SfAp参与调控白背飞虱的翅发育

SfAp participates in regulating wing development in the white-backed planthopper,Sogatella furcifera(Hemiptera:Delphacidae)

【目的】Apterous(Ap)属于LIM结构域家族的发育调控蛋白。本研究旨在探究Ap基因在白背飞虱Sogatella furcifera翅发育中的作用。【方法】基于白背飞虱基因组和转录组数据库,采用RT-PCR验证SfAp的cDNA序列并进行生物信息学分析;利用RT-qPCR检测SfAp在白背飞虱不同发育阶段(1-5龄若虫和雌雄成虫)和成虫不同组织(头、胸、腹、足、翅、体壁、脂肪体和肠道)中的表达量;通过RNAi技术靶向沉默3龄若虫SfAp后观察白背飞虱的生长发育情况,并统计RNAi后的存活率、总死亡率和翅畸形率,采用RT-qPCR测定翅发育信号通路关键基因(鞣化激素基因SfBurs-α和SfBurs-β、Hippo信号通路基因SfHippo和SfSal、Wnt信号通路基因SfWg、Hedgehog信号通路基因SfHh和SfDpp及SfHOW)的表达量。【结果】克隆获得了白背飞虱SfAp(GenBank登录号:PP901867)的开放阅读框,长1287 bp,编码428个氨基酸的蛋白质,其编码蛋白质预测的分子量为47.41 kD,理论等电点为8.99,SfAp含有典型的LIM保守结构域;SfAp与褐飞虱Nilaparvata lugens的NlAp亲缘关系最近,氨基酸序列一致性达84.86%。发育表达谱结果显示,SfAp在4龄若虫前表达量较高,随后逐渐下降,到5龄第3天若虫期表达量显著升高,成虫羽化后表达量下降;组织表达谱结果表明,SfAp在成虫胸部中表达量最高,其次是在腹部、脂肪体和肠道中高量表达。显微注射dsSfAp导致羽化成虫表现出翅不伸展及畸形,并且翅发育相关基因SfBurs-α,SfBurs-β,SfHippo,SfSal,SfWg,SfHh,SfDpp和SfHOW的表达也被显著抑制。【结论】SfAp通过影响翅发育信号通路关键基因的表达量,进而影响白背飞虱的翅伸展及发育过程。

【Aim】Apterous(Ap)is a developmental regulatory protein belonging to the LIM domain family.This study aims to elucidate the role of the Ap gene in the wing development of the white-backed planthopper,Sogatella furcifera.【Methods】Based on the genome and transcriptome databases of S.furcifera,the cDNA sequence of SfAp was verified by RT-PCR and subjected to bioinformatic analysis.RT-qPCR was used to detect the expression levels of SfAp in different developmental stages(1st-5th instar nymphs,and female and male adults)and various adult tissues(head,thorax,abdomen,leg,wing,integument,fat body and gut)of S.furcifera.The growth and development of S.furcifera were observed after silencing SfAp in the 3rd instar nymphs using RNAi,and the survival rate,total mortality rate,and wing deformity rate after RNAi were counted,while the expression levels of the key genes involved in wing development signaling pathways(bursicon genes SfBurs-αand SfBurs-β,Hippo signaling pathway genes SfHippo and SfSal,Wnt signaling pathway gene SfWg,Hedgehog signaling pathway genes SfHh and SfDpp,and SfHOW)were measured using RT-qPCR.【Results】The open reading frame of SfAp(GenBank accession no.:PP901867)of S.furcifera was cloned,with the length of 1287 bp encoding a protein of 428 amino acids.The encoded protein has the predicted molecular weight of 47.41 kD and the theoretical isoelectric point of 8.99.SfAp has the conserved typical LIM domain.SfAp was closely related to NlAp of Nilaparvata lugens,and their amino acid sequence identity was 84.86%.Developmental expression profile result revealed that SfAp exhibited high expression levels before the 4th instar nymph followed by a gradual decline,a significant increase on the 3rd day of the 5th instar nymph,and a subsequent decrease after adult eclosion.Tissue expression profile result showed that the expression level of SfAp in the adult thorax was the highest,followed by those in the abdomen,fat body,and gut.Microinjection of dsSfAp resulted in the impaired wing extension and wing deformation of the eclosed adults,and significantly suppressed the expression of the wing development-related genes SfBurs-α,SfBurs-β,SfHippo,SfSal,SfWg,SfHh,SfDpp and SfHOW.【Conclusion】SfAp influences wing expansion and development of S.furcifera by modulating the expression levels of the key genes involved in the wing development signaling pathway.