Molecular Cloning and Functional Analysis of NlTgo in the Rice Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

doi:10.16819/j.1001-7216.2016.6032

Abstract

Abstract:

Transcriptional regulator Tango (Tgo) plays crucial roles in insect development, including neurogenesis, hematopoiesis, sex determination and gut development. In this study, a full-length cDNA of Tango was cloned in Nilaparvata lugens. qRT-PCR and RNA interference (RNAi) were further used to analyze the expression pattern and function role, respectively. Our results showed that NlTgo contained a 2007-bp open reading frame (ORF), encoding 669 amino acid residues. Sequence alignment showed that NlTgo shared an identity of 68% with Pediculus humanus. Phylogenetic analysis suggested that NlTgo was closely related to the Tango proteins from Acyrthosiphon pisum, Pyrrhocoris apterus and Cimex lectularius. Expression profile revealed that NlTgo expression was higher in the first- and second-instar larvae than that in eggs. Furthermore, the expression level of NlTgo was higher in ovary than that in integument. Knocking down of NlTgo, in the 4th-instar nymph, was performed by double-stranded RNA (dsRNA) targeting NlTgo. It was found that the expression level of NlTgo, 4 days after injection, was significantly decreased by 77%, compared with control. Furthermore, nymphs died due to the abnormal molting, and the survival rate was only 23%, significantly lower than control group (98%). The results suggest that NlTgo is involved in the development of N.lugens and can serve as a potential target for controlling the brown planthopper.

Key words: Nilaparvata lugens, Tango, RNAi, molting, pest control

摘要：

转录因子Tango(Tgo)在昆虫神经元发生、血细胞生成、性别决定、肠道发育等过程中发挥重要作用。克隆了褐飞虱Tango基因(NlTgo),应用荧光定量PCR和RNAi探索了NlTgo在褐飞虱中的表达动态和生物学功能。结果表明,NlTgo的开放阅读框为2007 bp,推测编码669个氨基酸残基。多序列比对表明NlTgo与已知的Tgo高度同源,其中与人体虱(Pediculus humanus corporis) Tgo的一致性达68%。系统进化分析表明,NlTgo与豌豆长管蚜(Acyrthosiphon pisum)、始红蝽(Pyrrhocoris apterus)和臭虫(Cimex lectularius)的Tgo在系统发育树中形成一个类群。时空表达谱表明NlTgo在褐飞虱1龄、2龄时期高表达,在卵期表达量较低;在卵巢中表达量最高,在体壁的表达量较低。RNAi结果表明,注射dsNlTgo 后4 d,NlTgo 的表达量较空白对照组显著降低了77%,并导致若虫不能正常蜕皮而死亡。其中,处理5 d后褐飞虱的存活率仅为23%,显著低于注射dsGFP的对照组(98%)。结果显示,NlTgo与褐飞虱的生长发育有关,可作为防治褐飞虱的潜在靶标。

关键词: 褐飞虱, Tango, RNA干涉, 蜕皮, 害虫防治

CLC Number:

Long-fei CHEN, Pin-jun WAN, Wei-xia WANG, Qiang FU, Ting-heng ZHU. Molecular Cloning and Functional Analysis of NlTgo in the Rice Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)[J]. Chinese Journal OF Rice Science, 2016, 30(6): 653-660.

陈龙飞, 万品俊, 王渭霞, 傅强, 朱廷恒. 褐飞虱NlTgo基因的克隆及功能研究[J]. 中国水稻科学, 2016, 30(6): 653-660.

Figures/Tables 7

Table 1 Primers used for RT-PCR, qPCR and dsRNA synthesis.

