| As the closest living invertebrate relative of the vertebrates, amphioxus (subphylum Cephalochordata) occupy a key position in animal evolution and is regarded the best available proxy for the study of the last common ancestor of all vertebrates. In this study we reported the successful construction of BAC library of Chinese amphioxus Branchiostoma belcheri after optimizing a series of conditions in the library construction. The resulted library consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further formulated the library into characteristic DNA pools for two-round PCR screening for 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a valuable resource in comparative genomics study。The Pax gene family consists of tissue-specific transcriptional regulators and plays key regulatory roles in embryonic and sensory organ development in metazoans. Four BAC clones each containing the amphioxus Pax genes were sequenced and a total of 474 kb sequence were obtained. The BbPax1/9, BbPax2/5/8, BbPax3/7 and BbPax6 spans 59.7 kb, 68.1 kb, 13.1 kb, 38.2 kb of genomic DNA and is composed of 5, 9, 5 and 11 exons, respectively. The deduced proteins are 363, 444, 470 and 466 aa long, comparing to 363, 445, 464 and 483 aa of the corresponding BfPax genes. Between the two amphioxus species the amino acid identities of the four are 97.2 %, 98.9 %, 70.1 % and 97.0 % whereas the intron position are identical.Combining the survey of genomic assembly of 17 species and homologous search against...
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