摘要
为获得转基因高油酸油菜T-DNA插入拷贝数及整合位点相关信息,本研究应用地高辛标记的NPTII基因片段为探针,与经BamHI酶切的转基因甘蓝型油菜高油酸株系W-4的T2代单株的基因组DNA进行Southern杂交。结果显示:W-4的T2代单株的基因组含有一个T-DNA拷贝。用3到4个根据载体pCNFIRnos序列设计的嵌套特异性引物分别与简并引物组合进行TAIL-PCR反应,扩增得到转基因油菜T-DNA插入位点的左、右边界旁侧序列。经分析右边界旁侧序列长度为470bp,其中180bp为载体序列,290bp为W-4的基因组序列;左边界旁侧序列长度为641bp,其中365bp为W-4的基因组序列,276bp为载体序列。序列比对结果发现该转基因事件中,T-DNA左边界序列完全整合到油菜基因组中,仅有1个碱基由G转换成了A。而右边界则缺失了包括RBborder在内的62个碱基。结果表明:转基因高油酸油菜T-DNA的整合是一次无其他额外载体序列的整合。blast分析获得的与左右边界相连的油菜基因组序列,未检索到与之高度同源的序列,推测T-DNA插入位点可能位于油菜基因组非编码区。综上所述,本研究分析了转基因油菜W-4基因中T-DNA拷贝数、整合特点和旁侧序列,研究结果为转基因油菜的生物安全性评价以及转基因高油酸油菜的检测提供重要的基础信息。
To investigate the copy of T-DNA integrated into the genome of transgenic high oleic acid Brassica napus L.the southern blot was conducted using the genomic DNA from the T2 generation plants of the transgenic line W-4.The genomic DNA was digested with BamHI and then probed with a segment of NPT II gene labeled with Dig.The result showed that only one copy of T-DNA was integrated into the genome of W-4.For isolating the flanking sequence to both the right border and the left border of the T-DNA in the genome,the thermal asymmetric interlaced PCR（TAIL-PCR） was conducted using three or four nested specific primers designed based on the sequence of the vector pCNFIRnos and a short arbitrary degenerate primer（AD1 or AD2） respectively.The specific PCR products corresponding to both the right and left border flanking sequence were successfully amplified.The right border flanking sequence is 470 bp which includes a 290 bp genome sequence and a 180 bp vector sequence based on VecScreen.Further sequence alignment analysis showed that the 180 bp was identical to the RB border of pCNFIRnos but with a 62 bp deletion.The left border flanking sequence is 641 bp including a 365 bp genome sequence and a 276 bp vector sequence.The 276 bp was identical to the left border of pCNFIRnos except a change of G to A.So,the integration of the T-DNA in the transgenic line W-4 is a vector backbone-free integration.The obtained genomic sequences were done blast on web,but did not found highly homologous sequence.We conclude that the T-DNA may integrated at non-coding site of the genome.The results about the T-DNA copy,characteristic of integration and flanking sequence in the genome of the transgenic rapeseed W-4 may provide an important reference for the bio-safety of transgenic rapeseed evaluation and the detection of the transgenic rapeseed.
出处
《分子植物育种》
CAS
CSCD
2011年第1期17-24,共8页
Molecular Plant Breeding
基金
农业部"948"项目(Q04)
江苏省科技支撑计划(BE2009304)共同资助
关键词
转基因高油酸油菜
基因拷贝数
整合位点
Transgenic high oleic acid rapeseed
Transgenic copy
Integration site
