恒山黄芪毛状根遗传转化体系的建立及活性成分含量测定

Agrobacterium rhizogenes Mediated Transformation System of Hengshan Astragalus membranaceus and Determination of Active Components

以恒山黄芪叶片为试材,采用发根农杆菌侵染诱导产生毛状根的方法,研究了不同菌株对恒山黄芪叶片诱导毛状根的能力和恒山黄芪毛状根的遗传转化体系,包括恒山黄芪不同外植体、菌液浓度、共培养时间、抗生素类型和浓度、乙酰丁香酮浓度对毛状根诱导率的影响,并测定规模培养的毛状根中总黄酮和总皂苷的含量,建立稳定的恒山黄芪毛状根体系,以期为进一步建立积累活性成分的恒山黄芪毛状根液体培养体系提供参考依据。结果表明:以OD_(600)=0.6的发根农杆菌LBA9402为菌种,侵染恒山黄芪叶片30 min,转入含100μmol·L^(-1)乙酰丁香酮1/2MS培养基中共培养3 d,在含500 mg·L^(-1)头孢克肟的筛选培养基上对于恒山黄芪叶的诱导能力最好,毛状根诱导率达54.1%,经PCR扩增鉴定,发根农杆菌LBA9402的发根基因整合到了恒山黄芪叶的基因组中。恒山黄芪毛状根在液体培养扩增后,总黄酮和总皂苷含量均明显提高,分别是自然根中含量的1.72倍和2.31倍。

Hengshan Astragalus membranaceu leaves were used as the experimental materials.The experimental method was used to induce hairy roots by Agrobacterium rhizogenes infection.The ability of different strains to induce hairy roots from leaves and the genetic transformation system of hairy roots were studied, including effect of different explants, bacterial concentration, co-cultivation time, antibiotic type and concentration, and acetosyringone concentration on hairy root induction rate and the contents of total flavonoids and total saponins in hairy roots cultured on large scale were determined, a stable hairy root system of Hengshan Astragalus membranaceus were established, in order to provide reference for establishing a liquid culture system of hairy roots that accumulate active ingredients.The results showed that the induction rate of hairy root was higher when the leaves were used as the explant, soaked for 30 minutes by OD_(600)=0.6 LBA9402,co-cultured for 3 days, cultured in 1/2 MS medium containing 100 μmol·L^(-1) acetosyringone, selected on screening medium with 500 mg·L^(-1) cefixime.The induction rate of hairy root was 54.1%.It was confirmed by PCR analysis that the rooting gene in the LBA9402 was integrated into the genome of Astragalus membranaceus.The content of total flavonoids and total saponins of hairy roots increased significantly after liquid culture expansion, which were 1.72 times and 2.31 times that of natural roots, respectively.