Preliminary Functional Analysis Of The Of GmSWEET9 From Soybean Nectary

Angiosperms developed floral nectary that reward pollinating insects.SWEET9(Sugars will eventually be exported transporter)gene family has been proved to be a sugar efflux transporter,specifically expressed in Arabidopsis thaliana,Brassica rapa and Nicotiana attenuate,which is necessary for nectary to secrete nectar.Soybean is a self-pollinating crop with low natural outcrossing rate.The utilization of heterosis has been proved to be one of the effective ways to break through yield.Improving the natural outcrossing rate of soybean is the key to the efficient seed production technology of soybean hybrids.The production of soybean hybrids with the help of insect pollination directly affects the outcrossing rate and the production of hybrids.Therefore,it is of great importance to determine the nectarsecreting gene of soybean nectary and to clone and analyze the function of nectary-secreting nectar GmSWEET9 gene to further improve the natural outcrossing rate of soybean.In this study,two candidate genes related to nectar secretion from soybean nectary were identified by bioinformatics analysis,named GmSWT9-1 and GmSWT9-2.The full-length CDS sequences of these two genes were cloned and the introductory vector and the vector for genetic transformation of Arabidopsis thaliana were constructed by Gateway system.Analysis of the expression of GmSWEET9 candidate genes in different tissues showed that GmSWT9-1 and GmSWT9-2 were highly expressed in soybean flower organs(Active,Post-active stage),while other genes such as nodules,stems,leaves and pods were low expressed.The expression of GmSWT9-1 in six soybean organs was higher than that of GmSWT9-2,the results showed that GmSWT9-1 may have a significant effect on the secretion of sucrose from soybean nectar..Analysis of the expression level of the homologous gene AtS WEET9 of soybean GmSWEET9 gene in Arabidopsis thaliana showed that the expression level in inflorescence(9-12,13-14 stages)of Arabidopsis thaliana was higher than that in stem,leaf and pod.Soybean nectary formed quickly during the Active stage of flower,and degenerated 24 hours after flowering.Arabidopsis nectary existed in the 9th to 17th stages of flowering.The results showed that the expression patterns of GmSWT91 and GmSWT9-2 genes secreting nectar from soybean nectary were similar to those of AtSWEET9 homologous genes in Arabidopsis thaliana,both of which were highly expressed in flower organs,and the expression of candidate genes synchronized with nectary development.The results of subcellular localization showed that GmSWT9-1 and GmSWT9-2 was located in plasma membrane and Golgi apparatus,which reflected that sucrose transport in nectary was through plasma membrane carrier rather than exocytosis.Potassium iodide staining was performed on nectary of floral organs of transgenic lines overexpressing GmSWT9-1 and GmSWT9-2,respectively.The number of modified stomata of single lateral nectary was counted,and there was no significant difference with that of control wild type(Col 0).At the same time,the amount of nectar secreted from the epidermis of the nectary gland did not increase.Based on the above results,we speculate that the genetic mechanism and metabolic network of GmSWT9-1 and GmSWT9-2 regulating nectary secretion genes in soybean may be complex,and their specific biological functions still need to be further explored and analyzed.