Cloning And Functional Analysis Of GmABCA7 In Soybean

Soybean?Glycine max?is one of the most important oil crops in China and in the world.In recent years,the domestic demand for soybean is increasing,but traditional soybean breeding is insufficient to meet people's needs in China.From the perspective of long-term development,we need to cultivate high-yield soybean varieties with independent intellectual property urgently.The successful seed germination and seedling establishment is essential for plant growth and propagation.In agricultural production,early and even emergence of seedlings is the first step to obtain high yield of crops.Compared with the cereal crop seeds,including rice,wheat,and maize,soybean seeds contain much higher oil and fatty acid contents.Thus,seed ageing happens easily during storage,resulting in reduced seed germination and seedling emergence,thereby affecting the high and stable yield.Therefore,to speed up the soybean breeding process and cultivate more high-vigorous germplasm resources through genetic engineering and traditional breeding techniques will help solve the problem of soybean demand in China.The oil in soybean seeds is mainly in the form of triacylglycerol?TAG?,providing carbon source and energy for seed germination and seedling establishment.During germination and seedling development,TAG breaks down into fatty acids,which are then converted to succinate in peroxisomes through fatty acid?-oxidation and glyoxylate cycles,and ultimately produces sucrose through the gluconeogenic pathway.At present,research on oil seed germination was focused on plant hormones,plant growth regulators and illumination.However,there are few reports on the study of seed germination in terms of molecular mechanisms such as peroxisomes and fatty acid?-oxidation.Previous studies have reported that peroxisome-localized Arabidopsis ABC transporters were involved in the balance of intracellular lipid metabolism,and the related mutants such as atped3 cannot germinate normally.ABC transporters are an important class of transmembrane proteins that are widely present in organisms,and their structures and functions are highly conserved.Whether or not there is similarly localized peroxisome ABC transporter in soybean affecting fatty acid?-oxidation and seed germination has not been reported worldwide.We identified a new ABC transporter gene GmABCA7 from soybean cultivar Heinong 44 based on soybean transcriptome and genome database.GmABCA7 CDS sequence has a length of 2865 bp,which encodes 954 amino acids.GmABCA7 protein sequence from cultivar Heinong 44 has one amino acid substitution?R82H?compared to the sequence of Glyma17g10670 from cultivar Williams 82.Quantitative RT-PCR?qRT-PCR?data showed that GmABCA7 was expressed in various tissues?SAM,root,stem,leaf,flower,cotyledon,pod and seeds at different developmental stages?of soybean,with the highest expression in seeds.The gene was highly expressed between14 and 22 DAF?Days after flowering?and peaks at 18 DAF in developing seeds.These results suggested that GmABCA7 might be involved in the regulation of seed development in soybean.Soybean GmABCA7 and peroxisome-marker protein were co-expressed in tobacco leaf epidermal cells,it was found that GmABCA7 completely coincides with the fluorescent signal of the marker protein.We further isolated the protoplasts from these leaves,and GmABCA7 signal and the marker protein signal also showed similar signals as the tobacco leaves.These results indicated that GmABCA7 is localized at the peroxisomes.GmABCA7 was transformed into wild type?WT?in Arabidopsis by floral dip with agrobacterium,and three homozygous transgenic plants?OE-1,OE-3 and OE-4?were screened.Developing seeds at 14 DAF and dry seeds from WT and GmABCA7overexpression plants were observed under stereomicroscope.We found that there was no significant difference in seed size and 1000-seed weight between transgenic lines and WT.These results indicated that GmABCA7 might not affect the seed-related phenotypes.A large amount of lipids were stored in the oilseeds.To verify whether the overexpression of GmABCA7 in Arabidopsis affects the level of major fatty acids in the seeds,the contents of fatty acid were measured in seeds.We found that overexpression of GmABCA7 significantly reduced the levels of C18:1 and C18:2 in OE-1,while levels of C18:1 and C18:2 in OE-3 were significantly increased.The C18:3 levels had a significant increase in all three lines analyzed?OE-1,OE-3 and OE-4?.In comparison with WT,the C20:0 levels significantly increased in over-expressing lines?OE-1 and OE-4?.However,no significant change in total fatty acid content was observed.These results suggested that GmABCA7 overexpression altered the composition of fatty acids,but did not change the total content of fatty acid in seeds.Sugar,protein and starch are also the main storage substances in seeds.To determine whether overexpression of GmABCA7 affects the sugar and starch content of the seed,we examined the sugar and starch content of the GmABCA7 transgenic lines.The results showed that there was no significant change in the content of sugar,protein and starch in mature and dry transgenic seeds,except the content of fructose in OE-3and OE-4 increased significantly.In summary,overexpression of GmABCA7 had no significant effects on the accumulation of lipid,sugar,starch and protein in seeds.Since GmABCA7 is localized at peroxisomes,we further investigate whether GmABCA7 is involved in the fatty acid degradation metabolic pathway and affects seed germination.The WT and GmABCA7 transgenic seeds were grown on 1/2 MS medium,and the radicle protrusion was studied.We found that 20%more seeds from the three transgenic lines had prominent radicles at 24 h after sowing,while only 10%radicle protrusions were observed in the seeds of WT;compared to WT,germination rates of the transgenic lines were 12.96%to 18.52%higher.All the seeds germinated at48 h after sowing.These results suggested that overexpression of GmABCA7 promoted seed germination in Arabidopsis.Plant hormones abscisic acid?ABA?and gibberellin?GA₃?play important roles in the regulation of seed dormancy and germination process.We studied germination rates of seeds treated with exogenous ABA and GA₃.We found that germination rates of the transgenic seeds were higher than that of the WT seeds upon 0.3?M,0.5?M and 1.0?M of ABA treatments.Germination rates of the transgenic seeds were also significantly higher than that of the WT upon 0.3?M,0.5?M and 1.0?M of GA₃ treatment.These results indicated that ABA and GA₃ might not change the earlier germination phenotype of GmABCA7 transgenic seeds comparing to WT.To further investigate the function of GmABCA7 during seed development and germination,we examined succinate and malate levels of the gluconeogenic pathway precursors.The levels of succinate and malate in mature dry seeds were significantly higher compared to WT.This result indicates that GmABCA7 has a positive impact on the?-oxidation process during seed development,thereby promoting the accumulation of succinate and malate in seeds.Then,we soaked the seeds with distilled water at 37°C for 72 h to initiate seed germination.The succinate and malate levels in GmABCA7 overexpression lines were significantly up-regulated,probably due to the highly expressed GmABCA7 during germination in transgenic lines.These results suggested that overexpression of GmABCA7 could enhance?-oxidation in transgenic Arabidopsis seeds.In summary,overexpression of GmABCA7 affects?-oxidation of fatty acids and promotes seed germination in Arabidopsis thaliana.This provided a theoretical basis for studying the regulation of plant lipid metabolism by soybean ABCA transporters,and provided a new strategy for accelerating the breeding process,cultivating high-vigorous seeds and achieving high yield and stable yield of crops in agricultural production.