摘要
基因工程是改良百合性状的重要手段,建立高效稳定的遗传转化体系是百合转基因研究的基础。以百合地下茎鳞片为外植体,筛选并优化百合的直接和间接再生体系;把含枸杞GR（Glutathione reductase）基因和筛选基因NPTII的载体,利用农杆菌转化法对鳞片和愈伤组织进行转基因操作,采用正交试验,优化转化条件以建立适合不同受体的遗传转化体系。结果表明,各阶段最优培养条件分别为：鳞片诱导和膨大MS＋2mg/L 2,4-D（2,4-二氯苯氧乙酸）＋0.1mg/L NAA（萘乙酸）＋90g/L蔗糖;鳞片直接分化MS＋1.0mg/L 6-BA（6-苄氨基嘌呤）＋0.2mg/L NAA＋30g/L蔗糖,间接分化MS＋2.5mg/L 2,4-D＋0.4mg/L TDZ（噻重氮苯基脲）＋60g/L蔗糖;百合鳞片的Kana（卡那霉素）选择压为100mg/L,愈伤组织75mg/L。遗传转化体系条件为：鳞片,农杆菌OD600=0.6,预培养3d,侵染40min,As（乙酰丁香酮）200μmol/L,阳性植株转化率为17.50%;鳞片分化愈伤组织,农杆菌OD600,预培养5d,侵染40min,As 200μmol/L,阳性植株转化率为12.60%。
Genetic engineering is an important means to improve the traits of lily. To establish an efficiency and stable transformation system is the basis of transgenic research. The different regeneration systems of lily were screened. The vector containing the target gene GR and selection gene NPTII was screened by Agrobacterium tumefaciens. Establish efficiency genetic transformation system for callus and scales through orthogonal test. The results showed that the optimum culture conditions as follow. The medium of lily scale inducing was MS ＋ 2mg/L 2,4-D ＋ 0. lmg/L NAA ＋ 90g/L ＋ 0.2mg/L NAA ＋ 30g/L sucrose. Indirect differentiation medium was MS ＋2.5mg/L 2,4-D ＋0.4mg/L 0.4mg/L TDZ ＋ 60g/L sucrose. The Kana selection pressure for scales was 100mg/L and for callus was 75mg/L. The direct genetic transformation of the scales was Agrobacterium 0D6oo = 0.6, pre-cuhure for 3 days, infiltrating for 40min, As 200μmol/L. The indirect callus transformation was： Agrobacterium OD600, pre-incubated for 5 days, infiltrating for 40min, As 200μmol/L- The direct and indirect positive plant transformation rates were 17.50% and 12.60%.
作者
安婷
季静
王昱蓉
马志刚
王罡
李倩
杨丹
张松皓
AN Ting;JI Jing;WANG Yu-rong;MA Zhi-gang;WANG Gang;LI Qian;YANG Dan;ZHANG Song-hao(School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China;Tianda Tianfu Bio. Co. LTD, Tianjin 300072, China;UCSD, Biological Sciences, Molecular Biology, California 92093, USA;School of Chemical Engineering and Technology, Tianjin University , Tianjin 300072, China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2018年第1期25-31,共7页
China Biotechnology
基金
国家自然科学基金(31271793)
国家科技重大专项(2014Z1-0002)资助项目
关键词
百合
遗传转化
GR
再生体系
直接分化
Lily Genetic transformation GR Regeneration system Direct differentiation
