Gene Identification And Comparative Study Of Tung Oil GPAT Family

Vernicia fordii(Hemsl.)Airy Shaw)is an important industrial oil tree species,native to China,with high oil content of seeds.The oil made from tung tree seeds is called tung oil,and its main ingredient is ?-tung acid(18:3?9,11,13)with 3 conjugated double bonds,which is one of the most high-quality dry vegetable oils.3-Phosphoglyceryl acyltransferase(GPAT)is a key enzyme in the glycerolipid synthesis pathway that catalyzes the first acylation reaction and has an important limiting effect on the oil content of oil crop seeds.In this paper,based on the data of the tung tree genome and transcriptome,members of the VfGPAT were identified,and the differences in phylogenetic evolution,expression patterns,and heterologous expression function verification between different members were cloned and analyzed,with a view to Genetic improvement provides scientific guidance.The main findings are as follows:1.Cloning and bioinformatics analysis of the VfGPAT.Based on the Tung tree genome data,nine VfGPAT were identified,named VfGPAT1,VfGPAT3-1,VfGPAT3-2,VfGPAT3-3,VfGPAT5,VfGPAT6,VfGPAT8,VfGPAT9 and VfATS1.Phylogenetic analysis shows that the GPAT gene family of Tung oil tree can be divided into four branches.Different branch genes have different evolutionary styles and functional differentiation.Among them,VfGPAT3-1?3-2 and 3-3 is derived from the same gene ancestor Duplication events that occur during evolution.Protein structure analysis showed that the VfGPAT has a highly conserved triglyceride acyltransferase structure domain,of which VfGPAT8 has a typical dilysine motif(-KKXX-COOH)and VfGPAT9 has a hydrophobic pentapeptide motif.2.Subcellular localization analysis of GPAT protein of Tung trees.A yellow fluorescent protein fusion expression vector was constructed and impregnated into tobacco leaves.The results show that the VfGPAT1?5?6?8 and 9 protein is located in the endoplasmic reticulum of the cell,which is consistent with the place where TAG synthesis occurs in the Kennedy pathway;VfGPAT3-1?3-2?3-3 and VfATS1 proteins are located in the cell chloroplast It implies that the VfGPAT may also participate in unknown metabolic pathways.3.Analysis of the expression pattern of the VfGPAT.Using the Tung tree transcriptome data and qRT-PCR experiments,analysis of the VfGPAT expression pattern of the tung tree showed that the expression of different VfGPAT copies in different tissues of Tung tree and in different developmental stages of seeds may be functionally different.4.Heterologous expression of the VfGPAT.Construction of yeast expression vector,and transformation into double mutant yeast ZAFU1.The results showed that VfGPAT1?5?6 and 8 genes have GPAT enzyme activity and can supplement the defects of phosphatidyl ester biosynthesis in the double mutant.Construct plant heterologous expression vector,and transform into wild-type Col Arabidopsis plants.Transgenic T2 positive seedlings have been obtained,but the research on the growth of transgenic plants and the oil content of seeds needs further experimental exploration.