Identification And Expression Analysis Of Tea Plant SWEET Family Genes And Functional Analysis Of Low-Temperature Response Gene CsSWEET1a/16/17

Tea plants are perennial evergreen woody plant,mainly distributed in tropical and subtropical regions,low temperature is one of the most important environmental factors that restrict the growth of tea plants.Sugar is the main source of energy for plants to maintain life activities,and plays an important role in plant normal growth and reproductive development and in responding to biotic and abiotic stress.Plant are distribute sugar according to the demand if sugar in the sink organ,and the sugar from the source organ into sink organ needs to be combined with the carrier protein on the membrane to complete the trans-membrane transport,these membrane proteins that transport the sugar are called sugar transporters.When subjected to low temperature stress,the soluble sugar content increases in plant,and the sugar transporter maintains the balance of cell penetration potential by balancing and distributing sugar distribution,thereby improving the cold resistance of the plant.SWEETs(sugars will eventually be exported transporters)family is a monosaccharide transporter that contains seven transmembrane helixes and two MtN3 conserved domains,and is widely found in plants,animals and prokaryotes.Studies have shown that SWEETs sugar transporter plays an important role in plant growth and reproduction development,biotic stress and abiotic stress(such as low temperature).In this study,the sequence information of 13 CsSWEETs was analyzed.The expression patterns of CsSWEETs in different tissues,during flower development,and under different stress were analyzed by qRT-PCR,screen out the low temperature response genes CsSWEET1a,CsSWEET16 and CsSWEET17 for in-depth study.Their sugar transport activity was identified by transforming yeast hexose transporter deletion mutants,and the low temperature response genes CsSWEET1a,CsSWEET16 and CsSWEET17 were transgenic into Arabidopsis for functional analysis.The main research results are as follows1.CsSWEETs gene cloning and bioinformatics analysis.Using RT-PCR technology,eight CsSWEETs genes were cloned and identified from tea plant,and name according to their homology with the Arabidopsis AtSWEETs gene.Combining the five genes cloned in the previous study,the analysis found that 13 CsSWEETs genes all have the typical MtN3 domain and seven transmembrane structures of the SWEET family.The 13 CsSWEETs proteins were divided into four subfamilies by constructing a phylogenetic tree with amino acid sequences of members of the Arabidopsis,rice and grape SWEET family.2.Analysis of CsSWEETs gene expression and sugar transport activity.Tissue specific expression analysis found that CsSWEETla/1b/2a/2b/2c/3/9b/16/17 were expressed in all tissues,whereas the expression of CsSWEET5/7/9a/15 was not detected in some tissues such as mature leaves.The expression patterns of 13 CsSWEETs genes during flower development can be classified into three categories.Expression analysis of nine CsSWEETs genes in leaves in response to abiotic stresses,natural cold acclimation and Colletotrichum camelliae infection,it was found that eight CsSWEETs genes could respond to abiotic stress,while CsSWEET3 responded to C.camelliae infection.By transforming yeast hexose transporters deletion mutants,CsSWEET1a/lb/7/17 were found to have transport activity for glucose analogues and one or more hexose molecules3.CsSWEET16 improves the cold resistance of transgenic Arabidopsis.CsSWEET16 belongs to the subfamily IV,and its expression is suppressed by low temperature.GUS staining analysis of promoter-connected GUS lines revealed that under normal conditions,they are active in leaves,roots,flowers and fruits,but GUS activity in these tissues is reduced under low temperature conditions Subcellular localization showed that CsSWEET16 protein was localized in the vacuolar membrane Under low temperature conditions,compared with wild-type plants,the relative electrolyte leakage of transgenic lines overexpressing CsSWEET16 decreased,Fv/Fm values increased,soluble sugar and glucose content increased,and fructose content decreased.In addition,CsSWEET16 can restore the fructose accumulation phenotype of atsweetl6-1 mutant.These results showed that CsSWEET16 enhances the cold tolerance of Arabidopsis by regulating soluble sugar distribution inside and outside the vacuole.4.The C-terminal of CsSWEET17 affects sugar transport activity.CsSWEET17 belongs to the subfamily IV,its Arabidopsis homologous gene and CsSWEET16,which belong to the subfamily IV,are all localized in the vacuolar membrane,while the subcellular localization results show that CsSWEET17 is localized in the plasma membrane.Amino acid sequence alignment found that the C-terminal outside the transmembrane domain of CsSWEET17 had 67 more amino acids than AtSWEET17.After removing the C-terminal,the plasma membrane localization characteristics of CsSWEET17 were not affected,but the transport ability for fructose,glucose and mannose is weakened5.Overexpression of CsSWEET1a and CsSWEET17 can improve the abilities of transport sugar into plants.Different sugar concentration treatment found that CsSWEET1a-OE lines could not grow normally at high sugar concentrations,and yellowing the leaf color at low sugar concentrations CsSWEET17-OE lines had lower germination rates at high sugar concentrations than wild-type plants,while at low sugar concentrations they grew better than wild-type plants.In addition,the rosette leaves and seeds of the CsSWEET17-OE lines under normal soil culture were larger than the wild-type plants6.CsSWEETla and CsSWEET17 improve the freezing resistance of transgenic Arabidopsis.The expressions of CsSWEET1a and CsSWEET17 is induced by low temperature.Subcellular localization showed that both CsSWEETla and CsSWEET17 proteins were localized in the plasma membrane Experimental analysis of yeast two-hybrid and biomolecular fluorescence complementary found that CsSWEETla and CsSWEET17 have interaction.Under freezing conditions,compared with wild-type plants,the relative electrolyte leakage of transgenic lines overexpressing CsSWEET1a and CsSWEET17 was significantly reduced,and the expression of sucrose invertase AtCWINVs and AtVACINVs gene was suppressed.The results show that CsSWEETla and CsSWEET17 form homo/heterodimers in the plasma membrane and mediate partitioning of sugars between the cytoplasm and the apoplast,thereby regulating plant growth and freezing tolerance.