引物名称 Primer name	正向引物(5'-3') Forward sequence(5'-3')	反向引物(5'-3') Reverse sequence(5'-3')
基因克隆 Gene cloning
NlTgo	GGCTGTTGCGCATATGAAGG	ATGTCGGACAACTCCTGCTG
双链RNA合成 dsRNA synthesis
dsNlTgo	T7-ACCGACGAGGTCGAGTACAT	T7-GTGTGTAGGTGGGTGACCTG
dsGFP	T7-AGATTTGTATAGTTCATCCATGCCATGT	T7-AGAATGAGTAAAGGAGAAGAACTTTTCA
定量PCR qPCR
qNlTgo	GGAGGAGGATGGTTCACACT	GGTCCACAAACGTGAACTTG
qNl18s	CGCTACTACCGATTGAA	GGAAACCTTGTTACGACTT

Fig. 1. Alignment of amino acid residues of Tgo in Nilaparvata lugens and other insects. Insect species and GenBank accession numbers of Tango: Ph,Pediculus humanus corporis (XP_002430960.1); Cl, Cimex lectularius (XP_014259945.1); Cm, Callosobruchus maculatus (AFL70632.1); Ld,Leptinotarsa decemlineata (AKG92750.1); Pa,Pyrrhocoris apterus (AGI17574.1); Tc, Tribolium castaneum (TC004710); Ac, Acyrthosiphon pisum (XP_008180102.1).

Fig. 2. Phylogenetic relationship of Nilaparvata lugens Tgo and insect homologues. Insect species and GenBank accession numbers of Tango: Ph, Pediculus humanus corporis (XP_002430960.1); Cl, Cimex lectularius (XP_014259945.1); Cm, Callosobruchus maculatus(AFL70632.1); Ld, Leptinotarsa decemlineata (AKG92750.1); Pa, Pyrrhocoris apterus (AGI17574.1); Tc, Tribolium castaneum (TC004710); Ac, Acyrthosiphon pisum (XP_008180102.1); Dm, Drosophila melanogaster (FBgn0264075); Nv, Nasonia vitripennis (Nasvi2EG004242); Bm, Bombyx mori (BGIBMGA003472); Hs,Harpegnathos saltator (HSAL22140); Ag, Anopheles gambiae (AGAP009748); Am, Apis mellifera (GB44259); Aa, Aedes aegypti (AAEL010343).

Fig. 3. Relative expression level of NlTgo in different development stages of Nilaparvata lugens. Different lowercase letters indicate significant difference at the 0.05 level between different groups. n=3

Fig. 4. Relative expression level of NlTgo in different tissues of Nilaparvata lugens. Different lowercase letters indicate significant difference at the 0.05 level between different groups.n=3.

Fig. 5. Relative expression level of NlTgo at 4th day injection with dsRNA(A) and survival rate of Nilaparvata lugens after injection dsRNA of NlTgo(B). n=3. Different lowercase letters indicate significant difference at the 0.05 level between different groups.

Fig. 6. Phenotypic types of Nilaparvata lugens nymph after injection with dsNlTgo. Red arrow shows the edges of the split notum.

