Identification And Preliminary Functional Analysis Of Mycorrhizal-induced Gm Sweets Genes In Soybean.

Arbuscular mycorrhizal fungi(AMF)form mutualistic relationship with above 80 percent of terrestrial plants.The exchange of nutrients and carbon occurs during the symbiosis processes between AMF and plants,thus both host plants and fungi can supply more benefits for rewarding their partners reciprocally.Sugars Will Eventually be Exported Transporters(SWEET),a novel class of sugar transporters,functions for effluxing sucrose and monosaccharides,which might be the key to unload sucrose from the phloem to the sink organs,such as roots.Until now,sugar transporters which are responsible for exporting sucrose to the symbiotic interface are not found in mycorrhizal plants.In addition,there are few reports on the mycorrhizal-induced SWEET transporters in soybean.In the present study,mycorrhizal-induced GmSWEET genes were identified in soybean through gene expressions analysis under different phosphorus(P)treatments and AMF inoculation conditions,and the preliminary molecular and physiological function analyses were carried out in order to explore the important roles of GmSWEET genes in carbon allocation during mycorrhizal symbiosis.The results were as follows.1.The results from sand and soil culture experiments with different AMF inoculation treatment showed that P and AMF inoculation treatments significantly affected soybean growth.High P application dramatically promoted plant growth compared to low P application.Inoculation with two different AMF significantly improved plant growth.Realtime quantitative PCR analysis was performed on 29 candidate GmSWEET genes,and found that five and three GmSWEET genes were up-regulated by AMF inoculation in shoots and roots,respectively.2.Bioinformatics analysis showed that GmSWEETs gene family fell into 4 clades.Two mycorrhizal-induced genes,GmSWEET6 and GmSWEET15,belonged to Clade Ⅲ.GmSWEET6 and GmSWEET15 had high similarity,up to 87 percent when comparing their gene structures,protein structures and molecular weights.3.The prediction of cis-acting elements of GmSWEET6 promoter as well as GUS staining analysis showed that there were some elements related to the metabolism and synthesis of sugars,pathogen infection,hormone response and symbiosis with microorganism in the region of GmSWEET6 promoter.The results from GUS staining of the transgenic soybean plants containing GmSWEET6 promotor showed that GmSWEET6 was mainly expressed in leaves,roots and seeds under low P conditions,followed by pods.No detectable GUS staining in flowers and stems was observed.GUS staining was welldistributed in old leaves,but was not well-distributed in functional leaves,and very low in young leaves.4.The gene functional analysis of GmSWEET6 showed that over-expressing GmSWEET6 dramatically increased plant height of the transgenic plants,but obviously decreased biomass and P content of roots compared to the empty vector controls under AMF inoculation conditions.However,the opposite results were showed in RNA interference(RNAi)transgenic plants.Over-expression and knockdown of GmSWEET6 had no effect on biomass and P content of shoots,and AM colonization.5.The gene functional analysis of GmSWEET15 showed that over-expression of GmSWEET15 dramatically increased biomasses of shoots and roots,and root P content of the transgenic plants compared to the empty vector controls under AMF inoculation conditions,but decreased AM colonization.In summary,GmSWEET6 and GmSWEET15 were both mycorrhizal-induced SWEET genes.They possibly play a role in carbon allocation of plant and mycorrhizal symbiosis through affecting AM colonization in different ways.