农杆菌介导的AtMYB118基因转化橡胶树易碎胚性愈伤组织体系优化(英文)

Optimization of system for Agrobacterium tumefaciens-mediated AtMYB118 genetic transformation into friable embryogenic calli in Hevea brasiliensis

[目的]采用正交设计L25（56）优化农杆菌介导的AtMYB118基因对橡胶树易碎胚性愈伤组织的遗传转化体系,为橡胶树品种遗传改良提供参考.[方法]以75.0 mg/L卡那霉素筛选经过转化的愈伤组织;采用GUS瞬时表达检测方法评价6个因素即预培养时间、农杆菌菌液浓度（OD600）、乙酰丁香酮（AS）浓度、侵染时间、共培养温度和共培养时间对遗传转化的影响.[结果]6个因素显著影响橡胶树长期培养的易碎胚性愈伤组织的转化频率,易碎胚性愈伤组织遗传转化优化条件为：预培养0d,菌液OD600为0.7,侵染时间7 min,共培养时间5d,AS 200 μmol/L,共培养温度25℃.在此优化条件下可获得最高转化频率.经过4～6个月的筛选,获得17个GUS染色阳性的易碎愈伤组织系.经PCR和反转录PCR分析转化愈伤组织,进一步证实uidA、nptⅡ、AtMYB118基因已被整合到愈伤组织基因组中并表达.培养获得1539个胚状体,其中164个为转基因胚状体,转化频率为10.6％;最终获得4株转AtMYB118基因植株.[结论]优化获得可行有效的橡胶树易碎胚性愈伤组织遗传转化体系,可为其品种遗传改良提供技术支持.

[Objective] An orthogonal design L25 （56） was used to optimize Agrobacterium-mediated AtMYB118 gene genetic transformtion system of friable embryogenic calli in Hevea brasiliensis in order to provide references for genetic improvement of its clones.[Method]Kanamycin at a concentration of 75.0 mg/L was used to select transformed calli.The effects of six factors viz.,preculture time,Agrobacterium growth phase （OD600）,acetosyringone （AS） concentration,infection time,co-culture temperature and co-culture time on genetic transformation were evaluated by GUS transient expression detection.[Result]Six factors were found to significantly affect transformation frequency of long-term cultural friable embryogenic calli.The highest transformation efficiency was achieved by 0-day preculture,long-term cultural friable embryogenic calli as recipients infected by Agrobacterium cultures corresponding to OD600=0.7 for 7 min,followed by co-culture for 5 days in a co-culture medium containing 200 μmol/L AS at 25 ℃.After 4-6 months,17 GUS positive callus lines were achieved.Polymerase chain reaction （PCR） and reverse transcription-polymerase chain reaction （RT-PCR） analysis results of transgenic tissues further confirmed that uidA,nptⅡ and AtMYB118 genes had been inserted into genome of calli and expressed.At last,164 transgenic embryoids were obtained from 1539 cultured embryoids.A transformation efficiency of 10.6％ was achieved for longterm cultural friable embryogenic calli using this protocol.Four AtMYB118 transgenic plantlets were obtained.[Conclusion]The optimized genetic transformation system using friable embryogenic calli of rubber tree as recipients was effective and available,which would provide technological supports on genetic improvement of clones.