Isolation And Functional Analysis Of 2-methyl- 6-phytyl-1,4-benzoquinol Methyltransferase Gene In Soybean

Vitamin E is a small molecule that synthesized by photosynthetic organisms and plants,which has stronge ability of antioxidation and essential for animals and hunman.When the body lacks vitamin E, the reproductive system of human was shrinkaged and damaged, so vitamin E also known as tocopherol. Vitamin E can be classified into natural vitamin E and synthetic vitamin E according to the source.The natural vitamin E have several advantages compared with synthetic vitamin E as follow:It has no toxic side effects, food safety,high biological activity.In short, people on the demand for natural vitamin E increased year by year. Vitamin E in the human diet comes from the seeds of oil crops which have high total vitamin E content but lowγ- tocopherol andα-tocopherol content. Therefore, it has great significance using genetic engineering technique to change the component of vitamin E and increaseγ- tocopherol andα-tocopherol in the proportion of the total tocopherol in plant seeds.2 - methyl -6 -phytyl - 1,4 - benzoquinone methyltransferase (MPBQ-MT) catalyse 2 - methyl -6 - phytyl- 1,4 - benzoquinone (MPBQ) to synthesis 2,3 - dimethyl -6 -phytyl- 1,4 - benzoquinone (DMPBQ) by adding a methyl ,it is a key enzyme in the synthesis of vitamin E. Tocopherol cyclase (TC) catalyse MPBQ and DMPBQ to synthesisδ,γ- tocopherol respectively.γ-methyltransferase (γ-TMT) catalyseδ,γ-tocopherol to synthesisβ,α-tocopherol respectively,so,MPBQ-MT gene increase the content ofγ-tocopherol andα-tocopherol .Given the above background,this study aims to separate the soybean 2 - methyl -6 - phytyl 1,4-benzoquinone methyltransferase gene from soybean , analysis its functions, transform the gene into the model plant Arabidopsis thaliana and tobacco,lay a foundation of transforming the gene into Crops and increase the content ofα-tocopherol. The follows are the experiment result:1. The full-length cDNA of 2 - methyl -6 - phytyl 1,4-benzoquinone methyltransferase gene was isolated by RT-PCR from Soybean and analysised the sequence.2. A recombinant vector pZP212 correct-MPBQ MT was constructed and introduced into Arabidopsis thaliana and tobacco, six homozygous MPBQ-MT gene transgenic lines were obtained respectively.3. Transgenic Arabidopsis and transgenic tobacco plants were analysised by RT-PCR and Northern blot analysis respectively, GmMPBQ-MT gene expressed in all transgenic lines.4. The Vitamin E content of Transgenic Arabidopsis thaliana seeds and leaves and tobacco seeds were analysised by HPLC, the results showed that the total tocopherol contents have no difference with wild-type,theγ-tocopherol content increase and theα-tocopherol have no difference with wild-type.