褐飞虱clip丝氨酸蛋白酶基因NlCSP4的克隆及功能分析

doi:10.16409/j.cnki.2095-039x.2021.11.008

摘要/Abstract

摘要： Clip丝氨酸蛋白酶（clip-domain serine protease，CSP）是广泛存在于昆虫体内的一类结构保守、功能多样的蛋白质水解酶类，在昆虫生长、发育和免疫等诸多生理过程中发挥重要功能。本研究克隆并鉴定获得一个褐飞虱CSP编码基因NlCSP4（GenBank登录号：OK018340），其cDNA序列全长1911 bp，编码636个氨基酸，预测蛋白质分子量为69.29 kD，等电点为9.02。NlCSP4蛋白N端包含一段由25个氨基酸残基组成的信号肽和一段由44个氨基酸残基组成的clip结构域，C端具有CSP蛋白家族典型的Tryp_SPc结构域，且其中包含特异性保守的His-Asp-Ser催化中心三元件。系统发育分析表明NlCSP4与其他同属半翅目昆虫的CSP在NJ进化树上聚为一支，而与鳞翅目昆虫CSP亲缘关系较远。qRT-PCR分析显示，NlCSP4基因表达具有明显的时空特异性，其在雌成虫中的表达量最高，且高龄若虫期（4龄和5龄）表达量显著高于卵期和1～3龄若虫期；NlCSP4基因在褐飞虱雌成虫脂肪体、肠道和卵巢中均有表达，且在脂肪体中表达量最高；金黄色葡萄球菌（革兰氏阳性细菌）和金龟子绿僵菌（丝状病原真菌）诱导72 h内NlCSP4表达量均显著上调，但大肠杆菌（革兰氏阴性细菌）短时间（36 h内）诱导效果不明显。显微注射NlCSP4基因dsRNA可显著抑制褐飞虱5龄若虫NlCSP4的表达水平。生物测定试验表明，NlCSP4干扰后褐飞虱5龄若虫存活率显著下降，且对金龟子绿僵菌侵染的抵御能力显著降低。本研究结果初步证实了褐飞虱NlCSP4在宿主生长发育和免疫防御过程中发挥重要调控作用，可作为开发RNAi介导的褐飞虱防控技术中的一个重要潜在靶标。

关键词: 褐飞虱, Clip丝氨酸蛋白酶, 基因克隆, 基因表达, RNA干扰

Abstract: Clip-domain serine protease (CSP),a protein hydrolase with conserved structure and diverse functions,widely exists in different groups of insects and plays important physiological roles in insect growth,development and immunity.In this study,a CSP-encoding gene NlCSP4(GenBank accession No.OK018340) was successfully cloned and identified in the brown planthopper (BPH),Nilaparvata lugens.The full-length cDNA of NlCSP4 is 1911 bp,encoding 636 amino acids with the predicted molecular weight of 69.29 kD and isoelectric point of 9.02.NlCSP4 protein contains a predicted signal peptide consisting of 25 amino acid residues and a clip domain comprising 44 amino acid residues at the N-terminal region,and a typical Tryp_SPc domain at the C-terminus with three specific conserved catalytic sites,namely His,Asp and Ser.The phylogenetic analysis showed that NlCSP4 clustered together with other hemipteran CSPs but were quite distinct from those of lepidopterans in the neighbor-joining tree.The qRT-PCR results showed that the expression of NlCSP4 had obvious temporospatial characteristics.The transcript level of NlCSP4 in old nymphs (4^th and 5^thinstars) was significantly higher than those in the eggs and young nymphs (1^st,2^nd and 3^rd instars),and the highest expression level was observed in the female adults.NlCSP4 was expressed in the fat body,gut and ovary,with the highest expression level in the fat body of the female adults.The expression of NlCSP4 in BPH was significantly up-regulated after infection with the Gram-positive bacteria Staphylococcus aureus and the entomopathogenic fungus Metarhizium anisopliae within 72 h post injection.However,no obvious induction of NlCSP4 expression was observed when the BPH was challenged by the Gram-negative bacteria Escherichia coli in a short time (within 36 h post injection).Microinjection of NlCSP4 dsRNA could significantly inhibit the expression level of NlCSP4 in the 5^th-instar nymphs of BPH.The results of bioassay experiments showed that the survival rate and the ability to resist M.anisopliae infection in the 5^th-instar nymphs of BPH were both significantly decreased by RNAi-mediated knockdown of NlCSP4.Our preliminary results confirm that NlCSP4 plays key roles in host growth,development and immune defense,and can be used as an important potential target for RNAi-mediated control of BPH.

Key words: Nilaparvata lugens, clip-domain serine proteinase, gene cloning, gene expression, RNA interference

中图分类号:

Q966

邬伟, 程依情, 王正亮, 俞晓平. 褐飞虱clip丝氨酸蛋白酶基因NlCSP4的克隆及功能分析[J]. 中国生物防治学报, 2022, 38(5): 1202-1212.

WU Wei, CHENG Yiqing, WANG Zhengliang, YU Xiaoping. Cloning and Function Analysis of a Clip-domain Serine Protease Gene NlSCP4 in the Brown Planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)[J]. Chinese Journal of Biological Control, 2022, 38(5): 1202-1212.

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