关键词: 小胸鳖甲, 几丁质酶, 低温胁迫, 基因表达, 免疫组织化学

Abstract: 【Aim】 To understand the mechanisms underlying cold tolerance and to discover novel cold-tolerant genes in the desert beetle Microdera punctipennis. 【Methods】 The chitinase gene in M. punctipennis was cloned by screening cold stress transcriptome data and analyzed by bioinformatics. Its expression profiles in response to low temperature (4℃) stress and in different adult tissues were detected by real-time fluorescence quantitative PCR and immunohistochemistry. 【Results】 A chitinase gene named Mpcht19 (GenBank accession no.: KY126382) was cloned from M. punctipennis adult. Bioinformatics analysis showed that the protein MpCHT19 encoded by Mpcht19 belongs to the type IV insect chitinase family. It is hydrophilic, and the predicted molecular weight is 27.18 kD with no signal peptide. Phylogenetic analysis showed that MpCHT19 is similar to TcCHT19 of Tribolium castaneum with the amino acid sequence identity of 34.05%. The results of real-time fluorescent quantitative PCR showed that the expression of Mpcht19 was high in the fat body and hindgut with tissue specificity. In addition, its expression was also induced by low temperature of 4℃. Immunohistochemical analysis showed that MpCHT19 protein was expressed in the fat body and hindgut at 4℃. 【Conclusion】 The expression of Mpcht19 shows the tissue specificity, and is low temperature inducible. The results may help to further investigate the relationship between chitinase function and cold tolerance of M. punctipennis.

Key words: Microdera punctipennis, chitinase, cold stress, gene expression, immunohistochemistry