Construction Of Neurogenic Acid Saccharomyces Cerevisiae Cell Strain

Nervonic acid is one kind of material which can improve nerve cell regeneration and play a functional role in repair of damaged neural circuitry.It was widely used in the field of medicine and food.The application and utilization of nervonic acid was restricted seriously,because its resource was few and its cost was high.It is necessary to develop a rapid,simple and safe method for synthetizing nervonic acid.In this study,we first collected common 3 kinds of plants(Passiflora edulia,Brassica napus and Broccoli oleracea)with high levels of nervonic acid.And their ?-ketoacyl-Co A synthases gene was cloned by RACE technology.Meanwhile,a Saccharomyces cerevisiae expression vector containing the ?-ketoacyl-CoA synthase gene from different sources was constructed.And its expression vector was transferred into the Saccharomyces cerevisiae by electroporation methods.Positive recombinant strains were screened with G418 plates,and then positive clones were cultured for 48 h.Positive Saccharomyces cerevisiae strains containing expression vectors for KCS gene from different sources were obtained and a strain of ceramide Saccharomyces cerevisiae was successfully constructed.The results were as following:1.The length of the conserved region of the KCS gene of Passiflora edulia,Brassica napus and Broccoli oleracea,were 692 bp,693 bp and 692 bp respectively.The full lengths of cDNAs of KCS genes obtained from 3 kinds of plants by RACE technology,their lengths were 1756 bp,1886 bp,and 1785 bp respectively.2.The length of the open reading frame(ORF)of KCS gene of 3 kinds of plants were all 1521 bp.And they all encoded 506 a mino acids proteins,with molecular weights of 56304.90 Da,56403.06 Da and 56410.00 Da.The isoelectric points were 9.120,9.238,a nd 9.230.At p H 7.0,the charged were 16.945,19.275 and 19.108.3.The function of 3 kinds of plant's KCS genes was predicted by using bioinformatics analysis software.The results showed that the tertiary structure of proteins was mainly composed of ?-helices and ?-sheets.It was predicted that the protein had 3 phosphorylation sites of serine,threonine,tyrosine,none of them had a signal peptide.Through of the KCS protein sequences of 15 kinds of plants was analysed the cluster analysis in this study,it was found that KCS protein sequences of Passiflora edulia,Brassica napus and Broccoli oleracea and Raphanus raphanistrum,Thlaspi arrense,Lunaria annua,Arabidopsis thaliana,Crambe hispanica,Cardamine parriflora were classified into a large category.Brassica napus and Broccoli oleracea had the closed genetic relasionship,and classified into one category.Passiflora edulia and Raphanus raphanistrum had the closed genetic relasionship,and classified into one category.However,their genetic relationship with Brassica napus and Broccoli oleracea was relatively distant.4.The KCS recombinant plasmids of 3 kinds of plants were heterologously expressed in Saccharomyces cerevisiae and positive clones were selected.The positive clones which was cultured for 48 h were detected by gas chromatography,and found the KCS protein of 3 kinds of plants could transform C18:1 into C24:1.In this experiment,we used the KCS gene from Passiflora edulia,Brassica napus and Broccoli oleracea to successfully construct the Saccharomyces cerevisiae cell strain,which laid a foundation for large-scale production of nervonic acid in the Saccharomyces cerevisiae factories.