Expression And Functional Analysis Of Lipid Metabolism Key Gene Of Peanut In Saccharomyces Cerevisiae H1246and Synechococcus Sp.PCC7962

As the fourth largest oil crop in the world, peanut is an important source ofvegetable oil which is widely cultivated. Vegetable triacylglycerols are the maincomponents of vegetable oil, many of whose fatty acid have significant nutritional,healthy and medical value. The low ratio of oleic/linoleic acid content has directlyinflunced the storage and shelf life of peanut in our country, which is theimmediate cause of low international competitiveness of China’s peanut. Toimprove the relative levels of oleic acid and oil content of oil crops and increasethe oleic acid/linoleic acid (O/L) ratio has become a major objective of oil cropsbreeding. In this study we analyzed the oil content and compositions of fatty acidsof different peanut varieties, which is helpful for us to find good germplams tobreed new varieties. Four key genes involved in fatty acid or lipid synthesispathway, AhDGAT1-1, AhDGAT1-2, AhDGAT3-3and AhFAD6were cloned by us.Quantitative real-time RT-PCR (qRT-PCR) was employed to confirm theexpression patterns of the four novel genes in different peanut tissues, at differentstages of seed development, and under different stresses.The functions of thesefour genes were validatied by transforming into Saccharomyces cerevisiae orSynechococcus sp.PCC7962. The present study provides significant information touse in modifying oil deposition and improving abiotic stress resistance of peanutthrough molecular breeding.1. The total lipid content and fatty acid compositions of15peanut varieties(lines) were analyzed by us. We found that there were minor differences existedamong different varieties (lines). The variety “Hebei high oil” accounts for thehighest oil content,53.97%, whereas oil content of line “E12” was only lowerthan” Hebei high oil”0.19%. Oleic and linoleic acids are the main compositions ofpeanut seeds, and the content of inoleic acid is higher.The accumulation mode of oils and fatty acids during the developmental stages of peanut seeds were alsoinvestigated by us usingfour varieties (lines), Huayu19, E18, E11, and Luhua No.6.We found that seed oil content was low in the first period of lipid accumulation,but was characterized by a drastic increase during the initial four stages afterpegging. With the increase of oil content, stearic acid content gradually increased,whereas palmitic acid content decreased. The relative content of oleic and linoleicacids showed a reverse trend during seed development.2. Quantitative real-time RT-PCR (qRT-PCR) was employed to confirm theexpression patterns of the four novel genes in different peanut tissues, at differentstages of seed development, and under different stresses. The expression ofAhFAD6was higher in the leaves than in other tissues examined, but showed adownward trend during the stages of seed development. AhDGAT1-2transcriptwas more abundant in roots, seeds, and cotyledons, whereas the transcriptabundances of other two genes were higher in flowers than in other tissuesexamined. During seed development, transcript levels of AhDGAT1-1remainedrelatively low at the initial stage but increased gradually during later stages, withthe highest levels seen at50days after pegging (DAP). Levels of AhDGAT1-2transcript were higher at10and60DAPs and much lower at other stages, whereasAhDGAT3-3gene showed higher expression levels at20and50DAPs. In addition,AhDGAT transcripts were differentially expressed following exposure to abioticstresses or a stress-induced plant hormone. Thus, we infer that these four genesmay be involved in regulating some kinds of abiotic stresses in peanut.3. Δ12fatty acid desaturase (Δ12fatty acid desaturase, Δ12FAD) catalyzesoleic acid and palmitoleic acid thus generates linoleic acid and palm diene acid. Inthis study, one novel AhFAD6gene was identified and isolated from peanut. Theactivity of the AhFAD6gene was confirmed by heterologous expression in theSynechococcus sp.PCC7942, which is an excellent light pattern autotrophic cyanobacteria, containing only saturated fatty acids and monounsaturated fattyacids. Linoleic acid, normally not present in wild-type cyanobacteria, was detectedin transformants of AhFAD6gene.as substrate.4. Diacylglycerol acyltransferase (DGAT, EC2.3.1.20) catalyzes theacyl-CoAdependent acylation of sn-1,2-diacylglycerol to form TAG, which is theonly enzyme in the sn-glycerol3-phosphate pathway unique to TAG biosynthesis,and has been proposed to be the rate-limiting enzyme in plant storage lipidaccumulation. In this study, three novel AhDGATs genes were identified andisolated from peanut. The activity of three AhDGAT genes was confirmed byheterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruplemutant. The recombinant yeasts restored lipid body formation and TAGbiosynthesis, and displayed a preference to the incorporation of unsaturated C18fatty acids into lipids.