桑遗传转化体系的建立及槲皮素合成的研究

Establishment of transformation system in mulberry and biosynthesis of quercetin

目的:建立桑的遗传转化体系,并对其毛状根生长特性和次生代谢产物槲皮素含量进行探索。方法:利用卸甲型根癌农杆菌Agrobacterium tumefaciensC58C1侵染桑的黄化无菌苗,建立毛状根的诱导与培养体系;优化毛状根的扩大培养条件并测定其生长曲线,对桑毛状根T-DNA转化结果进行PCR检测;利用RP-HPLC检测槲皮素的含量。结果:用C58C1侵染无菌苗外植体,侵染10 min、分别预培养、共培养2 d及添加质量浓度为100 mg.L-1的乙酰丁香酮(AS)可得最高转化率;PCR检测结果表明,发根农杆菌Ri质粒的rolB,rolC基因片段已整合入桑的毛状根基因组中;C58C1侵染桑黄化无菌苗茎段10 d后,茎段伤口处陆续产生毛状根,30 d后幼茎上产生毛状根的外植体达92%;在1/2MS+0.05 mg.L-1IBA液体培养基中培养50 d后,由HPLC检测结果表明,毛状根中槲皮素含量相比于原植株增加了8.5倍。结论:成功建立桑科毛状根诱导与离体培养体系,为其他木本植物毛状根培养的研究提供了参考,并进一步为槲皮素等药用活性成分的工业化生产奠定了基础。

Objective：To establish the transformation system of mulberry,and test its ability of quercetin biosynthesis.Method： Hairy roots of mulberry were obtained through infecting etiolated seedlings with Agrobacterium tumefaciens strain C58C1.The culture condition of hairy roots was optimized.The transformation of T-DNA was examined by PCR assay and quercetin content was determined by HPLC.Result： When infecting stem cutting of etiolated seedlings via C58C1 strain,the optimal transformation conditions were as follows： 10 minutes＇ infection,two-days pre-culture and co-culture,additional hydroxylacetosyringone（As） 100 mg·L-1.The PCR examination result showed that rolB and rolC genes could be inserted into the hairy roots of mulberry.Hairy roots appeared in 10 days after infecting,the frequency of stems explants was up to 92% after 30 days culturing.After 50 days culturing in 1/2MS＋0.05 mg·L-1 IBA liquid medium,the content of quercetin increased by 8.5-fold.Conclusion： Hairy root culture system of Moraceae plants was established successfully for the first time.In addition,it also provides a foundation for further industrial production of active compounds such as quercetin.