Functional Analysis Of Linum Usitatissimum LuFAD8 Gene On Seed Oil Accumulation

Flax is one of the common oil-producing crops.It has recently gained popularity because of the high level of alpha-linolenic acid in its oil.Alpha-linolenic acid has been shown to reduce blood lipid,improve cardiovascular health,and prevent cancer.In plant cells,theω-3 fatty acid desaturase FAD8 could catalyze the conversion of linoleic acid to linolenic acid.Currently,the FAD8 gene has been isolated and cloned in a variety of plants such as rice.However,the biological function of LuFAD8 is still unclear in flax,especially the differences and similarities between each paralog of LuFAD8.In the present study,the full length coding domain sequence（CDS）of three paralogs of LuFAD8 were cloned from the flax’Longya 10’.The physicochemical properties,sequence structures of LuFAD8 proteins and their genetic relationship with other plants were analyzed.The expression patterns of three paralogs of LuFAD8 gene were studied by GUS staining.On this basis,this study conducted the recombinant vector 35S:LuFAD8-X–6HA and transformed into the Arabidopsis wild-type to explore the functions of three paralogs of LuFAD8 on oil accumulation in mature seeds and low temperature stress response.The main results were as follows:（1）Three LuFAD8 paralogs,LuFAD8-1,LuFAD8-2,and LuFAD8-3,were obtained from Phytozome website based on AtFAD8 protein.LuFAD8-1,LuFAD8-2,and LuFAD8-3were predicated to share 75.3%,72.9%,and 74.5%amino acid identity with AtFAD8,respectively.According to multiple sequence alignment,all of them had desaturated domain.These results suggested that LuFAD8 might have conserved functions as AtFAD8.Analysis of basic physicochemical properties revealed that the LuFAD8-1,LuFAD8-2 and LuFAD8-3 proteins were stable and weakly basic;phosphorylation sites were mostly occurred at serine residues,no signal peptide.（2）Subcellular localization results showed that the proteins enconed by LuFAD8paralogs were specially localized in the endoplasmic reticulum of N.benthamiana leaves,suggested that they may play a role in the endoplasmic reticulum.The p LuFAD8-X:GUS vectors were designed to transform into the Arabidopsis wild-type（Col-0）,and the transgenic plants were used for GUS staining signal investigation.According to the GUS staining signal results,the expression of LuFAD8-1 was lower than LuFAD8-2 and LuFAD8-3 in seeds,while the expression of LuFAD8-1 was higher than LuFAD8-2 and LuFAD8-3 in leaves.（3）Overexpression of either of LuFAD8 paralogs（Col-0 35S:LuFAD8-1–6HA,Col-035S:LuFAD8-2–6HA,and Col-0 35S:LuFAD8-3–6HA）in Arabidopsis wild-type（Col-0）had no effect on several agronomic traits,including seed weight,seed coat color,seed length and width.However,overexpression of either of LuFAD8 enhanced total fatty acid content in mature seeds.The contents of linoleic acid and linolenic acid in the seeds of Col-035S:LuFAD8-1–6HA increased significantly,while the contents of linoleic acid decreased significantly in the seeds of Col-0 35S:LuFAD8-2–6HA and Col-0 35S:LuFAD8-3–6HA,and the contents of linolenic acid were significantly increased.These results suggested that LuFAD8 enhance the accumulation of total fatty acid in seeds.LuFAD8-2 and LuFAD8-3could catalyze the conversion of linoleic acid to linolenic acid in seeds,while LuFAD8-1 did not play this catalytic role in seeds.（4）Col-0 35S:LuFAD8-2–6HA and Col-0 35S:LuFAD8-3–6HA had no significant change in the contents of total fatty acid and fatty acid components in leaves after low temperature treatment.However,the total fatty acid and linolenic acid were significantly increased in the leaves of transgenic plants Col-0 35S:LuFAD8-1–6HA,while linoleic acid was decreased after low temperature treatment.The results indicated that LuFAD8-1 could promote linolenic acid biosynthesis in the leaves under low temperature.In order to further explore the response mechanism of LuFAD8-1 to low temperature stress,physiological and biochemical indexes of Col-0 35S:LuFAD8-1–6HA transgenic plants were determined after low temperature treatment.The content of O^2-,H₂O₂,and MDA in the leaves of Col-035S:LuFAD8-1–6HA was significantly lower than that of Col-0,while the CAT activity was significantly higher than Col-0 after low temperature treatment.These results indicated that LuFAD8-1 could reduce the accumulation of O^2-and H₂O₂,increase the activity of CAT,and alleviate the degree of cell membrane damage under low temperature.In summary,this study analyzed the sequence structures and spatiotemporal expression patterns of three LuFAD8,as well as the preliminary role of three paralogs of LuFAD8 gene on seed oil accumulation and low temperature stress response.This research not only contributes to the understanding of the molecular regulation mechanism of oil accumulation in oil crops,but also gives genetic resources and ideas for the invention of oil crop germplasm resources and the cultivation of novel varieties using molecular breeding methods.