Functional Verification Of GPAT Gene From Ammopiptanthus Nanus

Ammopiptanthus Nanus mongolicus that can grow in a very harsh natural environment.It has a 18-chromosome diploid and is highly tolerant to stress and is a very good cold-tolerant plant.The fatty acid unsaturation of phosphatidylglycerol(PG)in plant chloroplast membranes is closely related to the temperature of membrane lipid transition and cold resistance of plants.Glycerol-3-phosphate acyltransferase(GPAT)is the first acyl esterification enzyme in the process of PG biosynthesis,which plays a key role in determining the degree of unsaturation of plant membrane PG.The GPGP gene AnGPAT gene was obtained by PCR and the AnGPAT gene was transferred into tobacco to obtain the transgenic T2 generation tobacco of AnGPAT gene.The transgenic tobacco of AnGPAT gene was inherited in this study.Verification was performed to compare the changes in cold resistance physiological indices of wild-type and AnGPAT genes,and to further identify the function of the AnGPAT gene.The main results of this study are as follows:1.The genomic DNA of wild type and transgenic tobacco seedlings was extracted,AnGPAT gene was obtained in transgenic tobacco by PCR amplification,and the AnGPAT gene was confirmed to have been transferred into tobacco by Southern blot genetic verification.2.The ELISA enzyme-linked immunosorbent assay was used to determine the GPAT enzyme content in wild-type and AnGPAT transgenic tobaccos under low temperature stress.The changes of GC-MS fatty acid contents indicated that the integrated AnGPAT gene tobacco was transformed with the increase of cold stress time.The GPAT enzyme content and unsaturated fatty acid content in GPAT were higher than those in wild type tobacco,which was consistent with the selectivity of GPAT substrate.The transgenic tobacco with AnGPAT gene showed higher cold resistance.3.To identify the tobacco phenotypes of wild-type and AnGPAT transgenic tobaccos,and to measure the aboveground and underground fresh weight,dry weight,and electrical conductivity of the wild-type and transgenic AnGPAT genes under low temperature stress,and the content of malondialdehyde.Changes in acid content and other related indicators revealed that the degree of injury to tobacco cells transfected with AnGPAT gene under low temperature stress was lower than that of wild-type tobacco,and the transgenic tobacco was more tolerant to cold,further verifying the cold resistance of AnGPAT gene.