Construction Of Micro Algae Integrate Exprssion Vector And Bioinformatics Analysis Of ACCase

As a renewable energy sources to replace conventional fossil fuels, biodiesel fuels have been becoming increasingly requirements to global fuels market. Manipulation of high-lipid production in microalgae via genetic engineering has emerged as one of attractive fields in producing microalgae-based biodiesel. Cyanobacteria are remarkable group of simple photosynthetic micro-organisms which belong to prokaryote. They grow rapidly with production of high-lipids content. Cyanobacteria are characterized by the features of the better adaptability and good manipulation of genetic engineering which have an extraordinary potential for cultivation as raw material of microalgae-based biodiesel. Acetyl-CoA carboxylase (ACCase) is known to play an important role in fatty acid synthesis, because the enzyme catalyses the irreversible carboxylation of acetyl-coenzyme A (acetyl-CoA) to produce malonyl-CoA in most organisms. ACCase catalyzes the first step reaction for de novo fatty acid biosynthesis by which it is known as the rate-limiting reaction.This study focuses on the construction of integrate expression vector and microalgae ACCase bioinformatics.(1) The cyanobacterial pAnFP integrate expression vector is constructed successfully and the gene accD which encodes CT-β subunit of ACCase in Anabaena sp. PCC7120has been cloned in the studies. The cyanobacterial Integrative expression vector pAnFP comprises of fragment F (including two flanking regions F1and F2), promoter PpsbA1, and neomycin phosphotransferase gene nptⅡ with its own promoter. The integrative fragments of F1and F2, and promoter psbA1are obtained from the genomic DNA of Anabaena. sp. PCC7120by PCR amplification. The gene nptⅡ is cloned from the pET30vector by PCR amplification. The gene accD is characterized as the CT-β domain gene of ACCase in Anabaena sp. PCC7120by DNA sequencing after cloning into pBS-T vector.(2) Some physiological and biochemical characters of ACCase subunits in various cyanobacteria are analyzed by bioinformatics methods to predicate their signal peptides, trans-membrane regions, hydrophobicity/hydrophilicity, secondary and tertiary structures. The phylogenetic trees of ACCase subunits from various organism species are also constructed. Both of BC and BCCP subunit are unstable proteins and they do not have significant coil structure; Instead, CT-a subunit and CT-β subunit are stable proteins and have significant coil structures in cyanobacterial ACCase. Signal peptides and trans-membrane regions have not been found in the above four subunits. However, these subunits have their own conservative domains, especially BC subunit are up to seven domains. The secondary structure are mainly a-helix and random coil-based, scattering with a small amount of extension chain in these subunits. It is fact that there exist differences of the phylogenetic relationships in the four subunits of ACCase in various organism species. However, the results of phylogenetic analysis coincided with the law of biological evolution; namely, the species in relationship were near with each other in evolution. Phylogenetic analysis showed that the evolutionary distance of the four ACCase subunits is the closest among cyanobacteria, indicating that there is a stable evolutionary pathway in cyanobacterial species which probably evolved from a common ancestor. The evolutionary relationship of the four ACCase subunits is closer among cyanobacteria, eukaryotic alga and oil plants. It suggested that these species were derived form a close evolutionary relationship of the ancestor species. The evolutionary distances of the four ACCase subunits is the farthest among microalgae, yeast and higher animals which indicates the fact that microalgae begin to evolve while they separated early from other species in the evolution.