Cloning And Functional Analysis Of Maize Sugar Transporter ZmSWEET15a Gene

The sugar transporter（SWEET）gene family is a new type of sugar transporter,which maintains the balance of hexose and sucrose in plants by regulating the distribution of carbohydrates.It is a key gene family affecting the growth and development of plants and physiological metabolism.In this study,q RT-PCR was used to analyze the expression pattern of ZmSWEET15a gene in different tissues,different sugar treatments and different developmental stages of leaves,and bioinformatics analysis was used to identify the physicochemical properties of ZmSWEET15a gene.Through prokaryotic expression,yeast heterologous expression and subcellular localization,the function of ZmSWEET15a gene was analyzed from the perspective of protein.The plant overexpression vector p CAMBIA3301-ZmSWEET15a was constructed and transferred into maize inbred line H8185 by agrobacterium-mediated method.The positive plants were obtained by phenotypic identification,molecular detection and agronomic traits investigation.The function of ZmSWEET15a gene was further analyzed from soluble sugar content,glucose metabolism-related enzyme activity and gene expression.The effect of ZmSWEET15a gene on stress in transgenic plants was analyzed by expression level and sugar content determination.The results are as follows:（1）q RT-PCR was used to analyze the expression pattern of ZmSWEET15a gene.The results showed that ZmSWEET15a was highly expressed in leaves,and the expression pattern was closely related to the development process of leaves.The expression of ZmSWEET15a was affected by sucrose induction.（2）The transgenic tobacco was treated with different exogenous sugars.GUS histochemical staining and GUS protein expression analysis showed that ZmSWEET15a gene promoter had obvious response to exogenous sucrose.（3）Bioinformatics analysis showed that the SWEET gene family was divided into four subfamilies.ZmSWEET15a belonged to Clade III and had seven conserved transmembrane domains,which was a conserved protein.（4）The optimal conditions for maize callus infection by agrobacterium-mediated method were as follows:maize inbred line H8185 was selected as the recipient material and a layer of sterile filter paper was used for co-culture.The concentration of glyphosate was 2mg/L and4mg/L,respectively.The optimal level of factors in differentiation medium was A₃（IBA concentration 1mg/m L）B₃C₁（6-BA concentration 1.6mg/m L）D₃（Vc concentration 1.5mg/m L）.（5）Subcellular localization indicated that ZmSWEET15a gene was localized in the plasma membrane.Heterologous expression of ZmSWEET15a in yeast showed that ZmSWEET15a could transport sucrose independently of p H.SDS and Western blotting showed that there was an obvious specific band at about 31 k Da,indicating that ZmSWEET15a protein was successfully induced.（6）47 T₂overexpression positive plants were obtained by PCR detection,and the seedling growth of overexpression plants was significantly stronger than that of the wild type.The investigation and analysis of agronomic traits of transgenic plants showed that the 100-grain weight of overexpressed lines T₂-2-7 and T₂-5-4 was significantly higher than that of the control,and the plant height of overexpressed lines T₂-4-5 and T₂-4-15 was significantly higher than that of the control.（7）Compared with the wild type,the leaf sucrose content of overexpressed plants decreased by 30.1%and glucose content decreased by 25.1%.The sucrose content of overexpressed plants was 1.38 times that of wild type,and the difference of fructose content was small.Overexpression of ZmSWEET15a decreased starch content in grains,but there was no significant difference in starch content in leaves.（8）It was found that SS,SPS and CWINV peaked at 21 d after pollination,and VINV and NINV peaked at 14 d after pollination,indicating that ZmSWEET15a affected the activities of sugar metabolism-related enzymes and sucrose synthase gene expression levels in transgenic plants at grain filling stage.Moreover,ZmSWEET15a regulated the expression of sucrose invertase genes Zm Ivr1,Zm Ivr2,Zm Incw1,Zm Incw4,sucrose synthase genes ZmSus1 and ZmSh1,and sucrose transporter genes ZmSUT2 and ZmSUT4.（9）The expression of ZmSWEET15a gene was significantly increased under cold stress,which was 6.9 times that of the wild type,and the sucrose content of the transgenic plants was significantly decreased,suggesting that ZmSWEET15a was closely related to the cold stress response.