Development of efficient Catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants.
Development of efficient Catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants.
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DOI：
10.1186/1472-6750-12-34
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发表时间：
2012-06-29
影响因子：
3.5
通讯作者：
Tang K
中科院分区：
文献类型：
--
作者：
Wang Q;Xing S;Pan Q;Yuan F;Zhao J;Tian Y;Chen Y;Wang G;Tang K
马达加斯加长春花（Catharanthus roseus）是一种珍贵的药用植物，产生多种萜类吲哚生物碱，如文多灵、阿吉姆利辛、蛇纹石、长春质碱、长春碱和长春新碱等，其中一些是治疗癌症和高血压药物的重要成分。然而，这些TIA在野生型植物中的产量低，并且由于高成本和它们的复杂结构，全化学合成在大规模上是不切实际的。代谢工程策略的最新发展提供了一个有前途的解决方案。为了提高C.因此，需要建立高效的遗传转化方法。建立了一种转基因方法。roseus的根癌农杆菌菌株EHA 105，其携带含有报告β-葡萄糖醛酸酶（GUS）基因和选择标记新霉素磷酸转移酶II基因（NTPII）的双元载体pCAMBIA 2301。以下胚轴为外植体，对影响转化的因素进行了系统的研究，确定了最佳的转化条件：80 W，A.在含100 μM乙酰丁香酮的1/2 MS培养基中共培养2d。通过在愈伤组织、芽和根诱导培养基中的一系列筛选，成功获得了稳定的转基因再生植株。GUS基因的表达通过组织化学、聚合酶链反应和基因组Southern印迹分析得到证实。为了验证所建立的遗传转化体系的有效性，将TIA生物合成途径中的限速基因DAT（编码脱乙酰文多啉-4-O-乙酰基转移酶）转入C. roseus中获得了9株独立的转基因植株。高效液相色谱分析结果表明，过量表达DAT提高了转基因植株中文多灵的产量。本研究建立了一种高效的农杆菌介导的转化体系。roseus植株的转化率为11%。据我们所知，这是第一份关于A.根癌农杆菌介导的转化和再生。罗斯更重要的是，C。本工作建立的转化体系在C. roseus转化中得到了成功的验证。利用TIAs生物合成途径中的关键基因DAT，在转基因植物中积累了更多的文多灵。
As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.
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影响因子：
3.8
作者：
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