Cloning And Functional Analysis Of Sugar Transporters PbSUT2,PbHT1 And PbSOT2 Genes In Pyrus Fruit

Sugar content,sugar types and proportions in fruit are critical factors to fruit sweetness and flavor quality factors,the accumulation character and sugar component in pear fruit of different varieties were different.Sorbitol and sucrose are the photosynthesis of leaf are transported to the sink tissue of fruit,this process requires a long distance transport that relies on the sugar transporters,the sugar unloaded into fruit is metabolized by the various metabolic enzymes and stored in fruit finally.However,the study about the mechanism of sugar transport and function of sugar transporters in pear fruit is very limited at home and abroad.Therefore,low-sucrose cultivar 'Yali'(Pyrus bretschneideri Rehd.cv.'Yali')and high-sucrose cultivar 'Hosui'(Pyrus pyrifolia Nakai.cv.'Hosui')were used for the materials in this study.The relation of sugar content in leaf and fruit,the cloning and functional analysis of sucrose transporter gene and hexose transporter gene,the cloning and expression pattern of sorbitol transporter gene,the effect of bagging on the sugar content and expression of sugar transporter were studied.The results will help to clarity the molecular mechanism of sugar transport and lay a theoretical basis for the genetic modification of fruit quality.Main results as follows:1.Components and soluble sugar content in fruit and leaf was determined by high-performance liquid chromatography(HPLC)techniques during the fruit development of cv.Yali and cv.Hosui.The results indicated that the main soluble sugars were fructose and sorbitol in 'Yali' mature fruit,sucrose and fructose in 'Hosui' mature fruit.Sucrose and sorbitol were main soluble sugars in 'Yali' and 'Hosui' leaf.Sorbitol was the main photosynthesis product transported in the phloem of 'Yali' and 'Hosui'.The correlation analysis demonstrated that the sucrose content in fruit had no significant correlation with the content of that in leaf during 'Yali' and 'Hosui' fruit development.2.In order to determine the role of sucrose-metabolizing enzymes for sucrose accumulation,the activities of the sucrose-metabolizing enzymes and soluble sugar composition investigated in 'Hosui'(Pyruspyrifolia Nakai)and 'Yali'(Pyrus bretschneideri Rehd.),characterized as low and high sucrose cultivars,respectively.Results show that sucrose content in 'Hosui' was thirteen times higher than in 'Yali' in mature fruit.Activities of sucrose synthase(SSII)and sucrose-phosphate synthase(SPS)in 'Hosui'increased sharply,but the activities of sucrose cleavage(SSI),soluble acid invertase(S-AIV),and neutral invertase(NINV)decreased at fruit maturity.Whereas SSII activity in'Yali' fruit was weak and SPS activity decreased at fruit maturity.Cell wall invertase(CWINV)activities were changed similarly in the two cultivars.SPS and SSII activity was significantly correlated with sucrose content in 'Hosui' fruit,and weakly correlated with sucrose content in 'Yali' fruit.3.The search in the Pyrus Genome Database using the MdSUT1 sequence as bait yielded the SUT homologs.Polymerase chain reaction(PCR)was carried out with special primers to clone the sucrose transporter,PbSUT2,in pear(Pyrus bretschneideri Rehd.cv.'Yali')fruit.The length of PbSUT2 was 1497 bp,PbSUT2 encoded a protein of 499 amino acid residues and localized in the plasma membrane of transformed onion epidermal cells.Phylogenetic analysis revealed that PbSUT2 belonged to the SUT4 clade.Fusion expression vector of pBI121-PbSUT2 was constructed and transformed into the Micro-Tom tomato.The phenotype of overexpression of PbSUT2 tomato plants included early flowering,higher fruit quantity and lower plant height.Overexpression of PbSUT2 in transgenic tomato plants led to increases in the net photosynthetic rate in leaves and sucrose content in mature fruit compared with wild-type tomato plants,and to decrease the contents of glucose,fructose and total soluble sugars in mature fruits.These results suggested that PbSUT2 affected sucrose content in sink and the flowering phase during