γ-TMT和FAD2基因在大肠杆菌与里氏木霉中的表达及鉴定
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摘要
Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In the major oilseed crops, γ-tocopherol, and the biosynthetic precursor to α-tocopherol, is the predominant form found on the leaves. It was suggested that the final step of the α-tocopherol biosynthetic pathway was catalyzed by γ-tocopherol methyltransferase.
The full-length γ-PfTMT was obtained from the total RNA of Perilla frutescens leaves by RT-PCR. Sequence analysis indicates that γ-PfTMT consisted the open reading frame of894nucleotides encoding the protein of34kD polypeptide. Our results demonstrated that the E. Coli BL21(DE3) expression of the γ-PfTMT resulted in the α-tocopherol contents (and y-tocopherol conversion yield) from18%in the reaction products. Transgenic Trichoderma reesei Rut-C30strains, over-expressing the γ-PfTMT was also generated by Agrobacterium tumefaciens-mediated transformation. The presence of hph and γ-PfTMT genes in the transformants were confirmed by PCR analysis. The expression of the γ-PfTMT gene of the transgenes was demonstrated by SDS-PAGE. Furthermore, we demonstrated that the Trichoderma reesei Rut-C30expression of the γ-PfTMT gene resulted in the tocopherol composition5.9-fold increase in α-tocopherol content by using high-performance liquid chromatographic (HPLC) method. The increase in the α-tocopherol content indicates that a regulatory function of the γ-PfTMT protein converts γ-tocopherol to α-tocopherol.
The full-length γ-BoTMT was obtained from the total RNA of Brassica oleracea leaves by RT-PCR. Sequence analysis indicates that γ-BoTMT consisted the open reading frame of1041nucleotides encoding the protein of39kD polypeptide. Our results demonstrated that the E. Coli BL21(DE3) expression of the γ-BoTMT resulted in the α-tocopherol contents (and y-tocopherol conversion yield) from23%of the reaction products by using HPLC method. Transgenic Trichoderma reesei Rut-C30strains, over-expressing the γ-BoTMT gene was also generated by Agrobacterium tumefaciens-mediated transformation. The presence of hph and γ-BoTMT gene in the transformants was confirmed by PCR analysis. The expression of the γ-BoTMT gene of the transgenes was demonstrated by SDS-PAGE.
Fatty acids are the main groups of components of plant membrane lipid and seed storage lipid, and the major source of energy in plant. According to bioinformation analysis of the cDNA sequence, the specific fragment of FAD2from immature maize embryos was isolated by RT-PCR. Results of sequence analysis indicate that FAD2fragment contains the open reading frame of1,236bp long coding for the46kD polypeptide. Transgenic Trichoderma reesei Rut-C30strains, over-expressing the FAD2gene from maize were generated by Agrobacterium tumefaciens-mediated transformation. The presence of hph and FAD2gene in the transformants were confirmed by polymerase chain reaction (PCR) analysis. The expression of the FAD2gene of the transgenes from Trichoderma reesei and E. coli BL21was demonstrated by SDS-PAGE.
In this study, we developed novel plasmids containing three plasmids designated pBI121-TMT, pCAMBIA1301S-FAD2and pCAMBIA1301S-FAD2-TMT that incorporate modified and improved expression omega-3and vitamin E content in seeds of the plant transformation. The FAD2and γ-PfTMT genes of each plasmid were driven by the constitutive CaMV35S promoter which are mostly used for driving trangene expressions in both monocot and dicot plant transformation. The binary vector pCAMBIA1301S-FAD2and vector pBI121-TMT contains FAD2, γ-PfTMT genes respectively, whereby the binary vector pCAMBIA1301S-FAD2-TMT contain both FAD2and y-PfTMT gene. All three plasmid vectors were introduced into A. tumefaciens EHA105by electroporation.
引文
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