|Place of Conferral||北京|
|Keyword||花色素苷 彩色棉花 表达载体构建 转基因烟草|
As the biggest colored cotton-producing area, Xinjiang is with advantaged resource of water, soil, light and heat, it is also a national quality cotton producing areas. The unitary of color type of cotton is a serious problem to the development of colored cotton. It is only the Green and brown cotton that have been approved and applied in agriculture. The gene of biosynthesis pathway of anthocyanin is well studied in the ameliorating of flower color. With the development of the plant transgenic technology, so it is possible to alter the type of colored cotton by genetic engineering. According to the four genes that we cloned and conserved from biosynthesis pathway of anthocyanin, we wanted to study their function on altering the flower color. And the function of the genes we studied will lay foundation for further studying colored cotton too. In this paper, we constructed two vectors containing these genes. They are plant expression vector pCAMBIA2300-OCS which contained kanamycin resistance selection marker gene NPTII and plant expression vector pCAMBIA3301 –OCS which contained Basta resistance marker gene bar. The tobacco was transformed using the engineered Agrobacterium containing plant expression vector for obtaining transgenic tobacco plants. At last we tested the transformed tobacco with GUS staining. The main content of this research were as follows: 1. Construction of plant expression vector containing kanamycin resistance gene. With BamHⅠ and SalⅠ,the cloning vector pGEAM-F3'H、pGEAM-F3'5'H and plant expression vector pCAMBIA2300-OCS were digested, then respectively ligated the two interested fragments and large fragment of the expression vector, and got the plant expression vector pCAMBIA2300-F3'H and pCAMBIA2300-F3'5'H. With KpnⅠ and XbalⅠ, the cloning vector pMD19-phDFR2、pMD19-phDFR3 and plant expression vector pCAMBIA2300FT-GFP were digested, then respectively ligated the two interested fragments and large fragment of the expression vector, and at last we got the plant expression vector pCAMBIA2300-PhDFR2-GFP and pCAMBIA2300-PhDFR3-GFP. 2. Construction of plant expression vector containing Basta resistance gene Plant expression vector pCAMBIA3301-OCS contained not only Basta resistance gene but also GUS selection marker gene. With EcoRⅠand PstⅠ,the expression vector pCAMBIA2300-F3'H and plant expression vector pCAMBIA3301-OCS were digested, then ligated the interested fragments and large fragment of the expression vector, at last we got the plant expression vector pCAMBIA3301-F3'H. The pCAMBIA2300phDFR3-GFP and plant expression vector pCAMBIA3301-OCS were digested with EcoRⅠ and PstⅠ,then ligated the interested fragment and large fragment of the expression vector, and we got the plant expression vector pCAMBIA3300-PhDFR3-GFP.With EcoRⅠ and XbalⅠ,the pCAMBIA2300phDFR2-GFP and plant expression vector pCAMBIA3301-phDFR3 were digested, then ligated the interested fragment and large fragment of the expression vector, and we got the plant expression vector pCAMBIA3300-PhDFR2-GFP. 3. Agrobacterium-mediated transformation and GUS staining We chose three plant expression vector pCAMBIA3301-F3’H, pCAMBIA3301-PhDFR2-GFP, and pCAMBIA3301-PhDFR3-GFP to transform tobacco mediated by Agrobacterium GV3101.At last, we got transgene tobacco. The GUS staining results of transgenic plants further proved the correctness and practicability of the construction of expression vector in this paper. The plant expression vector containing different selection marker were constructed successfully in this study, which provides the plant transformation with unprecedented flexibility and convenience towards different selection method. It laid the foundation for further studying the function of interested genes on controlling flower color and developing modification of flower color by genetic engineering. GUS screening and herbicide resistance genes wo uld provide convenience for the screening and agricultural production. The genetically modified vectors provide great convenience for constructing other over-expression vector and alternative plant expression vector for plant genetic and scientific.
|曾闻. 类黄酮生物合成相关基因表达载体的构建及烟草的遗传转化[D]. 北京. 中国科学院大学,2014.|
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