Construction Of Pharmaceutic Protein Gene Expressing Vectors And Their Transformation To Carrot And Tomato

Primers of thymosin gene were designed according to the nucleotide sequence with the endonuclease recognition site. pART7 vector and PCR product of thymosin gene were digested with EcoRI and BamHI and constructed to pART7-THY vector. pART27 and pART7-THY were digested with NotI and constructed to pART27-THY. Plant expressing vector pART27-THY was transferred into C58 Agrobacterium tumefaciens by electrotransformation and C58 Agrobacterium tumefaciens was used to infect carrot and tomato.The transformed hypercotyle explants of carrot seedling were planted on B5+ 1.0 mg/L NAA+ 0.5 mg/L BA medium supplemented with 50 mg/L Kanamycin and 500 mg/L Carbenicillin for screening. The result showed that there were 87 plants resistant to Kanamycin. PCR detection showed 14 plants were positive and the positive plant ratio was 16.1%. Among the 14 positive plants, one got flowering but dead soon after transplant and 3 showed abnormal. Southern blotting of 3 reliving plants indicated that they were all positive and one plant showed one copy and the other two showed three copies. In RT-PCR detection, mRNA of the plant with one copy was not expressed, the two plants with three copies have transcription.The transformed tomato cotyledon explants were planted on MS+ 2.0 mg/L ZT+ 0.05 mg/L IAA medium supplemented with 50 mg/L Kanamycin and 500 mg/L Carbenicillin. DNA was extracted using CTAB method. PCR detection showed 4 positive plants in 34 resistant to Kanamycin plants. Southern blotting indicated one positive plant. RNA was extracted using guanidinium wasothiocyanate method and mRNA of the plant was expressed by RT-PCR detection.The transformed carrot hypercotyle explants were planted on MS+ 0.1 mg/L 2,4-D and B5+ 0.1 mg/L 2,4-D supplemented with 50 mg/L Kanamycin and 500 mg/L Carbenicillin, respectively. Four weeks later, the explants on B5+ 0.1 mg/L 2,4-D were transferred to MS (tackle 1) and B5( tackle 2) medium, and the explants on MS+ 0.1 mg/L 2,4-D were transferred to MS (tackle 3) and B5( tackle 4) medium. Plantlets of tackle 1 and 2 were directly regenerated from callus and the plantlets of tackle 3 and 4 were regenerated by embroygeneswas. The number of regenerated plants of tackle 3 was the most and tackle 4 was the least. The embryos of tackle 3 developed soon after transferred to MS from B5 medium.The purified pART27-CT plasmid was used to transform carrot callus induced on the medium of MS+ 0.1 mg/L 2,4-D by bombardment. After transformation, the callus was transferred to MS+0.1 mg/L BAmedium to regenerate. 16 resistant plants were got by Kanamycin screening and 14 were showed positive in PCR detection. The positive plant ratio was 81.25%. The peculiar band of positive plants appeared bigger than plasmid positive ck because of consociating repeats of two CT genes. Southern blotting of leaf material for the 4 reliving plants indicated that 3 were positive.The freshy roots were harvested in the 3 positive plants and two of them showed positive in PCR detection. PCR Southern blotting of the two freshy roots and Southern blotting of the bigger one showed they were all positive. Northern blotting indicated the three plants were all negative. Although one plant got flowering, its pollen was abortive and no seed was formed.Plasmids of PR1B and PB1121 were digested by SacI and Smal and constructed to PR1BG. pART7 was digested by Xbal and fillied-in with Klenow Fragment I, PR1B-G was digested by SacI and filled-in, and then the two was constructed to P71BG. P71BG was digested by Smal and constructed to P7G. Digesting P71BG by NotI incompletely and harvesting the biggest band, digesting pART27-THY and pART27-CT by NotI completely and harvesting the smaller band, connecting the products with T4 ligase and obtained P71BG-THY and P71B-GCT. Digesting P7G by NotI incompletely and harvesting the biggest band, digesting pART27-THY and pART27-CT by NotI completely and harvesting the smaller band , and then constructed the two harvested two partes to P7G-THY and P7G-CT. 3-glucuronidase gene, PRlb secreting gene and t...