根癌农杆菌介导的葡萄风信子遗传转化体系

Agrobacterium-mediated genetic transformation system of grape hyacinth

【目的】以葡萄风信子亚美尼亚(Muscari armeniacum)的胚性愈伤组织为受体系统,建立高效稳定的葡萄风信子遗传转化体系,为葡萄风信子遗传改良及相关基因功能的研究奠定基础。【方法】通过GUS(β-葡萄糖苷酸酶)瞬时表达率,研究根癌农杆菌的菌液浓度、侵染时间、共培养时间和超声波预处理时间等4个因素对葡萄风信子GUS瞬时表达效率的影响,并在最佳条件下将GUS基因转入葡萄风信子,获得稳定表达转化植株。【结果】葡萄风信子胚性愈伤组织GUS染色结果表明,随着根癌农杆菌菌液浓度的增加及侵染时间、共培养时间和超声波预处理时间的延长,GUS瞬时表达效率均呈现先增后降趋势;当根癌农杆菌菌液OD600为0.5、侵染时间20min、共培养4d、80W功率超声波预处理3min时,其GUS瞬时表达效率最高,分别为14.43%,13.73%,10.85%和75.36%;在最佳转化体系下将GUS基因转入葡萄风信子,获得207株潮霉素抗性植株。GUS组织化学检测结果表明:在抗性植株的不同部位都呈现不同程度的蓝色,PCR检测有7株为阳性植株,阳性率为7%;反转录PCR检测显示,7株阳性植株中有3株扩增出GUS基因目的条带。【结论】初步建立了根癌农杆菌介导的葡萄风信子遗传转化体系,该体系能显著提高葡萄风信子GUS瞬时表达效率。

【Objective】Efficient and stable genetic transformation system of Muscari armeniacum was established based on the regeneration system of embryogenic callus to study of genetic modified and functional genes of grape hyacinth.【Method】By GUS（β-glucuronidase）transient expression rate,several aspects influencing the efficiency of genetic transformation such as Agrobacterium-tumefaciens concentration,infection time,co-culture time and sonication pretreatment time were studied.Stable transformed plants were obtained under the optimal conditions.【Result】GUSactivity analysis of grape hyacinth embryonic callus indicated that Agrobacterium-tumefaciens concentration,infection time,co-culture time and sonication pretreatment time of GUS transient expression rate increased firstly and then decreased.With Agrobacterium-tumefaciens suspension OD600 of 0.5,infection time of 20 min,co-culture time of 4d,and 80 W power sonication pretreatment of 3 min,the highest GUStransient expression rates were 14.43%,13.73%,10.85%,and 75.36%,respectively.Under the optimal conditions,the GUSgene was transformed into grape hyacinth and 207 hygromycin resistant plants were obtained.GUSactivity analysis showed varying degrees of blue color at different parts of the resistant plants,PCR detected showed 7PCR positive plants,and reverse transcription-PCR analysis identified 3transgenic plants.【Conclusion】An Agrobacterium-mediated genetic transformation system of grape hyacinth was established and GUStransient expression was significantly improved with Agrobacterium-tumefaciens.