白氏文昌鱼单个体基因组BAC文库的构建
收稿日期: 2009-08-27
修回日期: 2009-10-10
网络出版日期: 2010-01-15
基金资助
国家高技术研究发展计划项目(863计划)(编号：2008AA092602)资助
Construction of genome BAC library for single Branchiostoma belcheri individual
Received date: 2009-08-27
Revised date: 2009-10-10
Online published: 2010-01-15
隶属于头索动物亚门的文昌鱼是现存生物中最近似于脊椎动物亚门直接祖先的一个类群, 具有重要的进化地位, 是研究脊椎动物原始祖先的重要材料和模式动物。随着文昌鱼实验室连续繁殖的成功, 全基因组测序成为中国文昌鱼模式化急需完成的工作之一。文章从单条雄性白氏文昌鱼精巢组织中提取高质量的基因组DNA, 经EcoRⅠ限制性内切酶和EcoRⅠ甲基化酶酶切, 脉冲场电泳选择合适酶切DNA片段, 连接线性磷酸化的载体pCC1BAC, 转化大肠杆菌EPI300 E. coli, 构建了含有44 706个克隆的全基因组BAC( Bacterial artificial chromosome)文库, 该文库平均插入片段80 kb,具有9倍的基因组覆盖率, 基本能够满足功能基因等研究需要, 为中国文昌鱼全基因组测序打下基础。
张巨永，黄盛丰，王蔚，徐安龙，王义权 . 白氏文昌鱼单个体基因组BAC文库的构建[J]. 遗传, 2010 , 32(1) : 67 -72 . DOI: 10.3724/SP.J.1005.2010.00067
As one of the closest living invertebrate relatives of vertebrates, amphioxus (subphylum Cephalochordata) occupies a key position in animal evolution and is becoming the best available proxy and model animal for studying the last common ancestor of all chordates, especially vertebrates. As long-term continuous culturing of amphioxus in laboratory became reliable, for pushing this animal to be a more successful model system, whole-genome sequencing of one or more species derived from this branch will be another urgent issue needed to address. In the present study, we described the construction and characterization of a bacterial artificial chromosome (BAC) library, using a single individual of Chinese amphioxus (Branchiostoma belcheri). High quality genomic DNA extracted from the spermary was partially digested with EcoRⅠand EcoRⅠmethylase. Desirable DNA fragments were isolated by pulsed field gel electrophoresis (PFGE), ligated to linearized and phosphorylased carrier pCC1BAC, and then transformed to Escherichia coli EPI300. The constructed library consists of 44 706 clones with the average insert fragment size around 80 kb as estimated by PFGE. The representation of the library is about 9 equivalents to the amhioxus genome. These results indicate that the BAC library will be useful for functional genomic studies and facilitate the whole-genome sequencing of Chinese amphioxus.
Key words： amphioxus; Cephalochordata; Branchiostoma belcheri; genomic library; BAC
[1] Putnam NH, Butts T, Ferrier DEK, Furlong RF, Hellsten U, Kawashima T, Robinson-Rechavi M, Shoguchi E, Terry A, Yu JK, Benito-Gutiérrez E, Dubchak I, Garcia-Fernàndez J, Gibson-Brown JJ, Grigoriev IV, Horton AC, Jong PJ, Jurka J, Kapitonov VV, Kohara Y, Kuroki Y, Lindquist E, Lucas S, Osoegawa K, Pennacchio LA, Salamov AA, Sa-tou Y, Sauka-Spengler T, Schmutz J, Shin-IT, Toyoda A, Bronner-Fraser M, Fujiyama A, Holland LZ, Holland PWH, Satoh N, Rokhsar DS. The amphioxus genome and the evolution of the chordate karyotype. Nature, 2008, 453(7198): 1064–1071.
