农杆菌介导的ACO-RNAi载体对百合胚性愈伤的转化  

Genetic transformation of lily embryogenic callus with ACO-RNAi vector by Agrobacterium tumefaciens

作　　者：田菲菲[1,2] 刘雅莉[1,2] 杜灵娟[1,2] 权永辉[1,2] 

机构地区：[1]旱区作物逆境生物学国家重点实验室(西北农林科技大学)/农业部西北地区园艺作物生物学与种质创制重点实验室,陕西杨凌712100 [2]西北农林科技大学林学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2015年第3期105-112,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31170652)

摘　　要：【目的】建立高效稳定的百合遗传转化体系,以得到甁插寿命长、开花早的百合转基因品种。【方法】以OT百合"罗宾那"的胚性愈伤组织为供试材料,从共培养方式(固体或液体共培养)、农杆菌菌液浓度(OD600=0.4,0.6,0.8,1.0,1.2)、亚精胺浓度(0.5,1.0,1.5,2.0μmol/L)以及超声波处理(功率60 W,处理时间分别为0,1,2,3,4min)等方面对百合遗传转化体系进行优化。【结果】液体共培养能提高稳定表达率并减少褐化率,稳定表达率为13.00%,褐化率为55.00%;农杆菌菌液浓度(OD600)在0.8时为最适侵染浓度,此时稳定表达率为12.77%,褐化率为55.00%;侵染前向菌液中加入2.0μmol/L亚精胺能提高转化效率,稳定表达率为88.30%;当超声波功率为60W、处理时间为3min时,百合胚性愈伤的瞬时表达率和稳定表达率均最高,分别为88.90%和27.33%;潮霉素抗性植株经GUS组织化学染色、目标基因PCR检测及Southern blot分析,证明目的基因已整合到百合基因组中。【结论】转化条件的优化能够有效提高农杆菌介导的百合胚性愈伤组织的遗传转化率。【Objective】This study aimed to establish an efficient and stable genetic transformation system of lily to obtain genetically modified lily varieties with long service life and early flowering.【Method】Using embryogenic callus of OT lily'Robina'as material,the lily genetic transformation system was optimized based on co-culture methods(liquid or solid co-culture),bacterial concentrations(OD600=0.4,0.6,0.8,1.0,and 1.2),and spermidine concentrations(0.5,1.0,1.5,and 2.0μmol/L),and different sonication treatments(60 W power by 0,1,2,3,and 4 min respectively).【Result】Liquid co-culture improved the transformation efficiency(13.00%)and reduce browning rate(55.00%).Bacterial concentration of OD600 at 0.8was the optimum concentration with transient expression rate and browning rate of 12.77% and55.00%,respectively.Adding 2.0μmol/L spermidine to bacteria before infection improved the transformation efficiency with stable transformation rate of 88.30%.When the sonication power was 60 W and the processing time was 3min,the highest lily embryonic callus transient expression rate and stable expression rate of 88.90% and 27.33% were obtained,respectively.After GUShistochemical analysis,PCR detection and Southern blot analysis,it was confirmed that the target gene was successfully integrated into the lily genome of hygromycin resistant plants.【Conclusion】Optimizing the transformation conditions can effectively improve the Agrobacterium-mediated genetic transformation rate.

关 键 词：百合 转基因 农杆菌 超声波 亚精胺 

分 类 号：S682.29[农业科学—观赏园艺]