长春花再生体系的建立及g10h、dat基因遗传转化的研究
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摘要
药用植物长春花(Catharanthus roseus)中含有130余种萜类吲哚生物碱(Terpenoid indole alkaloids, TIAs),包括长春碱(Vinblastine)、长春新碱(Vincristine)等重要的抗肿瘤TIAs。但这些TIAs在天然长春花植株中含量较低,化学结构复杂使其化学人工合成十分困难,因此,仅从天然长春花植物中提取TIAs不能满足市场需求,使其价格较为昂贵。尽管通过毛状根和细胞培养系统能产生单萜类吲哚生物碱,但因含量太低且不能产生最有药用价值的双萜类吲哚生物碱—长春碱和长春新碱而不具有商业化应用前景。因此,利用代谢工程提高植物生物碱含量和改变生物碱组成成份对商业化生产生物碱是非常必要的。
本研究建立了适合于遗传转化的长春花再生体系,为了提高TIAs的含量,并在长春花中过量表达TIAs生物合成途径中的关键基因g10h和dat。本研究得到了如下结果:
(1)建立了高效的长春花下胚轴再生体系。
(2)构建了含TIAs生物合成关键酶基因g10h或dat的单元或双元植物表达载体(pCAMBIA2300-g10h, pCAMBIA2300-g10h-dat)。
(3)采用农杆菌介导法转化长春花下胚轴,获得了19株转g10h基因的长春花、25株共转g10h和dat基因的长春花。
(4) Real-time PCR分析结果表明外源DNA整合到长春花基因组中。
Catharanthus roseus contains a group of about 130 terpenoid indole alkaloids (TIAs). Many TIAs are true lead compounds for drug development, including two commercially important cytotoxic dimeric alkaloids; vinblastine and vincristine are utilized in a number of different cancer chemotherapies. Most of these TIAs are produced at low levels in the natural plants and are difficult to be chemically synthesized due to their complex structures. Hairy root culture system and cell culture system offer promising alternatives for the production of these valuable alkaloids. Although transgenic hairy roots and cell cultures can produce a number of TIAs, they seldom produce the most valuable dimeric alkaloids, vinblastine and vincristine,only monomeric alkaloid can be produced in cell and tissue cultures, but at levels too low for commercialization. Metabolic engineering offers one of the most promising approaches for improving the product composition and increasing alkaloids yield in plants.
In this study, an efficient and rapid somatic embryogenesis based plant regeneration systerm was standardized for large-scale genetic transformation in Catharanthus roseus. To increase TIAs production, key genes involved in the TIAs biosynthetic pathway, g10h and dat were overexpressed in transgenic Catharanthus roseus plants by using genetic engineering method. The results are as following.
(1) An efficient and rapid somatic embryogenesis based plant regeneration systerm from Catharanthus roseus hypocotyls was establishment.
(2) Monovalent or bivalent expression vectors (pCAMBIA2300-g10h, pCAMBIA2300-g10h-dat) were constructed which contained the rate-limiting enzyme genes, g10h, and/or dat.
(3) A series of transgenic Catharanthus roseus were obtained using agrobacterium-mediated transformation. 19 independent transgenic plants containing g10h alone, 25 independent transgenic plants containing g10h and dat together were obtained.
(4) Some transgenic plants were analyzed by real-time PCR. Results showed that foreign DNA fragments were randomly inserted into Catharanthus roseus genome.
本研究建立了适合于遗传转化的长春花再生体系,为了提高TIAs的含量,并在长春花中过量表达TIAs生物合成途径中的关键基因g10h和dat。本研究得到了如下结果:
(1)建立了高效的长春花下胚轴再生体系。
(2)构建了含TIAs生物合成关键酶基因g10h或dat的单元或双元植物表达载体(pCAMBIA2300-g10h, pCAMBIA2300-g10h-dat)。
(3)采用农杆菌介导法转化长春花下胚轴,获得了19株转g10h基因的长春花、25株共转g10h和dat基因的长春花。
(4) Real-time PCR分析结果表明外源DNA整合到长春花基因组中。
Catharanthus roseus contains a group of about 130 terpenoid indole alkaloids (TIAs). Many TIAs are true lead compounds for drug development, including two commercially important cytotoxic dimeric alkaloids; vinblastine and vincristine are utilized in a number of different cancer chemotherapies. Most of these TIAs are produced at low levels in the natural plants and are difficult to be chemically synthesized due to their complex structures. Hairy root culture system and cell culture system offer promising alternatives for the production of these valuable alkaloids. Although transgenic hairy roots and cell cultures can produce a number of TIAs, they seldom produce the most valuable dimeric alkaloids, vinblastine and vincristine,only monomeric alkaloid can be produced in cell and tissue cultures, but at levels too low for commercialization. Metabolic engineering offers one of the most promising approaches for improving the product composition and increasing alkaloids yield in plants.
In this study, an efficient and rapid somatic embryogenesis based plant regeneration systerm was standardized for large-scale genetic transformation in Catharanthus roseus. To increase TIAs production, key genes involved in the TIAs biosynthetic pathway, g10h and dat were overexpressed in transgenic Catharanthus roseus plants by using genetic engineering method. The results are as following.
(1) An efficient and rapid somatic embryogenesis based plant regeneration systerm from Catharanthus roseus hypocotyls was establishment.
(2) Monovalent or bivalent expression vectors (pCAMBIA2300-g10h, pCAMBIA2300-g10h-dat) were constructed which contained the rate-limiting enzyme genes, g10h, and/or dat.
(3) A series of transgenic Catharanthus roseus were obtained using agrobacterium-mediated transformation. 19 independent transgenic plants containing g10h alone, 25 independent transgenic plants containing g10h and dat together were obtained.
(4) Some transgenic plants were analyzed by real-time PCR. Results showed that foreign DNA fragments were randomly inserted into Catharanthus roseus genome.
引文
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