Study On The Role Of Fatty Acid Export Protein Bn3662 In Brassica Napus L.

Fatty acids(FAs)are building-blocks of most cellular lipids,which are essential throughout all the life of an organism.In higher plants,the biosynthesis of fatty acids occurs in plastids,and then most of them are transported to the endoplasmic reticulum for further modification and lipid assembly.However,the transport mechanism of fatty acids has not been elucidated.Rapeseed is an important oil crop in the world,and the rapeseed oil as a potential renewable biomass energy has a wide range of applications in industrial production.The content of fatty acids produced and transported by plastids limits the accumulation of triacylglycerol(TAG)in oil crop seeds,which may have a great impact on the content of TAG by increasing the flux of fatty acid biosynthesis in plant tissues.Therefore,it is of great theoretical significance and economic value to reveal the molecular mechanism of fatty acid transport in rapeseedCBrassica napus L.).In this paper,Bn3662 was isolated and identified from Brassica napus L,which is the homologous protein of AtFAX1(fatty acid export 1).Through the study of gene expression,protein localization and function,the role of Bn3662 in fatty acid transport in Brassica napus L.and its related molecular regulation mechanism were discussed.The main research results are summarized as follows:1.Molecular Identification and expression Analysis of Bn3662 in Brassica napus L.Sequence alignment and Phylogenetic tree analysis showed that the fatty acid transporter Bn3662 had high homology with Arabidopsis thaliana AtFAX1.According to topological prediction,Bn3662 is a transmembrane protein with four transmembrane domains and a chloroplast transporter peptide leading sequence composed of 55 amino acid residues.Furthermore,a GFP fusion expression vector driven by CaMV 35S of the Bn3662 was constracute and transformed into agrobacterium tumefaciens cells.With the method of injecting agrobacterium which harbored the vector into Nicotiana benthamiana epidermal cells,the GFP green fluorescence was found to be localized in the chloroplast under laser confocal microscope.The expression of Bn3662 in tissues of Brassica napus was analyzed by Real-time quantitative RT-PCR,and the results showed that Bn3662 gene performed high expression level in seeds 25 days after pollination.In addition,analysis the expression of Bn3662 in seeds of two rapeseed varieties(HFA and LFA,which have significant difference in oil content in seeds)at 15,20,25,30,40 and 50 days of development,and it was found that Bn3662 was highly expressed in 25 DAP seeds at the peak of oil synthesis.Moreover,the expression level was higher in high oil content variety HFA,which indicated that Bn3662 might affect the oil synthesis of rapeseed seeds and other tissues and organs.2.Bn3662 promotes the synthesis of fatty acids in rapeseed leavesThe overexpression vector of 35S-Flag-Bn3662 was constructed and transgenic plants were obtained by agrobacterium tumefaciens-mediated genetic transformation of Brassica napus L.The results showed that the content of fatty acids in leaves of transgenic rapeseed overexpressing Bn3662 was significantly higher than that of wild type.In addition,fatty acid composition analysis showed that the ratio of low unsaturated fatty acids such as palmitic acid(C16:0),stearic acid(C18:0),oleic acid(C18:1n9)in Bn3662 transgenic rapeseed leaves was increased.However,the ratio of unsaturated fatty acids such as C16:1,a-linoleic acid(C18:2n6),and a-linolenic acidcc18:3(C18:3n3)was decreased.This indicated that the overexpression of Bn3662 would promote the transport of fatty acids in the plastids,leading to the faster flow of fatty acids from the plastids to the outside,and thus the content of total fatty acids in the leaves increased significantly.3.Bn3662 affects the expression of enzymes related to glycolysis,fatty acids and lipid synthesis pathwaysPlant oil synthesis involves a series of multi-link biochemical reactions,including glycolysis pathway,fatty acid synthesis and lipid assembly process.In order to investigate the effect of Bn3662 on oil synthesis,the expression levels of the lipid synthesis-related enzymes genes were quantitatively analyzed by fluorescence real-time quantitative RT-PCR.The results showed that compared with the wild-type rapeseed,the expression of these key enzyme genes in the overexpression of Bn3662 transgenic rapeseed was up-regulated,indicating that Bn3662 promotes the biosynthesis and accumulation of fatty acids in the rapeseed cells by mediating the expression of a class of enzyme genes involved in the synthesis of fatty acids and triacylglycerol assembly process.4.Bn3662 is involved in regulating the expression of genes related to flowering induction in Brassica napus L.It was found that compared with wild type,Bn3662 overexpression transgenic rapeseed shows an early flowering phenotype(flowering 30-45 days earlier).Moreover,the tissue expression analysis also showed that Bn3662 was highly expressed in the floral organs of rapeseed plants.The results showed that the expression levels of FT,TSF,LFY,SOC1,AG and AP3,which regulated flowering time,were significantly increased in Bn3662 transgenic rapeseed.These genes belong to flowering integration genes and are downstream target genes of multiple signal regulatory pathways.How their expression is directly or indirectly affected needs to be further explored and analyzed.Meanwhile,in order to eliminate the influence of flowering time on the growth period and thereby affect the research on seed oil content,a seed-specific promoter BnNapinP-Bn3662 was constructed for rapeseed transformation,and the related research is still in progress.5.The interaction between Bn3662 and BnPP2CThe pGBKT7-Bn3662 vector was constructed and transformed into yeast strain Y187,one of the interacting protein phosphatase PP2C was screened from the cDNA library of rape seeds 25 days after pollination(25DAP)by yeast two-hybrid systems.Subsequently,the interaction was verified in vitro and in vivo by yeast two-hybrid technique,pull-down and Co-IP assay.It is speculated that the interaction between BnPP2C and Bn3662 may affect the phosphorylation level of Bn3662,thus affecting the biological function of Bn3662 in Brassica napus L.