Molecular Cloning Of Vitamin E Synthesis-related Enzyme Genes And Functional Identification Of HPPD In Arachis Hypogaea

Vitamin E is an important class of lipid-soluble compounds with antioxidant activities that plays a very important role in plant and animal.However,Vitamin E can only be synthesized by photosynthetic organisms,including higher plants and cyanobacteria.Vitamin E is an essential nutritional element for both humans and animals and therefore must be adequately provided by foods or other supplements.Plant derived oils represent the major sources of vitamin E in the human diet.Peanut(Arachis hypogaea L.)is an important oilseed crop,peanut oil is one of the main edible oil.In order to understand vitamin E metabolism and to increase the vitamin E content and activity finally in peanut.The place of vitamin E(a-tocopherol)synthesis,and the accumulation pattern of a-tocopherol in peanut embryo development were preliminary analyzed in this study.The first cloned vitamin E synthesis-related enzyme gene of geranylgeranyl pyrophosphate synthase(GGDS),geranylgeranyl reductase(GGDR),arogenate dehydrogenase(ADH)and 4-Hydroxyphenylpyruvate dioxygenase(HPPD)in peanut,also analyzed their structure feature,expression and evolution.We performed functional identification of HPPD via agroinfiltration in N.tabacum(CB-1)and N.benthamiana,to identify the functional of HPPD,and to determine whether the enzyme HPPD was a rate-limiting enzyme in peanut tocopherol biosynthesis.T0 positive transgenic seedlings were obtained.The major results are abstracted as follows:1.Hairy root induced via Agrobacterium rhizogenes in CB-1.The extraction of a-tocopherol for hairy root and control by direct solvent extraction,and detected by RP-HPLC.Our results indicate that the content of a-tocopherol in control was 19.29±2.1?g/g,but there is no detected ?-tocopherol in hairy root.Further demonstrated that the synthesis of a-tocopherol is light-demanding.2.We also extracted a-tocopherol from the different time of embryo development in Minhua 6.After dectected by RP-HPLC,we found some regularity.The most obvious feature is ?-tocopherol content reach the highest during the peanut embryo development over 20 days.The ?-tocopherol content increase 2.6 times from 10 days to 20 days later in embryo development,and decrease 1.2 times from 20 days to 30 days later in embryo development,and then into the steady change period 40,50 and 60 days later in embryo development.3.The full-length cDNA encoding GGDS,GGDR and ADH were isolated by modified rapid amplification of cDNA ends(RACE)in Minhua 6,and designated as AhGGDS,AhGGDR and AhADH.The cDNA of AhGGDS was 1,596 base pairs(bp)long containing an open reading frame(ORF)of 1,026 bp encoding a protein of 341 amino acids.The cDNA of AhGGDR was 1,573 base pairs(bp)long containing an ORF of 1,119 bp encoding a protein of 372 amino acids.The cDNA of AhADH was 1,155 base pairs(bp)long containing an ORF of 810 bp encoding a protein of 269 amino acids.Blastn results showed,all of these genes fragments possessed high homology with the corresponding sequences from Glycine max.Multi-alignment and phylogenetic tree analysis revealed,that these protein sequencese of AhGGDS,AhGGDR,and AhADH are highly homologous with other plants',and belonged to the same clades with Glycine max.Real-time fluorescent quantitative PCR(qRT-PCR)indicated that:(1)The expression level of AhGGDS in embryo was much higher than in other tissues,and the level significantly decreased with embryo development;(2)The expression level of AhGGDR in leaves was much higher than in other tissues,and the level significantly decreased at the period of embryo development over 60 days;(3)The expression level of AhADH in embryo was much higher than in other tissues,and compared with the expression level at the period of 20 days later in embryo development,up-regulated 1.7 times of 40 days later,down-regulated 5 times of 60 days later.The microarray hybridization of Minhua 6 peanut analysis indicated that:The expression levels of AhGGDS and AhGGDR were decreased slightly under different exogenous hormones treatments,drought and low temperature stress;The expression levels of AhADH was changed slightly under different exogenous hormones treatments,but quite different in response to low temperature and drought stress,showed of 30%up regulation under low temperature stress,33%down regulation under drought stress.4.The full-length cDNA encoding HPPD was cloned and isolated from Minhua 6,and designated as AhHPPD.The cDNA of AhHPPD was 1,575 base pairs(bp)long containing an open reading frame(ORF)of 1,323 bp encoding a protein of 440 amino acids.Blastn comparison showed,AhHPPD gene fragment possessed high homology with the sequence from other plants.Multi-alignment and phylogenetic tree analysis revealed,that the protein sequencese of AhHPPD is highly homologous with other plants',and belonged to the same clade with Glycine max.The HPPD genomic sequences were obtained by amplified genomic DNA from cultivar Minhua 6 and two wild species.The nucleic acid sequences alignment and phylogenetic tree showed that two copies of HPPD from Minhua 6 are derived from the wild species A.ipaensis(BB).There are two exons and one intron of 248bp in 1,181-1,429bp by compared the genomic sequences with the cDNA of HPPD.QRT-PCR indicated that the expression level of AhHPPD in peg was higher than in other tissues,and the level was significantly decreased during embryo development.The microarray of Minhua 6 analysis indicated that the expression levels of AhHPPD was changed slightly under different exogenous hormones treatments,but significant up regulated under low temperature and drought stress.Construction the over expression vector of 35S::AhHPPD to transform N.tabacum(CB-1)and N.benthamiana.The extraction of a-tocopherol from leaves of transgenic plants and wild-type by direct solvent extraction,and detected by RP-HPLC.The transgenic HPPD tobacco plants had a a-tocopherol content increase 2-3 folds than the wild-type.