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大豆胚尖遗传转化体系优化及抗逆基因GmPK转化研究
引用本文:张 洁,商 蕾,郑文永,邱丽娟,王冬梅.大豆胚尖遗传转化体系优化及抗逆基因GmPK转化研究[J].植物遗传资源学报,2012,13(2):271-277.
作者姓名:张 洁  商 蕾  郑文永  邱丽娟  王冬梅
作者单位:1. 河北农业大学生命科学学院,保定,071000
2. 中国农业科学院作物科学研究所,北京,100081
基金项目:河北省科技攻关计划项目(NO. 042401116D-1); 河北省科学技术研究与发展计划子课题(NO.09220103D);河北省教育厅项目(NO.2008460);
摘    要:采用GUS基因瞬时表达检测方法,通过正交试验以AS浓度、侵染菌液OD值、侵染时间、共培养时间和恢复培养时间5个因素在4个水平上进行分析,优化了农杆菌介导的大豆胚尖遗传转化体系,并在此基础上进行了抗逆基因GmPK的遗传转化。结果表明,采用共培养培养基中添加100μmol/L AS、侵染菌液OD600值0.9、侵染15h、共培养5d和恢复培养3d的转化条件最佳,GUS阳性率达74.59%,经PCR及RT-PCR进一步验证获得了转基因阳性植株。利用优化的最佳条件进行抗逆基因GmPK的转化,炼苗移栽成活的再生植株经PCR及PCR-Southern blotting验证,初步证明外源基因已经整合至大豆基因组,转化率为0.6%。

关 键 词:大豆  胚尖  遗传转化  根癌农杆菌  GmPK基因
收稿时间:2011/3/13 0:00:00
修稿时间:2011/11/6 0:00:00

Optimization of Soybean Embryonic Tips Genetic Transformation System and Transformation of Stress Resistance Gene GmPK
ZHANG Jie , SHANG Lei , ZHENG Wen-yong , QIU Li-juan , WANG Dong-mei.Optimization of Soybean Embryonic Tips Genetic Transformation System and Transformation of Stress Resistance Gene GmPK[J].Journal of Plant Genetic Resources,2012,13(2):271-277.
Authors:ZHANG Jie  SHANG Lei  ZHENG Wen-yong  QIU Li-juan  WANG Dong-mei
Institution:Institute of CropSciences,Chinese Academy of Agricultural Sciences,College of Life Science,Agticultural University of Hebei
Abstract:The Agrobacterium tumefaciens mediated genetic transformation system of soybean embryonic tips was optimized.The experiment was carried out by orthogonal test involving four levels of five terms(Acetosyringone(AS) concentration,Agrobacterium OD value,infection time,co-culture time and recovery culture time),and the transgenic events were ascertained by β-glucuronidase assay.The results indicated that the optimum transformation conditions were: 100 μmol/L AS,Agrobacterium OD of 0.9,infected for 15hours,co-cultured for 5days,recovery cultured for 3days.In the above condition,GUS staining positive rate reached up to 74.59%.And then,transgenic plants were obtained by PCR and RT-PCR.Using the optimized system,a stress-resistant gene GmPK was transformed.PCR and PCR-Southern blotting results showed that the GmPK gene was integrated into the genome of soybean.The transformation efficiency was 0.6%.
Keywords:Glycine max  Embryonic tips  Genetic transformation  Agrobacterium tumefaciens  GmPK gene
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