荒漠甲虫小胸鳖甲低温响应几丁质酶基因Mpcht19的克隆及表达模式分析

Molecular cloning and expression profiling of a cold stress-related chitinase gene Mpcht19 in the desert beetle Microdera punctipennis (Coleoptera: Tenebrionidae)

【目的】探索荒漠昆虫小胸鳖甲Microdera punctipennis低温响应的分子机理并挖掘其耐寒基因。【方法】根据低温转录组数据筛选并克隆小胸鳖甲几丁质酶基因,并进行生物信息学分析;通过实时荧光定量PCR和免疫组织化学法分析该基因在成虫不同组织中和4℃低温胁迫下的表达模式分析。【结果】从小胸鳖甲成虫克隆获得一个几丁质酶基因Mpcht19(Gen Bank登录号:KY126382)。生物信息学分析表明,该基因编码的蛋白Mp CHT19属于Ⅳ型昆虫几丁质酶,理论分子量约为27.18 k D,无信号肽,具亲水性。系统进化分析表明,Mp CHT19与赤拟谷盗Tribolium castaneumⅣ型几丁质酶Tc CHT19同源性最高,氨基酸序列一致性为34.05%。实时荧光定量PCR结果显示,Mpcht19在成虫脂肪体和后肠中高表达,具有组织特异性;该基因的表达还受4℃低温诱导。免疫组织化学分析结果显示,在4℃低温下Mp CHT19蛋白在脂肪体和后肠中表达。【结论】Mpcht19基因的表达具有组织特异性,并受低温诱导。研究结果有助于深入研究几丁质酶与小胸鳖甲耐寒性的关系。

【Aim】To understand the mechanisms underlying cold tolerance and to discover novel coldtolerant 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（ Gen Bank accession no. ： KY126382） was cloned from M. punctipennis adult.Bioinformatics analysis showed that the protein Mp CHT19 encoded by Mpcht19 belongs to the type IV insect chitinase family. It is hydrophilic,and the predicted molecular weight is 27. 18 k D with no signal peptide. Phylogenetic analysis showed that Mp CHT19 is similar to Tc CHT19 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 Mp CHT19 protein was expressed in the fat body and hindgut at 4℃. 【Conclusion 】Theexpression 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.