中国海洋大学 海洋生命学院, 山东 青岛 266003;中国水产科学研究院 黄海水产研究所, 山东 青岛266071;甘肃农业大学 动物科学技术学院, 甘肃 兰州730070;大连海洋大学 水产与生命学院, 辽宁 大连 116023
黄进强(1980-), 男, 博士, 主要从事鱼类功能基因的研究. E-mail: hjq163163@163.com
S917
国家自然科学基金项目项目(31130057, 31072202); 国家863计划项目(2012AA092203); 山东省泰山学者工程专项.
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China;Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;College of Animal Science and Technology, Gansu Agricultural University,
区FP-T), 通过基因组步移和和利用在线生物信息学软件对发现在转录起始点上游框Sox-5C/EBPc-MycNKX2-5V-Myb将所构建的表达载体注射于青鳉。将有荧光的胚胎培养为成鱼的标记、追踪和操作研究以及半滑舌鳎的性别控制等奠定了基础。
gene is an excellent candidate molecular marker for studying primordial germ cells because of its specific expression in germ cell lineages. PGCs can be labeled by a reporter gene driven by regulatory regions of the ) is an economically important flatfish in China, and the females of this species grow two to four times bigger than males. Sex reversal is common in this fish and females cannot guarantee egg production by natural spawning under artificial culture conditions. Therefore, transplantation of PGCs and surrogate broodstock production might be employed to improve the production of this fish. Promoter choice in transgenic research is important for regulating expression of a foreign gene. However, the function of ) regulatory regions in tongue sole remains unknown. We aimed to isolate the regulatory regions of the gene, examine its transcriptional activity in transgenic medaka fish, and identify the conditions required for manipulation of PGCs. Two DNA fragments, including a 5166-bp 5′ region and a 1655-bp 3′ region, were obtained by genome walking and polymerase chain reaction of genomic DNA. The 5′ region of contains the major promoter region, exon 1, intron 1 and exon 2. The transcription initiation site and a putative TATA box were identified and potential transcription factor binding sites were bioinformatically analyzed. Several potential transcription factor binding sites that may perform important function relating to gene transcription, such as SRY, Oct-1, Sox-5, CREB, GATA, AP-1, C/EBP, Sp-1, c-Myc, HNF, NKX2-5, and V-Myb were predicted. Based on the above information, the regulatory regions were inserted into a pEGFP-N3 vector lacking the CMV promoter. This vector expresses the green fluorescent protein(GFP). The recombinant p-GFP-T plasmid was injected into medaka fertilized eggs by microinjection. Microfluoroscopy revealed that the level of expression was 81% in the injected embryos. GFP-fluorescent medaka embryos were cultured to adult fish and the integration efficiencyof pCsvasa promoter can efficiently drive expression in medaka embryos, and lay the foundations for identifying, labeling and tracing PGCs, investigating germ cell biology, and manipulating the sex of half-smooth tongue sole.
黄进强,陈松林,邵长伟,刘洋,林帆,李亚亚,王娜.半滑舌鳎vasa调控区的克隆、表达载体构建及其驱动活性检测[J].中国水产科学,2015,22(1):1-8
HUANG Jinqiang, CHEN Songlin, SHAO Changwei, LIU Yang, LIN Fan, LI Yaya, WANG Na. Molecular cloning, construction of the expression vector and detection of transcription activity of vasa regulatory regions in half-smooth tongue sole (Cynoglossus semilaevis)[J]. Journal of Fishery Sciences of China,2015,22(1):1-8
