论文部分内容阅读
目的共转orca3/g10h双基因于长春花毛状根,提高抗癌生物碱量。方法通过构建p CAMBIA1304++orca3+g10h表达载体,利用发根农杆菌介导获得双转orca3/g10h基因长春花毛状根。利用RT-q PCR研究共转orca3/g10h基因长春花毛状根萜类吲哚生物碱(TIAs)生物合成途径中相关基因的转录差异。采用HPLC研究共转orca3/g10h基因长春花毛状根TIAs(包括长春碱、长春新碱和阿吗碱)量。结果转基因长春花毛状根中与TIAs生物合成相关基因asα、ggpps、g10h、str、tdc、cpr、sgd和dat转录水平均较非转基因长春花普通根高。HPLC结果表明,转基因长春花毛状根总TIAs量达到58.23 mg/g,是非转基因长春花普通根的27.5倍。长春碱和长春新碱的平均质量分数均较非转基因普通根高。其中,长春碱平均质量分数最高,为51.30 mg/g,是非转基因普通根的197.3倍。结论共转orca3/g10h基因能够有效提高长春花毛状根中TIAs量。
Objective co-transfer orca3 / g10h double gene in the hairy roots of Catharanthus rosea to increase the amount of anti-cancer alkaloids. Methods The pCMBIA1304 ++ orca3 + g10h expression vector was constructed and Agrobacterium rhizogenes was used to obtain the hairy roots of Catharanthus roseus orca3 / g10h gene. RT-q PCR was used to study the transcriptional differences of related genes in the biosynthetic pathway of hairy terpenoid indole alkaloids (TIAs) of orca3 / g10h gene. The amount of TIAs (including vinblastine, vincristine and arsine) in hairy root of the orca3 / g10h gene was analyzed by HPLC. Results The transcriptional levels of asα, ggpps, g10h, str, tdc, cpr, sgd and dat related to the biosynthesis of TIAs in the hairy roots of transgenic Prunella vulgaris were all higher than those of non-transgenic Changchun flowers. HPLC results showed that total TIAs of hairy roots of transgenic Prunus villosa reached 58.23 mg / g, which was 27.5 times of that of common non-transgenic vinca. The average mass fraction of vinblastine and vincristine was higher than that of non-transgenic common roots. Among them, the average content of vinblastine was the highest, which was 51.30 mg / g, which was 197.3 times of the non-transgenic common root. Conclusion Co-transfection of orca3 / g10h gene can effectively increase the amount of TIAs in hairy roots of Catharanthus roseus.