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%。
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 byβ-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 trans- formed. PCR and PCR-Southern blotting results showed that the GmPK gene was integrated into the genome of soy- bean. The transformation efficiency was 0.6%.
出处
《植物遗传资源学报》
CAS
CSCD
北大核心
2012年第2期271-277,共7页
Journal of Plant Genetic Resources
基金
河北省科技攻关计划项目(042401116D-1)
河北省科学技术研究与发展计划子课题(09220103D)
河北省教育厅项目(2008460)
转基因生物新品种培育重大专项(2008ZX08004-002-1-5
2009ZX08004-001B)
关键词
大豆
胚尖
遗传转化
根癌农杆菌
GmPK基因
Glycine max
Embryonic tips
Genetic transformation
Agrobacterium tumefaciens
GmPK gene
