| Tree peony,belonging to the section Moutan DC.,genus Paeonia and the family Paeoniaceae,is an excellent ornamental and medicinal plant.Tree peony has dark oval seeds characterized by abundant unsaturated fatty acids(UFAs)and high proportion of ?-linolenic acid(ALA).Paeonia rockii is an important member of the section Moutan DC.,which is the species with the highest UFAs content among all nine tree peony species.In order to study the molecular mechanism of seed FA biosynthesis in P.rockii,we constructed a cDNA library at different developmental periods of seeds via RNA-seq-based transcriptome sequencing.Several ESTs related with biosynthesis and regulation of fatty acid(FA)and triglyceride(TAG)were identified from the cDNA library.Based on the EST fragment sequences,we cloned their full-length cDNA sequences,analyzed the expression pattern of these genes,and elucidated their biological functions in TAG biosynthesis via heterogeneous overexpression in Arabidopsis.Our work could be useful to better understand the molecular mechanism of lipid biosynthesis in tree peony and to improve ALA content of tree peony and other oil crops through genetic engineering.The main results are as follows:1.Five genes encoding fatty acid desaturases(FADs)were cloned,named PrSAD,PrFAD2,PrFAD3,PrFAD6,and PrFAD7,and the lengths of their coding regions were 1191 bp,1155bp,1308 bp,1230bp,and 1353 bp,respectively.They encoded putative proteins with 396,384,435,409,and 450 amino acids,respectively.Five amino acids shared a conserved FA-acyltransferase domain.Phylogenetic analysis suggested that PrSAD,PrFAD2,PrFAD3,PrFAD6,and PrFAD7 were highly homologous to the corresponding amino acids in P.lactiflora.2.QRT-PCR results showed that five genes encoding fatty acid desaturases were constitutively expressed in tree peony,with higher expression levels in pistil,stamen,and seeds.Besides,PrSAD,PrFAD6,and PrFAD7 had higher expression levels in leaves.PrSAD,PrFAD2,and PrFAD3 exhibited higher transcript abundances at 10~80 days after pollination(DAP),which coincided with the highest rate of ALA accumulation in tree peony seeds.Transcript levels of PrFAD6 and PrFAD7 had no significant changes.3.To study the roles of five genes in FA biosynthesis,we checked the content and composition of FAs in mature seeds of transgenic Arabidopsis plants using GC-MS method.Compared with wild-type(WT)plants,overexpression of PrSAD,PrFAD2,and PrFAD3 in Arabidopsis respectively results in a significant increase in total FAs content.The levels of C16:0,C18:1,C18:2,C20:1,and C20:2 had a significant increase in PrSAD-overexpressing lines.Overexpression of PrFAD2 resulted in a remarkable reduction in the ratio of a-linolenic acid/linoleic acid(ALA/LA),whereas a marked increase in the ALA/LA ratio was found in transgenic lines overexpressing PrFAD3,compared with WT.PrFAD2-overexpressing transgenic lines displayed elevated levels of C16:0,C18:0,and C18:2,but decreased levels of C18:1,and C20:0,while an obvious increase in C16:0,C18:1,C18:3,and C20:1 content but decline in C18:2 and C20:0 levels were detected in Arabidopsis with PrFAD3 overexpression.Ectipic expressions of PrFAD6 or PrFAD7 have no effect on Arabidopsis seeds total FAs content.By comparison with WT,overexpression of PrFAD6 resulted in a significant increase in the levels of C18:2 and C22:1,but a marked decrease in C18:1,C20:2 levels,and ALA/LA ratio in seeds;overexpression of PrFAD7 resulted in a significant increase in the levels of C16:0,C18:1,C18:3,and ALA/LA ratio,but a significant decrease in C18:2,C20:0,and C20:2 in seeds.This study revealed that PrSAD,PrFAD2,and PrFAD3 play important roles in FA biosynthesis,PrFAD2,PrFAD3,PrFAD6,and PrFAD7 have effect on UFAs(especially ALA)accumulation,but the effect of PrFAD2 and PrFAD3 may dominant.Therefore,high ALA levels were closely related to the efficient PC-derivative pathway in P.rockii. |
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