Cloning And Functional Study Of Soybean Squalene Epoxidase GmSQE3 Gene

Soybean is called"the vitamin of the 21st century"by nutritionists.It is the main source of plant protein and oil.Soybean saponins are a kind of important secondary metabolites formed during the growth and development of Soybean^[1].In this study,"Heinong 48"was used as experimental material to clone the sequence of squalene epoxidase GmSQE3 gene,the key enzyme in the synthesis pathway of soybean saponins,from soybean,analyze the expression pattern of the gene,and successfully transform the gene into tobacco and soybean hairy roots,so as to initially identify the function of GmSQE3 gene.The main results of this study include:1.qRT-PCR results showed that the expression of GmSQE3 gene was not significantly induced by high salt and drought stress,and had a downward trend.The expression was significantly up-regulated by Me JA and GA₃,and the relative expression was the highest in leaves.The content of total saponins in soybean induced by Me JA and GA₃was significantly higher than that of untreated control.2.GmSQE3 gene CDS was cloned from soybean,with a total length of 1647 bp and encoding 548 amino acids.Bioinformatics analysis showed that GmSQE3 protein had nadb＿Rossmann and PLN02985 domains.It contains two transmembrane domains,mainly located in the endoplasmic reticulum.Phylogenetic analysis showed that GmSQE3 protein was closely related to the homologous protein in Lathyrus sativus（Mucuna pruriens）.3.The recombinant plasmid p CPB-GmSQE3 was constructed and transferred into Agrobacterium tumefaciens.GmSQE3 gene was stably transformed into tobacco by leaf disc method.Three GmSQE3 gene positive plants were identified by PCR;GmSQE3gene was genetically transformed into hairy roots of soybean by Agrobacterium tumefaciens mediated method.PCR detection showed that the positive transformation rate of hairy roots was 89%.RT-PCR and q RT-PCR results showed that GmSQE3 gene in transgenic hairy roots was successfully expressed at the transcriptional level.4.GmSQE3 transgenic plants were treated with high salt（200 mmol/L Na Cl）and drought（20%PEG6000）.The results of phenotypic analysis showed that the leaves of transgenic tobacco and wild-type tobacco showed yellowing and chlorosis.The results of physiological indexes showed that under high salt and drought stress,the activities of SOD and APX of transgenic plants were significantly lower than those of wild type,and the activities of cat and MDA were significantly lower than those of wild type,indicating that the overexpression of GmSQE3 gene could not improve the resistance of plants to high salt and drought stress.5.The content of total saponins in hairy roots of GmSQE3 transgenic soybean complex plants showed that under normal conditions,the content of total saponins in transgenic hairy roots was significantly higher than that of empty vector hairy roots（P<0.01）,indicating that the overexpression of GmSQE3 gene was positively correlated with the synthesis of soybean saponins;Salt and drought stress could significantly reduce the content of total saponins in transgenic hairy roots（P<0.01）,and Me JA and GA₃could significantly increase the content of total saponins in transgenic hairy roots（P<0.01）.5.The content of total saponins in hairy roots of GmSQE3 transgenic soybean complex plants showed that under normal conditions,the content of total saponins in transgenic hairy roots was significantly higher than that of empty vector hairy roots（P<0.01）,indicating that GmSQE3 gene overexpression was positively correlated with soybean saponin synthesis;Salt and drought stress can significantly reduce the content of total saponins in transgenic hairy roots（P<0.01）,and Me JA and GA₃can significantly increase the content of total saponins in transgenic hairy roots（P<0.01）.