Effect Of SlSWEETs On Sugar Metabolism Of Tomato Fruits And Functional Verification Of Four SlSWEETs Genes

SWEETs,a novel class of membrane-transporters in higher plants,mediate sugar through facilitated diffusion across cell membrane transportation into cells,which are of great significance for regulating carbon transportation and distribution in crop production.Currently,the physiological functions of only some of the members were identified in plant leaves and flowers organs,but in fruits,of which the most important sink organ,the mechanism of sugar supply and the accumulation of metabolic and regulation physiological function are still unclear.Based on the tomato genome data,this Dissertation identified the members of the SWEETs family for the first time in the important vegetable crop-tomato.The fruit-specific SlSWEETs genes were screened by real-time quantitative PCR analysis.Yeast hexose transport-deficient mutants verified the sugar transport characteristics of SlSWEETs proteins that were specially differentially expressed in tomato fruits.The interactions between the SlSWEETs proteins were analyzed by mbSUS system and BiFC system.By analyzing the promoter of SlSWEETs gene,the expression pattern of SlSWEETs under different hormone treatments was preliminarily studied.In addition,the SlSWEETs-RNAi silencing vector was constructed using Gateway technology to obtain transgenic lines.Our study explored the molecular mechanism of sucrose transport regulated by SlSWEETs and the relationship between sucrose accumulation and sugar pathway protein activity in tomato fruit maturation,provided theoretical basis and new ideas for the genetic improvement of cultivated tomato fruit yield and quality.The main results are as follows:1.Based on tomato genome data,29 SlSWEETs family members were identified in tomato.Sequence alignment found that members of the family of tomato SWEET proteins are a kind of membrane localized proteins consisting of seven typical alpha helix transmembrane domains(TMDs),which harboring two MtN3/saliva domains,also known as the PQ-loop-repeat,connected by a loop.Phylogenetic tree analysis revealed that tomato SlSWEETs family members can be divided into typical four major branches.Members of branches I,II and IV have hexose transport capacity,while members of branch III preferentially transport sucrose,but also have a certain hexose transport capacity.The expression patterns of SlSWEETs genes were analyzed by sugar,salt,and temperature stress treatments,and SlSWEETs genes that responded to stress were selected.Among them,SlSWEET1 b,-1c,-5b,-6a and-7a may participate in the transport of glucose,SlSWEET10 a,-10 b,-10 c,-11 a,-11 b,-11 d,-12 a and-12 c may participate in the transport of fructose,SlSWEET10 a,-10 b,-10 c,-11 a,-11 b,-11 c,-11 d and-12 c may play an important role in the transport of sucrose.SlSWEET1 c,-2a,-3,-7a and-14 function under high temperature stress,SlSWEET1 c,-1f,-3,-5a and-5b may induce transcription under low temperature stress,SlSWEET1 c,-3,-7a and-14 play an important role in salt stress response.On this basis,SlSWEETs gene expression profiles of cultivated tomato ‘Heinz 1706'(S.lycopersicum)and wild-type tomato LA1598(S.pimpinellifolium)were obtained by comparing the published database,and SlSWEET7 a,-11 b,-12 c,and-14 were selected.These four genes,specifically expressed in fruits,are presumed to play a more important role in fruit development and maturation.2.To further verify the function of SWEET in tomato fruit,four SlSWEETs-RNAi recombinant plasmid were constructed and transformed into Micro-TOM tomato via Agrobacterium-mediated transformation.Identification by PCR and real-time quantitative PCR analysis of the target gene were performed.All transgenic lines have been obtained.Through the determination of sugar content in mature leaves and fruits of transgenic lines,it was found that after gene silencing,the accumulation of sugar content in mature leaves increased,and the hexose content of fruits also increased,but the sucrose content did not change much.Results indicated that there is functional redundancy between the SlSWEETs family members.Analysis of the expression of key enzymes in sucrose metabolism in gene-silenced transgenic plants of slsweet7a-1 and slsweet14.The results showed that after gene silencing,the monosaccharide-related enzyme genes(fructose kinase,FK and hexokinase,HK)in slsweet7a-1 fruit with down-regulated expression,the expression levels of sucrose-associated enzyme genes(sucrose phosphate synthase SPSA2,cell wall invertase LIN,vacuolar invertase VI)and sugar transporter genes(SlSWEET12c and SlSWEET14)were up-regulated.Only the expression levels of sucrose synthase SS1 and sugar transporter SlSWEET12 c were up-regulated in slsweet14 fruit,while the expression levels of other sugar metabolic pathway genes were down-regulated.The expression of 4 membrane-located SlSWEETs regulated the sugar facilitated diffusion and resulted in the accumulation of glucose and fructose,while the content of sucrose maintained stable.These results indicated that the function of SWEETs were redundant to each other.When silenced the SWEETs,the other family members were regulated and expressed to maintain the stability of the sucrose level,and the improvement of fruit monosaccharide has played an important role in the formation of fruit quality and yield.3.The pDR195-SlSWEET7 a,-11 b,-12 c,-14 yeast expression vectors have been successfully constructed,and transformed into the yeast hexose deficient mutant EBY.VW4000.Four SlSWEETs proteins differentially expressed in tomato fruits were verified to have glucose and fructose transport properties in SD-ura selection media of different carbon sources(maltose,glucose,fructose,galactose,and trehalose),but for other hexoses,such as galactose and trehalose,do not show significant transport capacity.4.Utilizing the mating-based Split-Ubiquitin System(mbSUS),the four fruit differentially expressed SlSWEETs genes were linked into bait vector pMetYC_GW and prey vector pXN22_GW,respectively,and transformed into THY.AP4 and THY.AP5 yeast,respectively.Identification by screening medium,under the strict selection of yeast two-deficient SD-Leu-Trp and three-deficient SD-Leu-Trp-His selection medium,SlSWEET11 b and SlSWEET14 self-activated to form a homodimer,respectively,and SlSWEET7a/-11 b,SlSWEET11b/-14,SlSWEET12c/-11 b,SlSWEET12c/-14 and SlSWEET14/-11b(Nub-Cub)can form heterodimers.5.Utilizing the Bimolecular Fluorescence Complementation system(BiFC),the four fruit differentially expressed SlSWEETs genes were linked into the pXC_GW and pXN_GW vectors,respectively.After transient expression in tobacco leaves,it was found that SlSWEET11 b and SlSWEET12 c can self-activate to form homodimers,SlSWEET7 a,SlSWEET14,and SlSWEET11 b.And SlSWEET12 c can interact with each other to form a heterodimer.These results indicated that only 7-TMDs may be insufficient to form a pore large enough to allow for sugar transport and the oligomerization of small molecule carrier proteins or MtN3 domain is essential for function.6.The tomato fruit discs were treated with GA,IAA,ABA and MeJA,respectively.After processing 8h and 24 h,the expression of 4 SlSWEETs genes and the change of sugar content were analyzed.The results showed that the expression levels of SlSWEET11 b and SlSWEET14 were significantly up-regulated after treatment with IAA,ABA and MeJA,and the expression of SlSWEET14 was significantly increased after 24 hours of ABA treatment.There are elements that respond to MeJA but not to ABA in the promoter of SlSWEET14,suggesting that other genes on this pathway may have affected its expression.Incubation experiments confirmed that 4 SlSWEETs genes may be involved in hormone-related regulatory pathways to respond to the induction of auxin,abscisic acid and jasmonic acid.