关键词: 家蝇, 细胞凋亡, Caspase-1, Mdom-Caspase-1, 克隆, 表达量

Abstract: Members of the Caspase family play a central and evolutionary role in apoptosis, which remove the unwanted, damaged and dangerous cells during development to maintain homeostasis. In this study, by using the total RNA isolated from the whole body of the house fly, Musca domestica, the full-length sequence of Mdom-caspase-1 gene encoding the Caspase-1 was cloned by the methods of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE) to explore the function of caspase in insect growth and metamorphism. The sequence analysis showed that Mdom-caspase-1 encodes 289 amino acid residues and the 1 295 bp full-length cDNA contains an 870 bp open reading frame (ORF). The GenBank accession numbers of cDNA and amino acid of Mdom-caspase-1 are EU854472 and ACF71490, respectively. The deduced Mdom-caspase-1 protein (MW 32.83 kDa; pI 8.67) has 5 conserved cysteines QACQG, which is one of the typical characteristics of Caspase. The Mdom-caspase-1 protein is hydrophilic, while 8 regions containing 89 amino acids are hydrophobic. The protein secondary structure mainly consists of 11 α helices, 7 β folders and 17 turns. The phylogenetic tree was created from the alignments of the amino acid sequences, and the analysis revealed that Caspase-1 possessed distinct conservation in evolution and the amino acid sequence of Mdom-caspase-1 is 65%-77% identical to Caspase-1 from Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus. The results of RT-PCR revealed that Mdom-caspase-1 was expressed in every stage of M. domestica, and in five stages including the egg, 3rd instar larva, pre-pupa, pupa and the 5 d adult female the expression levels were obviously higher than those in other stages. The results suggest that the Caspase-1 gene may have some relationships with the growth and metamorphism of insects. These results would provide some valuable information to explore the molecular function of Caspase-1 in insects and develop the specific Caspase-1 inhibitors.

Key words: Musca domestica, apoptosis, Caspase-1, Mdom-Caspase-1, cloning, expression level