Cloning And Sequence Analysis Of Vitamin E Synthesis-related Key Enzyme Genes (VTE3 And HPT) In Arachis Species And Genetic Transformation Of γ-tmt

Vitamin E is an important class of lipid-soluble compounds with antioxidant activities that plays a very important role in plant, animal and human being. However, natural vitamin E is synthesized only by oxygenic photosynthetic organisms, which can not synthesized by human body. Plant oil is the main resource of the vitamin E in human nutritious. Peanut (Arachis hypogaea L.) is an important oilseed crop, which is also one of the important source of vitamin E human body needed. Based on EST sequences, we cloned vitamin E synthesis-related key enzyme gene of 2-methyl-6-phytyl-1, 4-benzoquinone methyltransferase (VTE3) and homogentisate phytyltransferase (HPT) in peanut by RT-PCR for the first time. Two full-length DNA of VTE3 were cloned from each of thirteen varieties of A. hypogaea and one was cloned from each of six wild species by PCR. Based on these VTE3 DNA sequences, we studied the origin of different type varieties of peanut and the genetic relationship between cultivated peanut and each wild species involved. What's more, theγ-tocopherol methyltransferase gene (γ-tmt) was introduced into peanut cultivar and T0 and T1 positive transgenic seedlings were obtained. The major results are abstracted as follows:(1) Two cDNA sequences of VTE3 were cloned from each cultivar of Fenghua 2, Lanna 1 and Lipudahuasheng, which are identical and were symbolized rVTE3-1 and rVTE3-2. Both of the sequences code 351 amino acids, which share 97.8% identity in nucleotide acid level and share 98.6% identity in amino acid level. Eight sites of SNP variations were identified between rVTE3-1 and rVTE3-2. Five amino acids differences were identified between the two relevant amino acid sequences.(2) One HPT cDNA sequence, 1269 bp in length and symbolized AhHPT, was cloned from cultivar Krapt.st.16. The largest open reading frame of AhHPT gene has 1230 bp in length and encoded a protein of 409 amino acid residues. Blast result showed that the deduced amino acid sequence of AhHPT is highly homologous with the HPT amino acid sequences from sixteen plants include Glycine max, Medicago sativa, Manihot esculenta, Arabidopsis thaliana, et al.(3) Two DNA sequences of VTE3, symbolized gVTE3-1 and gVTE3-2, were cloned from each of thirteen cultivars. The thirteen sequences of gVTE3-1 from the thirteen cultivars share 99.9% identity in nucleotide acid level and the thirteen sequences of gVTE3-2 are identical. A total of 36 sites of SNP variations and three sites of endonuclease recognition variations were identified between the intrones of sequences gVTE3-1 and gVTE3-2 from Fenghua 2, which share 96.6% identity in nucleotide acid level. One DNA sequences of VTE3 was cloned from each of six wild diploid species. The gVTE3 sequences of four wild diploid species from A genome and the sequences of gVTE3 from A. batizocoi share more than 99.9% identity in nucleotide acid level with only two bases differences. The sequence of gVTE3 from A. ipaensis and the sequence of gVTE3-2 from Fenghua 2 are identical. The sequence of gVTE3-1 from Fenghua 2 share no more than 98.7% identity in nucleotide acid level with the gVTE3 sequences of wild diploid species from A genome. The phylogenetic tree revealed that the gVTE3 sequences of four wild diploid species from A genome and the sequences of gVTE3 from A. batizocoi grouped together with the sequence of gVTE3-1 from Fenghua 2 while the sequence of gVTE3 from A. ipaensis grouped together with the sequence of gVTE3-2 from Fenghua 2.(4) 164 PPT-resistance plants were obtained, which were positive verified by PCR analysis. 560 T₀ individual plants and 356 T₁ individual plants from 34 plant strains were obtained. 185 T₁ individual plants were positive verified by PCR analysis.