Effect of ecdysone and validamycin on chitin synthase B gene expression in Mythimna separata

Author of the article:WANG XiaoXi；ZHAO GuiYing；YANG Hang；ZHANG YaNan；FAN Dong

Author's Workplace：Agronomy College, Northeast Agricultural University, Harbin 150030, China；Agronomy College, Northeast Agricultural University, Harbin 150030, ChinaAgronomy College, Northeast Agricultural University, Harbin 150030, China；Agronomy College, Northeast Agricultural University, Harbin 150030, China；Agronomy College, Northeast Agricultural University, Harbin 150030, China

Key Words：Mythimna separata; chitin synthase B; cloning; 20-hydroxy ecdysone; validamycin; expression

Abstract：[Objectives]  To obtain the full-length cDNA sequence of the Mythimna separata chitin synthase B (CHSB) gene, and investigate the effect of 20 E and validamycin on the mRNA expression levels of this gene in different developmental stages and tissues. [Methods]  A full-length cDNA sequence of the M. separata CHSB genet was obtained by high- throughput sequencing and its mRNA expression levels in different developmental stages and tissues quantified with RT-qPCR. mRNA expression levels after treatment with 20 E and validamycin were also measured with RT-qPCR. [Results]  The cDNA sequence of the M. separata CHSB gene is 4 617 bp with an open reading frame encoding a polypeptide of 1 538 amino acids, an estimated molecular mass of 175.629 ku, and an estimated pI of 5.96. It has 17 transmembrane helices, 4 tag sequences (CHS, CATMWHET, DGD, EDR and QRRRW), and one putative catalytic domain. Its cDNA sequence was designated MsCHSB and has been deposited in GenBank (accession No. KY348776). Sequencing indicated > 52% shared identity between MsCHSB and CHSB from other insect species, and 92% and 83% similarity with Mamestra configurata and Helicoverpa armigera CHSB. Real-time PCR indicated that MsCHSB mRNA transcripts were expressed in all developmental stages and in tissues but were most highly expressed in the early 3^rd larval instar and in the midgut. MsCHSB expression was significantly higher than that of the control group 6 h and 12 h after treatment with 10 µg/µL of 20 E. The relative expression of MsCHSB after validamycin treatment was significantly lower than in the control group, and this was most apparent 48 h after treatment with 20 µg/µL validamycin. [Conclusion]  A novel CHSB cDNA sequence was obtained from M. separata. The expression of MsCHSB was increased by 20 E and inhibited by validamycin. These results provided a foundation for further research on the biological function of CHSB in insects.