The Preliminary Functional Analysis Of The Sucrose Synthase Gene VvSS3 In Vitis Vinifera

Sucrose synthase(SS)is a key rate-limiting enzyme in the process of plant carbohydrate metabolism,which is widely existed in organisms to catalyze the reversible reaction of sucrose cleavge and synthesis:sucrose+uridine phosphate(?)fructose+Uridine diphosphate-glucose,playing an important role in the process of plant respiration,carbohydrate biosynthesis and utilization.In order to fully understand the sucrose synthase,this study carried out the characterization of members of grape sucrose synthase gene family,the analysis of different factors influencing the distributions of sucrose synthase activity,the construction of prokaryotic expression system of sucrose synthase,the measurement of transgenic tobacco phenotype traits and the selection of interacting proteins of grape sucrose synthase,to investgated the biological functions of sucrose synthase.We performed a systematic analysis of the grape genome and reported that there are five SS genes(VvSS1-5)in the grape genome.Using the comparison of gene structures,syntenic analyses and phylogenetic analysis,we found VvSSl/VvSS5,VvSS2/VvSS3 and VvSS4 originated from three ancient SS gene,respectively,which were generated by duplication events.Bioinformatics analysis of publicly available microarray data which was validated by quantitative real-time reverse transcription PCR(qRT-PCR)revealed distinct temporal and spatial expression patterns of VvSS genes in various tissues,organs and developmental stages,as well as in response to biotic and abiotic stresses.Taken together,our results will be beneficial for further investigations into the functions of SS gene in the processes of grape resistance to environmental stresses.Based on the known cDNA sequence of VvSS3,we cloned the CDS of grape VvSS3,and VvSS3-pET24a-BL21 recombinant expression system was constructed,and therecombinant cells were induced under the conditions of different IPTG concentration and different temperature.Then we conducted the incubation experiments in vitro.The results indicated that the VvSS3-pET24a-BL21 recombinant cells were able to express the target protein after the induction at 37℃,0.5mM IPTG for 8h.The incubation experiments in vitro further confirmed that sucrose synthase plays a major role in the sucrose cleavging during grape berry development.In this study,the prokaryotic expression system of grape sucrose synthase gene was successfully constructed,which was helpful to further study the function of sucrose synthase.To characterize its functions in plant growth,multiple tobacco overexpression transgenic lines of VvSS3 were generated via Agrobacterium-mediated transformation.The changes in the VvSS3 expression levels,enzyme activity,biomass,sugar content and ultrastructure in stem segments from the transgenic lines were carefully characterized.The results demonstrated that the levels of VvSS3 gene expression,enzyme activity,chlorophyll content,fructose and glucose increased significantly,while the sucrose level decreased significantly.Consequently,the cellulose content increased,whereas the lignin content decreased,suggesting that VvSS3 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis,suppresses lignin biosynthesis.Scanning electron microscopy was performed to study the cell wall morphology of stem,and surprisingly,we found that the secondary cell wall was significantly thicker in transgenic tobacco.In addition,the 15 candidate interacting proteins interacting with VvSS3 were screened by yeast two-hybrid technique.This systematic analysis demonstrated that VvSS3 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.We studied the berry weight,soluble sugar content and the cleavage and synthetic activity of sucrose synthase of berry during grape berry development.In additional,we also studied the effects of different ecological factors such as divalent cations,monovalent cations and hormones,and different ecological factors such as temperature,humidity and pH on the berry weight,sugar content and SS activity.The results showed that the sucrose content was lower during berry development,and the fructose content and glucose content was higher.The cleavage activity and synthesis activity of sucrose synthase increased with fruit development,and the cleavage activity was much higher than that of synthesis activity.NaCl,HgCl and Ca2+ significantly inhibited the SS activity,resulting in the decreased of sugar content.GA,JA and ETH promoted the SS cleavage activity and thus glucose and fructose content increased.ETH,K+and NH4+promoted SS activity and the sucrose content thus increased.The effects of temperature and humidity on SS activity were not obvious.PH significantly inhibited SS activity,resulting in decreased sugar content.Sucrose significantly increased SS cleavge activity,and glucose and fructose promoted SS synthetic activity.The results indicated that the different factors such as hormones,metal ions,temperature,humidity,pH and other factors regulate the distribution of cleavage and synthetic activity of the sucrose synthase by changing the direction of the sucrose synthase catalyzed reaction to the cleavage direction or the synthesis direction,thus regulating the sugar accumulation.