Construction of BAC contigs of silkworm, Bombyx mori

家蚕 Bombyx mori 的 BAC 重叠群的构建

• 批准号: 11694226
• 负责人: MITA Kazuei
• 金额: $ 3.71万
• 依托单位: National Institute of Radiological Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694226/
• 关键词: silkworm, Bombyx mori; BAC library; BAC contig; hybridization; EST; physical map; fingerprinting; フィンガープリント; BACコンティーグ; 遺伝子地図; カイコゲノム解析

The purpose of the project is the construction of the physical map of silkworm genome for the silkworm genome analysis. First, we succeeded in the construction of a high quality BAC library. The BAC library contains approximately 36,000 clones with an average insert size of 168 kb, which corresponds to 11-fold genome redundancy. The rate of chimera was estimated to be less than 3%. The BAC clones were arrayed into 96 384-well microtiter dishes and were gridded in duplicate in specific patterns onto two nylon membranes (High Density Rplica (HDR) filters). We are employing two methods for the construction of BAC contigs covering whole genome, (1)the EST maker-overlapping method and (2)the fingerprinting method. For the overlapping of EST markers, BAC clones containing EST sequences were screened by hybridization of HDR filters using ESTs as probes. The genome size of silkworm is estimated to be 530 Mb. Since the average size of BAC clones is 168 kb, more than 6,200 hybridization experiments using non-redundant EST probes are required to complete BAC contig construction. So far, about 2,500 hybridizations were over. The fingerprinting method based on restriction enzyme cutting patterns is more effective than EST marker-overlapping method for the regions with low gene density, where the EST-overlapping method is not available. For fingerprinting experiments, about 1/3 experiments were carried out so far. The construction of whole BAC contigs using tose two methods are progressing. For several loci of Z and W sex chromosomes, chorion locus of chromosome 2 and virus resistant gene locus of chromosome 17, the BAC contigs were constructed and analyses using the BAC clones revealed characteristic features of silkworm genome and interesting genes.

本项目的目的是构建家蚕基因组物理图谱，用于家蚕基因组分析。首先，我们成功地构建了高质量的BAC文库。BAC文库包含约36,000个克隆，平均插入大小为168 kb，相当于11倍的基因组冗余。嵌合体率估计不到3%。将BAC克隆排列到96个384孔的微量培养皿中，并以特定的图案一式两份地网格在两个尼龙膜(高密度Rplica(HDR)过滤器)上。我们正在使用两种方法来构建覆盖整个基因组的BAC重叠群，(1)EST标记重叠法和(2)指纹法。针对EST标记的重叠现象，以EST为探针，通过HDR滤膜杂交筛选出含有EST序列的BAC克隆。家蚕基因组大小估计为530Mb。由于BAC克隆的平均大小为168kb，因此需要使用非冗余EST探针进行6200多次杂交实验才能完成BAC重叠群的构建。到目前为止，大约有2500次杂交已经结束。对于基因密度较低的区域，基于限制性内切酶切割模式的指纹图谱方法比EST标记重叠方法更有效，而EST重叠方法是不适用的。对于指纹实验，到目前为止已经进行了大约三分之一的实验。用这两种方法构建完整的BAC重叠群的研究正在取得进展。对Z、W性染色体、2号染色体绒毛膜基因座和17号染色体抗病毒基因座构建了BAC重叠群，利用BAC克隆进行分析，揭示了家蚕基因组特征和感兴趣的基因。

项目成果

Hiroaki Abe: "Molecular structure of a novel gypsy-T-13-like retrotransposon and nested retrotransposable elements"Mol.Gen.Genet.. 268. 916-924 (2000)

Hiroaki Abe：“新型 gypsy-T-13 样逆转录转座子和嵌套逆转录转座元件的分子结构”Mol.Gen.Genet.. 268. 916-924 (2000)

Jieun Lee: "Characterization of JDP genes, an evolutionarily conserved J domain-only protein family, from human and moths"Biochim.Biophyz.Acta. (印刷中). (2000)

Jieun Lee：“来自人类和飞蛾的 JDP 基因（一种进化上保守的仅包含 J 结构域的蛋白质家族）的特征”Biochim.Biophyz.Acta（出版中）。

Two novel Pao-like retrotransposons of Bombyx mori and B. mandarina

家蚕和柑橘的两个新的 Pao 样反转录转座子

• 发表时间: 2001
• 期刊: Mol. Gen. Genet. 265
• 作者: Hiroaki Abe

Guo-Xing.Quan: "Isolation and expression of the ecdysteroid-inducible angiotensin-converting enzyme-related gene in wing discs"Insect Mol.Biol.. 31. 97-103 (2001)

郭兴.全：“翼盘中蜕皮激素诱导的血管紧张素转换酶相关基因的分离和表达”昆虫分子生物学. 31. 97-103 (2001)

Genomic sequence of 320 kb containing a kettin orthologue on the Z chromosome in Bombyx mori.

320 kb 的基因组序列，包含家蚕 Z 染色体上的 kettin 直系同源物。

• 发表时间: 2003
• 期刊: Mol Genet Genomics. 269
• 作者: Yoshiko Koike;Kazuei Mita;Masataka G.Suzuki;Susumu Maeda;Hiroaki Abe;Kazutoyo Osoegawa;Pieter J.deJong;Toru Shimada
• 通讯作者: Toru Shimada