遗传 ›› 2013, Vol. 35 ›› Issue (6): 771-777.doi: 10.3724/SP.J.1005.2013.00771

• 研究报告 • 上一篇    下一篇

piggyBac转座子在牛基因组的整合位点及特征分析

杜新华, 高雪, 张路培, 高会江, 李俊雅, 许尚忠   

  1. 中国农业科学院北京畜牧兽医研究所, 中国农业科学院肉牛研究中心, 农业部畜禽遗传资源与利用重点开放实验室, 北京100193
  • 收稿日期:2012-12-18 修回日期:2013-02-27 出版日期:2013-06-20 发布日期:2013-06-25
  • 通讯作者: 许尚忠 E-mail:simmenta@vip.sina.com
  • 基金资助:

    “十二五”转基因重大专项(编号:2011ZX08007-002-009), “十二五”国家科技支撑计划(编号:2011BAD28B04), 现代农业(肉牛、牦牛)产业技术体系岗位体系科学家项目(编号:CARS-38)和中国农业科学院基本科研业务费增量(编号:2013ZLD31)资助

Integration sites and their characteristic analysis of piggyBac trans-poson in cattle genome

DU Xin-Hua, GAO Xue, ZHANG Lu-Pei, GAO Hui-Jiang, LI Jun-Ya, XU Shang-Zhong   

  1. Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture; Beef Cattle Research Center, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
  • Received:2012-12-18 Revised:2013-02-27 Online:2013-06-20 Published:2013-06-25

摘要: piggyBac(PB)转座子作为一种遗传工具被广泛应用于多个物种的转基因及插入突变研究, 目前PB转座子在牛中的相关研究还较少。为了获得PB转座子在牛基因组中的整合位点, 总结其转座特征, 文章构建了PB[CMV-EGFP]和pcDNA-PBase二元转座系统, 利用细胞核电转技术共转染牛耳组织成纤维细胞, 经G-418筛选, 获得了稳定转染EGFP的转基因细胞系; 提取细胞基因组DNA, 利用基因组步移技术扩增PB转座子5′ Bac区插入位置的DNA序列; 通过与牛基因组序列进行BLAST比对, 得到PB转座子在牛基因组中的插入位点。文章共获得了8个有效的整合位点, 但仅有5个位点定位到染色体1、2、11和X染色体上。序列分析表明:在牛基因组中, PB转座子可特异性的插入到“TTAA”位置, 并整合到基因间的非调控区; 分析整合位点“TTAA”相邻一侧的5个碱基组成, 发现PB转座子5′端倾向于插入到GC(62.5%)碱基富集区。该研究表明, PB转座子可以在牛基因组中发生转座, 获得的整合位点信息为利用PB转座子在牛上开展遗传学研究提供了理论参考。

关键词: piggyBac, 基因, 基因组, 合位点

Abstract: As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were detected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on chromosomes 1, 2, 11, and X chromosome. We found that PB transposon was inserted into the “TTAA” location and integrated into the intergenic non-regulatory sites between two genes. Analysis of the composition of the five bases, which was close to the side of the PB integration sites “TTAA”, showed that PB 5′ tended to be inserted into region rich in GC (62.5%). From the study, we got that transposition occurred in cattle genome by PB transposons and the integration site information acquired from the research will provide theoretical references for bovine study by PB transposon.

Key words: piggyBac, transgenic, cattle genome, integration sites