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pCAMBIA1304⁺+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-qPCR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 In order to improve the content of terpenoid indole alkaloids(TIAs),orca3/g10h genes were introduced to the hairy roots in Catharanthus roseus.Methods Bivalent expression vector CAMBIA1304⁺+orca3+g10h was constructed and introduced into Agrobacterium rhizogenes strain and transformed into C.roseus to obtain transgenic hairy roots.RT-qPCR was used to study the transcriptional differences of relative genes referred to the biosynthesis pathway of TIAs.Then HPLC was used to study TIAs content in the transgenic hairy roots of C.roseus,including vinblastine,vincristine,and ajmalicine.Results The transcriptional level of genes that linked to biosynthesis of TIAs in the transgenic hairy roots of C.roseus,asα,ggpps,g10h,str,tdc,cpr,sgd,and dat,were all expressed higher than those of the nontransgenic roots.HPLC results showed that modified hairy root of C.roseus owned more total TIAs production,58.23 mg/g,the number was larger than that of common roots in C.roseus as many as 27.5 times.On the other hand,the average content of vinblastine and vincristine was also more than the common roots in C.roseus.Among them,vinblastine content was the most.The number of production got 51.30 mg/g,which was as many as 197.3 times of the common root of C.roseus.Conclusion Orca3/g10h double-gene transgenic hairy root of C.roseus can increase TIAs content efficiently.

浙江省自然科学基金项目（LY13C020004）