Genome-wide Identification,functional Analysis And Cold Stress Response Of Soybean Stearoyl-ACP Δ~9 Desaturase(GmSAD)

Soybeans are important global food crops and oil crops.In particular,soybean oil consumption accounts for 29.18% of the global vegetable oil market,and demand continues to increase.Analysis of soybean seed oil biosynthesis and regulation mechanism can provide theoretical guidance and key modification targets for genetic improvement of soybean oil yield and quality.Stearoyl-ACP Δ9 desaturase(SAD)is a key enzyme that determines the ratio of saturated and unsaturated fatty acids in plant cells.It catalyzes saturated stearoyl-ACP(C18:0)or palmitoyl-ACP(C16:0)produces the first double bond between C9-C10 of the fatty acid chain,thereby forming monounsaturated oleyl-ACP(C18:1)and palmitoleyl-ACP(C16:1).How many members of the soybean SAD family? Do members have different substrate selectivity? Are there any differences in the contributions of members to soybean seed oil biosynthesis accumulation? And are soybean SAD and members involved in other physiological life processes? No detailed reports on these scientific issues have been seen so far.In this paper,members of the soybean SAD family are identified by whole genome,and their protein structure and physical and chemical characteristics are analyzed.The spatiotemporal expression profile of each member of the soybean SAD is quantitatively detected,and the c DNA clones of each gene are isolated.The biological functions of soybean SAD members were identified by the over-expression in INVSc1 wild-type yeast,yeast mutant BY4389 over-expression,and the tobacco leaf tissue transient expression test system.In addition,the response of soybean SAD to cold stress was explored.The main research contents and results are as follows:1.Using the Arabidopsis SAD(AtSADs)protein sequence as the search sequence BLAST soybean genome database,a total of 5 SAD family members were identified.Bioinformatics tools were used to make preliminary predictions of the genetic structure and protein properties,structure,and degree of conservation of each member.It was found that these enzymes consists of homodimers and contain a conserved domain belonging to the ferritin family and the acyl-ACP dehydrogenase family.The number of introns of these five soybean GmSADs genes is quite different.The physical and chemical properties of GmSADs encoded proteins,such as length,relative molecular weight,and theoretical isoelectric point,also show different differences with the length of the gene coding sequence and the base ratio.Multiple sequence alignments and phylogenetic analyses have shown that members of the soybean SAD family are highly conserved.2.The expression of GmSADs in the root,stem,leaf,flower,pod and seed of the soybean variety Jack was investigated.The results showed that soybean GmSADs members had different expression patterns in different tissues and different developmental stages of soybean seeds.Higher transcription levels of GmSAD1 and GmSAD2 were detected in all tissues.GmSAD3 and GmSAD4 were not expressed in the tissues detected above,while GmSAD5 expressed at very high levels in the seeds,and gradually increased as the seeds matured.This coincides with the model of seed oil accumulation,indicating that different members have functional differences in evolution.GmSAD1,GmSAD2,and GmSAD5 participate in the growth and development of soybean seeds.And GmSAD5 is specifically expressed in developing seeds.3.Cloning the GmSAD1,GmSAD2 and GmSAD5 genes,and transferring the GmSAD1,GmSAD2 and GmSAD5 genes to tobacco leaves for heterologous expression analysis by Agrobacterium.The constructed plant expression vectors p CAMBIA1303-GmSAD1,p CAMBIA1303-GmSAD2 and p CAMBIA1303-GmSAD5 were transferred to Agrobacterium strain GV3101,respectively.Agrobacterium was used to infect tobacco leaves of N.benthamian by injection.PCR confirmed GmSAD1,GmSAD2 and GmSAD5 genes effectively expressed in tobacco leaves.Analysis of the fatty acid profile and total lipid content of transgenic tobacco leaves showed that GmSAD1,GmSAD2,and GmSAD5 can dehydrogenate saturated fatty acids into unsaturated fatty acids,have SAD function,and prefer to catalyze C18:0-ACP to produce C18:1-ACP,and promote the accumulation of total oil.4.Construct corresponding yeast expression vectors to verify overexpression in INVSc1 Saccharomyces cerevisiae and perform functional complementation verification in defective BY4389 Saccharomyces cerevisiae.Each recombinant yeast expression vectors p YES2.0-GmSAD1,p YES2.0-GmSAD2 and p YES2.0-GmSAD5 were transformed into INVSc1 yeast and defective BY4389 yeast,respectively.The expression was induced,and the fatty acid composition and content and total lipid content of the transgenic INVSc1 yeast and transgenic defective BY4389 yeast were analyzed,respectively.The results showed that GmSAD1,GmSAD2,and GmSAD5 all have SAD enzyme activity,and the stearoyl-ACP(C18:0-ACP)is used as the substrate to catalyze their dehydrogenation to form oleyl-ACP(C18:1-ACP).Expression in yeast can increase the total lipid content of transgenic yeast.5.Soybean seedlings were subjected to cold stress,and the expression and regulation of soybean GmSADs under these conditions were analyzed,so as to identify new functions of soybean SAD members under cold stress conditions.Soybean seedlings were cold-stressed at 4℃ for 0,4,8,12,and 24 h,respectively.The expression of GmSADs and the content of unsaturated fatty acids were detected.The results showed that under cold stress at 4℃,only the expressions of GmSAD1 and GmSAD2 were detected in soybean GmSADs,and the expression of GmSAD1 showed an increasing trend with the extension of cold treatment time.The expression of GmSAD2 was unstable.With the extension of cold stress time,the unsaturated fatty acid content of the seedlings increased first and then decreased,and the highest content was at 8 h,but the unsaturated fatty acid content of the treatment group was higher than that of the control group.In general,different GmSADs members have different functional characteristics.Only GmSAD1 and GmSAD2 participate in the regulation of soybean seedlings under low temperature stress,and under cold stress conditions,the unsaturated fatty acid content in plants all rises accordingly.