烟粉虱溶菌酶基因的鉴定及表达分析

doi:10.3864/j.issn.0578-1752.2016.13.009

摘要/Abstract

摘要： 【目的】鉴定烟粉虱（Bemisia tabaci）体内的溶菌酶基因，并对其序列特征、进化关系和表达模式进行分析，探索该基因在烟粉虱应对球孢白僵菌侵染过程中的作用，为进一步解析烟粉虱先天免疫过程提供理论依据。【方法】以第2代高通量测序的结果为依据，比对非冗余核苷酸数据库，筛选E值＜10^-5的基因为烟粉虱溶菌酶基因（lysozyme gene of Bemisia tabaci，BtLyz），利用ORF Finder预测BtLyz的开放阅读框，并得到氨基酸序列；利用Pfam和SMART预测BtLyz的蛋白质结构域；利用SinglP 4.1 Server预测BtLyz序列的信号肽；利用MEGA6.0软件进行BtLyz的氨基酸多序列比对；利用MEGA6.0软件，对BtLyz与其他昆虫的31个同源蛋白序列进行系统进化分析，并利用邻接法构建系统进化树来进一步分析鉴定该基因；采用实时荧光定量PCR（qRT-PCR）分析球孢白僵菌侵染烟粉虱卵、若虫、成虫后，在12、24、36、48、60 h时目标基因的时空表达模式。【结果】鉴定出烟粉虱4个溶菌酶基因，分别命名为BtLyz1、BtLyz2、BtLyz3和BtLyz4，基因序列全长分别为1 819、1 149、829和928 nt，分别编码159、160、148和160个氨基酸，且均含有信号肽。蛋白质结构分析、氨基酸序列比对和系统进化分析发现，BtLyz1和BtLyz4属于i型溶菌酶，BtLyz2和BtLyz3属于c型溶菌酶。BtLyz-1与豌豆长管蚜ApLyz-i1位于同一进化支上，BtLyz4与柑橘木虱DcLyz-i3和豌豆长管蚜ApLyz-i2位于同一进化支上。BtLyz2和BtLyz3与褐飞虱NlLyz-c1和异色瓢虫HaLyz-c3位于同一进化支上。与对照相比，卵期4个溶菌酶基因和若虫期BtLyz4的相对表达量没有显著变化；若虫期BtLyz1的相对表达量先上调再下调，而后又有所上调的波动趋势，24 h时上调表达最明显，为对照组的4.55倍；成虫期BtLyz1和BtLyz4的相对表达量均为先上调再下调，而后又有所上调的波动趋势，60 h时上调表达最明显，为对照组的11.31和4.21倍；若虫期BtLyz2和BtLyz3的相对表达量均为先上调再下调表达，在诱导24 h时上调表达最明显，为对照组的5.09和8.31倍，而后急剧下调，在60 h时下调表达最明显，为对照组的0.19和0.13倍；成虫期BtLyz2和BtLyz3的相对表达量均为先上调再下调的表达趋势，在24 h时上调最明显，为对照组的5.56和8.84倍。【结论】从烟粉虱体内鉴定了4个溶菌酶基因序列，其中2个为c型溶菌酶序列，2个为i型溶菌酶序列；在球孢白僵菌侵染烟粉虱不同虫态、不同时间后，卵期的相对表达量与对照相比没有显著变化；在若虫和成虫期，不同BtLyzs表现出不同的表达趋势。4个BtLyzs可能参与了烟粉虱对真菌侵染的免疫反应，有可能成为烟粉虱生物防治的潜在新靶标。

