| Fructans are linear or branched polymers of repeating fructose residues connected byβ(2→1) and/orβ(2→6) fructosylfructose linkages,optionally including one terminal glucosyl unit.They are water-soluble and non-oxidative.Fructans are widely distributed in nature occurring in bacteria,fungi and over 40,000 higher plant species.The best characterized fructan is the inulin type,which has linear fructose chains attached to sucrose withβ(2→1) fructosylfructose linkages.Many important nutritional and functional characteristics are attributed to inulin-type fructans.They can increase calcium absorbance,act as a low calorie sweetener or be used as a water-soluble dietary fiber in food ingredients.More importantly,inulin is not metabolized by humans and animals and can act as a prebiotic.It is generally believed that inulin-type fructan biosynthesis in plants occurs through the concerted action of two enzymes,1-SST(sucrose:sucrose 1-fructosyltransferase,EC2.4.1.99) and 1-FFT(fructan:fructan 1-fructosyltransferase,EC2.4.1.100).1-SST catalyzes the conversion of sucrose to the trisaccharide 1-kestose(DP3).The elongation of 1-kestose to fructan(of DP up to 200) is catalyzed by 1-FFT.During previous work,a coexpression vector was constructed including 1-SST and 1-FFT separately promoted by the zein promoter,which introduced fructan biosynthesis pathway into maize endosperms.This vector was transformed into maize bud using agrobacterium-mediated method.On this founation,transformants were selected on bialaphos and PCR assay.Transgenic lines that overexpressed target genes were confirmed by RT-PCR.Southern blot analysis suggested that these two enzymes are probably encoded by single-copy gene.Plant growth and kernel development were not affected.Transgenic lines that overexpressed target genes were chosen for determination of carbohydrate content,using TLC for separation and HPLC for precise quantitative analysis.Data showed that,transgenic maize plants produced inulin-type fructan in the endosperms,ranging from 0.05～2.0 mg/g kernels.The levels of glucose,fructose and sucrose in these transgenic lines were not significantly different from WT plants. To test the enzyme activity of 1-SST and 1-FFT,protein extracts of transgenic maize kernels were incubated with 100 mmol/L sucrose solution at 27℃and 3 h,and produced some fructan.These results suggested that the protein extracts had some level of fructosyltransferase activity,they can use sucrose as substrate to pruduce fructan.In this study,we confirmed that the 1-SST and 1-FFT transformed into maize plants were active through carbohydrate analysis and measurement of enzyme activity,which means that the complete plant fructan biosynthesis pathway has been cloned into maize.We had obtained new type of maize lines which can be used to produce functional food in the future. |
