| 姚昱帆，张小雨，李长友，孟祥谦，于乾龙，李洁，万方浩，褚栋，郑桂玲,2026,苹果蠹蛾胚胎细胞系的建立及其颗粒体病毒的离体复制[J].环境昆虫学报,(3):677-684 |
| 苹果蠹蛾胚胎细胞系的建立及其颗粒体病毒的离体复制 |
| Establishment of embryonic cell lines of Cydia pomonella (Lepidoptera: Tortricidae) and in vitro replication of Cydia pomonella granulovirus |
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| DOI： |
| 中文关键词: 苹果蠹蛾 细胞系 线粒体 COI基因 颗粒体病毒 离体复制 |
| 英文关键词:Cydia pomonella cell line mitochondrial COI gene granulovirus in vitro replication |
| 基金项目:国家重点研发计划（2021YFD1400200） |
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| 摘要点击次数: 205 |
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| 中文摘要: |
| 【目的】昆虫细胞系是生物学、生理学和病毒学等研究领域的重要工具，苹果蠹蛾 Cydia pomonella是一种具有毁灭性危害的入侵性害虫，目前国内尚未有苹果蠹蛾细胞系的报道。【方法】本研究以苹果蠹蛾卵为材料建立胚胎细胞系，从中筛选对苹果蠹蛾颗粒体病毒（Cydia pomonella granulovirus, CpGV）敏感的细胞系，研究细胞系的核型和生长等生物学特性，通过实时荧光定量PCR（qPCR）和透射电子显微镜技术分析CpGV在细胞系中的离体复制。【结果】本研究成功建立了8个苹果蠹蛾胚胎细胞系，可在含10%胎牛血清的TNM-FH培养基中稳定传代；筛选获得1个对CpGV敏感的细胞系QAU-Cp-E-11-L，QAU-Cp-E-11-L细胞为贴壁生长，圆形、梭形和棒状细胞的比例分别为90.0%、7.5%和2.5%；扩增的线粒体细胞色素氧化酶Ⅰ亚基（COI）基因片段长度为576 bp，经Blast比对与苹果蠹蛾COI基因核苷酸序列的相似性为100%，证实该细胞系源自苹果蠹蛾；QAU-Cp-E-11-L细胞染色体数目为23~440条，染色体异倍化现象明显；QAU-Cp-E-11-L在培养168 h 时细胞数量达到最大值2.27 × 106 cells/mL，群体倍增时间为33.81 h；CpGV接种QAU-Cp-E-11-L细胞，qPCR检测到CpGV在细胞内复制，12 d达到最高值2.48 × 108 copies/μg DNA，通过透射电子显微镜可以观察到CpGV包涵体在细胞内形成，随着感染时间的增加，细胞质内病毒包涵体数量增多。【结论】本研究建立的苹果蠹蛾细胞系丰富了昆虫细胞系资源，可用于颗粒体病毒与宿主相互关系的深入研究。 |
| 英文摘要: |
| 【Aim】Insect cell lines serve as essential in vitro models for biological, physiological, and virological research. The codling moth, Cydia pomonella, is a highly destructive invasive pest; no cell lines of C. pomonella has been reported in China currently.【Methods】Using eggs as starting material, embryonic cell lines of C. pomonella were established. From these, a cell line sensitive to Cydia pomonella granulovirus (CpGV) was screened, and its biological characteristics, including karyotype and growth were studied. The in vitroreplication of CpGV this cell line was analyzed by real-time fluorescence quantitative PCR (qPCR) and transmission electron microscopy (TEM).【Results】The results showed that eight embryonic cell lines of C. pomonella were successfully established and could be stably passaged in TNM-FH medium containing 10% fetal bovine serum (FBS). One CpGV-sensitive cell line, designated QAU-Cp-E-11-L, was obtained. The cells grew adherently, the round, spindle-shaped, and rod-shaped cells accounted for 90.0%, 7.5%, and 2.5% of the total population, respectively. The amplified mitochondrial cytochrome oxidase I subunit (COI) gene fragment was 576 bp in length. BLAST analysis revealed 100% nucleotide sequence identity with the COI gene of C. pomonella, confirming that the cell line derived from C. pomonella. The chromosome number of QAU-Cp-E-11-L cells varied from 23 ~ 440, indicating pronounced aneuploidy. The cell line QAU-Cp-E-11-L reached maximum density (2.27×106 cells/mL) at 168 h with a population doubling time of 33.81 h. Following infection of QAU-Cp-E-11-L cells with CpGV, qPCR detected CpGV replication in the infected cells, with the viral copy number reaching a maximum of 2.48×108 copies/μg DNA at 12 days post-infection (dpi). TEM observations results showed that the formation of CpGV occlusion bodies (OBs) in the cells, and the number of OBs in the cytoplasm increased with the inoculation time.【Conclusion】Hence, this study will enrich the resources of C. pomonella cell lines and provide critical tools for investigating granulovirus-host interactions. |
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