Construction Of Thaumatin Gene Plant Expression Vector And Genetic Transformation Of The Smallanthus Sonchifolius

Sweet protein thaumatin exists in the gelatinous aril of fruit of Thaumatococcus danielli Benth in the tropical rainforest of West Africa. Thaumatin has many advantages such as high sweetness intensity, low calories, non-toxicity, and nice taste. It also can be degraded into amino acids which required by human body. Therefore, it is expected to be the substitute for nutritious carbohydrate in the foodstuff industry and become a new sweetener in the future. However, T. deniellii can’t fruit normally outside its place of origin, and it is difficult to conduct its mass production due to unsuccessful introduction of variety. Thus the thaumatin becomes very expensive commodity. The rapid development of biotechnology inspires people to use the method of gene recombination to develop sweet protein. By cloning and sequencing the genes of sweet protein and taste-modifying protein, those proteins were sucessfully expressed in other hosts such as bacteria, fungi, and some plants. In this test, based on the predecessors’ experiences, the plant expression vector pBI121- Thaumatin was constructed and the gene of thaumatin was transfered into Smallanthus sonchifolius mediated by Agrobacterium tumefaciens EHA105. In addition, the presence of the transgenes was assessed by PCR. The transgenic plants were tested to preliminarily study the transformation and expression of thaumatin in the plant and lay a foundation for further transforming the horticulture plant. The experiment results are as follows:(1) Artificial synthesis and clone of thaumatin geneIn the experiment, according to the reported thaumatin gene sequence, the entire thaumatin gene was artificially synthesized by overlap extension PCR. Then the gene was transformed to E. coli DH5αby connecting the gene to T vector pMD19-T, and the clone vector pMD-Thaumatin was obtained after identification of colony PCR, double digests, and sequencing. (2) Construction and identification of thaumatin gene plant expression plasmid pBI121- Thaumatin.By using DNA recombination technology, the thaumatin gene was inserted into the plant expression plasmid pBI121 and verified by PCR, enzyme cutting and electrophoresis identification.(3) Transferring pBI121- Thaumatin plasmid to Agrobacterium tumefaciensThe recombination plasmid pBI121- Thaumatin was led to Agrobacterium tumefaciens EHA105 with direct transformation method. The positive clone was selected with Rif and Kan flat plate and single colony. Through bacteria solution PCR identification, it is proved that the plasmid was transferred to Agrobacterium tumefaciens EHA105.(4) Construction of plant regeneration systemThe leaves of sterile seedling Smallanthus sonchifolius were used as explants, and MS as basic culture medium. The medium MS+6-BA1.0mg/L+NAA0.1mg/L was selected to form callus, MS+6- BA1.0mg/L+IAA0.1mg/L to induce the differentiation of adventitious shoots, and 1/2MS+NAA0.3mg/L for rooting cultivation. Then the complete regeneration plant is obtained.(5) Transformant selection and plant regenerationAfter being precultured for 3-4 days, the explants were dipped in the bacteria solution of Agrobacterium tumefaciens for 5-15 minutes and cultured for 2-3 days. Then transfer them to the media containing MLPN and Kan for differentiation and selection. And the transformants transferred to exogenous genes germinated and took root. Thus the transgenic plants were obtained.(6) PCR test of transgenic plantAfter extracting the genome DNA of transgenic plant, PCR thaumatin gene, proving that the gene is led to Smallanthus sonchifolius, with transformation rate of 36.4%.