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Construction a Recombined Vector That Expressed the accA Gene of α-CT Subunit of Acetyl-CoA Carboxylase and Its Over-expression in Dunaliella Bardawil

Author: RenNi
Tutor: LiuRenHai；LiuGuangFa
School: Xiamen University
Course: Hydrobiology
Keywords: Dunaliella bardawil accA lipid contents
CLC: Q943.2
Type: Master's thesis
Year: 2009
Downloads: 178
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Abstract


It is effective to relieve the pressure from conventional energy shortage and environment and establish the sustainable energy system by exploring and using biomass.The exploitation and utilization of biomass energy are effective stratagems,which not only relieve the pressure from conventional energy shortage and environment,but also establish the sustainable energy system.Among numerous organisms,microalgae are considered as excellent candidates for biomass energy producing,because of their larger biomass,faster growth rate and higher percentage of lipid components.In this paper,Dunaliella bardawil,a halophilous algea,was used as a transgentic bioenergy producer.The accA gene which encodedα-CT subunit of acetyl coenzyme A carboxylase(ACCase,a key enzyme of lipids synthesis) was transformed into its chromosome.It is expected that a large number of mutated accA protein was expressed,and the capability of producing lipids was improved obviously.In this study,the accA gene of ACCase enzyme of the E.coli was cloned, then inverted into a prokaryotic expression plasmid pGEX-6P-1-accA,and then the recombined plasmid expressed in E.coli strain.Results showed that the expressed mutated accA protein accounted for 29.4%of the bacteria’s total protein,and most of them existed as insoluble protein through SDS-PAGE analysis.The shuttle vector p-accA-CAT with a reporter gene CAT and a resistance gene of chloramphenicol,not only clones largely in prokaryotes cells but also could over-express in eukaryotic ones.The recombined shuttle vector was transformed into D.bardawil through ultrasound,electroporation and gene gun methods.Results of PCR and DNA sequence analysis showed that the target gene has been genetically integrated into the host cell.The accA protein of transgenic D.bardawil was detected through SDS-PAGE and Western Blot methods.The result of protein scan showed that the target protein was 15.8%of the total dissoluble protein.Comparison with the growth rates of wild-type algaes and transformed ones under the pressure of chloramphenicol through gene gun method of transformating,result indicated that the integration of transgenic gene not obviously impacted the normal growth of transformed strain,although transgenic accA gene could inherit and express stably in D.bardawil.The transgenic accA could inherit and express stably in D.bardawil,but the lipids percentage of transformed D.bardawil determined by Soxhlet procedure did not increased markedly,comparing with wild-type strain. However,the results of this study have certain theoretical significance and practical value.It has broken a new path for high-fat algae species cultivating,and provided certain bases for further research.

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