Cloning And Functional Identification Of SEIPIN Family Gene Of Soybean

Soybean is one of the important oil crops in the world.Soybean oil is mainly composed of five fatty acids,among which oleic acid,linoleic acid and linolenic acid are the main unsaturated fatty acids in soybean oil,which may play a role in the prevention and treatment of certain cancers and heart diseases.The content of unsaturated fatty acids is directly related to the quality of soybean oil.With the improvement of people's living standard,people need to consume more and better soybean oil.In addition,soybean oil as an energy material,can be used in industrial production.Therefore,it is an important breeding target to cultivate soybean varieties high in oil and unsaturated fatty acids.Berardinelli-Seip congenital lipodystrophy 2(SEIPIN)plays an important role in the formation of lipid droplets in human,yeast and Arabidopsis thaliana.However,the function of the SEIPIN family gene has not been investigated in soybeans.In this study,four SEIPIN family genes of soybean were cloned,and GmSEIPIN1 A,GmSEIPIN2A and GmSEIPIN2 B genes of the family were transformed into yeast,Arabidopsis thaliana and soybean root by transgenic technology,which verified the function of SEIPIN family genes.In order to improve the oil content of soybean by genetic engineering and create new high-oil soybean varieties,effective genetic resources are provided.The main results of this experiment are as follows:1.According to the similarity of amino acid sequences of four soybean SEIPIN family proteins with At-SEIPIN1 and At-SEIPIN2 proteins,the soybean SEIPIN protein family was divided into GmSEIPIN1 and GmSEIPIN2.At the same time,SEIPIN proteins of soybean and other legumes was divided into 2 subfamilies according to the results of phylogenetic tree analysis,which was different from three subfamilies of Arabidopsis thaliana.There is one SEIPIN1 protein in Arabidopsis and only one SEIPIN1 protein in other legumes including alfalfa,chickpeas and peanuts.Wild beans and soybeans evolved into two.There is one SEIPIN2 protein in Arabidopsis thaliana and two SEIPIN2 proteins in legumes.2.The results of fluorescence real-time quantitative PCR showed that the three genes of SEIPIN family were highly expressed in the young embryo of soybean,but not in the root and leaf.The prediction results of transmembrane structure showed that GmSEIPIN1 A had two transmembrane domains and typical characteristics of SEIPINprotein.GmSEIPIN2 A and GmSEIPIN2 B have three transmembrane domains.The results of subcellular localization of GmSEIPIN1 A,GmSEIPIN2A and GmSEIPIN2 B showed that all three proteins were located in the endoplasmic reticulum.3.In this study,the functions of soybean genes GmSEIPIN1 A,GmSEIPIN2A and GmSEIPIN2 B were identified.Through the repair experiment of yeast SEIPIN deletion mutant,we found that GmSEIPIN1 A,GmSEIPIN2A and GmSEIPIN2 B have different effects on the size of lipid droplet(LD).Among them,GmSEIPIN1 A and GmSEIPIN2 B significantly increased the size of LD.The oil content of yeast mutants mutated to GmSEIPIN1 A,GmSEIPIN2A and GmSEIPIN2 B by efficient gas phase technology was determined,and the results showed that all three genes could improve the oil content of yeast and increase the proportion of unsaturated fatty acids.In order to verify the function of GmSEIPIN1 A,GmSEIPIN2A and GmSEIPIN2 B genes in seed oil production,we obtained overexpression of 3 genes in Arabidopsis thaliana.Compared with wild types,the oil content of overexpressed Arabidopsis seeds increased significantly.In addition,the 1000-seed weight of Arabidopsis thaliana dry seeds also increased significantly,with the highest increases of 45.82%,23% and 27.97% respectively.To verify the function of GmSEIPIN2 B in soybean.We overexpressed the GmSEIPIN2 B gene using the soybean hair root system.The results showed that the oil content in the hair root of transgenic soybean increased by 1.08 times than that of the control group.In summary,our results indicate that the soybean GmSEIPIN2 A,GmSEIPIN2A and GmSEIPIN2 B genes regulate the synthesis of LD and increase the oil content.