栉孔扇贝Fosmid文库的构建及基因组结构特征分析

Fosmid Library Construction and Genomic Structure Analysis in Zhikong Scallop(Chlamys farreri)

研究构建第一个栉孔扇贝的fosmid基因组文库,该文库包含133 851个克隆,插入片断平均长度为40 kb,覆盖栉孔扇贝基因组的4.3倍。栉孔扇贝DNA在fosmid传代中表现得较为稳定,没有发现插入片段的丢失或重排。所有的克隆进行了超级池和二级池的构建,并筛选了2个基因和7个微卫星,结果阳性克隆数介于2到8之间。由此可见,该文库可以很好的用于目的基因或标记的筛选,将有利于物理作图和基因的图位克隆研究。2 016个克隆进行双末端测序,获得3 646条序列。序列总长2 286 986 bp,大约占栉孔扇贝基因组的1.84‰。共得到2 500条串连重复序列,其中微卫星序列371条,小卫星序列1 816条,卫星序列313条。共得到317条散布重复序列,总长97 412 bp,占测序总长的4.26%。查找到的散布重复序列共有4种类型:DNA转座子、LTR反转座子、LINE反转座子和滚环转座子,其中LINE反转座子的数目最多。BLAST比对共有1 383条序列与数据库现有的序列有明显的相似性(E<e-5),占测序总数的37.93%。BLASTN比对有明显相似性的1 036条序列中,与nr数据库序列相似的有113条,与EST数据库序列相似的有923条。BLASTX比对有明显相似性的序列有347条,占序列总数的9.52%。

Zhikong scallop （ Chlamys farreri ） is one of the most commercially important bivalves in China, but study on its genome is underdeveloped. The first Zhikong scallop fosmid library was constructed in this study. It consists of 133 851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equiv- alents. Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between 2 and 8 positive clones, and none of those tested was absent from the library. The fosmid library will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of Zhikong scallop genome. In the present study, 2 016 individual clones were sequenced, resulting in 3 646 sequences. A total of 2 286 986 bp of genomic sequences was generated, representing approximately 1.84%o of the Zhikong scallop genome. Using Tandem Repeats Finder（TRF） software to analyze the sequences, a total of 2 500 tandem repeats were found, including 371 microsatellites, 1,816 minisatellites and 313 satellites, RepeatMasker analysis showed that a total of 317 interspersed repetitive sequences were found, which account for 4.26% of the total length. There are 4 types in these sequences. DNA transposons, LTR retrotransposons, LINE retrotransposons and rolling circles. Among them, LINE retrotransposons is the most frequent, followed by LTR retrotransposons, DNA transposons and rolling circles. A total of 1 383 sequences were found to significantly hit to sequences in GenBank database using BLASTN and BLASTX（E〈 e^-5）. BLASTN searches on the nr and EST databases of GenBank and BLASTX searches on the nr database resulted 113, 923 and 347 significant hits, respectively.