Cloning And Expression Analysis Of BnGPAT9 Gene From Brassica Napus

Glycerol-3-phosphate acyltransferase(GPAT;EC2.3.1.15)catalyzes the first step in the synthesis of various types of glycerolipids,including membrane lipids and triacylglycerol(TAG).Rapeseed oil is an important storage material,according to the study,each 1% increase in rapeseed oil was theoretically equivalent to a 2.5% increase in rapeseed production.In plant,GPAT9 is one of the important genes in plant glycerolipids metabolism,but the genetic research is not detailed in Brassica napus.We study focus on the following 3 aspects of GPAT9 gene.1.We cloned the BnGPAT9 gene(GenBank:KX344511.1)of B.napus,the sequence analysis showed that the gene was 1131 bp,encoding a protein of 376 amino acids with the molecular weights of 43.25 kDa,the instability index of 46.46 and the isoelectisc point of 8.97.BnGPAT9 encoded a protein of three TMHs without SignalP.The protein second structure of BnGPAT9 has 38.03% alpha helix,21.28% beta sheet,9.04% turn and 31.65% coil.BnGPAT9 possesses LPCAT1-like in Lysophospholipid acyltransferase superfamily and GPAT9 motifs.Homology analysis showed that BnGPAT9 protein was highly homologous to other GPAT9 proteins from different plants,and it was highly conserved in the process of biological evolution.2.qRT-PCR results showed that BnGPAT9 expressed specially in 1-4 week-seeds after flowering and in the stem,flower,leaf,root and silique wall.Abiotic stress results showed that the expression of BnGPAT9 was enhanced by PEG-drought and high temperature,but was inhibited by low temperature,6-BA,ABA and salt.Z pattern was found under waterlogging.3.Successful build pBI121-BnGPAT9 recombinant overexpression vector,and is transformed into agrobacterium.Using soil agrobacterium-mediated transformation of arabidopsis thaliana,it was expressed in arabidopsis thaliana.As a result,the oil content of the seeds is significantly higher than that of the wild type.Fatty acid composition analysis showed that BnGPAT9 gene has strong selectivity for oleic acid substrate,can increase oleic acid content and reduces linoleic acid and erucic acid content.