农杆菌介导大豆子叶节遗传转化体系的优化  

Optimization of Cotyledonary-node Agrobacterium-mediated Soybean Transformation System

作　　者：翟锐[1] 高乐[1] 丁雪妮[1] 廖文林[1] 郑明洁[1] 陆智文 智海剑[1] 

机构地区：[1]南京农业大学大豆研究所/国家大豆改良中心/作物遗传与种质创新国家重点实验室/农业部大豆生物学与遗传育种重点实验室,江苏南京210095

出　　处：《大豆科学》2015年第5期768-775,共8页Soybean Science

基　　金：转基因专项(2008ZX08004-004);国家自然科学基金(31171574;31101164);现代农业产业技术体系(CARS-004)

摘　　要：利用农杆菌菌株EHAl05，对17个栽培大豆品种的大豆子叶节进行了侵染，从大豆基因型、氯气灭菌时间、外植体状态、菌株活力、侵染浓度、侵染时间、共培养时间等方面进行了优化。结果表明：氯气灭菌14～18h，可在不影响大豆种子活力的同时最大限度的减少污染；暗处理1d的外植体活力高于光照处理5～7d的外植体；菌液浓度（OD600nm）在0．8～1．0且侵染浓度OD600nm）为0．6～0．8时GUS瞬时表达率最高；适宜的侵染时间和共培养天数分圳为30min和4d。在上述优化研究基础上，形成一套综合的转基因体系，该体系的最高转化率可达3．33％。不同处理下的GUS瞬时表达率以及17个大豆品种的丛生芽诱导率综合评价显示，适宜转化的基因型为TL-1、HC-3、HC-6、Wil-liams82。In the present study, cotyledon nodes obtained from 17 soybean cuhivars were infected with Agrobacterium tume- facines EHA105. Subsequently, we optimized the soybean genotype, chlorine slerilization lime, explant state, vitality of bac- terial strain, concentration of infection liquid, infection time and co-cultivation time. As a result, when the chlorine steriliza- tion time was 14-18 h, the minimal pollution and the highest seed vigor was observed. The vitalily of overnight-treated explants in the dark were better than those germinated in the light for 5-7 d. The highest GUS transient rate was achieved when the bac- terial concentration at OD600nm was 0. 8-1.0 and the infection liquid at OD600nm was 0. 6-0.8. We also found that the best infec- tion and co-cultivation time were 30 rain and 4 d, respectively. Based on the above optimized research, we established a com- prehensive transgenic system which resulted in the maximum transformation efficiency of 3.33%. For the comprehensive eval- uation about the transient GUS expression under different treatments and the shoot induction rate of 17 soybean varieties, TL- 1, HC-3, HC-6 and Williams 82 were the ideal genotypes for transformation.

关 键 词：大豆 子叶节 农杆菌中文 转基因体系优化 

分 类 号：S565.1[农业科学—作物学]