Identification And Characterization Of The Key Enzyme Genes In The Lipid Biosynthesis From Jatropha Curcas L.

Jatropha curcas L. is an oil-bearing shrub widely distributed in many countries and areas. Its seed kernels contain up to 62% oil, and linoleic acid accounts for 47%. 4 cDNA sequences, designated Jcω6, Jc ω3, Jcd8 and Jcdagat, putatively encoding a Δ12-fatty acid desaturase, a Δ15-fatty acid desaturase , a Δ8-fatty acid desaturase and a DAGAT, were isolated from this plant using RT-PCR and RACE methods. Sequence analysis indicated that the Jcω6 had an open reading frame of 1152 bp encoding 383 amino acids of 44. 5 kDa; the Jcω3 had an open reading frame of 1341 bp encoding 446 amino acids of 51 kDa; the Jcd8 had an open reading frame of 1344 bp encoding 447 amino acids of 51 kDa; the Jcdagat had an open reading frame of 1566 bp encoding 522 amino acids of 71 kDa; The deduced amino acid sequences showed high identity to several known related enzymes from higher plants. Bioinformatics analysis on these genes and their deduced amino acid sequences implied all enzymes invovled in this work were membrane-bound enzymes. Among them, the Jc ω3 was a chromplastid desaturase, and had two transmembrane regions;the Jew6 was a desaturase in ER and had six transmembrane domains; the Jcd8 was located to ER and had 3 transmembrane regions; the last one, Jcdagat also was ~an* membrane-bound enzyme and existed 9" transmembrane domains.Heterologous expression in S. cerevisiae strain INVScl done for Jcco6and Jcdagat demonstrated that the Jcco6 exhibited high A l2-fattyacid desaturase activity by the accumulation of C18:2 A9' 12;the Jcdagatexhibited high diacylglycerol acyltransferase activity by theincreased accumulation of TAG in the mutant and wild transgenic yeast.Southern blot and temporal expression analysis suggested that the Jcdl2 was a single-copy gene with a housekeeping function in Jatropha curcas (Panzhihua, China), the Jcdagat was a multi-copy gene with specific expression in different tissuse in different developing stages and the Jcd8 also was a single-copy gene with distinct specific expression in developing tissues.