Cloning And Function Analysis Of Genes Related To Lipid Biosynthesis In Idesia Polycarpa

Idesia polycarpa is a very important woody oil plant,whose fruit has a high oil content which has the reputation of “oil depot on trees”.It has a variety of uses in food,health care,medicine and chemical industry.Breeding excellent varieties is the premise of exploitation and utilization of I.polycarpa.However,due to the lack of theoretical research on the genes related to oil synthesis and the complete genetic transformation system of I.polycarpa fruit,the selection and breeding of I.polycarpa,an excellent oil tree,has been severely restricted.In-depth understanding the molecular mechanism and gene function of lipid biosynthesis of I.polycarpa fruit is a great significance for the development of I.polycarpa resources.It is not only conducive to genetic improvement of I.polycarpa by molecular breeding technology,but also can provide reference for the study of gene function of other oil plants.In this study,I.polycarpa fruits were used as experimental materials.The dynamic changes of oil content and fatty acid composition in I.polycarpa fruits at different development stages were analyzed,and the genes related to oil biosynthesis in I.polycarpa fruits were excavated by transcriptome sequencing data.Then the genes related to lipid synthesis were successfully cloned and the genes related to linoleic acid biosynthesis were selected.The prokaryotic expression vectors and plant expression vectors were constructed,and their gene functions were verified by substrate reaction and Agrobacterium-mediated genetic transformation of Arabidopsis thaliana.Finally,the genetic transformation receptor system of leaves callus regeneration system was established,which laid a foundation for the genetic improvement of I.polycarpa.The main results were as follows:(1)The dynamic change rules of oil content and fatty acid composition in different development stages of I.polycarpa fruits were revealed.During the development of Jatropha fruits,the oil content gradually increased in an S-shaped growth pattern,and there was an intermittent period between 80 and 90 DAP,during which the fruit color changed.The proportion of linoleic acid content in fatty acid components gradually decreased,but its final value was still above 64%.At 70 DAP and 130 DAP,there were significant differences in oil content and major fatty acid components in fruits(2)Genes related to lipid biosynthesis were identified by transcriptome sequencing.Transcriptome sequencing datas revealed 33 candidate genes which potentially involved in lipid biosynthesis.Among them,18 genes were significantly differentially expressed in 70 and 130 DAP,Ip ACCase、Ip KAS I、Ip KAS II、Ip SAD,Ip FAD2 and Ip FAD3 are key genes of fatty acid biosynthesis in I.polycarpa fruits,Ip DGAT2 and Ip GPAT are key genes of TAG biosynthesis in I.polycarpa fruits.(3)The full-length sequences of Ip SAD、Ip FAD2、Ip FAD3、Ip GPAT and Ip DGAT2 which was related to the synthesis of unsaturated fatty acids in I.polycarpa fruits were obtained.The phylogenetic tree results showed that they were closely related to Populus trichocarpa and Populus euphratica.Bioinformatics analysis of their predicted proteins suggested that these 5 functional genes played a key role in the synthesis of oleic acid,linoleic acid and lipid in I.polycarpa.(4)Functional analysis of Ip FAD2 and Ip DGAT2 were carried out,respectively.The recombinant plasmid of prokaryotic expression vector could normally express the same size and bioactive protein as the predicted protein under the induction of IPTG.In Ip FAD2 and Ip DGAT2 transgenic A.thaliana plants,the LA and lipid contents were increased by13.50～29.86% and 27.70～39.09% compared with wild-type seeds,respectively.These results showed that the expression levels of Ip FAD2 and Ip DGAT2 determined LA content and lipid content in I.polycarpa。(5)The genetic transformation receptor system of leacallus regeneration system was established.The following optimum medium were selected.The optimum medium for callus induction from leaf was MS + sucrose 30 g/L + 6-BA 1.0 mg/L + 2,4-D 2.0 mg/L +agarophyte 6 g/L(p H=5.8),callus induction rate was 36.67%;The optimum medium for callus subculture was MS + sucrose 30 g/L + 6-BA 1.5 mg/L + NAA 0.5 mg/L +agarophyte 6 g/L(p H=5.8),increment coefficient of callus was 4.46;The optimum medium for adventitious buds induction was MS + sucrose 30 g/L + 6-BA 2.0 mg/L + NAA 0.2mg/L + TDZ 0.1 mg/L + agarophyte 6 g/L(p H=5.8),adventitious bud induction rate was22.22%;The optimum medium for adventitious buds rejuvenation was MS + sucrose 30 g/L+ 6-BA 1.5 mg/L + NAA 0.1 mg/L + TDZ 0.08 mg/L + agarophyte 6 g/L(p H=5.8),increment coefficient of adventitious bud was 4.45.The maximum lethal concentration of Cef to callus was 200 mg/L,and the minimum lethal concentration of Kan was 50 mg/L.