Construction Of The Plant Expressing Vectors With Restructuring Sweet Protein Genes Brazzein And MBLII And Regeneration System Of The Pepino

Pepino(Solanum lycopersicum L.), is an herbaceous subshrub that has long been growing in its native Andean South America and has a close relationship with tomatoes and potatoes. It is usually cultivated for its edible fruits, which are juicy and scented, and with high protein, low sugar and low fat. They are rich in minerals and trace elements (such as calcium, selenium, molybdenum and zinc). The fruits are good for anti-cancer, cardiovascular disease and anti-caries and are also an ideal fruit for diabetics. Today, pepino is a kind of species that can increase economic profit, and has a considerable potential in the international market for future exploitation. However, most people do not like it because of the low sweetness. For this problem, in this study, the full-length fruit-specific promoter E8 with the sequence of ethylene responsivenessang was cloned by the molecular biology technique; The plant expression vector with the herbicide resistance gene bar was constructed by the plant sweet protein gene drived by E8 promoter; The construction of invitro regeneration system of the pepino was completed primitively. This experiment will build up a foundation for the following study of expressing sweet proteins gene in the Pepino fruit and for improving its flavor. The research has drawn five conclusions as following:1. Determination of the quality of pepino fruit from Zhangyi town of Wuwei city and the mean results are as following : the sugar content is 2.71 g/100g; the soluble solids content is 6.78; the Vitamin C content is 87.11 mg/100g; The fruit firmness was 5.62. The Vitamin C were higher and soluble sugar were lower than many other fruits. This was probably related to the low sweetness and poor palatability.2. Using gene splicing-by-overlap extension (SOE) technique (SOE), the full-length fruit-specific promoter E8 fragment was cloned from the template of tomato genomic DNA. The sequence analysis shows that the homology of E8 promoter is 99.12 % between alignment reported in GenBank (DQ317599.1) and cloned in this study.3. The recombining Brazzein gene is amplified from pET30a-Bra and connected to the cloning vector then the sequences are analysed. The sequence analysis shows that the homology of the restructuring Brazzein gene was 100 % between the original restructuring Brazzein gene and the subcloned gene in this study. The plant expression vector of pCA-E8-Bra were constructed based on the vector pCAMBIA3300 with the herbicide resistance gene bar and were constructed by the restructuring plant sweet protein gene Brazzein which was driven by E8 promoter. The plant expression vector pCA-E8-Bra was successfully introduced into Agrobacterium tumefaciens C58C1.4. According to the structure and function prediction of Nucleic acid sequences and amino acid sequence of MBLII,insert the peptide LP4/2A between the MBLâ…¡gene encoding A chain and B chain fragment which was amplified from pMD18-Mab by adopting gene splicing-by-overlap extension (SOE) technique to construct a recombinant MBLâ…¡gene. It was connected to the cloning vector then the sequences were analysed. The sequence analysis showed that the homology of MBLâ…¡gene was 100 % between alignment reported in GenBank (D83997) excepet for the connecting peptide LP4/2A in this study. The plant expression vector of pCA-E8-Mab were constructed based on the vector pCAMBIA3300 with the herbicide resistance gene bar and were constructed by the reconstructed MBLâ…¡gene which was driven by E8 promoter. The plant expression vector pCA-E8-Mab was successfully introduced into Agrobacterium tumefaciens C58C1.5. As explants, the leaf blade and stalk with single bud were used to the construction of invitro regeneration system of the pepino. Based on the MS medium, the density and the combinations of the hormones were obtained. The induction medium consists of MS + 0.2 mg/L 2,4-D + 1 mg/L 6-BA+3% (w/v) sugar; regenaretion medium consists of MS + 0.1 mg/L IAA + 2 mg/L 6-BA + 1 mg/L ZT + 1 mg/L GA 3 % (w/v) sugar; The Rooting medium consists of 1/2 MS + 1 mg/L NAA + 1 mg/L GA 1.5 % (w/v) sugar. All the analysis shows that the Mabinlinâ…¡genes reconstruction scheme and the reported one are quite different in this study, and it is a new try. The effect of selected LP4/2A linking peptide has been validated by domestic and foreign experts, and the efficiency of its self cleavage is high, the residual of amino acid is less. It does not affect the location of both side genes, and the product balance is good. This is the first report on the internal transformation for a gene. On the other hand, the study finds the expression sweet proteins gene in the Pepino fruit by E8 promoter, expectation of retaining the quality characteristics of pepino fruit with low sugar, low fat, rich in Vitamin, Selenium, Molybdenum and other characteristics, etc.. This experiment will build up a foundation for the following study of improving the flavor and increasing the sweetness in Pepino fruit.