基于颠茄发根的外源基因表达系统的建立

 用携带植物高效表达载体pCAMBIA1304 ⁺ “解除武装”的重组C58C1工程菌转化颠茄无菌苗真叶, 发根诱导率达100%。PCR检测证实发根型质粒pRiA4和表达型质粒pCAMBIA1304 ⁺均能整合到颠茄发根基因组内, 共转化率达30%。建立了基于颠茄发根的高效外源基因表达系统, 为将托品烷类生物碱东莨菪碱的生物合成关键酶基因导入颠茄, 实现其次生代谢工程及开展分子育种奠定了基础。

Establishment of the Exogenous Gene Expression System Based on Atropa belladonna Hairy Roots

The disarmed Agrobacterium tumefaciens strain C58C1 harboring the plasmid pRiA4 was engineered by introducing the plant expressing vector pCAMBIA1304~ . The bacteria-free well-growing true leaves of A. belladonna were infected with the engineered recombined C58C1. The hairy roots were induced from the wounded sites of the leaves at the frequency of 100%. Then, genomic PCR was applied to detect rolB, rolC, and antihygromycin gene in different monoclonal hairy root lines. All the three genes were simultaneously detected in 30% hairy root clones. The results demonstrated that exogenous genes could be expressed in hairy roots of A. belladonna at a relatively high frequency by the method in the present studies. These were fundamental works for tropane alkaloids metabolic engineering in hairy roots and molecular breeding of A. belladonna.