根癌农杆菌介导的怀地黄遗传转化研究  

Studies on genetic transformation of Rehmannia glutinosa mediated by Agrobacterium tumefaciens

作　　者：王丰青[1] 田云鹤[1,2] 谢彩侠[3] 杜家方[1] 李烜桢[1] 张留记[4] 何华勤[2] 张重义[1,2] 

机构地区：[1]河南农业大学农学院,河南郑州450002 [2]福建农林大学中药材GAP研究所,福建福州350002 [3]河南中医学院药学院,河南郑州450046 [4]河南省中医药研究院,河南郑州450004

出　　处：《中草药》2014年第17期2541-2546,共6页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81274022;81473299);中国博士后科学基金资助项目(2013M541977);河南省教育厅科学技术研究重点资助项目(14A360007)

摘　　要：目的研究适于怀地黄Rehmannia glutinosa遗传转化的激素质量浓度和潮霉素质量浓度。方法以地黄无菌苗叶片为外植体,以根癌农杆菌介导的叶盘法进行转化,分析了潮霉素和乙酰丁香酮(AS)对抗性愈伤诱导率和再生芽分化效率的影响。结果潮霉素的质量浓度对抗性愈伤和抗性芽的产生影响很大,影响抗性愈伤诱导的临界质量浓度为9 mg/L,影响抗性芽分化的临界质量浓度为6 mg/L,适宜于进行地黄遗传转化的质量浓度为12 mg/L。侵染培养基和共培养培养基中添加100μmol/L的AS可以极显著提高转化效率。PCR扩增和β-葡萄糖苷酶(GUS)染色结果表明,外源基因成功整合到地黄基因组中。结论建立了有效的怀地黄稳定遗传转化再生体系,为地黄的分子生药学研究和遗传改良奠定基础。Objective To determine the optimal concentration of hormone and hygromycin for seedling regeneration of Rehmannia glutinosa. Methods Using the young leaf of sterile plantlet from R. glutinosa as explants, we conducted the transformation mediated by Agrobacterium tumefaciens, analyzed the efficiency of hygromycin and acetosyringone（AS） on resistant callus induction and plant regeneration. Results The concentration of hygromycin had greatly affected the production of resistant callus and seedlings. The critical concentration of hygromycin on the resistant callus induction and shoot regeneration were 9 and 6 mg/L, respectively. The optimal concentration of hygromycin for the genetic transformation of Wen 85-5 was 12 mg/L. Adding 100 μmol/L AS could greatly improve the transformation efficiency of R. glutinosa. It was confirmed by PCR detection of hpt gene and GUS staining that the foreign gene was integrated into the genome of R. glutinosa. Conclusion The stable genetic transformation system of R. glutinosa is established, which lays the foundations for the research on molecular pharmacognosy and genetic improvement.

关 键 词：地黄 根癌农杆菌 遗传转化 潮霉素 乙酰丁香酮 

分 类 号：R282.21[医药卫生—中药学]