Genomic structure and expression analysis of the gene encoding a silkworm basic Kunitz-type chymotrypsin inhibitor.
Kunitz-type chymotrypsin inhibitor CIb1 of silkworm Bombyx mori is a basic peptide consisting of 62 amino acid residues. To elucidate the mechanisms of transcriptional regulation of CIb1 gene expression, we cloned it for genomic structure analysis. CIb1 cDNA was used as a probe to screen a BAC sub-library. One positive clone containing the upstream sequences was isolated and the sequence result showed that CIb1 gene consists of three exons spaced by two introns. In the 5'-flanking region, consensus TATA and CCAAT boxes were identified. Other binding sites for transcription factors such as NF-kappaB, GATA, C/EBP, COUP-TF/HNF-4, RORalpha1, SRY, and HOXA3 were also detected. Southern blot analysis suggested a single copy of CIb1 gene in the silkworm genome. Northern blot analysis indicated that the expression of CIb1 gene is transcriptionally regulated during development and is apparently tissue-specific. The CIb1 mRNA was detected in fat body, ovary, trachea, and skin. We furthermore investigated the CIb1 expression profiles after LPS and E. coli injection. The fluctuations of CIb1 transcript in challenged larvae confirm our proposal that CIb1 is an immune responsible gene. According to our data, we discussed the transcriptional factors putatively responsible for the physiological role of CIb1 in the silkworm hemolymph.
He
Ningjia
N
Institute of Insect Genetic Resources, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Higashi, Japan.
Yakiyama
Masatoshi
M
Fujii
Hiroshi
H
Banno
Yutaka
Y
Yamamoto
Kohji
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D012831:Bombyx; D002874:Chromosome Mapping; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D016678:Genome; D006458:Hemolymph / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides; D010940:Plant Proteins; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics*
D018076:DNA, Complementary; C053865:Kunitz-type protease inhibitor, plant; D008024:Ligands; D010455:Peptides; D010940:Plant Proteins; D012333:RNA, Messenger; D014361:Trypsin Inhibitors; D002918:Chymotrypsin