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【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0 d,菌液OD600为0.7,侵染时间7 min,共培养时间5 d,AS 200μmol/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】 The objective of this study was to optimize the genetic transformation system of AtMYB118 gene mediated by Agrobacterium tumefaciens to the embryogenic calluses of rubber tree by orthogonal design L25 (56), and to provide a reference for the genetic improvement of rubber tree varieties. 【Method】 The transformed callus was screened with 75.0 mg / L kanamycin. Six factors including pre-culture time, Agrobacterium tumefaciens concentration (OD600), and acetosyringone (AS) were evaluated by GUS transient expression assay Concentration, infection time, co-culture temperature and co-culture time on genetic transformation. 【Result】 Six factors significantly affected the frequency of transformation of the fragile embryogenic callus of long-term cultivated rubber tree. The optimal conditions for the genetic transformation of the fragile embryogenic callus were as follows: pre-culture 0 d, OD 600 of the bacterial liquid 0.7, infection time 7 min, co-culture time 5 d, AS 200 μmol / L, co-culture temperature 25 ℃. The highest conversion frequency is achieved under this optimization. After 4 to 6 months of screening, 17 GUS-positive friable callus lines were obtained. The transformed calli were analyzed by PCR and reverse transcriptional PCR to further confirm that the uidA, nptII and AtMYB118 genes have been integrated into the callus genome and expressed. A total of 1539 embryoid bodies were obtained, of which 164 were transgenic embryoids with a transformation frequency of 10.6%. Finally, 4 transgenic AtMYB118 plants were obtained. 【Conclusion】 Optimizing the genetic transformation system of viable embryogenic callus of viable rubber tree could provide technical support for the genetic improvement of its varieties.