tomato plant growth and development.4.To obtain the full-length cDNA sequence of hexose transporter protein gene,RT-PCR was carried out using a pair of primers designed on the basis of amino acid sequence which was obtained by Blastping with the LeHT1 gene sequence as bait from the Pyrus Genome Database.The clone was obtained by PCR amplification and its length was 1557 bp,named PbHTl.PbHTl gene encoded 519 amino acids and was located the cell plasma membrane by formulating with transforming the GFP fusion expression vector into the onion skin test.Bioinformatics analysis shows that,PbHTl encoded a protein with typical features of MFS members,in the middle of a hydrophilic molecule central cytoplasmic loop and the 12 transmembrane domains.To verify the functioning of PbHT1 gene,the fusion vector of pBI121-PbHT1 was transformed into the Micro-Tom tomato.The results showed that the phenotype of overexpression of PbHTl tomato plants included the decrease of plant height,more fold of leaf,later flowering phase and the numbers increase of flower phenotypic flowers.Overexpression of PbHT1 in transgenic tomato plants led to increases in the net photosynthetic rate in leaves,and hexose and soluble sugar content in mature fruit compared with wild-type tomato plants,but sucrose content there was no significant difference in transgenic and wild type tomato fruit.These results above suggested that PbHT1 gene may promote the transport and accumulation of hexose in tomato fruit.5.The candidate gene sequence was obtained from pear genome database using apple MdSOT5 gene as bait.The gene was cloned by PCR amplification with the special primers.This clone,named PbSOT2,encoded a 537-aa protein with a calculated molecular mass of 57.92 kDa.The analysis of Amino acid sequence and phylogenetic tree showed PbSOT2 had 12 transmembrane domains and belonged to the MFS carriers;it was high homology with the other species.The results of subcellular localization prediction and subcellular localization test were consistent.PbSOT2 was sub-cellularly targeted to the plasma membrane.PbSOT2 gene was expressed during the fruit development,and the expression of PbSOT2 was highest during the rapid enlargement phase of 'Yali' and 'Hosui' pear fruit(100 days after full blooming).In addition,the sorbitol content in fruit fluctuated within certain limits,but its proportion of total sugars decreased continuously.The activities of NAD-SDH and NADP-SDH were higher in fruit of the period of young fruit and fruit enlargement period.This work shows that PbSOT2 may play a role in the fruit enlargement and accumulation of hexose during fruit development.NAD-SDH and NADP-SDH activities increased during the immature stage,which corresponded with the rapid accumulation of hexose.6.The bagging treatment of effects on fruit sugar content and expression of sugar transporters were studied,the results showed that the total sugar content in mature bagging fruit of both 'Yali' and 'Hosui' pear were significantly lower than that of control fruit,sucrose content of bagging 'Hosui' pear fruit was also significantly lower than in the control fruit.Expression level of PbSUT2,PbHT1 and PbSOT2 genes were determined by fluorescence quantitative PCR(qRT-PCR),the results showed that expression level of PbSUT2 gene in bagging fruit of ' Yali' and 'Hosui' pear was lower than that of control fruit,the effect of bagging on PbHT1 expression has no obvious regularity in both 'Yali' and'Hosui' pear.Expression level of PbSOT2 gene in bagging fruits is higher than in control before 'Hosui' fruit mature,however,the expression level of PbSOT2 gene in bagging fruits is lower than in control in 'Hosui' fruit mature period;And the effect of bagging on PbSOT2 expression level has no obvious regularity in both 'Yali' and 'Hosui' pear.All the results above suggested that the decrease of sucrose content in bagging fruit may be related to the decrease of sucrose PbSUT2 gene expression which was regulated by sugar content,the influences of bagging on expression of sugar transporter genes were different in pear fruit of different varieties.