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褐飞虱组织解剖及NlEF7基因的雄虫组织特异性表达研究
Male tissue-specific expression of the brown planthopper, Nilaparvata lugens (St?l), NlEF7 gene
王渭霞;何佳春;魏 琪;赖凤香;万品俊;傅 强
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DOI:10.7679/j.issn.2095-1353.2022.104
作者单位:中国水稻研究所,水稻生物学国家重点实验室,杭州311401;中国水稻研究所,水稻生物学国家重点实验室,杭州311401
中文关键词:褐飞虱;内生殖器;消化道;唾液腺;显微解剖;原位杂交
英文关键词:Nilaparvata lugens; internal genitalia; alimentary canal; salivary glands; microdissection; in situ hybridization
中文摘要:
【目的】 探究褐飞虱 Nilaparvata lugens雄虫特异基因NlEF7的组织表达特性,为褐飞虱多组织的解剖分离以及基因表达的组织原位杂交提供参考。【方法】 选择不同发育时期的褐飞虱雌成虫和雄成虫,在显微镜下解剖获取唾液腺、肠、雌虫生殖器和雄虫生殖器等组织并提取RNA利用定量PCR进行NlEF7表达分析。利用地高辛标记NlEF7探针,通过多聚甲醛固定样本和NBT/NBCT显色,进行了组织RNA原位杂交检测。【结果】 获得了唾液腺、肠道、内生殖器的细致解剖显微实图,展示了褐飞虱内生殖器的形态变化,据此可确认雌虫的交配状况。定量PCR和原位杂交分析结果表明,NlEF7基因在雄虫内生殖器和脂肪体中特异性表达,且在睾丸中表达量最高。利用完整的虫体进行固定和原位杂交后解剖观察,几乎没有杂交信号。对虫体进行破坏性处理或分离组织样本再进行固定和杂交有助于提高杂交信号的强度。【结论】 NlEF7基因在褐飞虱雄虫内生殖器和脂肪体中特异性表达。
英文摘要:
[Objectives]  To investigate the tissue-specific expression of the male-specific gene Nilaparvata lugens NlEF7 and thereby provide a reference for multi-tissue dissection and in situ hybridization techniques for this species. [Methods]  Female and male adult N. lugens of different developmental stages were selected and dissected under a microscope to obtain salivary glands, intestines, female and male genitalia and other tissues. RNA was then extracted to analyze NlEF7expression in different tissues with quantitative PCR. After fixing samples with paraformaldehyde, RNA in situ hybridization detection was then performed using a digoxigenin-labeled NlEF7 probe and NBT/NBCT color development technology. [Results]  Detailed anatomical microscopic images of salivary glands, intestines and internal genitalia were obtained. The latter show morphological changes in the internal genitalia of female N. lugens that can confirm a female’s mating status. The results of quantitative PCR and in situ hybridization indicate that the NlEF7 gene was specifically expressed in the internal genitalia and fat body of males, and that expression was highest in the testis. There was almost no hybridization signal after fixation and in situ hybridization of the intact body. Destructive treatment of insects, or separation of tissue samples, followed by fixation and hybridization can help increase the intensity of the hybridization signal. [Conclusion]  The NlEF7 gene is specifically expressed in the internal genitalia and fat body of adult male N. lugens.
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