Preliminary Cloning The 3-ketoacyl-CoA Synthase (KCS) With RACE Technique And Establishment Of Regeneration System In Linumusita Tissimum

Very long chain fatty acids(VLCFAs) play a comprehensive role in Flax.They are essential biological components of triacylglycerols(TAGs),membrane lipids, and sphingolipids.They also serve as precursors of wax layer compounds. Biosynthesis of VLCFAs is catalyzed by the fatty acyl-CoA elongase.The first step of biosynthesis which is catalyzed by 3-ketoacyl-CoA synthase(KCS) is the rate-limiting step.At present,the study of the fatty acyl-CoA elongase focuses on the KCS.In this study,firstly,a DNA fragment was got by degenerate primer,designated FKCS.A cDNA encoding the 3-ketoacyl-CoA synthase was isolated and sequenced from the leaves tissue of flax(Linum fusitatissimum) for the first time,by 3'rapid amplification of cDNA ends(RACE) technique based on the DNA fragment. Analysis on the cDNA sequence of FKCS,we obtained the open reading frame(ORF) from NCBI.The ORF contained 822 bp bases,flanked by a complete 3'untranslated region with 252 bp bases.Because of the failure of 5'RACE,we only got a putative 273 amino acid of FKCS located at the carboxyl terminal.Sequence analysis indicated that the sequence of amino acids of FKCS is more similar to five members of KCS family in Arabidopsis.The phylogenetic tree based on the amino acid sequences showed that genetic relationship between FKCS and KCS-1 was closest in Arabidopsis.Therefore,FKCS is possibly a novel member of KCS family.The cloning and sequencing of FKCS establishes a good foundation for the future work on the biological functions of FKCS in the flax(Linum usitatissimum).For the future study of FKCS,MS was found to??the best basal medium in regeneration of flax in which hypocotyl was used as explant.0.5mg/L 6-BA was effective to induce callus and shoot.An optimized protocol was established as following:a MS medium supplemented with 300mg/L casein acid hydrolysates and 0.5mg/L6-BA was beneficial to callus induction and shoot regeneration,then a half-strength MS medium is helpful to shoots elongation and rooting.