Effect Of Transgenic Inhibition Of PEPC On The Oil Content

Due to environmental pollution, worsening shortage of oil supplies and other global problems, there is an urgent need for a green renewable resources to replace traditional fossil fuels. Biodiesel is attracting more and more attention because it is one of green resources, and can be fabricated from extensive source of raw materials. Microalgae is one of the raw material in the preparation of biodiesel with high light utilization efficiency, more biological capacity, lower culture cost, never take arable land, and many other advantage.Phaeodactylum tricornutum is one of unicellular marine diatom, which has 20% to 30% oil content of dry weight. Currently its whole genome sequencing has been completed, and the nuclear transformation system has also been established.Therefore Phaeodactylum tricornutum is a good model organism to study the fat metabolic pathways of microalgae.Phosphoenolpyruvate carboxylase(PEPC) is a key enzyme in plants to regulate lipid and protein synthesis pathways. Inhibition of PEPC enzyme activity will lead to an enhancement of lipid synthesis while Reduce the amount of protein synthesis, and vice versa. In the Cyanophytaa and Chlamydomonas reinhardtii, there are also reports of similar regulatory effect of PEPC enzyme. Phaeodactylum tricornutum has two kinds of PEPC, PEPC1 and PEPC2.This experiment in accordance with the sequences of pepc1 and pepc 2 gene in Phaeodactylum tricornutum published in the NCBI, and designed antisense expression vector of PEPC1 and PEPC2.Then transformed the recombinant plasmid to wildtype Phaeodactylum tricornutum with particle bombardment. GC-MS, Real-Time PCR were used to detect the conversion of growth rate, fatty acid composition and content, as well as the relative m RNA expression of pepc gene, to research inhibiting pepc 1(pepc 2) genes whether affect the oil content of Phaeodactylum tricornutum. Specific results are as follows:1. We cloned and constructed the sequence before gene promoter(P1-1), the 5 ’end of the extension sequence(P1-2), the 3’ end of the complementary sequence(P1-3), the intermediate coding sequence( P1-4) of PEPC1, and the sequences before the gene promoter(P2-1), the 5 ’end of the extension sequence(P2-2), the sequences across intron(P2-3), the 3’ end of the complementary sequence(P2-4), the coding sequence(P2-5) of PEPC2,to build antisenseexpression vector respectively(P1-1/Bar, etc. 9). After screen we obtained P1-2/Bar, P1-3 / Bar, P1-4/Bar, P2-2/Bar, P2-3/Bar, P2-5/Bar total 30 algal strains.2. The growth curve and specific growth rate analysis showed that the conversion of growth rate in different algae strains has significant difference. In pepc1 gene transformed strains the lowest growth rate strain belong to P1-2/Bar, the highest one belong to P1-4/Bar, specific growth rate were 77.43% and 122.77% of wildtype respectively; while transformed strains of pepc2 gene, the lowest and the highest growth rate of algae strains both belong to P2-5/Bar, the specific growth rate 114.70% and 75.03% respectively of the wildtype.3. Real-Time PCR results showed that compared with the wildtype, each transformed strains have different degrees of relative m RNA expression reduced. pepc 1 gene transformed strains were reduced in a range of 1.36% to 48.58%, among the lowest relative m RNA expression strain belong to P1-3/Bar transformed strains, the relative m RNA expression levels decreased 48.58%; pepc 2 gene transformed strains were reduced in a range of 12.80% to 57.93%, of which the lowest relative m RNA expression strain belong P2-2/Bar transformed strains, the relative m RNA expression levels decreased 57.93%.4. Fatty acid analysis showed that, compared with the wildtype, most of the transformed strains has a total oil content increased. Total oil content of pepc 1 gene transformed strains has a increase in the range of 2.67% to 74.98%, the highest enhance of total oil content belong to P1-3/Bar transformed strains, the total oil content has a increase of 74.98%; total lipid of pepc 2 gene transformation group has a increase in the range of 5.51% to 108.34%, the highest oil content strain belong to P2-2/Bar transformed strains, total oil content has a increase of 108.34%. Fatty acids focus on C14: 0, C16: 0, C16: 1, C16: 2, and C20: 5, C16: 1 has a highest concentrations. The relative content of saturated fatty acids was 35.80% to 24.60%, and the relative content of unsaturated fatty acids was 64.2% to 75.40%.The relative content of C16: 0 to C18: 0 was 62.84% to 83.12%. Of transgenic alga biodiesel quality indicators do not meet all criteria simultaneously.In this study, we built antisense expression vector different sequences of pepc and pepc 2 gene respectively to inhibit the expression of PEPC1(PEPC2) in Phaeodactylum tricornutum, and observed the oil content of some transformed strains has been significantly improved. The results will provide a theoretical basis for the development of microalgae biodiesel.