Cloning And Sequence Analysis Of Vitamin E Synthesis-related Key Enzyme Genes And Genetic Transformation Of γ-TMT In Peanut

Vitamin E as a general term for varieties of tocopherols and tocotrienols is a kind offat-soluble natural antioxidant ingredients and mainly exists in the plant oil. It can only besynthesized in photosynthetic organisms, human and animal can only acquire it through eating.Peanut as an important oil crop also is one major source of vitamin E. In this study, genesencoding key enzymes of4-hydroxyphenylpyruvate dioxygenase(HPPD) and γ-tocopherolmethyltransferase(γ-TMT) in biosynthesis of vitamin E was cloned by using in silico cloningcombined with PCR, and their DNA sequences was also cloned from different cultivatedpeanut and diploid wild species. Through the alignments of DNA sequences of HPPD orγ-TMT, we studied for the wild parents of A. hypogaea. Besides, transgenic peanut harboringγ-TMT of Arabidopsis thaliana was obtained through Agrobacterium-mediated genetictransformation. Five major results were obtained as the follows:(1) Cloning and sequence analysis of HPPD in peanutFull length of cDNA sequences of4-hydroxyphenylpyruvate dioxygenase in peanut(designated as AhHPPD) which is1579bp length and contains a1323bp open reading framewas cloned by using in silico cloning. AhHPPD encodes a protein of440amino acid residueswhich has a conserved N-terminal and C-terminal domain of HPPD and is localized inchloroplasts. Its similarity to HPPDs reported in other species is between61.0%and78.0%.As primer was designed based on the electronic cloning results, two different DNA sequencesof HPPD named as was cloned from9peanut cultivars(Arachis hypogaea L) and DNAsequences named gHPPD-A and gHPPD-B was cloned from the wild species A.duranensisand A.ipaensis respectively. Sequence analyses shows that there is a different intron in each ofthe four DNA sequences, but their exon sequences are of the same length as the electroniccloning result; sequence of AhgHPPD-1and AhgHPPD-2are obviously different, but each ofthem are the same in9different cultivars; Cluster analysis of homologous sequences showedthat AhgHPPD-1and gHPPD-A are clustered into one group with a99%homology,AhgHPPD-2and gHPPD-B are clustered into another with a100%homology.(2) Cloning and sequence analysis of γ-TMT in peanutThe γ-TMT of peanut (named AhTMT) was cloned from Fenghua2. AhTMT contains1167bp and has a1059bp length ORF, which encodes a protein of352amino acid residues.The protein named AhTMT has a conserved SAM domain and is localized in chloroplasts. Its similarities to γ-TMTs reported in other species are between61.75%and72.80%. DNAsequences of AhTMT were also cloned from six different cultivars and four diploid wildspecies. Sequence analysis shows that there are no intron in AhgTMTs; the similarity betweenAhgTMTs of six different cultivars are100%; compared with A.ipaensis(BB), A.batizocoi(BB),A.duranensis(AA) and A.kuhlmannii(AA), the identity of AhgTMT in cultivated peanut is100%,99.91%,99.74%and95.63%, respectively.(3) Construction of expression vector containing γ-TMT of peanutThe plant expression vector named pGBTMT was constructed, which contains the targetgene AhTMT and selection marker gene bar both of which are drived by35SCaMV promoter.(4) Acquirement of transgenic peanut harboring γ-TMT of Arabidopsis thalianaTransgenic peanut harboring γ-TMT of Arabidopsis thaliana was obtained throughAgrobacterium-mediated genetic transformation：116seedings with PPT resistance wasassayed, and some of them were transplanted in field after propagation.43transformed plantstransplanted in field and41plants survived after transplantation and had been harvested, ofwhich36plants showed positive by PCR detection. Investigation of the morphologicalcharacter showed that petiole of plant NO.12shortened and blossoming type of plant NO.31changed to alternative.