Cloning,Expression And Functional Analysis Of A Cold Related Gene GPAT In Forsythia

Low temperature is the main factor limiting the distribution and growth of trees and crops,and the damage caused by the sudden drop of temperature is huge,which causes great economic losses.Forsythia to Oleaceae Forsythia species,It is divided into Forsythia mandshurica and Forsythia suspense.Fatty acid unsaturation and cold sensitivity are closely related in the plasma membrane of higher plants.Glycerol-3-acyltransferase(GPAT)is the first acyl esterified enzyme in the biosynthesis of PG(Phosph-atidglglycerol),which directly determines the unsaturation of plant membrane PG.In cold resistant plants,GPAT preferentially chose 18:1 as substrate.In cold sensitive plants,GPAT preferred 16:0 as substrate.This leads to the saturation of the two biofilm and its fluidity at low temperature,which affects its cold tolerance.In this study,GPAT gene was cloned from two materials of Forsythia,Forsythia suspensa and Forsythia mandschurical,which were named as FsGPAT and FmGPAT.(1)Sequence analysis showed that the length of two gene sequences were 1347bp that encoded 448 amino acids.Forsythia GPAT gene has the conserved domain GPAT N domain and LPLAT domain of GPAT gene family.(2)Forsythia GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsisthaliana(ATS1,At1g32200).ATS1 is the only one located in the chloroplast Arabidopsis thaliana GPAT gene family.This study showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant,consistent with the predicted results.Forsythia GPAT is samed with Sesamum indicum and Ipomoea nil nearly 74%.(3)Connecting the FsGPAT gene coding region with the expression vector pCAMBIA 1302,Construction of eukaryotic expression vector pCAMBIA 1302-FsGPAT,loaded in Agrobacterium LBA4404,Agrobacterium mediated transformation of Tobacco callus to obtain transgenic Tobacco with FsGPAT gene.In addition,the PCR gene was amplified by transgenic Tobacco,which proved that FsGPAT gene had been integrated into the genome of transgenic Tobacco.(4)The expression of GPAT in different tissues showed that the expression of GPAT increased with the decrease of temperature,which indicated that the expression of GPAT was related to the cold resistance of plants.This study will provide an important reference for further study on molecular mechanism of cold resistance of Forsythia suspense,and lay a theoretical foundation for improving the cold resistance of crops or trees by transgenic means.