褐飞虱NlTgo基因的克隆及功能研究

doi:10.16819/j.1001-7216.2016.6032

摘要/Abstract

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

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

中图分类号:

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.

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.

图/表 7

表1 RT-PCR、qPCR和dsRNA合成引物

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

图1 褐飞虱与其他物种Tgo氨基酸序列比对昆虫名称及Tango的GenBank登录号:Ph-人体虱(XP_002430960.1); Cl-温带臭虫(XP_014259945.1); Cm-四纹豆象(AFL70632.1); Ld-马铃薯甲虫(AKG92750.1); Pa-始红蝽(AGI17574.1); Tc-赤拟谷盗(XP_008190739.1); Ac-豌豆长管蚜(XP_008180102.1)。

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).

图2 褐飞虱与其他昆虫的Tango的进化树昆虫名称及Tango的GenBank登录号:Ph-人体虱(XP_002430960.1); Cl-温带臭虫(XP_014259945.1); Cm-四纹豆象(AFL70632.1); Ld-马铃薯甲虫(AKG92750.1); Pa-始红蝽(AGI17574.1); Tc-赤拟谷盗(XP_008190739.1); Ac-豌豆长管蚜(XP_008180102.1); Dm-果蝇(FBgn0264075); Nv-蝇蛹金小蜂(Nasvi2EG004242); Bm-家蚕(BGIBMGA003472); Hs-印度跳蚁(HSAL22140); Ag-冈比亚按蚊(AGAP009748); Am-西方蜜蜂(GB44259); Aa-埃及伊蚊(AAEL010343)。

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).

图3 褐飞虱不同发育阶段NlTgo的相对表达量样本量n=3。不同小写字母代表不同组之间存在显著差异(P<0.05)。

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

图4 褐飞虱不同组织间NlTgo的相对表达量样本量n=3。不同小写字母代表不同组之间存在显著差异(P<0.05)。

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.

图5 注射dsRNA后第4天NlTgo相对表达量的变化(A)和干扰NlTgo后对褐飞虱存活率的影响(B) 样本量(n=3)。不同小写字母代表不同组之间存在显著差异(P<0.05)。

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.

图6 干扰NlTgo后褐飞虱表型变化红色箭头表示背板裂开的边缘。

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

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