摘要
【目的】探明褐飞虱Nilaparvata lugens过氧化物酶基因NlPOD1的分子特性、表达模式和生物学功能。【方法】基于褐飞虱转录组数据,利用PCR技术克隆获得NlPOD1基因全长cDNA序列;利用生物信息学手段分析其序列特征;通过qRT-PCR技术检测NlPOD1在褐飞虱不同发育时期(卵、1-5龄若虫和初羽化雌雄成虫)和5龄若虫不同组织(头部、脂肪体、血淋巴和肠道)中的表达水平,以及不同微生物(大肠杆菌Escherichia coli、金黄色葡萄球菌Staphylococcus aureus和金龟子绿僵菌Metarhizium anisopliae)(1×10^(7)/mL)注射感染褐飞虱5龄若虫不同时间后的诱导表达模式;利用RNAi技术沉默褐飞虱5龄若虫NlPOD1基因,并通过生物测定分析NlPOD1基因沉默和接种金龟子绿僵菌(1×10^(8)/mL)后褐飞虱的存活率及致死中时(LT_(50))。【结果】获得褐飞虱NlPOD1全长cDNA序列(GenBank登录号:MZ682107),其开放阅读框长2049 bp,编码682个氨基酸,含有一个典型的动物亚铁血红素过氧化物酶结构域(An_peroxidase domain),且N端包含一段由29个氨基酸残基组成的信号肽。系统发育分析表明,NlPOD1与半翅目其他昆虫的POD亲缘关系较近,其中与茶翅蝽Halyomorpha halys POD亲缘关系最近。发育表达谱结果表明,NlPOD1在褐飞虱不同发育阶段均有表达,其中卵期表达量最低,5龄若虫中表达量最高;组织表达谱结果表明,NlPOD1在褐飞虱5龄若虫不同组织中均有表达,且肠道和血淋巴中的表达量显著高于头部和脂肪体中的;微生物诱导表达模式表明,与注射PBS的对照组相比,大肠杆菌诱导48 h内各时间点NlPOD1在褐飞虱5龄若虫中的表达量显著上调,而在金黄色葡萄球菌和金龟子绿僵菌诱导下,NlPOD1表达量均呈现先上调后回稳的态势。RNAi分析结果显示,显微注射ds NlPOD1可显著抑制NlPOD1的表达水平。通过RNAi抑制NlPOD1表达后褐飞虱5龄若虫存活率不变,但注射ds NlPOD1联合金龟子绿僵菌感
【Aim】To clarify the molecular characteristics,expression patterns and biological function of the peroxidase gene NlPOD1 from the brown planthopper,Nilaparvata lugens.【Methods】Based on the transcriptome data of N.lugens,the full-length cDNA sequence of NlPOD1 was cloned by PCR,and its nucleotide and protein sequences were subsequently characterized using bioinformatics tools.The expression patterns of NlPOD1 across different developmental stages(egg,1 st-5th instar nymphs and newly emerged female and male adults),in different tissues(head,fat body,hemolymph and gut)of the 5th instar nymphs,and in the 5th instar nymphs at different time post injection of different microbes(Escherichia coli,Staphylococcus aureus and Metarhizium anisopliae)at the concentration of 1×107/mL were determined by qRT-PCR.The target gene NlPOD1 in the 5th instar nymphs of N.lugens was further silenced by RNAi,and the survival rates and the median lethal time(LT_(50))values of the nymphs after target gene silencing and infection with M.anisopliae(1×10^(8)conidia/mL)were determined by bioassay.【Results】The full-length cDNA sequence of NlPOD1(GenBank accession no.:MZ682107)was successfully cloned from N.lugens.Its open reading frame(ORF)is 2049 bp in length,encoding 682 amino acids with a typical animal heme peroxidase domain(An_peroxidase domain)and a predicted signal peptide consisting of 29 amino acid residues at the N-terminus.The phylogenetic analysis showed that NlPOD1 is closely related to the PODs of other hemipteran insects,and has the highest homology with the POD of Halyomorpha halys.Developmental expression profiling showed that NlPOD1 was expressed in various developmental stages of N.lugens,with the lowest and highest expression levels in the eggs and the 5th instar nymphs,respectively.Tissue expression profiling revealed that NlPOD1 was expressed in different tissues of the 5th instar nymphs of N.lugens,with significantly higher expression levels in the gut and haemolymph than in the head and fat body.The expressio
作者
李慕雨
王彦丹
王正亮
俞晓平
LI Mu-Yu;WANG Yan-Dan;WANG Zheng-Liang;YU Xiao-Ping(Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2022年第8期958-966,共9页
Acta Entomologica Sinica
基金
浙江省杰出青年基金项目(LR19C140001)
国家自然科学基金项目(31972347,31601698)。
