摘要
目的:确定抗除草剂转基因大豆外源基因拷贝数和其插入位点侧翼序列。方法:采用绝对定量PCR法测定转EPSPS基因大豆中外源基因拷贝数,内参照基因标准曲线选用大豆凝集素(Lectin)基因为标准品,外源基因标准曲线以含EPSPS基因的阳性质粒为标准品。采用基因组步移技术和巢式PCR方法确定抗除草剂转基因大豆插入位点旁侧序列。结果:抗除草剂转基因大豆外源基因的拷贝数为1。CaMV35S上游扩增887bp,NOS下游扩增1 340bp。结论:明确了EPSPS外源基因在转基因大豆中为单拷贝,转基因大豆插入位点附近大豆基因组发生了DNA重排。
Objective:To determine the copy number of foreign gene and the flanking sequence of insertion site.Method:Absolute quantitative technique was used to analyze the copy number of transgenic soybean with the EPSPS gene.A conserved soybean housekeeping gene(lectin) was used as an internal control the plasmid containing the foreign gene EPSPS was used as the standard samples.Genome walking technique and nested PCR method were used to determine the flanking sequence of insertion site.Result:The copy number of foreign gene in herbicide resistant transgenic soybean was single copy.A 887bp fragment of CaMV35S upstream and a 1 340bp fragment of NOS downstream were amplified.Conclusion:The copy number of EPSPS gene was single copy.The genome DNA rearrangement was occurred in the insertion site of genetically modified soybean.
出处
《生物技术》
CAS
CSCD
北大核心
2011年第6期31-35,共5页
Biotechnology
基金
国家转基因重大专项子课题(2008ZX08012-001)
哈尔滨市科技创新人才专项资金(2010RFQXN101)资助
关键词
抗除草剂转基因大豆
旁侧序列
拷贝数
基因组步移
接头PCR
herbicide resistant transgenic soybean
flanking sequence
copy number
genome walking
adapter PCR
