| The coccolithophore is a globally important unicellular marine phytoplankton. Emiliania huxleyi is the most prominent coccolithophore and has attracted the attention of scientists from fields as diverse as geology, biogeography, paleoclimatology, ecophysiology, material science,and medicine. E. huxleyi is much of interest to those in biotechnology due to its capable of production abundant bioactive metabolites. A group of secondary metabolites known as polyketides that E. huxleyi synthesizes possess a wealth of pharmacologically important activities, including antimicrobial, antifungal, antiparasitic, antitumor and agrochemical properties. It was attractive that specific virus infection contributes to the accumulation of ceramide in E. huxleyi hosts’ cells. This biosynthetic pathway of ceramide appears to function during lytic infecton, which resulted in the apoptosis of host cell. Ceramide, an intracellular signal molecule, plays an important regulatory role in the cell differentiation, proliferation,apoptosis, aging and other activities. As a new biologically active substance, The unique physiological function and drug effect of ceramide makes it become a kind of high added value and has huge market potential of active material with a wide range of applications in the chemical engineering, pharmaceutical health care products, food and cosmetic. There is very small amount of ceramide in cell, generally from 0.01% to 0.2% biomass dry weight. Although the content of ceramide in E. huxleyi reached only 0.091% of cell dry weight after being infected by E. huxleyi specific virus, the unique structural features make its activity much higher than that from any other biological sources. Transgenesis in microalgae is a complex and fast-growing technology, as well a powerful tool for the manipulation of these organisms. Therefore, it is of great importance to improve the key link of sphingolipid biosynthesis pathway of E. huxleyi by microalgae genetic engineering. To this day, there is no report on the genetic transformation system construction and its application of E. huxleyi.In this work, we perform the experments using the strain of E. huxleyi EhBOF92 and its specific virus EhV99B1 isolation. A genetic transformation system for the transformation of coccolithophore E. huxleyi was designed and developed. On this basis, the gene coding serine palmitoyl transferase SPT-LCB2 subunit from EhV, was subcloned into the expression vector and transformed into E. huxleyi to investigate the effect of EhV99B1-LCB2 on synthesis of ceramide in cell. SPT is the first rate limiting enzyme in de novo sphingolipid synthesis and LCB2 is the catalytic subunit in the N-terminal domain of SPT. The main results were shown as follows:(1) Screening of antibiotic resistance gene and cloning: It focused on the sensitivities characteristics of E. huxleyi to seven kinds of antibiotics and one kind of herbicide. For antibiotic verification, growth experiments were conducted to understand the growth behaviour of E.huxleyi in unselective and selective media. The result showed that E. huxleyi was insensitive to amplicillin, kanamycin and streptomycin; and medium sensitive to chloromycetin and novobiocin; and hypersensitive to G418, puromycin and Glufosinate-ammonium. Growth of cultures was inhibited immediately in the presence of G418, puromycin and glufosinate-ammonium respectively, as opposed to control cultures. G418 was chosen as the most suitable selective antibiotics and the corresponding resistance gene “neo” as the selectable marker for E. huxleyi genetic system. The resistance gene neo was amplified from a commercially available plasmid pSELECT and verified by sequencing.(2) Promoter and reporter gene search. The gene coding green fluorescent protein-gfp,amplified from a plasmid pGFP and verified by sequencing and was employed as reporter gene.Based on the systematic functional analysis by bioinformatic method, the promoter of fucoxanthin chlorophyll a/c-binding protein-fcp was prefered to use from E. huxleyi genome database. fcp promoter gene fragment was amplified from E. huxleyi genome by PCR and its sequence was analyzed through tools from PLACE(http://www.dna.affrc.go.jp/PLACE/) and Plant CARE(http://bioniformatics.psb.ugent.be/webtools/plantcare/html/) database. It showed that the fcp promoter gene contains various cis-acting elements with potentially strong promoter activity.(3) Expression vector construction and its transformation. gfp gene, fcp promoter gene and neo gene were inserted into the basic vector pUC18, and recombinant expression vectors pUC18-gfp-fcp and pUC18-fcp-neo were constructed, respectively. Electroporation method was used to co-transform binary recombinant vectors pUC18-gfp-fcp and pUC18-fcp-neo into E.huxleyi cells. The optimal parameters for electroporation were as followings: the cells were counted and harvested in exponential phase; concentration of plasmid DNA was 10 μg/mL;electric buffer at pH 7.2 containing 0.080 mol/L KCl, 0.005 mol/LCaCl2, 0.2 mol/Lmannitol, 0.2mol/Lsorbitol and 0.01 mol/L Hepes; the electric field strength was 2000V/cm and the electric shock time was 2ms per time for two times. Fluorescence microscopy and real-time fluorescent quantitative PCR inspection showed that the algae cell was transformed successfully with ratio of 0.07%. fcp promoter could activate gfp gene and neo gene to gain a high performance transcription and expressed in transformed E. huxleyi cells.(4) The influence of EhV99B1-SPT on the synthesis of ceramide. EhV99B1-LCB2 gene was amplified by PCR with specific primers and subcloned into the eukaryotic expression vector pUC18-fcp-neo to get the recombinant plasmid pUC18-fcp-neo-LCB2. A positive plasmid wastransformed into E. huxleyi cell under the optimal electroporation condition. PCR analysis indicated that the target gene was successfully introduced into E. huxleyi. A high-performance liquid chromatograph(HPLC) method was used to measure the content of ceramide both in wild type and in modified E. huxleyi cells. The concentration of ceramide in modified E. huxleyi was45.25 fg/cell, which was about 1.2 times higher than that in wild type E. huxleyi. The results demonstrated that EhV99B1-LCB2 can promote the synthesis of ceramide in host cell.A stable transformation of the microalga E. huxleyi would allow the further generation of gain-of-function mutants e.g. virus genes contributing to ceramide systhesis or knock-out mutants e.g. genes that catalyze the hydrolysis of ceramide(such as ceramidase). This work may lay a primary foundation for the realization of ceramide industrilization in the future. |
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