| Lycopene is an important intermediate product of the carotenoid biosynthetic pathway in plants and possesses strong antioxidant function.Human and most of animals cannot synthesize lycopene on its own and must uptake from diet.Therefore,it is very important to study the molecular mechanism of lycopene accumulation in germplasm with high lycopene content.In our previous study,we found the lycopene content in mature fruits of autumn olives(Elaeagnus umbellata Thunb.)was 5-8 times than tomato fruits.By analyzing the temporal and spatial expression patterns of related enzyme genes in carotenoid metabolism during fruit ripening of autumn olives,we found the upstream synthetic gene phytoene synthase(EutPSY)manuscripts was significantly upregulated during fruit ripening,which was approximately 3 times higher than that of green fruit.Hence,we speculated that EutPSY might play an important role for high lycopene accumulation in autumn olive fruits.Furthermore,fruit-specific EutPSY was overexpressed in tomato,and two individual lines were obtained.In this study,the crucial role of EutPSY in the high accumulation of lycopene in the autumn olive fruits was determined.The main results were as follows:1.The contents of lycopene and ?-carotene in ripe transgenic and widetype tomato fruits was determined by High performance liquid chromatography(HPLC).The results showed thatlycopene and ?-carotene contents in the ripe EutPSY-overexpressed fruits were significantly higher than that of the wild-type fruits(P<0.01 and P<0.05,respectively).According to the results,it was confirmed that overexpressed EutPSY gene in tomato could promote lycopene accumulation in the mature fruits significantly.2.The qRT-PCR technique was applied to detect the expression of carotenogenic genes,ethylene synthetic,perceptive and responsive genes as well as fruit ripening-related genes in the transgenic and nontransgenic fruits.The results showed that overexpression of the exogenous gene EutPSY enhanced downstream SlLCY-b and SlCYCB gene expression in a feedforward manner,thereby promoted lycopene metabolic flux into the ?-branched pathway.However,overexpressed EutPSY gene inhibited the expression of SlLCY-e,and reduced?-branched carotenoid accumulation.Four ethylene biosynthetic genes(SlACS2,SlACS4,SlACO1 and SlACO3),ethylene perceptive genes SlNR/ERT3 and SlERT4),and ethylene responsive genes SlE4 and SlE8 were also significantly down-regulated(P <0.01),indicated that some inner interaction might be invloved between EutPSY expression and ethylene biosynthesis and signaling transduction.Moreover,the expression of SlPG2 a gene,a member of polygalacturonase gene family,was significantly down-regulated,and the fruit ripening date was delayed in transgenic plants(P <0.01),which confirmed that overexpressed EutPSY could inhibit tomato fruit ripening.3.Full-length genomic DNA sequence of EutPSY was isolated and analyzed.The EutPSY gDNA was 2964 bp in length,and contained 5 introns and 6 exons.4.1072 bp EutPSY promoter fragment was isolated by chromosome walking technique.Based on software PLANTCARE,ethylene response element(ERE)and gibberellin response element(GARE-motif)were found to distribute in the EutPSY promoter.Hence,three fragments with different length of the EutPSY promoter were amplificated and constructed in binary expression vector pCambiaA2-GUSA-1381 z,including A1(0-427 nt),A2(0-973 nt)and A3(0-1072 nt).Then,recombinant vectors were transformed into Arabidopsis thaliana by floral-dip method,and some resistant lines were obtained,but needed further study. |
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