References 29

[1]	Atchley W R, Fitch W M.A natural classification of the basic helix-loop-helix class of transcription factors.Proc Natl Acad Sci USA, 1997, 94(10): 5172-5176.
[2]	Massari M E, Murre C.Helix-loop-helix proteins: Regulators of transcription in eucaryotic organisms.Mol Cell Biol, 2000, 20(2): 429-440.
[3]	Kadesch T.Consequences of heteromeric interactions among helix-loop-helix proteins.Cell Growth Differ, 1993, 4(1): 49-55.
[4]	Reyes H, Reisz-Porszasz S, Hankinson O.Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor.Science, 1992, 256(5060): 1193-1195.
[5]	Jan Y N, Jan L Y.HLH proteins, fly neurogenesis, and vertebrate myogenesis.Cell, 1993, 75(5): 827-830.
[6]	Weintraub H.The MyoD family and myogenesis: Redundancy, networks, and thresholds.Cell, 1993, 75(7): 1241-1244.
[7]	Crews S.Control of cell lineage-specific development and transcription by bHLH-PAS proteins.Genes & Dev, 1998, 12(5): 607-620.
[8]	Sonnenfeld M, Ward M, Nystrom G, et al.The Drosophila tango gene encodes a bHLH-PAS protein that is orthologous to mammalian Arnt and controls CNS midline and tracheal development.Development, 1997, 124(22): 4571-4582.
[9]	Ghaffar M B, Pritchard J, Ford-Lloyd B.Brown planthopper (N. lugens Stål) feeding behaviour on rice germplasm as an indicator of resistance. PLoS One, 2011, 6(7): e22137.
[10]	李进波, 万丙良, 夏明元, 等. 抗褐飞虱水稻品种的培育及其抗性表现. 应用昆虫学报, 2011, 48(5): 1348-1353.
	Li J B, Wan B L, Xia M Y, et al.Breeding of the brown planthopper resistant rice varieties.Chin Bull Entomol, 2011, 48(5): 1348-1353. (in Chinese with English abstract)
[11]	Cheng X, Zhu L, He G.Towards understanding of molecular interactions between rice and the brown planthopper.Mol Plant, 2013, 6(3): 621-634.
[12]	Yu R, Xu X, Liang Y, et al.The insect ecdysone receptor is a good potential target for RNAi-based pest control.Int J Biol Sci, 2014, 10(10): 1171-1180.
[13]	Li K L, Wan P J, Wang W X, et al.Ran involved in the development and reproduction is a potential target for RNA-interference-based pest management in Nilaparvata lugens. Plos One, 2015, 10(11): e0142142.
[14]	王彦华, 王鸣华. 近年来我国水稻褐飞虱暴发原因及治理对策. 农药科学与管理, 2007, 25(2): 49-54.
	Wang Y H, Wang M H.Factors affecting the outbreak and management tactics of brownplanthopper in China recent years.Pest Sci & Admin, 2007, 25(2): 49-54. (in Chinese)
[15]	Long S K, Fulkerson E, Breese R, et al.A comparison of midline and tracheal gene regulation during drosophila development.PLoS One, 2014, 9(1): e85518.
[16]	Larkin M A, Blackshields G, Brown N P, et al.Clustal W and Clustal X version 2.0.Bioinformatics, 2007, 23(21): 2947-2948.
[17]	Tamura K, Peterson D, Peterson N, et al.MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.Mol Biol Evol, 2011, 28(10): 2731-2739.
[18]	Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.RNA, 2001, 9(3): 299-308.
[19]	Yuan M, Lu Y, Zhu X, et al.Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens(Hemiptera: Delphacidae) using reverse-transcription quantitative PCR. PLoS One, 2014, 9(1): e86503.
[20]	Wang W X, Li K L, Chen Y, et al. Identification and function analysis of enolase gene NlEno1 from Nilaparvata lugens (Stal) (Hemiptera:Delphacidae). J Insect Sci, 2015, 15(1): 66.
[21]	Li K L, Wan P J, Wang W X, et al.Ran involved in the development and reproduction is a potential target for RNA-interference-based pest management in Nilaparvata lugens. PLoS One, 2015, 10(11): e0142142.
[22]	Tang Q Y, Zhang C X. Data Processing System (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research.Insect Sci, 2013, 20(2): 254-260.
[23]	Peyrefitte S, Kahn D, Haenlin M.New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes.Mech Dev, 2001, 104(1/2): 99-104.
[24]	Probst M R, Fan C M, Tessier-Lavigne M.Two murine homologs of the Drosophila single-minded protein that interact with the mouse aryl hydrocarbon receptor nuclear translocator protein.J Biol Chem, 1997, 272(7): 4451-4457.
[25]	Fire A, Xu S, Montgomery M K, et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391(6669): 806-811.
[26]	Bitra K, Tan A, Dowling A, et al.Functional characterization of PAS and HES family bHLH transcription factors during the metamorphosis of the red flour beetle, Tribolium castaneum. Gene, 2009, 448(1): 74-87.
[27]	Ohshiro T, Saigo K.Transcriptional regulation of breathless FGF receptor gene by binding of TRACHEALESS/dARNT heterodimers to three central midline elements in Drosophila developing trachea.Development, 1997, 124(20): 3975-3986.
[28]	Crews S T, Fan C M.Remembrance of things PAS: regulation of development by bHLH-PAS proteins.Curr Opin Genet Dev, 1999, 9(5): 580-587.
[29]	Choi Y J, Kwon E J, Park J S, et al.Transcriptional regulation of the Drosophila caudal homeobox gene by bHLH-PAS proteins. Biochim Biophys Acta, 2007, 1769(1): 41-48.