褐飞虱自噬相关基因NlATG13的表达与功能分析

doi:10.16380/j.kcxb.2022.08.005

摘要/Abstract

摘要： 【目的】自噬参与多种细胞生理过程，自噬相关蛋白ATG13是ATG1/13复合物的组成部分, 在启动自噬中发挥着重要作用。本研究旨在分析ATG13在褐飞虱Nilaparvata lugens中发挥的功能，评估其作为害虫防治靶标的潜力。【方法】基于褐飞虱转录组数据，利用RACE克隆褐飞虱NlATG13的cDNA全长序列；应用生物信息学技术分析NlATG13的核苷酸和氨基酸序列特征。利用RT-qPCR检测其在褐飞虱不同发育阶段(1-5龄若虫和雌雄成虫)和不同组织(5龄若虫头、胸、中肠和脂肪体以及刚羽化雌成虫的卵巢)中的表达模式。通过显微注射dsNlATG13至3龄若虫进行RNAi敲减NlATG13的表达，探究其对褐飞虱生存和中肠细胞自噬的影响；利用RT-qPCR检测RNAi 4 d时3龄若虫中糖原合成与代谢通路相关基因NlGSK3, NlGS和NlGP的表达量。【结果】克隆得到NlATG13的cDNA全长序列(GenBank登录号: MF805752)，其开放阅读框长1 203 bp，编码一个含400个氨基酸的蛋白质(GenBank登录号: AWW05678.1)。系统发育分析表明，在纳入分析的物种中，NlATG13蛋白与半翅目温带臭虫Cimex lectularius和茶翅蝽Halyomorpha halys的ATG13蛋白进化关系较为接近。发育表达谱结果表明NlATG13在褐飞虱3和5龄若虫中的表达量显著高于在1-2龄若虫、雌成虫和雄成虫中的；组织表达谱结果表明NlATG13在5龄若虫头部和脂肪体中的相对表达量较高，在胸部的表达量最低。RNAi结果显示，与dsGFP对照组相比dsNlATG13处理组中褐飞虱中肠细胞中存在明显的糖原颗粒积累，NlGS, NlGSK3和NlGP的表达量均无显著变化，组织ATP含量显著降低；褐飞虱的存活率显著降低，处理第10天时褐飞虱存活率下降到41.4%，而dsGFP对照组的存活率保持在85.6%的较高水平。【结论】 RNA干扰NlATG13基因对褐飞虱的生存和中肠细胞自噬具有显著的抑制效果，NlATG13基因具有作为褐飞虱防治靶标的潜力。

关键词: 褐飞虱, NlATG13, RNA干扰, 基因克隆, 自噬

Abstract: 【Aim】 Autophagy participates in various physiological processes of cells. The autophagy-related protein ATG13 is a component of the Atg1/13 complex and plays an important role in initiating the autophagy. This study aims toanalyzethefunctionofATG13inthebrownplanthopper, Nilaparvata lugens, and to evaluate its potential as a pest control target. 【Methods】 Based on the transcriptome data of N. lugens, the full-length cDNA sequence of NlATG13 in N. lugens was cloned via RACE method. The nucleotide and amino acid sequence characteristics of NlATG13 were analyzed with bioinformatics technology. RT-qPCR technology was used to detect the expression patterns of NlATG13 in different developmental stages (1st-5th instar nymphs, and female and male adults) and different tissues (the head, thorax, midgut and fat body of the 5th instar nymphs and the ovary of newly emerged female adults) of N. lugens. The expression of NlATG13 was knocked down by RNAi through microinjection of dsNlATG13 into the 3rd instar nymphs to explore its effect on the survival, and autophagy in the midgut cells of N. lugens. RT-qPCR was used to detect the expression levels of NlGSK3, NlGS and NlGP related to glycogen synthesis and metabolism in the 3rd instar nymphs after RNAi for 4 d. 【Results】 The full-length cDNA sequence of NlATG13 (GenBank accession no.: MF805752) was cloned. It contains an open reading frame of 1 203 bp in length, encoding a protein of 400 amino acids (GenBank accession no.: AWW05678.1). The phylogenetic analysis showed that NlATG13 protein was closely related to ATG13 proteins of Cimex lectularius and Halyomorpha halys among the analyzed species. Developmental expression profiling showed that the expression levels of NlATG13 in the 3rd and 5th instar nymphs were significantly higher than those in the 1st-2nd instar nymphs, female adults and male adults of N. lugens. Tissue expression profiling revealed that the relative expression level of NlATG13 was higher in the head and fat body, but the lowest in the thorax of the 5th instar nymphs of N. lugens. RNAi results showed that in the dsNlATG13 treatment group, glycogen granules were accumulated in midgut cells, the expression levels of NlGS, NlGSK3 and NlGP had no significant change, the ATP content in tissues was significantly decreased and the survival rate of N. lugens significantly decreased as compared to those in the dsGFP control group. The survival rate of N. lugens on day 10 post dsNlATG13 treatment was 41.4%, while that in the dsGFP control group remained at a higher level of 85.6%. 【Conclusion】 RNA interference targeting NlATG13 gene has a significant inhibitory effect on the survival and autophagy of midgut cells in of N. lugens, and NlATG13 gene could be used as a potential target in controlling N. lugens.

Key words: Nilaparvata lugens, NlATG13, RNA interference, gene cloning, autophagy

吴建艮, 矫启启, 俞飞飞, 郑园园, 陈桐桐, 郝培应, 俞晓平. 褐飞虱自噬相关基因NlATG13的表达与功能分析[J]. 昆虫学报, 2022, 65(8): 967-976.

WU Jian-Gen, JIAO Qi-Qi, YU Fei-Fei, ZHENG Yuan-Yuan, CHEN Tong-Tong, HAO Pei-Ying, YU Xiao-Ping. Expression and functional analysis of the autophagy-related gene NlATG13 in the brown planthopper, Nilaparvata lugens(Hemiptera: Delphacidae)[J]. Acta Entomologica Sinica, 2022, 65(8): 967-976.

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褐飞虱自噬相关基因NlATG13的表达与功能分析

Expression and functional analysis of the autophagy-related gene NlATG13 in the brown planthopper, Nilaparvata lugens(Hemiptera: Delphacidae)

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