关键词: 烟粉虱, 溶菌酶, 生物信息学分析, 实时荧光定量PCR

Abstract: 【Objective】The objective of this study is to provide a basis for understanding the important role of lysozyme genes in Bemisia tabaci infected by the fungus Beauveria bassiana , identify lysozyme genes and analyze their sequence features, evolutionary relationships and expression pattern, and to provide a theoretical foundation for charifying innate immunity in B. tabaci. 【Method】Target genes were screened from the results of the second-generation high-throughput sequencing against the redundancy nucleotide databases when the E value ＜10^-5. The open reading frame and amino acid sequence of BtLyzs were found using the ORF Finder software; the protein domains of BtLyzs were predicted using Pfam and SMART software; the signal peptide sequence of BtLyzs was predicted using SignalP 4.1 Server; the amino acid sequence alignment of BtLyzs was performed using the MEGA6.0 software; BtLyzs were characterized by using phylogenetic analysis with homologous genes of 31 other insects, a neighbor-joining of phylogenetic tree was constructed using the MEGA 6.0 software for further analysis and identification of the genes. The expression pattern of screened genes were determined at egg, nymph and adult stages of B. tabaci which were infected by the fungus Be. bassiana after 12, 24, 36, 48, 60 h using the quantitative real-time PCR (qRT-PCR). 【Result】 Four lysozyme genes were identified and designated as BtLyz1, BtLyz2, BtLyz3 and BtLyz4, which were 1 819, 1 149, 829, 928 nt, respectively. They were predicted to encode proteins of 159, 160, 148, 160 amino acids, respectively. Amino acid sequence alignment, phylogenetic analysis and protein tertiary structure prediction showed that BtLyz1 and BtLyz4 belong to i-type lysozymes, BtLyz2 and BtLyz3 belong to c-type lysozymes. BtLyz-1 formed a clade specific with Acyrthosiphon pisum of ApLyz-i1, BtLyz4 formed a clade specific with Diaphorina citri of DcLyz-i3 and Acyrthosiphon pisum of ApLyz-i2; BtLyz2 and BtLyz3 formed a clade specific with Nilaparvata lugens of NlLyz-c1 and Harmonia axyridis of HaLyz-c3. Compared to the control, the relative expression of all four genes in egg and BtLyz4 in nymph were not induced significantly by fungi-infected; the transcription of BtLyz1 nymph stage underwent the fluctuation of up-regulation, down-regulation, and second up-regulation and peaked at 24 h, it was increased 4.55 folds compared to the control; the transcription of BtLyz1 and BtLyz4 adult stage underwent the fluctuation of up-regulation, down-regulation, and second up-regulation and peaked at 60 h, they were increased 11.31 and 4.21 folds compared to the control. The transcription of BtLyz2 and BtLyz3 nymph stage underwent the fluctuation of up-regulation and down-regulation and peaked at 24 h, they were increased 5.09 and 8.31 folds compared to the control, then down-regulated obviously at 60 h, they were reduced 0.19 and 0.13 folds compared to the control. The transcription of BtLyz2 and BtLyz3 adult stage underwent the fluctuation of up-regulation and down-regulation and peaked at 24 h, they were increased 5.56 and 8.84 folds compared to the control. 【Conclusion】 Four BtLyz genes were identified in B. tabaci. Among them, two are c-type and two are i-type lysozymes. Transcriptional levels of four BtLyzs genes in B. tabaci were induced through different developmental stages and different time points in fungi-treated individuals compared to the control, they were not induced significantly in eggs, and showed different expression trends in nymphs and adults. The four BtLyzs genes probably might participated in the innate immune responses to fungus infection, and could be a new potential target for biocontrol of B. tabaci.

Key words: Bemisia tabaci, lysozymes, bioinformatic analyses, quantitative real-time PCR

于洁，王登杰，雷仲仁，王海鸿. 烟粉虱溶菌酶基因的鉴定及表达分析[J]. 中国农业科学, 2016, 49(13): 2534-2543.

YU Jie, WANG Deng-jie, LEI Zhong-ren, WANG Hai-hong. Identification and Expression Analysis on Lysozyme Gene of Bemisia tabaci[J]. Scientia Agricultura Sinica, 2016, 49(13): 2534-2543.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2016.13.009

https://www.chinaagrisci.com/CN/Y2016/V49/I13/2534

参考文献

[1] Fio?ka M J, Ptaszyńska A A, Czarniawski W. Antibacterial and antifungal lysozyme-type activity in Cameraria ohridella pupae. Journal of invertebrate pathology, 2005, 90(1): 1-9.

[2] Hultmark D, Striner H, Rasmuson T, Boman H G. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. European Journal of Biochemistry, 1980, 106(1): 7-16.

[3] Abraham E G, Nagaraju J, Salunke D M, Gupta H M, Dutta R K. Purification and partial characterization of an induced antibacterial protein in the silkworm, Bombyx mori. Journal of invertebrate pathology, 1995, 65(1): 17-24.

[4] Javar S, Mohamed R, Sajap A S, Lau W H. Expression profiles of lysozyme-and prophenoloxidase-encoding genes in Spodoptera species challenged with entomopathogenic fungus, Metarhizium anisopliae (Metchnikoff) Sorokin using qRT-PCR. Invertebrate Reproduction & Development, 2015, 59(4): 230-236.

[5] Boucias D G, Hung S Y, Mazet I, Azbell J. Effect of the fungal pathogen, Beauveria bassiana, on the lysozyme activity in Spodoptera exigua larvae. Journal of insect physiology, 1994, 40(5): 385-391.

[6] Gillespie J P, Burnett C, Charnley A K. The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium anisopliae var. acridum. Journal of Insect Physiology, 2000, 46(4): 429-437.

[7] Lee-Huang S, Huang P L, Sun Y, Huang P L, Kung H F, Blithe D L, Chen H C. Lysozyme and RNases as anti-HIV components in β-core preparations of human chorionic gonadotropin. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(6): 2678-2681.

[8] Beck G, Habicht G S. Immunity and the invertebrates. Scientific American, 1996, 275(5): 60-66.

[9] Jollès J, Schoentgen F, Croizier G, Croizier L, Jollès P. Insect lysozymes from three species of Lepidoptera: their structural relatedness to the C (chicken) type lysozyme. Journal of molecular evolution, 1979, 14(4): 267-271.

