Screening Of SCN Induced Promoters And Function Analyze Of Legume-specific WRP1 And Exo70J Proteins From Soybean

Soybean (Glycine max) is one of the most important food and economic crops in the world and plays a key role to food security strategic layout of our country. Accordingly, the yield and quality of soybean are closely related with the life of all the people. In recent years, global climate changes has getting worse. Cold, heat, drought and flood disasters occurred more frequently in our country, which can also lead to increased susceptibility of diseases caused by microbial pathogens and other pests. As a result, soybean yield and quality are under the common threat of both biotic and and abiotic stresses. For improved development of soybean production in China, therefore, it is important to analyze the mechanisms of plant response to stress, to use genetics and molecular biology to identify the function of important genes, and ultimately to utilize biotechnology to improve the disease resistance, stress tolerance and quality of soybean.In this study, we have isolated and analyzed the promoters of a group of nematode-induced genes (NIGs) for their response to SCN. Using promoter-GUS fusion constructs, we have confirmed a substantial number of NIGs that are highly responsive to SCN infection, suggesting their possible roles soybean-SCN interactions. These NIG promoters will be useful as tools in the research and for genetic engineering of soybean SCN resistance.In this study, we have also functionally analyzed soybean GmWRPI and GmExo70J proteins using the amiRNA (artificial microRNA) and VIGS (Virus-induced gene silencing) approaches. Major findings from these experiments include:1. Using promoter-GUS fusion constructs indicates the induced condition of NIG promoter and transgenic hairy soybean roots, we have analyzed 32 putative NIGs for their expression through assays of the fused GUS reporter gene at five and ten days after SCN inoculation. We have identified two of the 32 genes to be specific SCN-inducible based on their promoter-GUS assays. The expression levels of these NIG genes increase in soybean roots after infection by SCN, suggesting their possible involvement in soybean-SCN interaction.2. We have obtained soybean transgenic hairy roots expressing GmExo70J, GmWRPl-amiRNA via the agrobacterium rhizogenes mediated procedure. These hairy roots were examined for nodule formation after inoculation with rhizobia bacteria. When compared with the control roots expressing the empty vector, the transgenic roots expressing the amiRNA for GmWRP1, GmExo70J7 and GmExo70J9 had reduced numbers of nodules. These results indicate that GmWRP1 and some GmExo70J proteins are required for the development of soybean nodules. To further study these genes, we have generated transgenic hairy roots containing the promoter-GUS constructs for the three genes. Through GUS reporter gene assays, we have found that the activity of GmExo70J7 and GmExo70J9 promoters is suppressed at three days after inoculation of rhizobia bacteria, suggesting a possible negative feedback mechanism of the regulation of the expression of the genes during nodulation.3. We have found that GmExo70J7 and GmExo70J8 are required for delaying the leaf senescence of soybean. Using virus-induced gene silencing (VIGS), we down-regulated the transcript levels of the two GmExo70J genes. The leaves of GmExo70J7- and GmExo70J8-silenced plants displayed earlier leaf senescence when compared with control plants, indicating that GmExo70J7 and GmExo70J8 play a negative role in the leaf senescence.