The Cloning And Preliminary Study On The Function Of EIN3-1, EIN3-2Gene Related To Phytophthora Sojae Resistance In Soybean

Soybean Phytophthora root and stem rot come from Phytophthora sojae Kaufmann&Gerdemann.It is a devastating disease that is spread all over the world, its a serious threat to soybean production. In China, the incidence of soybean root rot has become increasingly serious trend. Changes in P. sojae is very high, and disease-resistant varieties will lose their growth and changes in resistance and toxicity of the new race population. In the complication of development of the soybean genome and molecular biology aspects of soybean resistance mechanism has made great progress.The transcription factor of EDST3belongs to a small family of transcription factors, and it has been reported increasing resistance in tomato Arabidopsis thaliana, tobacco, and rice. In previous study, Enriched mRNA encoding EST, an increase in the cDNA library in abundance during infection with Phytophthora sojae was constructed by suppression Subtractive hybridization （SSH） cDNA bound microarrays from leaf tissues of high resistant soybean, and an EST homologous to AtEIN3showed differential abundance. Use RT-PCR to cloneThe full-length cDNA sequences of EIN3-1and EIN3-2genes from’Suinong10’total RNA. Experimental results were as follows:1. GmEIN3-1sequence of length （GenBank accession number XM₀03519439.2） and GmEIN3-2（GenBank accession number XM₀03545435.2） gene were separated from’Suinong10’by RT-PCR. Sequence analysis showed that GmEIN3-1comprised2486bp, containing a1845bp open reading frame which encoded a614residue, and GmEIN3-2comprised2576bp, containing a1833bp open reading frame which encoded a610residue. The EIN3-1encoding amino acids had a conserved domain from168-305and three nuclear localization signals, and the EIN3-2encoding amino acids had a conserved domain from168-305and two nuclear localization signals.2. Real-time PCR were studied for the expression pattern of EIN3-J and EIN3-2, and the results showed that the gene was induced by ET, P. sojae, ABA, and GA3. The mRNA abundance was significantly affected by ET and P. sojae.3. GFP fusion expression vector of GmEIN3-2was constructed and transformed into onion epidermal cells using Gene gun mediated transformation. The results showed GmEIN3-2was localized in the nucleus.4. EIN3-1and plant EIN3-2expression construct and transformed into transgenic tobacco by Agrobacterium mediation to overexpress carrier. A total of eleven PPT resistant EIN3-I-overexpression plants and six PPT resistant EIN3-2-overexpression plants were selected by PCR amplification. Transgenic tobacco was palyed with Phytophthora nicatianae, and it can very resistanced if it compared with control.5. EIN3-1and EIN3-2are transfer to soybean ’Dongnong50’; A total of20T2EIN3-1-overexpression plants were positive examined by PCR, and6T3EIN3-2-overexpression plants, of which one T2EIN3-2-overexpression plants was tested by Southern blot. The resistance of transgenic soybean paints playted with P. sojae is very if compared with control.