摘要
【目的】构建piggyBac(PB)转座子表达载体系统,研究PB转座子在绒山羊基因组中的整合位点。【方法】构建pB-CMV-EGFP-Neo转座载体和pcDNA-Trans辅助载体,利用lipofectamine 2000介导共转染绒山羊胎儿成纤维细胞,经G418筛选获得稳定转染的转基因细胞系。提取转基因细胞基因组DNA,利用反向PCR检测piggyBac转座子整合位点。【结果】构建了转座系统并成功介导外源基因在绒山羊成纤维细胞染色体中整合,获得了转基因细胞系;反向PCR检测显示在绒山羊基因组中有21个PB转座子整合位点;整合位点TTAA毗邻一侧的5个碱基组成中,PB转座子3′倾向于插入到富含AT(58.35%)碱基的区域,PB转座子5′倾向于插入到富含GC(57.8%)。【结论】PB转座子可在绒山羊基因组中发生高效转座,获得的整合位点信息可为利用PB转座子开展绒山羊研究提供理论参考。
【Objective】 The objective of the experiment was to construct piggyBac(PB) transposons vectors and integration site in Cashmere goat genome of PB was also studied.【Method】 Donor plasmid of PB-CMV-EGFP-Neo and helper-dependent plasmid of pcDNA-Trans were constructed and transferred into Cashmere goat fetal fibroblasts by lipofectamine 2000.Cell clones stably transfected were obtained after screening by G418.Transgenic cell genome was obtained and the integration site of PB transposons were detected by r-PCR.【Result】Transposition system was constructed and the integration of foreign gene in Cashmere goat fibroblasts was successfully mediated.Transgenic cell line was also obtained.There were 21 integration sites of PB transposon in Cashmere goat genome after r-PCR detection.Analysis of the composition of the five bases,which was close to the side of the PB integration sites TTAA,showed that PB 3′ tended to insert into region rich in AT(58.35%) and the PB 5′ tended to insert into region rich in GC(57.8%).【Conclusion】 The transposition of foreign PB transposons in Cashmere goats was highly efficient.The integration site information which is acquired from this research will provide theoretical references for Cashmere goats study by PB transposons.
出处
《中国农业科学》
CAS
CSCD
北大核心
2012年第5期958-965,共8页
Scientia Agricultura Sinica
基金
国家转基因生物新品种培育重大专项课题研究计划(2008ZX08008-002)
作者简介
白丁平，E-mail：bdpnx@163．com。
通信作者陈玉林，E-mail：myxy11@yahoo．com．cn
