灰飞虱中IKK相关基因的鉴定及其在水稻条纹病毒侵染中的功能

Identification of the IKK related genes and their antiviral roles in response to Rice stripe virus infection in Laodelphax striatellus(Hemiptera:Delphacidae)

【目的】本研究旨在鉴定灰飞虱Laodelphax striatellus中的IκB激酶(IκB kinase,IKK)相关基因,并调查其在灰飞虱抗病毒中的作用,以进一步深入理解传毒介体应对植物病毒的先天免疫机制。【方法】通过生物信息学鉴定了灰飞虱基因组中IKK相关基因;以无毒及水稻条纹病毒(rice stripe virus,RSV)侵染的灰飞虱为材料,利用RT-PCR方法检测IKK相关基因在无毒灰飞虱各个龄期(卵、1-5龄若虫、雄成虫和雌成虫)及成虫不同组织(肠道、唾液腺、血淋巴、脂肪体、卵巢和精巢)中的表达量;利用qRT-PCR方法检测无毒以及RSV侵染后的灰飞虱IKK相关基因在各个龄期及成虫不同组织中的表达量;通过对3龄若虫注射IKK基因dsRNA进行RNA干扰后,利用qRT-PCR检测带毒灰飞虱中表示病毒含量的RSV外壳蛋白(CP)基因转录水平。【结果】在灰飞虱基因组中鉴定到了两个IKK相关基因即IKKα(GenBank登录号:MK903504)和TANK结合激酶1(TANK-binding kinase1)基因TBK 1(GenBank登录号:MN124506)。IKKα开放阅读框长2379 bp,编码792个氨基酸;TBK 1开放阅读框长1551 bp,编码516个氨基酸。两个基因编码的蛋白都具有1个保守的丝氨酸/苏氨酸激酶结构域和1个泛素折叠结构域。RT-PCR结果表明,IKKα和TBK 1在无毒灰飞虱各个龄期及成虫不同组织中均有表达。qRT-PCR分析结果表明,IKKα和TBK 1在RSV侵染后的灰飞虱各个龄期及成虫不同组织中的表达水平与其在无毒灰飞虱中的表达量之间存在明显差异。进一步将RSV侵染后的灰飞虱3龄若虫中的IKKα和TBK 1干扰后,带毒灰飞虱中的RSV含量显著上升。【结论】本研究结果表明,NF-κB信号途径中的两个重要基因IKKα和TBK 1在灰飞虱中广泛表达,且在灰飞虱抵御RSV的侵入过程中可能具有重要功能。这些结果为今后进一步深入研究NF-κB免疫通路在灰飞虱抗病毒中的功能提供了丰富的基础信息。

【Aim】This study aims to identify the IκB kinase(IKK)related genes in the small brown planthopper,Laodelphax striatellus,and to investigate its antiviral role in this insect,so as to further understand the innate immune mechanisms in insect vectors responsive to plant viruses.【Methods】The IKK related genes in the L.striatellus genome were identified and characterized using bioinformatics approach.The healthy and the rice stripe virus(RSV)infected planthopper were used as the materials.The expression levels of IKK related genes in various developmental stages(egg,1-5 instar nymphs,male and female adults)and adult tissues(gut,salivary gland,hemolymph,fat body,ovary and testis)of the RSV-free planthoppers were detected by RT-PCR.The relative expression levels of IKK related genes in the above different developmental stages and adult tissues between the RSV-free and RSV-infected planthoppers were assayed by qRT-PCR.In addition,the transcription levels of coat protein(CP)gene indicating the accumulation levels of RSV were determined after RNAi by injection of the double-stranded RNA of IKK related genes in the 3rd instar nymph of RSV-infected planthoppers.【Results】Two IKK related genes,IKKα(GenBank accession number:MK903504)and TANK-binding kinase 1 gene TBK 1(GenBank accession number:MN124506),were identified in the genome of L.striatellus.The open reading frame(ORF)of IKKαis 2379 bp in length,encoding 792 amino acids,while the ORF of TBK 1 is 1551 bp in length,encoding 516 amino acids.The proteins coded by both genes have a conserved serine/threonine kinase domain and a ubiquitin folding domain.The RT-PCR results confirmed that IKKαand TBK 1 were ubiquitously expressed in various developmental stages and different adult tissues of RSV-free L.striatellus.Significant expression differences of IKKαand TBK 1 in various developmental stages and different adult tissues between the RSV-free and RSV-infected planthoppers were observed.The accumulation level of RSV was significantly increased after RNAi of IKKαand TBK 1 in the 3rd instar nymphs of the RSV-infected planthoppers,respectively.【Conclusion】The results indicate that IKKαand TBK 1,the important genes of NF-κB signaling pathway,are widely expressed in L.striatellus and may play important roles in L.striatellus in response to RSV invasion.These results will contribute to the further investigation on antiviral immune pathway in insect vectors against plant virus.