Bioinformatics And Gmole1 Gene Function Analysis Of Soybean Oil Body Protein Gene Family

Glycine Max oil is one of the important indicators of soybean quality.Glycine Max oleosin is a protein closely related to the formation and accumulation of oil in Glycine Max seeds.Oleosin is generally classified into H and L according to their molecular weight.The H type oleosin in Glycine Max is twice of L type.The " proline knot" structure in the amino acid sequence of the oleosin makes the oleosin highly conserved in the hydrophobic area,which makes the oleosin can anchor the oil body and maintain the stability of the oil body.Oleosin can regulate component content of fatty acid in oils and fats.Fatty acids can also be metabolized by enzymes on the oleosin to provide energy for seed germination.Therefore,the analysis and study of the structure and function of the members of the Gmole gene family were analyzed and studied to find the genes related to the component content of fatty acid,which can provide a theoretical basis for the improvement of Glycine Max quality.Main results of this study are as follows:In NCBI,Blast obtained 12 members of the Gmole gene family,which were distributed on chromosomes 4,5,6,10,16,17,19 and 20 respectively,and no genetic clusters were formed.The hydrophobicity index is near 1,indicating that the Gmole is hydrophobic protein.The family members are divided into two categories according to their molecular weight:(1)H includes Gmole1,Gmole2 and Gmole8;(2)L includes Gmole3~Gmole7 and Gmole9~Gmole12.The primary structure analysis showed that there was a highly conserved proline knot in the conserved regions of amino acid sequences.The second-level structure and structural domain prediction analysis showed that the oleosin was embedded in the oil body and formed a U like needle structure in combination with the oil body.By constructing phylogenetic tree,cluster analysis showed that the Gmole gene was highly conserved and the relationship between members was far away.RNA was extracted from Glycine Max(Jiyu 72),and Gmole1(813bp),Gmole2(706bp)and Gmole8(808bp)were cloned by RT-PCR.From the beginning of the soybean pod period to full maturity,a sample was taken every 5 days as a maturity,and the relationship between the expression of Gmole1,Gmole2 and Gmole8 genes in the seed maturation process and the component content of fatty acid was analyzed by PCR.It was found that the expression level of Gmole1 was positively correlated with the content of C16:0,C18:0,C18:1 and C22:0,and was negatively correlated with the changes of linoleic acid(C18:2)and linolenic acid(C18:3),and the level of Gmole2 and Gmole8 was not related to the component content of fatty acid.A primer clone Gmole1 with EcoR? and Hind III cleavage sites was designed to construct plant expression vector of pCAMBIA-3301-Gmole1.The three-parent hybrid method is transferred to agrobacterium.Agrobacterium tumefaciens mediated transformation of Arabidopsis thaliana by inflorescence impregnation was screened with 1% concentration of herbicide and PCR was verified to be positive.The comparison of the germination state and physiological and biochemical indexes of transgenic Arabidopsis found that under the light conditions,the transgenic Arabidopsis and the wild type seeds were close to the final germination rate,but under the dark conditions,the transgenic Arabidopsis seeds were higher than the wild Arabidopsis seeds,indicating that the transgenic Gmole1 was beneficial to the seed germination.The content of palmitic acid(C16:0)in Arabidopsis seeds with Gmole1 gene increased by 10%,linolenic acid(C18:3)decreased by 9%,and the total soluble protein content increased slightly,but it was not significant,indicating that Gmole1 could change the proportion of palmitic acid(C16:0)and linolenic acid(C18:3)in seeds.