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.
In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, 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 x 10(5) 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 A. pernyi, 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 A. pernyi (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.
Li
Yu-Ping
YP
Shenyang Agricultural University, China.
Xia
Run-Xi
RX
Wang
Huan
H
Li
Xi-Sheng
XS
Liu
Yan-Qun
YQ
Wei
Zhao-Jun
ZJ
Lu
Cheng
C
Xiang
Zhong-Huai
ZH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020224:Expressed Sequence Tags; D015723:Gene Library; D017344:Genes, Insect; D007093:Imidazoles / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007605:Juvenile Hormones / Q000378:metabolism*; D008675:Metamorphosis, Biological / Q000235:genetics*; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics* / Q000378:metabolism; D011679:Pupa
D007093:Imidazoles; D019476:Insect Proteins; D007605:Juvenile Hormones; C063652:1-benzyl-5-(2,6-dimethyl-1,5-heptadienyl)imidazole