Promoter Cloning And Functional Analysis Of Soybean GmExo70J

Legumes,whose economic and nutritional value are second only to the grass family,are an important food crop and economic crop in the world.Their roots can fix atmospheric nitrogen through symbiosis with rhizobia in soil to improve soil fertility.Legumes are grown on 15%of the world's arable land,providing about 33%of human dietary nitrogen in the world and 60%in developing countries.Currently,legumes are also increasingly recognized as a potential source of biofuels.Despite their importance,our understanding of their unique traits such as the evolution of symbiotic nitrogen fixation and related processes is still very limited.During the analysis of the soybean WRKY family,we have recently found an EXO70 protein subfamily(GmExo70J)unique to legume plants.Some of the GmExo70J proteins are localized in the Golgi apparatus through the N-terminal transmembrane domain.In addition,silencing of some GmExo7OJ genes could lead to premature senescence of soybean leaves and reduction in the number of root nodules.In this study,the tissue-and development-specific expression patterns of 12 soybean GmExo70J genes were further analyzed using the GUS reporter gene in transgenic Arabidopsis thaliana carrying GmExo70J promoter-GUS vector.In addition,the responses of GmExo70Jpromoters to dark and heat stress were also determined.The promoters of GmExo70J7 and GmExo70J8 were also dissected to identify the core elements responsible for the tissue-specific expression patterns.Finally,the responses of GmExo70J7 and GmExo70J9 genes to rhizobia and the changes of soybean nodul in gene after the suppression of GmExo70J7 and GmExo70J9 gene expression were examined.Major results from these experiments are as follows:1.The expression of GmExo70J genes is high in the tissues where the exocytosis is active,such as the meristem,the elongation area,the vascular tissues of seedling roots,anthers,sepals,pistil stigma at the flowering stage.The results suggested that GmExo70J proteins played an important role in the growth and development of soybean.2.The expression of some GmExo70J genes responded to dark stress and heat stress.Under the dark stress,even though the expression levels of some GmExo70J genes displayed slight increased during the first two days,their overall levels of expression were generally reduced,based on significantly reduced GUS staining.Under the heat stress,the expression levels of some GmExo70J genes generally increased sharply and then declined,as indicated from the intensity of GUS staining.3.The dissection of the promoter of GmExo70J7 and GmExo70J8 showed their core sequences were between 128bp?315bp and 153bp-543bp upstream of their translation initiation codons,respectively.4.The role of GmExo70J genes in soybean nodulation was also analyzed.The expression of GmExo70J7 and GmExo70J9 were significantly down-regulated after the infection of Rhizobia.Furthermore,the expression of the N-20 and ENOD55-2 nodulin genes associated with nodulation were significantly down-regulated after the suppression of GmExo70J7 and GmExo70J9 gene expression using artificial microRNA-mediated gene silencing.These results suggest that the legume-specific GmExo70J proteins are involved in the process of legume nodulation.