Identification Of Sulfate Transporter Gene Family In Soybean And Functional Analysis Of GmSULTR1;2b

Sulfur is the fourth nutrient element following the nitrogen,phosphorus and potassium necessary for the plant.In plant,sulfur-containing organic compounds are involved in many important physiological and biochemical processes,play important roles to withstand envi-ronmental stresses,and in growth and development.Sulfur assimilation includes absorption and activation of sulfate,synthesis of cysteine and sulfur-containing secondary metabolites.Soybean(Glycine Max(L.)Merr.)is one of important economical crops,that is the main source of vegetable protein and oil for humankind.The amino acid content of soybean is not well balanced,with very low content of sulfur-containing amino acids,and therefore with limited nutritional value.Study on molecular mechanism and regulation of sulfur metabolism,it hopes to improve the content of sulfur-containing amino acids by genetic engineering reg-ulating SO42-flow in sulfur metabolic pathway of distribution.In this study,we identified 28 SULTR genes in soybean genome by bioinformatics anal-ysis.The identity of protein sequence between soybean and Arabidopsis thaliana is 53%to 77%.And the similarities of protein sequence between soybean and Arabidopsis is 69%to 87%.These genes are divided into 4 sub-families,SULTR 1?SLUTR4,by cluster analysis-soybean and Arabidopsis protein sequences.There are 6 genes in SULTR 1,7 genes in SULTR2,13 genes in SULTR3,2 genes in SULTR4.These putative genes located at 15 of 20 chromosomes.The genetic distance between SULTR1 and SULTR2 is 0.691,and the ge-netic distance between SULTR4 and each of other 3 sub-families is even greater than 1.The length of genomic DNA sequence of 28 putative SULTR genes range from 4232 bp to 10003 bp,CDS sequence range from 1794 bp to 2603 bp,the deduced proteins sequences are from 457 to 702 amino acids,the molecular weights are from 42.99 to 76.98 kDa,and the predicted isoelectric points are from 9.06 to 10.5.All of putative SULTR proteins contains 12 trans-membrane structures,N-terminal polar amino acid-rich conserved motif and C-terminal STAS conserved domain.We cloned 5 genes of GmSULTRl;2a,GmSULTRl;3a,GmSULTR2;3,GmSULTR3;4d,GmSULTR4;2 and analyzed their sequences.We constructured the plant expression vectors of 4 genes for transient expression of onion epidermal cells.Proteins of GmSULTR1;2a,GmSULTR1;3a,GmSULTR2;3 and GmSULTR3;4d localized in the plasma membrane.In addition,we constructured the yeast expression vectors of GmSULTR2;3 and transformed in CP154-7A yeast mutant.The transformed gene could complement the defect of CP 154-7A that cannot import the SO42-.It is suggested that the GmSULTR2;3 protein can tranport SO42-.We analyzed the expression pattens of several SULTR genes in different tissue or under infection of Bradyrhizobium japonicum,Phytophthora sojae and rust(Phakopsora pach-yrhizi)fungus,under cold,drought and flooding sterss using DNA microarray data in the GEO and EMBL-EBI database.The results showed that the GmSULTR1;2b gene specifically expressed in root.The GmSULTR1;lb gene expressed highly in root,flower and leaf.GmSULTR2;1b highly expressed in root,leaf and seed coat parenchyma,and GmSULTR2;2b expressed in leaf,seed coat aleurone and parenchyma.Seven genes in SULTR3 expressed in different parts of the seed,however,GmSULTR3;4a specifically expressed in root,GmSULTR3;5a expressed in root,pollen and seed coat hourglass.Interestingly,the expres-sion of GmSULTR2;3,GmSULTR3;4a and GmSULTR3;5a were up-regulated during infec-tion of B.japonicum,it is suggested that the three genes may be responsible to transport the SO42-to nodule.And the expression of at least seven genes changed in varying degrees,when with infection of P.sojae after 6 to 12h.During infection of rust,at least 6 genes were down-regulated in resistant variety PI4590258,but there were no change in non-resistant varietie Williams.Moreover,the expression level of many genes in SULTR2 and SULTR3 up-regu-lated or down-regulated,during suffering the abiotic stresses of flooding,drought or cold.Under sulfur deficiency conditions,the expression of GmSULTR1;1b and GmSULTR1;3a genes increased significantly in root after suffering 5 days,however,GmSULTR1;2a and GmSULTR1;2b highly expressed in root after suffering 1 day sufur deficiency,the expression of the two genes decreased with the extension of time suffering sufur deficiency.Under 0.2 mM metal cadmium treatement,the expression of GmSULTR1;1b increased significantly,and GmSULTR1;3a increased slightly after 3 days,GmSULTR1;2b has no significant changed.However,the expression of GmSULTR1;2a decreased.Taken together,the soybean SULTR genes are in response to environmental stresses to different extents.GmSULTR1;2b gene is specifically expressed in root,and can in response to environ-mental stresses.Full length cDNA of GmSULTR1;2b was isolated from soybean(cv.N2899),containing a 1980bp ORF,which was deduced to encode a peptide of 659 amino acids.The deduced protein sequence possesses 12 trans-membrane structures,N-terminal polar amino acid-rich conserved motif and C-terminal STAS conserved domain.And it was localized in the plasma membrane,could complement the defect of CP 154-7 A that cannot import the SO42-.To further investigate function of GmSULTR1;2b,we constructed plant expression vector pMDCS3-GmSULTR1;2b.And it was transformed into tobacco using Agrobacterium-mediated method.The results showed that the content of SO42-in the overexpression trans-genic tobacco plants roots,the content of total sulfur in all overexpression transgenic plants,the content of sulfur-containing amino acid and soluble protein in overexpression transgenic tobacco leaves improved compared to control tobacco plants.In addition,photosynthetic rate and chlorophyll content also increased in overexpression transgenic tobacco.To be measured the biomass and seeds thousand-grain weight in tobacco plants,compared to control plants,the biomass significantly increased,and the thousand-grain weight of seeds also increased almost 8%.Altogether,it is indicated that GmSULTRl;2b is participation in SO42-absorption,and it can contribute to the assimilation of sulfur metabolism,improves sulfur amino acid content and yield of plants overexpressing GmSULTRl;2b.