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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
曾闻
学位类型硕士
导师李晓波
2014-05-27
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物有机化学
关键词花色素苷 彩色棉花 表达载体构建 转基因烟草
摘要

新疆是我国种植天然彩色棉最多的地区,具有发展棉花生产得天独厚的水、土、光、热资源条件,是国家优质棉基地。而目前彩色棉花发展遇到的一个问题是色泽单一,已经通过审核并应用生产的只有绿色和棕色2种。在花色改良方面,花色素苷合成途径中的相关基因研究的比较充分,由于近年来植物基因工程的应用蓬勃发展,因而可以通过关基因工程的办法改良彩色棉花。根据实验室已经保存的4个花色素苷生物合成途径中的基因,验证其在花色改良方面的功能,为最终能够应用于改良彩色棉花奠定基础。本研究将目的基因分别构建到含有卡那霉素抗性基因的植物表达载体pCAMBIA2300-OCS以及除草剂抗性基因的植物表达载体pCAMBIA3301-OCS中,然后通过遗传转化烟草获得转基因植株,最后通过GUS染色检测,研究内容如下:
1.含有卡那霉素抗性基因的植物表达载体的构建对目的基因所在的克隆载体pGEAM-F3’H、pGEAM-F3’5’H以及植物表达载体pCAMBIA2300-OCS进行BamHⅠ-SalⅠ双酶切,将目的基因片段和载体大片段分别进行连接,得到了植物表达载体pCAMBIA2300-F3’H和pCAMBIA2300-F3’5’H。对目的基因所在的克隆载体pMD19-phDFR2、pMD19-phDFR3以及pCAMBIA2300FT-GFP进行kpnⅠ-XbalⅠ双酶切,将目的基因片段和载体大片段分别进行连接,得到了植物表达载体pCAMBIA2300-PhDFR2-GFP和pCAMBIA2300-PhDFR3-GFP。
2.含有除草剂抗性基因的植物表达载体的构建
含有除草剂抗性基因的植物表达载体pCAMBIA3301不仅具有除草剂抗性、还有GUS标记基因。对2300F3’H和3301-OCS进行EcoRⅠ-PSTⅠ双酶切,胶回收,连接后得到了pCAMBIA3301F3’H。对pCAMBIA3301-和2300PhDFR3-GFP进行EcoRⅠ-PstⅠ双酶切,胶回收,将目的基因片段和载体大片段进行连接,得到了pCAMBIA3301-PhDFR3-GFP。对pCAMBIA3301-PhDFR3-GFP和pCAMBIA2300-PhDFR2-GFP进行EcoRⅠ-Xbal的双酶切,胶回收,将目的基因片段和载体大片段进行连接,得到了pCAMBIA3301-PhDFR2-GFP。
3.农杆菌介导的遗传转化以及GUS染色
我们选择了3种植物表达载体,pCAMBIA3301F3’H、pCAMBIA3301-PhDFR2-GFP和pCAMBIA3301-PhDFR3-GFP,利用农杆菌GV3101介导的方法对烟草遗传转化,最后获得了转基因烟草。而转基因植物的GUS染色结果进一步证明了本研究所构建载体的正确性和实用性。本研究成功构建了不同筛选标记的植物表达载体,为适合不同筛选剂的植物转化提供极大的便利。为进一步验证目的基因在控制花色调控以及通过基因工程开展花色修饰方面都奠定了基础。GUS筛选基因和除草剂抗性基因不仅方便了筛选工作而且有利于农业生产,为构建其他基因超表达载体提供了极大便利,为植物基因工程科研工作提供了一个较好的备选植物表达载体。

其他摘要

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.

文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/3440
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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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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