Identification Of Members Of SWEET Gene Family Of Peach And Their Function In Sucrose Transport

Peach(Prunus Persica L.Batsch)is an important fruit in the Rosaceae family.As an important seasonal fruit in summer and autumn,peaches are characterized by thin skin,solid flesh,delicious flavor,and rich nutrition.Sucrose and glucose are the main soluble sugars in peach fruit.SWEET(Sugars Will Eventually be Exported Transporters),a new type of sugar transporter identified in recent years,can selectively transport monosaccharides or disaccharides through the plasma membrane or subcellular membranes,and thus plays a key role in plant development.This protein is a novel diffused protein family independent of energy-mediated sugar transport in higher plants and is of important signify for carbohydrate transportation and allocation.So far,the physiological functions of some plant members have only been identified in leaf and flower organs,while the functions of sucrose supply and metabolic accumulation in some "reservoir" tissue such as fruit organs,have been rarely reported.In this study,we analyzed the evolutionary and structural characteristics of the Pp SWEETs gene family based on peach genome data available by bioinformatics method.Functional prediction of known genes and real-time quantitative PCR analysis were combined to screen candidate genes involved in the sucrose transport.Subcellular localization was performed by transient expression in tobacco.The function and characteristic of Pp SWEET11 and Pp SWEET12 were prelimainarily revealed using sucrose transport-deficient yeast strain(SUSY)and Arabidopsis atsweet11/12 double mutants transform systems.The results not only provided a theoretical basis,but also give an new insight into regulation of peach fruit flavor quality.The main findings are as follows:1.A total of 19 Pp SWEETs genes were identified from the peach genome database based on PFAM(PF03083)using bioinformatics method.these genes were classed into six branches according to phylogenetic relationship.Six SWEET genes(Prupe.2G307800,Prupe.2G307700,Prupe.8G017400,Prupe.8G017500,Prupe.4G072300,Prupe.4G155700)were included in Clade I,five(Prupe.5G125100,Prupe.3G034900,Prupe.8G076100,Prupe.5G175500,Prupe.3G283400)were included in CladeⅡ,and another six SWEET genes(Prupe.5G146400,Prupe.5G146500,Prupe.1G220700,Prupe.8G253500,Prupe.2G245600,Prupe.6G355900)were included in CladeⅢ,Prupe.2G118600 and Prupe.1G133300 were in CladeⅣ.Further analysis revealed that all the members contained Mt N3/Saliva domains and at least one transmembrane α-helix,which indicated that all the members belonged to the Sweet gene family.2.Based on phylogenetic analysis of multiple species,Prupe.5G146400 and Prupe.8G253500 are highly homologous with At SWEET11 and At SWEET12,respectively,and clustered with the two proteins with sucrose transportation ability.The two candidate genes were named Pp SWEET11 and Pp SWEET12,and they belongs to SWEET protein Clade Ⅲ members.Gene expression analysis showed that high expression of Pp SWEET11 and Pp SWEET12 were detected in both mature leaves and fresh leaves of peach,but their expression levels were low in fruit,suggesting that these two candidate genes may be related to sucrose transport from source to sink in peach.3.The results of subcellular localization using PHB-X-YFP fluorescence label carrier and tobacco showed that Pp SWEET11 and Pp SWEET12 proteins only showed strong fluorescence signal in the cell membrane of tobacco cells,which proved that Pp SWEET11 and Pp SWEET12 proteins were located on the cell membrane of peach and belonged to one of the membrane proteins with the basic characteristics of sucrose transporter.4.In order to validate Pp SWEET11 and Pp SWEET12 of sucrose transport and features,the two candidate genes were transformed into sucrose transport defect type yeast strains SUSY.The results showed that the yeast strains can grow in the selective medium containing glucose as the sole carbon source,and also grow in the selective SD medium devoid of uracil containing sucrose as a sole carbon source.The observation proved that Pp SWEET11 and Pp SWEET12 has the role of sucrose transporter in vitro.5.Starch accumulation in transgenic plant was measured by starch staining method.The dark stain was observed in leaves of Arabidopsis atsweet11/12,indicating the presence of more starch accumulation.However,light stain was observed in rosette leaves of atsweet11/12 plant overexpressed with Pp SWEET11 and Pp SWEET12.The results demonstrated that the two candidate genes compensated for the phenotypic defects of atsweet11/12 double mutants,reduced the accumulation of starch in rosette leaves,suggesting that both candidate genes Pp SWEET11 and Pp SWEET12 had the function of sucrose transport to promote sucrose efflux.Overally,the study suggests that Pp SWEET11 and Pp SWEET12 have the biological function of sucrose transport in vivo and in vitro.It is supposed that the two genes play an important potential role in phloem loading of transport sucrose from leave source to fruit sink.But further work is required to investigate into the specific mechanism in future.