蜕皮激素和有效霉素对粘虫几丁质合成酶B基因表达的影响

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

【目的】 克隆粘虫Mythimna separata几丁质合成酶B基因的全长cDNA序列,研究该基因的时空表达特性,分析蜕皮激素(20-hydroxy ecdysone, 20 E)和有效霉素(Validamycin)对该基因表达水平的影响。【方法】 本试验通过高通量测序法获得粘虫几丁质合成酶B基因的cDNA全长序列,利用RT-qPCR技术分析粘虫几丁质合成酶B基因在不同发育阶段和不同组织的特异性表达及蜕皮激素和有效霉素对其表达的影响。【结果】 基因cDNA全长4 617 bp,包含一个完整开放阅读框,编码1个1 538个氨基酸组成的多肽,分子量为175.629 ku,理论等电点为5.96,包含17个跨膜螺旋,4个几丁质合成酶的标签序列CATMWHET,DGD,EDR和QRRRW及1个催化结构域。该基因命名为MsCHSB,GenBank登录号为KY348776。氨基酸序列比对表明,该基因与其他昆虫的几丁质合成酶B基因同源性高于52%,其中与蓓带夜蛾Mamestra configurata和棉铃虫Helicoverpa armigera的几丁质合成酶B基因同源性最高,分别为92%和83%。RT-qPCR技术表明粘虫在不同发育阶段和组织中均有mRNA的特异性表达,其中3龄第1天和中肠中MsCHSB基因相对表达量最高。注射10 μg/μL浓度的蜕皮激素6 h和12 h后,表现为对该基因的诱导效应,与对照组差异显著;有效霉素处理后该基因相对表达量均被显著抑制,其中注射20 μg/μL浓度的有效霉素48 h后,抑制作用最为明显。【结论】 本试验得到了一条新的粘虫几丁质合成酶B基因cDNA序列全长。蜕皮激素对MsCHSB基因的表达有一定的诱导作用,有效霉素对MsCHSB基因的表达有一定的抑制作用,该结果为进一步研究昆虫几丁质合成酶B打下了基础。

[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 3rd 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.