Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (12): 2338-2346.doi: 10.3864/j.issn.0578-1752.2022.12.006

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning and Function Analysis of a Serine Protease Inhibitor Gene Nlserpin2 in Nilaparvata lugens

WU Wei(),XU HuiLi,WANG ZhengLiang(),YU XiaoPing()   

  1. College of Life Sciences, China Jiliang University/Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, Hangzhou 310018
  • Received:2021-12-16 Accepted:2022-01-21 Online:2022-06-16 Published:2022-06-23
  • Contact: ZhengLiang WANG,XiaoPing YU E-mail:906287591@qq.com;zhengliang.w0234@163.com;yuxiaoping19630306@163.com

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Abstract:

【Objective】The objective 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.【Method】Based on the transcriptome data of N. lugens, 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 across different developmental stages (egg, 1st-5th instar nymphs, female and male adults), in different tissues (fat body, gut and ovary) of the newly emerged female adults, and in the 5th instar nymphs at different times post infection of an entomopathogenic fungus Metarhizium anisopliae were determined by qRT-PCR. The effects of Nlserpin2 knockdown on the survival rate and the resistance to M. anisopliae infection of the N. lugens nymphs at 5th instar were evaluated based on RNAi technique.【Result】The Nlserpin2 (GenBank accession number: KC355239) was successfully cloned from N. lugens. 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 serpin, and has the highest homology with Sogatella furcifera serpin. The qRT-PCR results showed that the expression of Nlserpin2 had obvious spatio-temporal characteristics. The expression level of Nlsperpin2 in adults was significant higher than that in other developmental stages, and the highest expression level was observed in male adult. Significant higher expression level was detected in the high-instar nymphs (4th-5th instar) when compared to that in the eggs and the low-instar nymphs (1st-3rd instar), and the lowest expression level was observed in the 3rd instar nymph. Nlserpin2 was expressed in the gut, fat body and ovary of the female adults, and its expression level in the gut was significantly higher than that in the fat body and ovary. The expression of Nlserpin2 was significantly upregulated at 2 and 3 days post infection with M. anisopliae, but gradually stabilized with the increase of infection time. RNAi results showed that the expression level of Nlserpin2 could be significantly inhibited by microinjection of dsNlserpin2. Inhibition of Nlserpin2 expression caused significant decrease in the survival rate and the capability to resist M. anisopliae infection of the 5th instar nymphs of N. lugens. Compared with the control group, the corrected survival rates of Nlserpin2-interfered N. lugens nymphs were significantly decreased by 28.4% and 31.0% at 5 and 8 days post infection with M. anisopliae, respectively.【Conclusion】Nlserpin2 plays important roles in the pathogen defense of N. lugens, which can be used as a potential target for RNAi-mediated control of N. lugens and provides the gene of interest for genetic improvement of entomopathogenic fungi with a hypervirulent to N. lugens.

Key words: Nilaparvata lugens, serine protease inhibitor (serpin), spatio-temporal expression, inducible expression, RNA interference (RNAi), Metarhizium anisopliae

Table 1

Information of primer sequences used in this study"

引物名称 Primer name 序列 Sequence (5′-3′) 用途 Purpose
Nlserpin2-F ATGAGCTCTGCATTTGTTACA cDNA克隆
cDNA cloning
Nlserpin2-R TCAAGGTTCCATTAGTCTTCCA
qNlserpin2-F TCAAAGACGCCTACAGGCAA 实时荧光定量PCR分析
qRT-PCR analysis
qNlserpin2-R AACGGATACAGCCAATCCGA
qNl18S-F GTAACCCGCTGAACCTCC
qNl18S-R GTCCGAAGACCTCACTAAATCA
dsNlserpin2-F GGATCCTAATACGACTCACTATAGGGGGTTGGACTCGTTTCAC dsRNA合成
dsRNA synthesis
dsNlserpin2-R GGATCCTAATACGACTCACTATAGGTCAACGCTACCTGATGGA
dsGFP-F GGATCCTAATACGACTCACTATAGGGATACGTGCAGGAGAGGAC
dsGFP-R GGATCCTAATACGACTCACTATAGGGCAGATTGTGTGGACAGG

Fig. 1

cDNA sequence and the coded amino acid sequence of Nlserpin2 Signal peptide sequence is marked with black box; Reactive central loop is underlined; The glycosylation sites are shaded; Asterisk indicates termination codon"

Fig. 2

Phylogenetic tree of serpins in N. lugens and other insects by neighbor-joining method (1000 replicates)"

Fig. 3

Relative expression levels of Nlserpin2 at different developmental stages (A), different tissues of female adult (B) and different time points after M. anisopliae infection (C) in N. lugens"

Fig. 4

Relative expression levels of Nlserpin2 in the 5th instar nymphs of N. lugens after RNAi The asterisk above the bars indicates a significant difference (**P<0.01, t-test)"

Fig. 5

The corrected survival rates of the 5th instar nymphs of N. lugens after dsRNA injection and M. anisopliae infection The asterisk indicates a significant difference (*P<0.05, **P<0.01, t-test)"

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