中国农业科学

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褐飞虱丝氨酸蛋白酶抑制剂基因Nlserpin2的克隆及其功能分析

邬伟,徐慧丽,王正亮,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
  • 发布日期:2022-01-26

Cloning and function analysis of a serine protease inhibitor gene Nlserpin2 in the Nilaparvata lugens #br#

WU Wei, XU HuiLi, WANG ZhengLiang, YU XiaoPing   

  1. Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018
  • Online:2022-01-26

摘要: 【目的】克隆鉴定褐飞虱(Nilaparvata lugens)丝氨酸蛋白酶抑制剂基因Nlserpin2,探明其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Nlserpin2全长cDNA序列;利用生物信息学手段分析其核酸和蛋白质序列特征;通过实时荧光定量PCR(qRT-PCR)技术检测其时空表达规律和病原真菌诱导表达模式;利用RNAi技术探明Nlserpin2基因沉默对褐飞虱存活率及抵御金龟子绿僵菌(Metarhizium anisopliae)侵染能力的影响。【结果】Nlserpin2 cDNA序列(GenBank登录号: KC355239)全长1 209 bp,编码402个氨基酸,含有serpin蛋白超家族所具有的典型serpin结构域和RCL区,且N端包含一段由27个氨基酸残基组成的信号肽。系统进化树分析表明,Nlserpin2与半翅目其他昆虫的serpin聚为一支,其中与白背飞虱(Sogatella furciferaserpin亲缘关系最近。qRT-PCR分析表明,Nlserpin2表达具有明显的时空特异性,其在褐飞虱雄成虫中表达量最高,3龄若虫期表达量最低,肠道中表达量显著高于脂肪体和卵巢组织。Nlserpin2的表达量在金龟子绿僵菌诱导2 d和3 d后显著上调。RNAi分析结果显示,显微注射dsNlserpin2可显著抑制Nlserpin2的表达水平。Nlserpin2干扰后褐飞虱5龄若虫的存活率以及对金龟子绿僵菌侵染的抵御能力均显著降低。【结论】Nlserpin2在褐飞虱生长发育和防御病原真菌侵染过程中发挥重要作用,可作为应用RNAi技术防控褐飞虱的潜在靶标和褐飞虱高毒力病原真菌遗传改良的资源基因。


关键词: 褐飞虱, 丝氨酸蛋白酶抑制剂, 时空表达, 诱导表达, RNA干扰

Abstract: ObjectiveThe aim of this study is to clone a serine protease inhibitor gene Nlserpin2 and clarify its expression patterns and biological functions in the brown planthopper (BPH), Nilaparvata lugens.MethodBased on the transcriptome data of BPH, the full-length cDNA of Nlserpin2 was cloned by PCR, and its nucleotide and protein sequences were subsequently characterized using bioinformatics tools. The expression patterns of Nlserpin2 in different developmental stages, different tissues and in response to an entomopathogenic fungus Metarhizium anisopliae were determined by qRT-PCR. The target gene Nlserpin2 was further silenced by RNAi, and the surviaval rate and the changes in the resistance to the M.anisopliae infection of BPH were determined by bioassay.ResultThe Nlserpin2 (GenBank accession number: KC355239) was successfully cloned from BPH. The open reading frame (ORF) is 1 209 bp in length, encoding 402 amino acids with a conserved serpin domain and a reactive center loop (RCL) that typically existed in the members of the serpin superfamily. A signal peptide consisting of 27 amino acid residues was also predicted at the N-terminus. The phylogenetic analysis showed that Nlserpin2 is clustered together with other hemipteran Nlserpin2, and has the highest homology with Sogatella furcifera serpin. The qRT-PCR results showed that the expression of Nlserpin2 had obvious temporospatial characteristics. The lowest and highest expression levels were observed in the 3rd nymphs and the male adults, respectively. The transcript level of Nlserpin2 in the gut was significantly higher than that in the fat body and ovary. The expression of Nlserpin2 in BPH was significantly upregulated at 2 and 3 days post infection with M. anisopliae. RNAi results showed that the expression levels of Nlserpin2 could be significantly inhibited by microinjection of dsNlserpin2. Inhibition of Nlserpin2 expression caused significantly decreased in the survival rate and the capability to resist M. anisopliae infection of the 5th instar nymphs of BPH. ConclusionNlserpin2 plays important roles in the growth, development and pathogen defense of BPH, which can be used as a potential target for RNAi-mediated control of BPH and provided the gene of interest for genetic improvement of entomopathogenic fungi with a hypervirulent to BPH.


Key words: Nilaparvata , lugens, serine protease inhibitor,  , spatio-temporal expression, inducible , expression, RNA , interference