Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination

• Main Author: Yu-Ping Li, Run-Xi Xia, Huan Wang, Xi-Sheng Li, Yan-Qun Liu, Zhao-Jun Wei, Cheng Lu, Zhong-Huai Xiang
• Format: Article
• Language: English
• Published: Ivyspring International Publisher 2009-01-01
• Series: International Journal of Biological Sciences
• Online Access: http://www.biolsci.org/v05p0451.htm

<p>In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, <i>Antheraea pernyi</i>, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 &#215; 10⁵ cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from <i>A. pernyi</i>, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in <i>A. pernyi</i> (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.</p>