昆虫学报 ›› 2020, Vol. 63 ›› Issue (7): 779-787.doi: 10.16380/j.kcxb.2020.07.001

• 研究论文 •    下一篇

褐飞虱丝氨酸蛋白酶抑制剂基因Nlserpin4的克隆及表达模式分析

王正亮#, 朱杭锋#, 潘海波, 俞晓平*   

  1. (中国计量大学生命科学学院, 浙江省生物计量及检验检疫技术重点实验室, 杭州 310018)
  • 出版日期:2020-07-20 发布日期:2020-07-29

Cloning and expression profiling of the serine protease inhibitor gene Nlserpin4 in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

WANG Zheng-Liang#, ZHU Hang-Feng#, PAN Hai-Bo, YU Xiao-Ping*   

  1.  (Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
  • Online:2020-07-20 Published:2020-07-29

摘要: 摘要: 【目的】克隆和分析褐飞虱Nilaparvata lugens丝氨酸蛋白酶抑制剂基因Nlserpin4,并探明其时空表达谱和病原真菌诱导表达模式。【方法】基于褐飞虱转录组和全基因组序列数据,利用PCR技术克隆得到褐飞虱Nlserpin4基因的全长cDNA序列;利用生物信息学手段分析其核苷酸和蛋白质序列特征;通过qRT-PCR技术检测其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(脂肪体、肠道、血淋巴和剩余虫体)中的时空表达谱,以及金龟子绿僵菌Metarhizium anisopliae注射感染褐飞虱5龄若虫不同时间后的诱导表达模式。【结果】克隆获得褐飞虱Nlserpin4基因全长cDNA序列(GenBank登录号: MN822802),其开放阅读框长1 227 bp,编码408个氨基酸,蛋白的相对分子质量和等电点分别为45.91 kD和6.23。氨基酸序列分析表明,Nlserpin4蛋白无糖基化位点,N端包含一段由23个氨基酸残基组成的信号肽,C端具有serpin蛋白家族典型的RCL区,且含有能被靶标蛋白酶识别的活性裂解位点。系统发育分析表明,Nlserpin4与半翅目其他昆虫的serpin亲缘关系较近,其中与蔗黄伪毛蚜Sipha flava serpin4的亲缘关系最近。qRT-PCR分析表明,Nlserpin4基因表达具有明显的时空特异性,其在成虫中的表达量显著高于在其他龄期的,且在雄成虫中表达量最高;Nlserpin4基因在褐飞虱5龄若虫脂肪体、肠道、血淋巴和剩余虫体中均有表达,且在剩余虫体组织中表达量最高;病原真菌金龟子绿僵菌诱导48 h内Nlserpin4表达量均显著下调,但随着诱导时间的增加,Nlserpin4表达量呈回升趋势。【结论】褐飞虱Nlserpin4基因在褐飞虱不同发育阶段、不同组织以及病原真菌金龟子绿僵菌诱导不同时间下差异表达。研究结果为深入研究Nlserpin4在褐飞虱生长发育和免疫调节中的功能奠定了基础。

关键词: 褐飞虱, 金龟子绿僵菌, 丝氨酸蛋白酶抑制剂, 基因克隆, 时空表达, 诱导表达

Abstract: 【Aim】 To clone and characterize the serine protease inhibitor gene Nlserpin4 from the brown planthopper (BPH), Nilaparvata lugens, and to determine its spatio-temporal expression profiles and its expression pattern induced by entomopathogenic fungi. 【Methods】 Based on the transcriptome and whole genome data of BPH, the full-length cDNA of Nlserpin4 from BPH was cloned by PCR, and its nucleotide and protein sequences were subsequently characterized using bioinformatics tools. The expression patterns of Nlserpin4 across different developmental stages (egg, 1st-5th instar nymphs and newly emerged female and male adults), in different tissues (fat body, gut, hemolymph and carcass) of the 5th instar nymphs, and in the 5th instar nymphs at different time post injection of the entomopathogenic fungus Metarhizium anisopliae were determined by qRT-PCR. 【Results】 The full-length cDNA of Nlserpin4 (GenBank accession no.: MN822802) was successfully cloned from BPH. The open reading frame (ORF) of Nlserpin4 is 1 227 bp in length, encoding 408 amino acids with the predicted molecular weight of 45.91 kD and isoelectric point (pI) of 6.23. The amino acid sequence analysis revealed that Nlserpin4 protein has no putative N-glycosylation site, but contains a predicted signal peptide consisting of 23 amino acid residues at the N-terminal region and a typical RCL region of the serpin family at the C-terminal region with an active cleavage site which can be recognized by target protease. The phylogenetic analysis showed that Nlserpin4 is closely related to the serpins of other hemipteran insects, with the highest homology with Sipha flava serpin4. The qRT-PCR results showed that the expression of Nlsperpin4 had obvious temporospatial characteristics. The expression level of Nlsperpin4 in adults was significantly higher than those in other developmental stages, and the highest expression level was observed in male adult. Nlsperpin4 was expressed in the fat body, gut, haemolymph and carcass, with the highest expression level in the carcass of the 5th instar nymphs. The expression of Nlserpin4 in BPH was significantly down-regulated after infection with M. anisopliae within 48 h post injection, but the expression level of Nlserpin4 was gradually increased with the increase of infection time. 【Conclusion】 The Nlserpin4 of BPH is differentially expressed in different developmental stages, different tissues and different time after infection with the entomopathogenic fungus M. anisopliae. The results of this study provide a theoretical basis for further studying the functions of Nlserpin4 in the growth, development and immune regulation of BPH.

Key words: Nilaparvata lugensMetarhizium anisopliae, serine protease inhibitor, gene cloning, spatio-temporal expression, inducible expression