| With the improvement of living standards,people's demand for lipids is also increasing.Lipids produced by animals and plants can only meet people's daily needs,and people's demand for unsaturated fatty acids still unable to meet.In addition,the rapid consumption of fossil fuels brings ecological damage,resource shortage and other problems.While the current new bioenergy,such as ethanol,can partially alleviate people's demand,but it consumes a lot of cultivated land resources and water resources.Therefore,the development of new lipid production resources is particularly important for the sustainable development of society.Synechocystis PCC 6803 is a kind of unicellular cyanobacteria with a natural transformation system.The whole genome has been sequenced.It has the characteristics of fast growth speed,short growth cycle,simple culture conditions,no occupation of arable land and high level of lipid accumulation.And it has become a model cyanobacteria of photosynthesis molecular biology research.In this paper,a potential key node in the fatty acid metabolism pathway of Synechocystis sp.PCC 6803 was studied.Acetyl-Co A is catalyzed by acetyl-Co A carboxylase to generate malonyl-Co A,and malonyl-Co A is loaded onto the acyl carrier protein under the action of malonyl transferase to finally generate malonyl-ACP as donor of carbon chain during fatty acid extension.Two key enzymes are involved in this step: acyl carrier protein(ACP)and malonyl-Co A : ACP transferase(MCAT).Through Gen Bank,we found the gene encoding ACP was ssl2084,and the gene encoding MCAT was slr2023.In this study,ssl2084 and slr2023 were cloned and overexpressed in Synechocystis PCC 6803,laying a foundation for further research on the fatty acid metabolism pathway of Synechocystis PCC 6803.The main research contents and results are as follows:(1)The ssl2084 gene fragment cloned from the total DNA of Synechocystis PCC 6803 was sequenced and compared for bioinformatics analysis.The results showed that the ssl2084 sequence had a 234 bp open reading frame and encoded 77 amino acids.The molecular weight of acyl carrier protein is about 8.6 k Da,and the isoelectric point is 3.94.It is a hydrophilic protein without signal peptide and transmembrane domain.It has four potential phosphorylation sites.The largest component of the protein structure is ?-helix,which has the binding site of phosphopantylmercaptoethylamine.This proves that it is a stable protein.(2)To verify the effect of overexpressing ssl2084 gene on fatty acid biosynthesis in Synechocystis PCC 6803,the ssl2084 gene fragment was ligated with the recombinant expression vector p5S1285 UD after fusion with the super strong promoter pcpc560 to form the recombinant plasmid ssl2084(+).Then ssl2084(+)was introduced into Synechocystis PCC 6803 and the positive transformants were screened.The correctness of transformants was confirmed after identification at the DNA and protein levels.GC-MS was used to analyze the fatty acid content and composition of the mutant strain under normal culture conditions.The results showed that the total fatty acid content of the mutant was 24.41±0.95 mg / g(DCW),which was 1.5 times that of the wild type.At the same time,the contents of C12: 0,C16: 0 and C18: 0 in the mutant significantly increased,reaching 1.50 mg/g,8.74 mg/g and 0.60 mg/g,respectively.The results showed that overexpression of ssl2084 gene in Synechocystis PCC 6803 could increase the content of total fatty acids and some medium and long chain fatty acids.(3)In order to combine genetic engineering and biochemical regulation to improve the ability of producing fatty acids,low temperature was used as the stress condition,and the expression level of ssl2084 gene in mutant strains at 10 ?,15 ?,20 ?,25 ? and 30 ? was detected by RT-PCR.The results showed that the expression level of ssl2084 gene was highest after stress at 20 ? for 24 h in mutant strains.The GC-MS analysis of the fatty acid content and composition of the mutant strain at 20 ? showed that the content of C12: 0,C16: 0 and C18: 0 further increased compared with the wild type,reaching 1.70 mg/g,11.85 mg/g and 2.31 mg/g,respectively.This shows that the mutant can further increase the content of medium and long chain fatty acids at 20 ?.(4)The slr2023 gene fragment cloned from total DNA of Synechocystis PCC 6803 was sequenced and compared for bioinformatics analysis.The results show that the slr2023 sequence contains 932 bases and encodes 293 amino acids.The molecular weight of the protein is about 32 k Da,and the isoelectric point is 4.87.It is a hydrophilic protein with no signal peptide sequence,no transmembrane domain,and 28 potential phosphorylation sites.Advanced structure predictions show that the protein is an unstable protein.(5)To verify the effect of overexpressing slr2023 gene on fatty acid biosynthesis of Synechocystis PCC 6803,the slr2023 gene fragment was ligated with the recombinant expression vector p5S0168 UD after fusion of the super strong promoter pcpc560 to form recombinant plasmid slr2023(+).We introduced slr2023(+)into Synechocystis PCC 6803 and screened for positive transformants.After identification of the DNA level and protein level,the correctness of the transformants was determined.The detection of mutant fatty acids will be carried out in subsequent experiments. |
PDF Full Text Request