植物表达载体在三分三发根中的高效表达

Efficient expression of plant expressing vector in Anisodus acutangulus hairy roots

目的建立基于发根培养的三分三Anisodus acutangulus遗传转化体系及在三分三发根中表达外源基因的方法。方法由种子直接萌发得到无菌苗,建立了三分三的无菌苗培养系统。将植物高效表达载体pCAMBI-A1304+导入经过“disarmed”改造的根癌农杆菌C58C1(携带pRiA4质粒),获得了携带外源基因表达载体的重组C58C1工程菌。取生长良好的幼嫩真叶作为外植体,在乙酰丁香酮的诱导下用重组C58C1进行感染,诱导发根。选取在无激素培养基上生长迅速,分支多的发根单克隆进行继代培养。经过3次继代培养,PCR(polymerase chain reaction,聚合酶链式反应)检测用于验证发根型质粒pRiA4和表达型质粒pCAMBIA1304+是否整合到三分三发根基因组内。结果建立了基于发根培养的三分三遗传转化体系,发根诱导频率达到100%,PCR检测表明pRiA4和pCAMBIA1304+质粒共转化率达到32%。结论该方法可以用于在三分三发根中高效表达外源基因,为实现基于发根的三分三产托品烷类生物碱的代谢工程奠定了基础。

Objective To establish the genetic transformation system of Anisodus acutangulus based on hairy root cultures and the method of expressing exogenous genes in A. acutangulus hairy roots. Methods The bacteria-free seedlings of A. acutangulus were obtained from the sterilized seeds that were used for genetic transformation. The disarmed Agrobacterium tumefaciens strain C58C1 harboring the plasmid pRiA4 was engineered by introducing the plant expressing vector pCAMBIA1304^＋. The young leaves of A. acutangulus were infected with the engineered C58C1 in the treatment of AS to induce the hairy roots. The independently transformed hairy roots which well-grown on hormone-free medium were subcultured for three times to erase bacteria. Then, genomic PCR was applied to detecting whether pRiA4 and pCAMBIA1304^＋ had integrated into host genome of A. acutangulus hairy roots. Results The genetic transformation system of A. acutangulus was established, the hairy roots were induced from the wounded sites at the frequency of 100%. Both of the two plasmids were simultaneously detected in 32% （cotransformation frequency of pRiA4 and pCAMBIA1304^＋ in hairy roots） hairy root clones. Conclusion The results demonstrate that exogenous genes can be expressed in hairy roots of A. acutangulus at a relatively high frequency by the method in the present study. And this is a fundamental work for metabolic engineering tropane alkaloids in hairy roots of A. acutangulus.