Abstract
In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein.
Highlights
Serine proteases (SPs) constitute nearly one-third of all the known proteolytic enzymes and modulate the bioactivity of target proteins in a timely manner through proteolytic cleavage [1]
serine protease inhibitors (SPIs) genes from D. melanogaster and other insects were used to blast against the silkworm genome
Genes were identified and named as BmSPI1,BmSPI80 (Table 1 and Table S1), 41 of which had previously been submitted to GenBank, including 34 serpins, 3 Kazal-type, 2 Kunitz-type, 1 whey acidic protein (WAP)-type and 1 amfpi-type genes
Summary
Serine proteases (SPs) constitute nearly one-third of all the known proteolytic enzymes and modulate the bioactivity of target proteins in a timely manner through proteolytic cleavage [1]. SPIs are named as trypsin inhibitors (TIs), chymotrypsin inhibitors (CIs), elastase inhibitors (EIs) and subtilisin inhibitors (SIs) according to their different inhibitory target proteases. Ramesh et al (1988) purified two Kunitz-type inhibitors from the hemolymph of M. sexta and found that they could inhibit serine proteases including trypsin, chymotrypsin, and plasmin [16]. At least 16 B.mori hemolymph SPIs from kunitz family and serpin family show inhibitory activity to chymotrypsin and are named as CI-1,CI-13. CI-8 of serpin family has receptor protein in the midgut and shows inhibitory activity to protease in the digestive juice [24]. In order to reveal more unknown SPIs, this article systematically analyzed the classifications, domain organization, expression characters and immune response of SPIs on a genome-wide scale in the silkworm
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