Establishment Of The Agrobacterium Rhizogenes-mediated Transformation Of Soybean And Function Of GmFRD3under Al Stress In Soybean

Approximately30%of total land area around the world consists of acid soils,and almost70%of the world’s potentially arable lands are acidic（pH<5）. Aluminum(Al) toxicity is an important factor in limiting crop production.High concentrations of solublealuminium (Al3+) inhibit root elongation and restrict their ability to acquire water andnutrients. Many plant species relies on the release of organic anions from roots toresisitant Al. The gene controlling this trait are members of two gene families calledthe aluminium activated malate transporter (ALMT) family and multidrug and toxiccompound exudation (MATE) family.Soybean relies on the release of citrate fromroots to resisitant Al. The gene named GmFRD3of soybean belongs to MATEfamily.The function of GmFRD3on aluminium tolerance is not investigated inSoybean.To investigate the mechanism of aluminium tolerance in Soybean,we establisha high efficient Agrobacterium rhizogenes-mediated transformation and get the cDNAsequence of GmFRD3from jiyu70. The construction and function were analyzed toinvestigate the role of GmFRD3in the aluminium tolerance in Soybean. The results ofthis experiment are presented below:1.A high efficient Agrobacterium rhizogenes-mediated transformation isestablished by using K599. Using the method described here, one can expect thatalmost all of the A. rhizogenes-infected plants develop hairy roots.On average,30-60%. of the hairy roots are transgenic root.2. GmFRD3and its promoter were cloned from the root tipes of Soybean byRT-PCR.The ORF of GmFRD3is1674bp encoding a polypeptide of558amino acids.The protein of GmFRD3has12transmembrane domains and6intracellular loops.The ORF of the promoter is1543bp.we transformed it in tobacco leaves,the resultshows the promter has activity. 3. The green fluorescent protein（GFP） fused expression vectors of GmFRD3was constructed and transformed into Arabidopsis protoplast.The results indicatedGmFRD3is localized in the cell membrane.4. We analyzed the expression patterns of GmFRD3by quantitative real-timePCR. The results indicated GmFRD3had no tissue-specific expression characteristics butexpressed higher in roots than in other tissues and the expression value increased begain at4hand peak at8h.The result shows that GmFRD3respond to aluminium intensively.5.The coding region of GmFRD3was cloned into over-expression vectorpCAMBIA3301,transformed it and the vector into Soybean to analysis its function bystaining Hematoxylin and measuring the rate of citrate release.Compared with thecontrol (WT and the Vector), overexpression of GmFRD3genes not only enhancedcitrate exudation but also improved Al tolerance in transgenic hairy roots.The resultshow that GmFRD3plays an important role in resisitant to Al in Soybean.