[10] Vogel H, Altincicek B, Glöckner G, Vilcinskas A. A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella. BMC genomics, 2011, 12: 308.

[11] Altincicek B, Vilcinskas A. Metamorphosis and collagen- IV-fragments stimulate innate immune response in the greater wax moth, Galleria mellonella. Developmental & Comparative Immunology, 2006, 30(12): 1108-1118.

[12] Zhang Y, Huang J, Zhou B, Zhang C L, Liu W B, Miao X X, Huang Y P. Up-regulation of lysozyme gene expression during metamorphosis and immune challenge of the cotton bollworm, Helicoverpa armigera. Archives of insect biochemistry and physiology2009, 70(1): 18-29.,

[13] Li B, Calvo E, Marinotti O, James A A, Paskewitz S M. Characterization of the c-type lysozyme gene family in Anopheles gambiae. Gene, 2005, 360(2): 131-139.

[14] Tateishi K, Kasahara Y, Watanabe K, Hosokawa N, Doi H, Nakajima K, Adachi H, Nomoto A. A new cell line from the fat body of Spodoptera litura (Lepidoptera, Noctuidae) and detection of lysozyme activity release upon immune stimulation. In Vitro Cellular & Developmental Biology-Animal, 2015, 51(1): 15-18.

[15] Brown J K, Frohlich D R, Rosell R C. The sweetpotato or silverleaf whiteflies: biotypes of Bemisia tabaci or a species complex? Annual review of entomology, 1995, 40(1): 511-534.

[16] Faria M, Wraight S P. Biological control of Bemisia tabaci with fungi. Crop protection, 2001, 20(9): 767-778.

[17] 王登杰, 吴圣勇, 雷仲仁, 王海鸿. 感染球孢白僵菌的烟粉虱若虫免疫应答转录组分析. 应用昆虫学报, 2015, 52(1): 47-55.

Wang D J, Wu S Y, Lei Z R, Wang H H. Transcriptome analysis of Bemisia tabaci nymphs infected with Beauveria bassiana. Chinese Journal of Applied Entomology, 2015, 52(1): 47-55. (in Chinese)

[18] 王登杰, 臧连生, 张烨, 王海鸿, 雷仲仁. 球孢白僵菌对烟粉虱后代生命表参数的亚致死影响. 中国农业科学, 2014, 47(18): 3588-3595.

Wang D J, Zang L S, Zhang Y, Wang H H, Lei Z R. Sublethal effects of Beauveria bassiana Balsamo on life table parameters of subsequent generations of Bemisia tabaci Gennadius. Scientia Agricultura Sinica, 2014, 47(18): 3588-3595. (in Chinese)

[19] Iketani M, Morishima I. Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori. Insect biochemistry and molecular biology, 1993, 23(8): 913-917.

[20] Bao Y Y, Qu L Y, Zhao D, Chen L B, Jin H Y, Xu L M, Cheng J A, Zhang C X. The genome-and transcriptome-wide analysis of innate immunity in the brown planthopper, Nilaparvata lugens. BMC genomics, 2013, 14: 160.

[21] Elmogy M, Bassal T T M, Yousef H A, Dorrah M A, Mohamed A A, Duvic B. Isolation, characterization, kinetics, and enzymatic and nonenzymatic microbicidal activities of a novel c-type lysozyme from plasma of Schistocerca gregaria (Orthoptera: Acrididae). Journal of Insect Science, 2015, 15(1): 57.

[22] Paskewitz S M, Li B, Kajla M K. Cloning and molecular characterization of two invertebrate-type lysozymes from Anopheles gambiae. Insect molecular biology, 2008, 17(3): 217-225.

[23] 罗晨, 姚远, 王戎疆, 阎凤鸣, 胡敦孝, 张芝利. 利用mtDNA COⅠ基因序列鉴定我国烟粉虱的生物型. 昆虫学报, 2002, 45(6): 759-763.

Luo C, Yao Y, Wang R J, Yan F M, Hu D X, Zhang Z L. The use of mitochondrial cytochrome oxidase Ⅰ (mt COⅠ) gene sequences for the identification of biotypes of Bemisia tabaci (Gennadius) in China. Acta Entomologica Sinica, 2002, 45(6): 759-763. (in Chinese)

[24] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Methods, 2001, 25: 402-408

[25] Vilcinskas A, Mukherjee K, Vogel H. Expansion of the antimicrobial peptide repertoire in the invasive ladybird Harmonia axyridis. Proceedings of the Royal Society of London B: Biological Sciences, 2013, 280(1750): 20122113.

[26] Vogel H, Badapanda C, Knorr E, Vilcinskas A. RNA-sequencing analysis reveals abundant developmental stage-specific and immunity-related genes in the pollen beetle Meligethes aeneus. Insect molecular biology, 2014, 23(1): 98-112.

[27] Irwin D M, Gong Z M. Molecular evolution of vertebrate goose-type lysozyme genes. Journal of molecular evolution, 2003, 56(2): 234-242.