Cloning And Expression Analysis Of DGAT And PEPC Gene From Paeonia Suffruticosa Andr

Paeonia suffruticosa Andr.is a perennial deciduous plant,which belongs to Paeoniaceae,widely distributed in Shandong,Henanand so on.Its seed oil contains various unsaturated fatty acids and has been a kind of new resources food by the Chinese Ministry of Health.In recent years,molecular biotechnology has been widely applied in vegetable lipid biosynthesis researches,and the lipid synthesis-related genes and their function have been one of the research focuses.In this study,we designed their degenerate primers according to the conservative region of Diacylglycerol acyltransferase gene and Phosphoenolpyruvate carboxylase gene in GenBank respectively,cloned their full length sequences from seed of Paeonia suffruticosa by the method of RT-PCR and RACE,and analyzed their bioinformatics characteristics.The expression levels of two genes were analyzed in different organs,developmental stages and stages after harvest by the method of qRT-PCR.Here are the main results.1.Cloning and Bioinformatics Analysis of DGAT Gene from Paeonia suffruticosa Andr.The Diacylglycerol acyltransferase gene PaDGAT1?The accession number in GenBank:MG214258?was cloned from the seed of Paeonia suffruticosa by the method of RT-PCR and RACE.The full-length cDNA of PaDGAT1 gene was 2028bp,whose open reading frame was 1554 bp encoding 517 amino acids.The PaDGAT1 protein was a hydrophobic alkaline protein with molecular weight of 58.86kD and the academic isoelectric point of 8.62.Prediction of secondary structure showed that the protein was mainlymade up of the random coil and extended strand.Amino acids sequences alignment and phylogenetic tree analysis indicated that the amino acid sequence of PaDGAT1 protein belongs to DGAT1 subfamily,which was closed to DGAT1 from Olea europaea.2.Cloning and Bioinformatics Analysis of PEPC Gene from Paeonia suffruticosa Andr.The Phosphoenolpyruvate carboxylase gene PaPEPC?The accession number in GenBank:MH106701?was cloned from the seed of Paeonia suffruticosa by the method of RT-PCR and RACE.The full-length cDNA of PaPEPC gene was 3524 bp with an open reading frame of 2898 bp encoding 965 amino acids.The PaPEPC gene encoding protein was a hydrophilic protein with molecular weight of 110.31 kD and the academic isoelectric point of 5.85.Prediction of secondary structure showed that the protein was mainlycomposed of the?-helix.Amino acids sequences alignment and phylogenetic tree analysis indicated that the amino acid sequence of PaPEPC protein belongs to C₃-PEPC protein,which was closed to PEPC from Gossypium arboreum.3.Expression Analysis of PaDGAT1 Gene from Paeonia suffruticosa Andr.The qRT-PCR analyses of PaDGAT1 gene showed that the expression level of the gene was the highest in bud,and was lower in leaf,stem and undeveloped ovary.During the seed developing,the expression level of PaDGAT1 gene showed a trend of increasing firstly,then decreasing and increasing once more.The expression level of the gene was the highest at 28 d,after that,the level came down gradually,and was increased again at 70 d with a higher level at 85 d.After harvest,the expression of the PaDGAT1 gene was increased with the highest level in the seed under room temperature after harvest for 7 d,then it was decreased by degrees.4.Expression Analysis of PaPEPC Gene from Paeonia suffruticosa Andr.The qRT-PCR analyses of PaPEPC gene showed that the expression level of the gene was the highest in bud,and was the lowest in leaf.During the seed developing,the expression level of PaPEPC gene was increased firstly at the early satages with a highest level at 28 d,then it began to decrease gradually.After harvest,the expression of the gene showed a trend of increasing firstly,decreasing after that,and the expression level was the highest when seeds were stored for 7 d under room temperature.