[2] Huang SF, Yuan SC, Guo L, Yu YH, Li J, Wu T, Liu T, Yang MY, Wu K, Liu HL, Ge J, Yu YC, Huang HQ, Dong ML, Yu CL, Chen SW, Xu AL. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordi-nary innate complexity and diversity. Genome Res, 2008, 18(7): 1112–1126.
[3] Holland LZ, Albalat R, Azumi K, Benito-Gutierrez E, Blow MJ, Bronner-Fraser M, Brunet F, Butts T, Candiani S, Dishaw LJ, Ferrier DEK, Garcia-Fernàndez J, Gib-son-Brown JJ, Gissi C, Godzik A, Hallbook F, Hirose D, Hosomichi K, Ikuta T, Inoko H, Kasahara M, Kasamatsu J, Kawashima T, Kimura A, Kobayashi M, Kozmik Z, Ku-bokawa K, Laudet V, Litman GW, McHardy AC, Meule-mans D, Nonaka M, Olinski RP, Pancer Z, Pennacchio LA, Pestarino M, Rast JP, Rigoutsos I, Robinson-Rechavi M, Roch G, Saiga H, Sasakura Y, Satake M, Satou Y, Schubert M, Sherwood N, Shiina T, Takatori N, Tello J, Vopalensky P, Wada S, Xu A, Ye Y, Yoshida K, Yoshizaki F, Yu JK, Zhang Q, Zmasek CM, Jong PJ, Osoegawa K, Putnam NH, Rokhsar DS, Satoh N, Holland PWH. The amphioxus ge-nome illuminates vertebrate origins and cephalochordate biology. Genome Res, 2008, 18(8): 1380.
[4] 张士璀, 郭斌, 梁玉君. 我国文昌鱼研究50年. 生命科学, 2008, 20(1): 64–68.
[5] 王义权, 张秋金, 吕小梅, 钟婧, 孙毅. 文昌鱼的实验室繁育及子二代获得. 动物学研究, 2006, 27(6): 631–634.
[6] Zhang QJ, Sun Y, Zhong J, Li G, Lü XM, Wang YQ. Continuous culture of two lancelets and production of the second filial generations in laboratory. J Exp Zool (Mol Dev Evol), 2007, 308(4): 464–472.
[7] Nohara M, Nishida M, Manthacitra V, Nishikawa T. An-cient phylogenetic separation between Pacific and Atlantic cephalochordates as revealed by mitochondrial genome analysis. Zoolog Sci, 2004, 21(2): 203–210.
[8] Kumar S, Hedges SB. A molecular timescale for vertebrate evolution. Nature, 1998, 392(6679): 917–920.
[9] Weiss KM, Smith FH. Out of the veil of death rode the one million! Neandertals and their genes. Nature, 2007, 29(2): 105–110.
[10] Green RE, Krause J, Ptak SE, Briggs AW, Ronan MT, Simons JF, Du L, Egholm M, Rothberg JM, Paunovic M, Paabo S. Analysis of one million base pairs of Neanderthal DNA. Nature, 2006, 444(7117): 275–276.
[11] Wang W, Xu HL, Lin LP, Su B, Wang YQ. Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-Pax genes. Genes Genet Syst, 2005, 80(3): 233–236.
[12] Zhong J, Zhang JY, Mukwayaa E, Wang YQ. Revaluation of deuterostome phylogeny and evolutionary relationships among chordate subphyla using mitogenome data. J Genet Genomics, 2009, 36(3): 151–160.
[13] Xu QS, Ma F, Wang YQ. Morphological and 12S rRNA gene comparison of two Branchiostoma species in Xiamen waters. J Exp Zool (Mol Dev Evol), 2005, 304(3): 259–267.
[14] 王蔚. 白氏文昌鱼BAC文库的构建及Pax基因家族的进化分析[学位论文]. 厦门大学, 2006.
[15] Shigekawa K, Dower WJ. Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells. Botechniqueis, 1988, 6(8): 742–751.
[16] Zimmer R, Verrinder-Gibbins AM. Construction and characterization of a large-fragment chicken bacterial arti-ficial
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
