白背飞虱酚氧化酶原PPO基因特性及其免疫应答  

Characteristics and Immune Response of Prophenoloxidase Genes in Sogatella furcifera

作　　者：张道伟[1] 康奎 余亚娅 匡富萍 潘碧莹 陈静 唐斌 ZHANG DaoWei;KANG Kui;YU YaYa;KUANG FuPing;PAN BiYing;CHEN Jing;TANG Bin(College of Biology and Agriculture,Zunyi Normal University/Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin,Zunyi 563006,Guizhou;School of Basic Medical Science,Zunyi Medical University,Zunyi 563006,Guizhou;College of Life and Environmental Science,Hangzhou Normal University,Hangzhou 310036)

机构地区：[1]遵义师范学院生物与农业科技学院/赤水河流域动物资源保护与应用研究特色重点实验室,贵州遵义563006 [2]遵义医科大学基础医学院,贵州遵义563006 [3]杭州师范大学生命与环境科学学院,杭州310036

出　　处：《中国农业科学》2020年第15期3108-3119,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31560511);贵州省科学技术基金(黔科合JZ字[2014]2014号)。

摘　　要：【目的】酚氧化酶(phenoloxidase,PO)是昆虫体内的免疫蛋白,在昆虫体内起重要的调节作用,主要以无活性的酚氧化酶原(prophenoloxidase,PPO)形式存在。本研究在转录组测序获得白背飞虱(Sogatella furcifera)3条PPO序列的基础上,通过分析白背飞虱体内不同SfPPO的发育和组织表达模式,并进一步抑制SfPPO的表达以及病原菌诱导,探讨SfPPO在白背飞虱体内的生物学功能。【方法】以白背飞虱3条SfPPO序列为研究对象,利用生物信息学方法分析其蛋白结构以及与其他昆虫之间的同源性。并以注射绿色荧光蛋白(green fluorescent protein,GFP)的dsRNA作为对照组,通过注射合成的dsSfPPO后采用实时荧光定量PCR(qRT-PCR)检测基因表达抑制效果;此外,为了探究SfPPO在白背飞虱生长发育和免疫应答方面的作用,利用qRT-PCR检测SfPPO在不同发育阶段及组织中的表达情况,以及注射大肠杆菌(Escherichia coli)和枯草芽孢杆菌(Bacillus subtilis)后3个SfPPO的诱导表达情况。【结果】SfPPO1、SfPPO2-1、SfPPO2-2的开放阅读框长度分别为2079、999、2070 bp,分别编码692、332、689个氨基酸,预测蛋白分子量分别为79.84、37.67、79.53 kD,等电点分别为6.43、9.56、6.20。生物信息学分析表明,白背飞虱的3个PPO蛋白具有较高的同源性,且与褐飞虱(Nilaparvata lugens)的亲缘关系最近;SfPPO1、SfPPO2-1、SfPPO2-2均在成虫第3天表达量最高,其中SfPPO2-1和SfPPO2-2的发育表达模式相似;SfPPO1和SfPPO2-1在表皮和脂肪体内表达量较高,而SfPPO2-2在翅和脂肪体内表达量较高,在头、足、中肠、马氏管中表达量相对较低;与注射dsGFP组相比,注射靶标基因的dsRNA后都能够显著沉默目的基因的表达,而且当SfPPO2-1表达被沉默后,SfPPO1出现显著高表达,说明不同的PPO之间可能具有补偿功能;大肠杆菌诱导后12 h,SfPPO2-1和SfPPO2-2表达量显著升高,SfPPO1表达量则在诱导24 h后显著升高;枯草芽胞杆菌�【Objective】Phenoloxidase(PO)is an immune protein and plays an important regulatory role in insects.It exists as the form of inactive prophenoloxidase(PPO).In this study,based on the three PPO sequences of Sogatella furcifera by transcriptome sequencing,the biological functions of SfPPOs were further explored by analyzing the developmental and tissue expression patterns of different SfPPOs,suppressing the expression of SfPPOs and inducing by pathogenic bacteria.【Method】Taking three SfPPO sequences as research objects,the protein structure and homology with other insects were analyzed by bioinformatics method.In addition,dsRNA injected with green fluorescent protein(GFP)was used as a control group,and the inhibitory effect of target gene was detected by quantitative real-time PCR(qRT-PCR)after injecting with dsSfPPO.In order to investigate the role of SfPPO in the growth and immune response of S.furcifera,qRT-PCR was used to detect the expression of SfPPO at different developmental stages and in different tissues,as well as the induced expression of three SfPPOs after injecting with Escherichia coli and Bacillus subtilis.【Result】The open reading frames(ORF)of SfPPO1,SfPPO2-1,and SfPPO2-2 are 2079,999,and 2070 bp in length,respectively,which encoding 692,332,and 689 amino acids,respectively.The predicted protein molecular weights are 79.84,37.67,and 79.53 kD,and the isoelectric points are 6.43,9.56,and 6.20,respectively.Bioinformatics analysis showed that the three PPO proteins of S.furcifera had high homology and were closely related to Nilaparvata lugens.SfPPO1,SfPPO2-1 and SfPPO2-2 were all highly expressed on the 3rd day of adult,and the developmental expression patterns of SfPPO2-1 and SfPPO2-2 were similar.The tissue expression results showed that SfPPO1 and SfPPO2-1 were highly expressed in the epidermis and fat body,while SfPPO2-2 was highly expressed in the wing and fat body,and the expressions of all the three genes were relatively low in head,foot,midgut and Malpighian tubule.Compared with the

关 键 词：白背飞虱 酚氧化酶原 RNA干扰 实时荧光定量PCR 诱导表达 免疫应答 

分 类 号：S435.112.3[农业科学—农业昆虫与害虫防治]