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Preliminary Cloning the 3-ketoacyl-CoA Synthase (KCS) with RACE Technique and Establishment of Regeneration System in Linumusita Tissimum

Author: ZhangYanPing
Tutor: HouSuiWen
School: Lanzhou University
Course: Cell Biology
Keywords: Flax β-ketoacyl -CoA synthase acyloxy FKCS RACE technology Regeneration System
CLC: S565.9
Type: Master's thesis
Year: 2009
Downloads: 141
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Abstract


Flax long chain fatty acids having a wide range of physiological functions , it is involved in seed glycerides , the biofilm membrane lipid and sphingolipid synthesis , and to provide for the biosynthesis of epidermal wax precursor substance . The extension of the long chain fatty acid synthesis by the fatty acyl -CoA enzyme catalysis, the synthesis of the first step reaction - condensation reaction is the rate-limiting step catalyzed by β - ketoacyl -CoA synthase (KCS) . Fatty acid elongase gene research has focused on the KCS gene . This experiment, first using degenerate primers flax β - ketoacyl -CoA synthase gene DNA fragment was obtained and expected β - ketoacyl -CoA synthase gene named FKCS. Then this DNA fragment as a basis, using 3'RACE reaction to FKCS 1082bp of the cDNA fragment cloned from flax blade , and The the NCBI ORF analysis shows that it contains a length of an open reading frame of 822bp and 252 bp of the 3'UTR . Since this experiment 5'RACE did not give the corresponding objective fragment , only by 3'RACE reaction cDNA obtained speculated out FKCS 273 amino acids located in FKCS to the C-terminus . Sequence homology analysis showed FKCS protein encoded by its amino acid sequence with high homology Arabidopsis in the five kinds of β - ketoacyl acyl -CoA synthase . The phylogenetic tree analysis showed FKCS Arabidopsis KCS family in kinship recent KCS- 1 . Arabidopsis KCS-1 primarily with the synthesis of long chain fatty acids , suggesting flax in FKCS also concerned with the synthesis of long chain fatty acids , FKCS may flax a novel β - ketoacyl -CoA aggregate enzymes. Cloning and sequencing of FKCS laid the foundation for the future study of its biological functions in the flax . Order to facilitate later the study FKCS gene function , flax hypocotyls explants successfully established flax rapid propagation regeneration system : MS 300mg / L hydrolyzed casein protein 0.5mg / L 6- BA favor callus adventitious buds generated : promoting rooting , do not add any plant hormones 1/2MS medium more conducive to root growth .

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