论文部分内容阅读
【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,ACh E)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ACh E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ACh E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ACh E的积累,且主要分布在细胞质中;ACh E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ACh E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ACh E的积累。这些发现为揭示ACh E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。
【Objective】 Nilaparvata lugens is one of the important pests in rice. The aim of this study was to investigate the relationship between cell apoptosis and N-acetylcholinesterase (AChE) in the midgut of N. lugens induced by rice variety resistance. 【Method】 Primary cells were isolated from the midgut of nymphs of 4-year-old N. lugens and treated with different resistant rice seedlings juice for the first generation of subcultured cells. TUNEL staining was used to detect the cell apoptosis. Chemical and Fluorescent Quantitative PCR techniques were used to determine the subcellular localization of ACh E and to detect its expression level. 【Result】 The green fluorescence of apoptotic nuclei was undetectable in control (untreated) cells, whereas the positive color after immunohistochemistry was very light, indicating the presence of a background level of ACh E in the cells. In the susceptible rice variety TN1 seedling juice-treated cells, the apoptosis rate of the cells was 8%, and 65% of the cells in the resistant rice B5 seedling juice-treated cells were apoptotic; whereas the resistant rice TKM-6 seedling juice-treated 85% of cells in the cell undergo apoptosis. The results of immunohistochemistry showed that there was accumulation of ACh E in all the apoptotic cells, and mainly distributed in the cytoplasm. ACh E did not migrate into the apoptotic body in the late stage of apoptosis, but concentrated in the vicinity of the nucleus . Fluorescent quantitative PCR showed that the expression of ACh E in the seedlings of TKM-6, B5 and TN1 seedlings was 29.9, 18.4 and 8 times higher than that of the control cells, respectively. The results were consistent with the results of immunohistochemistry on the cell level. 【Conclusion】 The results of this study confirmed that there was a positive correlation between resistance of rice cultivars and the apoptosis rate of midgut, and the accumulation of ACh E in the cytoplasm of apoptotic cells. These findings provide some reference information for revealing the function of ACh E in the interaction between BPH and rice, promoting the breeding of new resistant rice varieties and developing new BPH control measures.