Construction of BAC contigs of silkworm, Bombyx mori

本项目的目的是构建家蚕基因组物理图谱，为家蚕基因组分析提供基础。首先，我们成功地建立了一个高质量的BAC库。BAC文库含有约36，000个克隆，平均插入片段大小为168 kb，相当于11倍基因组冗余。估计嵌合体率小于3%。将BAC克隆排列到96个384孔微量滴定皿中，并在两个尼龙膜（高密度Rplica（HDR）过滤器）上以特定图案一式两份网格化。我们采用两种方法构建覆盖全基因组的BAC重叠群，（1）EST标记重叠法和（2）指纹法。对于EST标记的重叠，以EST为探针，通过HDR滤膜杂交筛选含有EST序列的BAC克隆。家蚕的基因组大小估计为530 Mb。由于BAC克隆的平均大小为168 kb，因此需要使用非冗余EST探针进行超过6，200次杂交实验来完成BAC重叠群构建。到目前为止，大约有2,500个杂交完成。对于EST重叠法无法检测的基因密度较低的区域，基于酶切图谱的指纹分析方法比EST重叠法更有效。对于指纹识别实验，迄今为止进行了约1/3的实验。使用这两种方法构建完整BAC重叠群的工作正在进行中。对家蚕Z、W性染色体上的多个基因座、2号染色体上的绒膜基因座和17号染色体上的抗病毒基因座构建了BAC重叠群，利用BAC克隆进行分析，揭示了家蚕基因组的特征和感兴趣的基因。

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.