反义托品酮还原酶Ⅱ基因对三分三的遗传转化及生物碱含量测定

Genetic Transformation of Antisense Tropinone Reductase Ⅱ Gene into Anisodus acutangulus and Alkaloid Content Determination

[目的]通过反义托品酮还原酶Ⅱ基因提高托品酮还原酶I(TRI)的表达水平,从而增加莨菪碱在三分三中的含量,为大规模开发生产托品烷生物碱提供理论基础。[方法]将携带反义托品酮还原酶Ⅱ基因的重组工程菌C58C1-p1304+-antTRⅡ采用叶盘法对三分三进行遗传转化。潮霉素筛选转基因发根,PCR检测转基因发根基因组中是否同时含有rolB、rolC和Hygr抗性基因。PCR阳性转基因发根扩大培养后,提取生物碱,并采用高效液相色谱法进行生物碱含量测定。[结果]共有9个转基因发根的基因组同时检测到rolB、rolC和Hygr抗性基因,且这9个转基因发根中的莨菪碱含量较对照有不同程度的提高。[结论]反义托品酮还原酶II基因对托品烷生物碱代谢途径起到一定程度的正调控作用。

[Objective] The research aimed to increase the contents of hyoscyamine in Anisodus acutangulus by using the antisense tropinone reductase Ⅱ gene（ant-TR Ⅱ）,to improve the expression level of tropinone reductase I（TRI）,which provided the theory basis for the large-scale development of tropane alkaloids.[Method] The recombinant bacteria C58C1-p1304＋-antTRⅡ which carried ant-TR II gene was transformed into Anisodus acutangulus by using the leaf disc method.The hygromycin was used to screen the transgenic hairy root.PCR was used to detect if the genome of transgenic hairy root simultaneously contained rolB,rolC and Hygr resistance genes.After PCR positive transgenic hairy root was screened to culture,the alkaloid was extracted,and HPLC was used to measure the alkaloids content.[Result] rolB,rolC and Hygr resistance genes in the genomes of nine transgenic hairy roots were simultaneously detected.The hyoscyamine content in the nine transgenic hairy roots had the different degrees of increase when compared with the control.[Conclusion] ant-TR Ⅱ gene had the certain positive regulation effect on the metabolic pathway of tropane alkaloids.