| The contents of natural pigments such as yellow pigments, brown pigments andother flavonoids are closely related with wheat flour whiteness. Yellow pigmentsinclude carotene, lutein and flavone, among which the content of carotene showsnegative correlation with wheat flour whiteness. Carotenoid as one of isoprenoids inwheat flour is synthesized by a series of enzymes involved in mevalonate pathway. Inthis pathway, farnesyl pyrophosphate synthase (FPPS), a key branch point enzyme,catalyzes isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP)by isoprene1-4order condensation to firstly produce geranyl pyrophosphate (GPP)with10carbons, and then condensate with another isopentenyl pyrophosphate (IPP)to produce an important much branch point intermediate product, namely, farnesylpyrophosphate (FPP).In this thesis, according to the highly conservative motifs of farnesyl pyrophosphatesynthases (FPPS) from other plant sources, the primers for the middle sequenceamplification of wheat farnesyl pyrophosphate synthase gene was designed, and thenthe full length farnesyl pyrophosphate synthase gene with a1065bp open readingframe coding354amino acid residues was cloned with RACE method from high flourwhiteness wheat cultivar Jinan13and low flour whiteness wheat landrace Yumairespectively. Through the analysis of amino acid sequence, it's found that twodifferent amino acid residues exist in farnesyl pyrophosphate synthases betweenwheat cultivar Jinan13and landrace Yumai. To testify the function of wheat farnesylpyrophosphate synthase gene cloned and investigate the catalytic effect of distinctamino acid residues, prokaryotic gene expression vectors for wild types and mutantL278H and E295D from cultivar Jinan13NO.produced by site-directed mutagenesiswere constructed, and then overexpressed in Eschetichia coli BL21DE3. The proteinwas purified by Hi-Trap column, and then kinetic parameters of wheat solublefarnesyl pyrophosphate synthase purified were measured. |
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