| The inulin-type fructans accumulates in Jerusalem artichoke(Helianthus tuberosus L.)tubers.During the storage of tubers,with the germination of tubers,the content of carbohydrates decreased significantly,which led to the decline of quality.In order to further improve our understanding of tuber germination,we studied the characteristics of sugar metabolism during tuber germination.The metabolism of fructan and sucrose in different tissues of Jerusalem artichoke tuber germination under indoor and outdoor conditions were studied.The relative expression levels of fructan synthesis gene Ht1-FFT(Fructan:fructan 1-fructosyltransferase)and Ht1-SST(Sucrose:sucrose 1-fructosyltransferase)and degradation gene Ht1-FEH Ⅰ(Fructan exohydrolase Ⅰ)Ht1-FEH Ⅱ and Ht1-FEH Ⅲgenes during germination were carried out to explore the gene expression patterns.Moreover,the activities of fructan metabolism enzymes(1-FEH、1-FFT and 1-SST)and sucrose metabolism enzymes VI(Vacuolar invertase),NI(Alkaline/Neutral invertase),SS(Sucrose synthase)and SPS(Sucrose 6-phosphate synthase)and sugar contents in different periods under two treatments of Jerusalem artichoke tubers were determined in order to elucidate the regulation mechanism of sugar metabolism during the germination process.The genes of fructan metabolism in Jerusalem artichoke have been cloned,but no corresponding sucrose invertases in Jerusalem artichoke have not been reported.Thus,we cloned and identified the sucrose invertases HtINVs cDNA sequences from Jerusalem artichoke.It will be helpful to better elucidate the mechanism of sugar metabolism in the germination of Jerusalem artichoke tubers.The results obtained were listed as follows:1.Jerusalem artichoke were germinated under two conditions.Ht1-FEH Ⅱ and Ht1-FEH Ⅲ expression was increased 5 days after planting indoors,whereas Htl-FEH Ⅱexpression was increased 72 days after planting in the field in T(Tuber without BE/S)and BE/S(Bud eyes plus shoots).Ht1-SST and Ht1-FFT expression was generally decreased in indoor-grown T.The enzyme activities of 1-FEH and 1-FFT were stable during germination in both indoor-and field-grown T and BE/S,however 1-SST activity decreased in indoor-grown T,while 1-FEH and 1-FFT activities was increased during germination time in BE/S of both indoor-and field-grown tubers.In both environments,the total soluble sugar content decreased gradually in T after germination.Besides,at the end of germination,the sucrose and fructan contents was decreased and fructose content was increased in the field.The enzyme activities of VI and NI had no significant change except in the later stage of germination.SS and SPS activities did not change significantly in T and BE/S in indoor-grown artichokes,but SS activity was gradually increased and SPS activity was gradually decreased in field-grown artichokes,alongside sucrose degradation.Compared to T,BE/S generally had higher enzyme activities of 1-FEH and 1-FFT,which promoted inulin hydrolysis.2.The reported coding sequences of INVs from the other plants were used to BLASTN the Jerusalem artichoke EST database.Based on these to design the specific primers,five sequences were isolated from Jerusalem artichoke.All Ht INVs contained three conserved amino acid motifs(NDPNG(A),RDP and WECP(V)D)that are essential for INV family.According to the amino acid sequence and isoelectric point analysis results,HtINV-1 and HtINV-2 are predicted to be vacuolar invertase(vacINV),while HtINV-3,HtINV-4 and HtINV-5 are cell wall invertase(cwINV).By heterologous expression in P.pastoris X-33,it was determined that HtINV-5 was fructan exohydrolase Htl-FEH I.However,recombinant HtINV-1 and HtINV-2 could not be successfully transferred into yeast cells due to low linearization concentration.Recombinant proteins HtINV-3 and HtINV-4 were found to have the ability to hydrolyze sucrose to produce reducing sugar and do not react with inulin,which are sucrose invertase. |
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