Cloning And Functional Analysis Of E2 And E3 Genes Encoding Enzymes Involved In Soybean Ubiquitylation System

Most of the plant growth and development are regulated by the ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway (UPP), the main proteolysis mechanism ubiquitous in all eukaryotes, consists of two major components:the ubiquitination machinery attaching a ubiquitin or a polyubiquitin chain to a substrate, and the 26S proteasome responsible for the degradation of protein substrates tagged by the polyubiquitin chain. Ubiquitin attachment is a multistep reaction involving three enzymes referred to as El (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin-protein ligase). With more than 1,300 genes predicted to encode for E3 components in Arabidopsis, the RING-type E3s account for more than 36%. Their known biological functions include photomorphogenesis, gametogenesis, floral development, seed development, hormone signaling, responses to biotic and abiotic challenges.Soybean (Glycine max (L.) Merrill) is one of the most important sources of vegetable protein and edible oil world-widely. However, environmental stresses such as drought, high salt and cold affect its growth and productivity extremely. Recent data suggest a linkage between protein ubiquitination and stress responses in plants. In this study, a cDNA clone encoding a novel RING protein, designated as GmRFP1, was isolated and characterized from soybean. As GmRFPl transcript was affected by stress treatments, we further focused on its functional analysis. It may be of great importance in stress-resistance breeding of soybean.The mostly results obtained in this study are as follows: 1. By RT-PCR and RACE approach, a cDNA clone encoding a novel RING protein, designated as GmRFPl, was isolated and characterized from soybean. GmRFPl was 1671 bp and contained a 1179 bp of ORF encoding a protein of 392 amino acids with a molecular mass of-43 kDa. The protein contained a RING-H2 motif and an N-terminal transmembrane domain. Sequence alignment indicated that GmRFPl protein shared more than 83.3% identity in the RING domains with other RING proteins. The GmRFPl transcript expression analysis showed that GmRFPl was observed constitutively in various tissues, indicating that it could be a house-keeping gene. We then explored the expression of GmRFP1 under different stresses. Through QRT-PCR approach it was found that the GmRFP1 transcript was up-regulated by abscisic acid (ABA) and salt stress, but down-regulated by cold and drought treatments.2. We then expressed and purified both wild type and mutant version of GmRFPl in E. coli. In vitro assays showed that the purifed GmRFP1 induced the formation of polyubiquitin chains while mutation within the RING finger region abolished the ubiquitination activity. To our knowledge, GmRFPl is the first functionally characterized E3 ligase gene encoding for RING finger protein in soybean, and RING domain is required for its activity. It may play unappreciated roles in ABA signaling and stress responses via mediating the ubiquitination and degradation of target proteins through the ubiquitin-proteasome pathway.3. To further address function of GmRFPl, we constructed the sense vector and antisense vector of GmRFPl gene. By Agrobacterium mediated transformation, transgenic tobacco plants were generated. The sense and antisense transgenic tobaccos were both confirmed by semi-quantitative RT-PCR and western blot. Sense transgenic seedlings over-expressing GmRFPl gene resulted in enhanced growth rate than controls under ABA stress and decreased tolerance to low temperature treatment; however, antisense repression of GmRFP1 enhanced the tolerance of transgenic plants to cold stress, suggesting GmRFPl might be a negative regulator of ABA signal transduction and cold responses. A proteomic approach was used to analyze differential expression of proteins among the leaves of the control, the over-expression of GmRFP1 and the repressing expression of GmRFP1 transgenic tobacco. Total 6 proteins were identified from NCBI database. These proteins were as follows:Annexin VCaB42, Thioredoxin H-type 1, Glucose-6-phosphate dehydrogenase, Ribosomal protein S15, Transcription elongation factor GreA and Maturase K. The result indicated that a decrease in the level of GmRFP1 protein leads to an increased level of Annexin VCaB42, Thioredoxin H-type 1, Glucose-6-phosphate dehydrogenase and Ribosomal protein S15.4. We isolated and characterized two E2s genes in soybean. They both contained a 447bp of ORF encoding a protein of 148 amino acids and a conserved Cys at the position 85. GmUBCc571 was expressed in leaves, stems, shoot apical meristem and flowers, but not found in roots and seeds; GmUBCc171 was observed in all detected organs, indicating that this gene could be a house-keeping gene. Through QRT-PCR approach it was found that GmUBCc571 transcript was up-regulated by drought stress and increased twice under ABA, cold and salt treatments. GmUBCc171 was not affected by ABA stress and and was up-regulated by cold and drought stress; under salt treatment GmUBCc171 increased twice. We further expressed and purified both GmUBCc571 and GmUBCc171 proteins in E. coli. In vitro assays showed that they were E2 ubiquitin-conjugating enzymes and could interacted with GmRFP1 E3 ubiquitin-protein ligase.