of C.rheinistidis CC425 under nitrogen deficiency.4.The CRISPR-Cas 9 vector of AMXs were successfully constructed,and the plasmid vector was transformed into C.rheinistidis CC425 by Agrobacterium mediated method with liquid-medium co-culture 5 d.After resistance screening and PCR identification,Hpt positive transformants were obtained,these transformants were amplified by PCR and sent to biotechnology companies for high-throughput sequencing. |
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