Cultivation And Functional Analysis In Transgenic Rice Of WRI1 And OsFAD3

Omiga-3 fatty acid desaturase(FAD3)is a rate-limiting enzyme in fatty acid metabolic pathways,it can catalyze formation of alpha-linolenic acid from linoleic acid.Because lacking of this enzyme,alpha-linolenic acid can be obtained only from food.Alpha-linolenic acid is an essential nutrient and has important significance in human health.It can regulate blood fat,prevent myocardial infarction,inhibit thrombotic and inflammatory reaction.It also plays important roles in regulation of the allergic reaction,vision improvement,weight loss and so on.WRI1 is a transcription factor in regulating oil metabolism,and it has been proved to be an efficient regulatory factor in regulating fatty acid metabolism in arabidopsis.Using genetic engineering technology,we expect to get the high-yield rice of alpha-linolenic acid varieties of both WRI1 gene and OsFAD3 gene,which can co-regulate fatty acid metabolism in rice.1.The 302 Os FAD 3 gene clones were got by using agrobacterium-mediated transformation of the OsFAD3 gene into the rice genome,and finally 13 marker-free OsFAD3 plant strains were got.17 marker-free OsFAD3 homozygous plant strains,which came from 8 independent clones,were got by self-cross separation and screening.The assay results showed that the linoleic acid contents of positive strains dramatically decrease as compared to wild type rice,while the average contents of alpha linolenic acid raised more than a dozen times.The least increases 9.6 times,while the highest up to 15.4 times.It suggested that OsFAD3 can effectively convert linoleic acid into alpha linolenic acid,thus increasing the content of unsaturated fatty acid in rice.2.The amount of fatty acid were analyzed by constructing 3 AtWRI1 gene expression vectors which were respectively drived by SbeIIb starch branching enzyme gene promoter,Ubiquitin constitutive promoter and pLatex endosperm-specific promoter.In order to analyze the fatty acids contens and compare the actual effect of different promters to AtWRIl gene.Now 17 out of 23 independent clones from the strains of T0 with SbeIIb-AtWRI1 vector,were identified as a single copy.The assay of the contents of fatty acid in T0 generation seeds showed that palmitic acid,oleic acid and linoleic acid contents were obviously improved compared with wild type rice,and the total TAG is also significantly improved that the highest even increased more than 3 times.The strains of T0 with Ubi-AtWRI1 and Latex-AtWRI1 vector were got at the same time,and the screening and detection are still in progress.3.The phylogenetic tree were constructed by comparing the homologous sequences,and found three higher homology rice genes with arabiopsis WRI1 gene,but only one gene could express at the transcriptional level by RT-PCR analysis.This gene contains only one AP2/EREBP domain,and the further functional verification is still in research.