AChE在水稻抗性诱导的褐飞虱凋亡中肠细胞中的定位及表达

doi:10.16380/j.kcxb.2016.10.001

摘要/Abstract

摘要： 【目的】褐飞虱Nilaparvata lugens是水稻的重要害虫之一。本研究旨在了解水稻品种抗性诱导的褐飞虱中肠细胞凋亡与细胞中乙酰胆碱酯酶（acetylcholinesterase, AChE）的关系。【方法】从4龄Ⅰ型褐飞虱若虫中肠组织分离原代细胞，用不同抗性的水稻幼苗汁液处理第一代继代细胞，利用TUNEL染色法检测细胞的凋亡情况，再分别利用免疫组织化学和荧光定量PCR技术对AChE进行亚细胞定位和检测其表达水平的变化。【结果】在对照组(未处理)细胞中，检测不到代表凋亡细胞核的绿色荧光，而免疫组化后阳性反应颜色很浅，表明细胞中存在AChE的本底水平表达。感虫水稻品种TN1幼苗汁液处理细胞中，细胞的凋亡率为8％，抗性水稻B5幼苗汁液处理的细胞中有65%的细胞发生凋亡；而抗性水稻TKM-6幼苗汁液处理的细胞中则有85％的细胞发生凋亡。免疫组化的检测结果表明，所有凋亡的细胞中都存在AChE的积累，且主要分布在细胞质中；AChE在凋亡后期并不迁入凋亡小体中，而是集中在细胞核附近。荧光定量PCR检测表明，TKM-6, B5和TN1幼苗汁液处理的细胞中AChE的表达量分别为对照细胞的29.9, 18.4和8倍，该结果与细胞水平上免疫组化的检测结果一致。【结论】本研究结果证实了水稻品种抗性与其诱导的中肠细胞凋亡率呈正相关，且凋亡细胞的细胞质中存在AChE的积累。这些发现为揭示AChE在褐飞虱与水稻的互作中的功能、促进抗性水稻新品种的选育及开发新的褐飞虱防治措施提供了一定的参考信息。

关键词: 褐飞虱, 乙酰胆碱酯酶, 中肠细胞凋亡, 细胞免疫组化, 荧光定量PCR

Abstract: 【Aim】 The brown planthopper, Nilaparvata lugens, is one of the most destructive pests of rice. This study aims to investigate the relationship between acetylcholinesterase (AChE) and the apoptosis in midgut cells of N. lugens induced by host rice resistance. 【Methods】 Primary cells were isolated from the midgut of the 4th instar nymph (biotype Ⅰ) of N. lugens. The first-generation subculture cells were treated with juice from rice seedlings with different resistance levels. Subcellular localization and expression of AChE were detected by using cell immunohistochemistry and real-time fluorescence quantitative PCR techniques, respectively. 【Results】 No green fluorescent signals representing apoptotic nuclei were detected in the untreated control cells, but these cells exhibited few signals in immunohistochemistry reaction, suggesting a basal expression level of AChE within the cells. The cells treated with the juice from seedlings of the susceptible rice variety TN1 and two resistant rice varieties B5 and TKM-6 displayed apoptosis with the apoptotic rate of 8%, 65% and 85%, respectively. Immunohistochemistry reaction revealed that all apoptotic cells showed stronger signals representing overexpression and accumulation of AChE in the cytoplasm. AChE assembled around the nuclei instead of shifting to apoptotic bodies in the late apoptotic stage. The data from real-time fluorescence quantitative PCR showed 29.9-, 18.4- and 8-fold over-expression of AChE in cells treated with the juice from seedlings of TKM-6, B5 and TN1, compared with that in the control cells, which was consistent with the results from the immunohistochemistry detection. 【Conclusion】 The results confirm that host rice resistance is positively correlated with apoptosis in midgut cells of N. lugens, and AChE is overexpressed in apoptotic cells. These findings provide some valuable information for understanding the interaction mechanism of N. lugens and host rice, promoting breeding of novel resistant rice varieties and developing effective control strategies.

Key words: Nilaparvata lugens; acetylcholinesterase (AChE), midgut apoptosis, cell immunohistochemistry, real-time fluorescence quantitative PCR

王斯奇, 陈俊, 章娟娟, 杨之帆. AChE在水稻抗性诱导的褐飞虱凋亡中肠细胞中的定位及表达[J]. 昆虫学报, 2016, 59(10): 1033-1042.

WANG Si-Qi, CHEN Jun, ZHANG Juan-Juan, YANG Zhi-Fan . Location and expression of AChE in apoptotic midgut cells of Nilaparvata lugens (Hemiptera: Delphacidae) induced by host rice resistance[J]. Acta Entomologica Sinica, 2016, 59(10): 1033-1042.

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链接本文: http://www.insect.org.cn/CN/10.16380/j.kcxb.2016.10.001

http://www.insect.org.cn/CN/Y2016/V59/I10/1033

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AChE在水稻抗性诱导的褐飞虱凋亡中肠细胞中的定位及表达

Location and expression of AChE in apoptotic midgut cells of Nilaparvata lugens (Hemiptera: Delphacidae) induced by host rice resistance

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