黏虫几丁质酶MsCHT7基因的克隆、表达及蜕皮激素的调控

Cloning, Expression and Ecdysone Regulation of Chitinase MsCHT7 from the Oriental Armyworm, Mythimna separata Walker

几丁质酶是几丁质水解的关键酶,在昆虫生长发育中具有重要作用。本试验通过高通量转录组测序获得黏虫几丁质酶基因cDNA序列MsCHT7(GenBank登录号MG551526)。cDNA全长3360 bp,包含一个长度为2970 bp的开放阅读框,编码989个氨基酸,具有1个糖苷水解酶18家族高度保守序列,2个Glyco_18催化结构域和1个几丁质结合结构域ChtBD2。通过基因表达水平研究发现,黏虫MsCHT7基因在不同发育阶段和不同组织中均有表达,且具有差异性。预蛹期和表皮MsCHT7基因表达量最大,蛹期最后1 d和成虫表达量较高,且幼虫各龄期最后1 d表达量均高于第1 d表达量。20E(20-羟基蜕皮酮)处理幼虫后,不同时间点MsCHT7基因表达量存在显著差异,在1～24 h基因表达量随时间逐渐升高,24 h时最高,之后表达量迅速降低。4种不同浓度20E处理后,MsCHT7基因表达量存在一定差异,当浓度为10μg/μL时,MsCHT7基因表达量最大。通过生物学观察发现,不同浓度20E处理后5龄幼虫蜕皮时间均提前。由于MsCHT7基因表达量受蜕皮激素调控,MsCHT7可能在黏虫蜕皮过程中发挥一定作用。

Chitinase is a key enzyme in chitin hydrolysis and plays an important role in insect growth and development. In this study, a novel chitinase gene cDNA sequence MsCHT7 （GenBank accession No. MG551526） was obtained from Mythimna separata by high-throughput transcriptome sequencing technology. The full-length cDNA is 3360 bp and encodes an amino acid sequence with 989 residues. The amino acid sequence includes a highly conserved sequence of family 18 glycoside hydrolases, two Glyco18 catalytic domains and a chitin binding domain ChtBD2. qRT-PCR method was used to reveal stage-and tissue-dependent expression patterns of MsCHT7. The results showed that MsCHT7 expressed specifically at different developmental stages and in different tissues. The expression of MsCHT7 was the highest at prepupal stage and in integuments and higher in last-day pupa and adult stage. The expression levels were higher on the last day than on the first day of each instars. After treatment with 20E （20-hydroxyecdysone）, the expression levels of MsCHT7 at different time points showed significant differences. The expression levels of MsCHT7 increased with time and reached the peak at 24 h, then significantly decreased. The MsCHT7 expression varied with four different 20E concentrations. When the concentration was 10 μg/μL, the expression level was the highest. Treatment with different 20E concentrations advanced molting of fifth instar larvae. Because expression of MsCHT7 was regulated by ecdysone, MsCHT7 might play a role in M. separata molting.